Records ofthe Western Australian Museum Supplement No. 69: 1-9 (2006). Interspecific differences in the efficiency and photosynthetic characteristics of the symbiosis of JJ solarpowered" Nudibranchia (Mollusca: Gastropoda) with zooxanthellae Ingo Burghardt1 and Heike Wagele2 1 Lehrstuhl fur Spezielle Zoologie, Ruhr-University Bochum, Germany , [email protected] 2Institut fUr Evolutionsbiologie und Okologie, University of Bonn, Germany [email protected] Abstract - In this study the photosynthetic performance and efficiency of the symbiosis of different species of the Cladobranchia (Nudibranchia) with zooxanthellae (Dinophyceae) of the genus Symbiodinium Freudenthal, 1962 is investigated. The species studied include members of the Aeolidoidea (Aeolidiella alba Risbec, 1928, Limenandra nodosa Haefelfinger and Stamm, 1958, Phestilla lugubris (Bergh, 1870) Phyllodesmium briareum (Bergh 1896), Spurilla australis Rudman, 1982), one member of the former Arminoidea (Dermatobranchus sp.) and one of the Dendronotoidea (Melibe cf. bucephala Bergh, 1902). Long term cultivation under starving conditions indicates a symbiotic relationship with zooxanthellae in all investigated species by means of Pulse Amplitude Modulated Fluorescence. The data taken by a Diving­ PAM (submersible Pulse Amplitude Modulated Fluorometer) indicate interspecific differences in the ability to retain photosynthetic active zooxanthellae and suggest different stages in the evolution of this symbiosis represented by different species of Nudibranchia. The efficiency of the symbiotic relationship can be correlated with morphological adaptations, especially the branching system of the digestive gland. Interspecific differences in the photosynthetic characteristics suggest different involved types or strains of Symbiodinium, depending on the specific food source and its distribution in geographic range and depth. Key words: Nudibranchia, mutualistic symbiosis, Pulse Amplitude Modulated Fluorescence, zooxanthellae, Symbiodinium, photosynthesis. INTRODUCTION feeding on soft corals, in some cases also hard The mutualistic symbiosis of different clades of corals. Some nudibranchs, e.g., Melibe Rang, 1829, the Nudibranchia with zooxanthellae (unicellular do not feed on cnidarians and the source of the dinoflagellates of the genus Symbiodinium zooxanthellae is still not verified. The advantages of Freudenthal, 1962) was already described by this mutualistic relationship were already discussed Rousseau (1934, 1935). Assignment to the genus by different authors. The incorporation of Symbiodinium with the known 5 clades (see zooxanthellae offers a cryptic appearance (Rudman LaJeunesse 2001, Rodriguez-Lanetty 2003, Ulstrup 1987, Wagele and Johnsen 2001, Burghardt and and van Oppen 2003) was verified by investigating Wagele 2004, Burghardt et al., in press) and several nudibranchs belonging to the family photosynthetic products to the involved nudibranch Facelinidae by molecular means (Melkonian pers. (Hoegh-Guldberg and Hinde 1986, Hoegh­ comm.). Other taxa ("zoochlorellae" like e.g., Guldberg et al. 1986, Rudman 1991, Wagele and Carteria Diesling 1866, Prochloron (Lewin 1975» Johnsen 2001, Burghardt and Wagele 2004, cultivating a symbiotic relationships with marine Burghardt et al., in press). These additional invertebrates (e.g., didemnid tunicates) are in nutrients allow them to survive periods of food nudibranchs only known from the temperate shortage or even without their documented food species Aeolidia papillosa (Linnaeus, 1761) although (Burghardt and Wagele 2004, Burghardt et al., in this symbiosis does not seem to be stable press). (McFarland and Muller-Parker 1993). In the past, zooxanthellae inside nudibranchs The zooxanthellae are housed inside the cells of were mainly detected by histological and the digestive gland. Generally the nudibranchs take ultrastructural investigations (Kempf 1984, Rudman up Symbiodinium through their prey, mostly by 1981a,b, 1982a,b, 1991; Marin and Ros 1991; Wagele 2 I. Burghardt, H. Wiigele and Johnsen 2001). Wiigele and Johnsen (2001) and f.illlol quanta m,2 S,l) in the laboratory at the Ruhr­ Burghardt et al. (in press) have already discussed University Bochum. Dermatobranchus sp. was kept that the existence of Symbiodinium inside the in the shade (under a roof, ~200 f.illlol quanta m 2S·l) digestive gland cells does not necessarily prove a on Pulau Bunaken (Bunaken Islands, Indonesia). All symbiotic relationship with the host. Wiigele and specimens were kept without their documented food Johnsen (2001) were the first who introduced the under starvation conditions to prevent the uptake of now established method of using a PAM ("Pulse fresh zooxanthellae. A Pulse Amplitude Modulated Amplitude Modulated Fluorometer") to distinguish Fluorometer (Diving-PAM, Walz, Germany) was digested from photosynthetically active used to detect in vivo photosynthetic activity of zooxanthellae in situ inside seaslugs. Burghardt and zooxanthellae in the investigated nudibranchs by Wiigele (2004) and Burghardt et al. (in press) measuring the fluorescence emitted by photosystem performed long term experiments with IT (PSIT) of chlorophyll a. This allows distinguishing "solarpowered" nudibranchs (Wiigele and Johnsen between active photosynthetic zooxanthellae and 2001) of the genus Phyllodesmium Ehrenberg, 1831 digested ones inside the nudibranch (Wiigele and and Pteraeolidia ianthina (Angas, 1864) and showed Johnsen 2001; Burghardt and Wiigele 2004; intra- and interspecific differences in the efficiency Burghardt et al., in press). of the symbiosis by means of PAM data. Measurements of the maximum fluorescence In this study we present new data from different yield (<Pile.mJ were taken in darkness. <Pile.max was nudibranch species belonging to different subtaxa of plotted versus time in diagrams in order to show the Cladobranchia (Aeolidoidea, 11Arminoidea" and how long the zooxanthellae are able to stay Dendronotoidea) and discuss the efficiency and photosynthetically active inside the nudibranch and photosynthetic characteristics of their symbiosis with to detect possible interspecific differences. Symbiodinium by presenting the results of long term Except for Dermatobranchus sp. and Phