Acclimation behaviour and bio-chemical changes during anemonefish (Amphiprion sebae) and sea anemone (Stichodactyla haddoni) symbiosis J. Balamurugan, T. T. Ajith Kumar, R. Kannan & H. D. Pradeep
Symbiosis ISSN 0334-5114 Volume 64 Number 3 Symbiosis (2014) 64:127-138 DOI 10.1007/s13199-014-0310-2
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Author's personal copy Symbiosis (2014) 64:127–138 DOI 10.1007/s13199-014-0310-2
Acclimation behaviour and bio-chemical changes during anemonefish (Amphiprion sebae) and sea anemone (Stichodactyla haddoni) symbiosis J. Balamurugan & T. T. Ajith Kumar & R. Kannan & H. D. Pradeep
Received: 7 May 2014 / Accepted: 22 December 2014 / Published online: 7 January 2015 # Springer Science+Business Media Dordrecht 2015
Abstract Anemonefishes are known to exhibit an obligate symbiotic relationship with a limited number of sea anemone species. This has raised queries about the adaptive mechanisms of these fishes involved. The present study was carried out to understand the role of visual and chemical cues used for host recognition and the bio-chemical changes that occur during acclimation by the fishes. The experiments used the fishes Amphiprion sebae (a common anemone associate), Terapon jarbua (a control fish that does not associate with sea anemone) and Stichodactyla haddoni (an anemone commonly hosting anemonefishes). The results suggested that fish settlers use visual, tactile and chemical cues to select and distinguish their hosts. Furthermore, the acclimation times of A. sebae with the anemone host decreased exponentially during repeated trials. This fish apparently secreted a protective mucus. The epidermal mucus of A. sebae possessed unique glyco-proteins compared with T. jarbua. Chemical analyses showed that A. sebae and S. haddoni produced similar chemical substances, but T. jarbua produced a different kind of glycoprotein. This study helps to explain how anemonefishes are able to live with their host anemone, whereas other fishes are not. Electronic supplementary material The online version of this article (doi:10.1007/s13199-014-0310-2) contains supplementary material, which is available to authorized users. J. Balamurugan (*) : T. T. A. Kumar Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai 608 502, Tamilnadu, India e-mail:
[email protected] R. Kannan Department of Botany, N. G. M. College, Pollachi 642 001, Tamilnadu, India H. D. Pradeep Fishery Survey of India, Port Blair, India
Keywords Host imprinting . Habitat selection . Anemonefish . Sea anemone . Jarbua terapon . Epidermal mucous . Acclimation
1 Introduction The symbiosis between sea anemones and anemonefishes are highly specific in that the latter require a suitable habitat for settlement (Fautin and Allen 1997). Anemonefishes are able to live among the stinging tentacles without being harmed. Early studies on this phenomenon found that fishes acquire protection from being stung through a behavioural process called acclimation (Davenport and Norris 1958; Schlichter 1976; Mariscal 1971). However, a few anemonefish species do not require this acclimation process to gain protection from being stung by their hosts (Miyagawa and Hidaka 1980: Miyagawa 1989; Elliott et al. 1994, 1995). Anemonefishes can swim through the nematocyst-studded actinian tentacles with impunity, but fishes of other species are stung, captured and eaten (Mariscal 1966). Anemonefishes are host selective during settlement of the planktonic larvae of the anemone and they are usually not stung during initial contact (Elliott et al. 1995), but they avoid some species of non-symbiotic anemones, as they are not protected from being stung. Studies have attempted to elucidate the mechanisms and the chemistry underlying the protection of the anemonefish from stinging by the nematocysts of the host sea anemone. The consensus is that fish have a protective mucous coat that acts as chemical camouflage or macromolecular mimicry preventing not-self recognition by the sea anemone and subsequent nematocyst discharge (Davenport and Norris 1958; Mariscal 1969, 1970a,b; Schlichter 1975, 1976; Lubbock 1980, 1981; Miyagawa 1989; Mebs 1994; Elliott
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et al. 1994; Elliott and Mariscal 1996). Anemonefishes generally have a comprehensive protection from all symbiotic anemones (Miyagawa 1989; Fautin 1991). For example, Balamurugan et al. (2013) reared six different anemonefishes with S. haddoni including some that were not the natural species that inhabited this host. Fishelson (1972) revealed that the skin of animals acted as a mediator between the interior of the organism and its environment. Researchers have focused attention on the problems of host recognition by newly settled juveniles (Fricke 1974; Miyagawa 1989; Elliott et al. 1995; Arvedlund and Nielsen 1996). The changes to the fish surface mucous coat occur during acclimation, have not been determined (Brooks and Mariscal 1984). Therefore, the present study set out to examine epidermal changes of an anemonefish during settlement, and to compare these to that of a non-symbiotic fish. We also explored, the role of visual, tactile and chemical cues in host recognition imprinting by the anemonefish.
2 Methods 2.1 Experimental design Sebae clownfish, Amphiprion sebae (sub-adult, n=15; TL 5.8 ±0.3 cm and adult, n=15; TL 3.9±0.2 cm) and Haddon’s anemone, Stichodactyla haddoni (n=10; oral disk diameter 7.6±0.3 cm) were procured from the traders at Chennai, Tamil Nadu, India. It was confirmed that the organisms were collected from Gulf of Mannar region, Southeast coast of India. Individuals of Jarbua Terapon fish, Terapon jarbua (n=20; TL 3.6±0.3 cm) were collected from the Vellar estuary, opposite to the Centre of Advanced Study in Marine Biology. All collected animals were quarantined separately in three different cement tanks (same species per tank) for 15 days prior to the experiment using filtered seawater at the Marine Ornamental Fish Hatchery, Centre of Advanced Study in Marine Biology, Annamalai University. Water quality and environmental parameters such as temperature (29–31 ° C), salinity (28–30 psu), pH (7.7–7.8), dissolved oxygen (5.2–5.4 ppm), light intensity (800– 1200 lux) and photoperiod (13 h L: 11 h D) were maintained at constant levels. Animals were fed with minced boiled mussels and shrimps (to avoid microbial contaminations), twice in a day and unutilized feeds were removed after an hour. All tanks were maintained in a closed culture system with the supply of regular aeration (with aerators) and regular water exchange. After acclimatization, the animals were transferred to the experimental glass tanks (3 ft×2 ft), holding 130 l of seawater for behavioural studies.
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2.2 Behavioural studies on visual and chemical cues S. haddoni individuals showing five different colours (brownish white (BrW), greenish white (GrW), green (Gr), greenish brown (GrBr) and whitish/bleached (W)) were selected and transferred to an experiment tank. The anemone’s colours were mapped and individuals were placed adjacent to each other at a distance of 5 cm (Fig. 1) in a single tank. After placing the anemones in the tank (allowing them to settle), individual sub-adults (n=15) and adults of A. sebae (n=15) were allowed to associate with the sea anemones (n=5). Each fishes were introduced separately into the tank without keeping other fishes inside and five trials per individual fish were made. The probability of host selection was calculated after complete acclimation of each fish with the respective host colour. Anemone positions were shuffled randomly to examine the specificity of substrate selection and association of the fishes (Fig. 2). In total, 150 trials were made to find out the most probable selection and imprinting of this anemonefish towards this host anemone. A further twenty-five trials were made in the presence of artificial sea anemones (greenish brown colour), consisting of moulded models made of silicone rubber. Twenty-five trials were also performed in the presence of both natural and artificial anemones to find out the association of fish using the tactile cues (Fig. 3). Probability was calculated (fi = ni / n) based on host selection by the fish where fi = probability of frequencies, ni = number of observed individuals, and n = total number of attempts. The time taken by A. sebae to associate with the respective sea anemones was noted in minutes for each trial, using a stopwatch. 2.3 Data analysis of Behavioural Experiments The data obtained from those studies on association of fish with differently coloured anemones were subjected to statistical analysis. All the statistical tests adapted were performed using the standard statistical tool, SPSS v16.0 (Norusis 2009). In order to understand the impact of variation in the colour of host anemone during fish association, analysis of variance (ONEWAY ANOVA) was conducted. The dependent
Fig. 1 Schematic diagram of experimental setup to test the visual cues of anemonefish in host recognition by random placing of anemones adjacent to each other with 5 cm distance and fish in 30 cm away from host
Author's personal copy Acclimation behaviour and bio-chemical changes during anemonefish
Fig. 2 Host recognition by anemonefish based on visual cues, positions of anemones were shuffled randomly in a distance of 15 cm each other and F-fish to ensure the selection
variables included the number of associations of fish with respect to differently coloured host anemones (independent variables). In the ANOVA, the fish group’s sub-adult and adult were not considered as they were redundant. Further, the Chisquare test was performed to test the frequency distribution of host colours during fish association, where the number of association was considered as the dependable variable and the colour of anemone as independent variable. The observed and expected values of frequency distribution (in percentage) were illustrated in rows and columns using the software SPSS. The Wilcoxon signed rank test was used to ranking the most preferable host colours (two non-parametric tests) by fish group (sub-adult and adult). In addition, the multiple regression analysis (Enter method) was used to find out the correlation between the number of association (dependent variables) and the time of association (dependent variables) with the host anemone colours (set values). 2.4 Histology of skin Hatchery bred A. sebae (TL 3.6±0.3 cm) were obtained from the Marine Ornamental Fish Hatchery, Centre of Advanced study in Marine Biology, Annamalai University. The animals were not allowed to associate with any host anemones before the experiment. A. sebae (n=3) and T. jarbua (n=3) were exposed up to 20 min to the sea anemone S. haddoni in a
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small tank (1 ft×1 ft) with low water level just above the tentacles to prevent the fish escaping from anemone into the water column. After 20 min exposure, the fishes were collected and sacrificed after anesthetization (using clove oil). Their skins were excised using scissors and forceps and then stored in 5 % formalin for 48 h before processing for histology. The samples were marked as S1 (skin of A. sebae prior to exposure to sea anemone), S2 (skin of A. sebae after exposure to anemone), S3 (T. jarbua before exposing to anemone) and S4 (T. jarbua after exposure to anemone). Skins were fixed in Bouin’s solution, routinely embedded in paraffin and skin section was done using a microtome (WESWOX-OPTIK MT10909A) with a thickness of 5 μm. Sections were stained with hematoxylin and eosin (HE), followed by microscopic observation (40×). Histological methods employed were those used for fishes by Hibiya (1982). 2.5 Epidermal mucous collection The epidermal mucous was collected from A. sebae (n=3), T. jarbua (n=3) and S. haddoni (n=3) using the methodology of Subramanian et al. (2008). Specimens were marked as L1 (A. sebae associated with anemone), L2 (A. sebae without anemone association), L3 (T. jarbua) and L4 (S. haddoni). Each fish was transferred into a beaker containing 10 ml of 100 mM NaCl. The fishes were moved back and forth to slough off the epidermal mucous. The fishes were then replaced into their tanks. The collected mucous from each sample was lyophilized and dissolved in 2 ml of Ringers buffer (10 mM HEPES, 112 mM NaCl, 3.4 mM KCl, 2.4 mM NaHCO3 and pH 7.4) and stored in −70 ° C prior to analyses. 2.6 Protein estimation Mucous protein contents were analysed using the Biuret test (Raymont et al. 1964). In summary, 100 μl of supernatant from each mucous sample was mixed with Biuret reagent and allowed to stand for 30 min. Absorbance values were recorded at 590 nm followed by the determination of protein concentration from a standard protein (Bovine Serum Albumin) concentration-absorbance curve. 2.7 Carbohydrate content
Fig. 3 Schematic diagram of experimental setup to test the chemical cues of anemonefish in host recognition between natural and artificial anemones (anemones position were shuffled randomly during experiment), F- fish
The polysaccharides of the mucous content were tested following the Phenol-Sulphuric acid method (Dubios et al. 1956). Briefly, 100 μl of supernatant from each sample was mixed with 1 ml of 5 % phenol and 4 ml of concentrated sulphuric acid. The mixture was incubated for 30 min. Absorbance value was noted at 490 nm followed by the determination of carbohydrate concentration from a standard (Glucose) concentration-absorbance curve.
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Table 1 One-way analysis of variance (ANOVA) on number of association of fish (dependable variables) with different colored host anemone (independent variables). The data were mentioned as Mean±SD Colours of anemone
Mean
SD±
F-Value
P-Value
GrBr Gr BW GrW W
11.67c 8.29b 3.33a 2.50a 0.00
3.38 2.81 1.03 1.00 0.00
20.661
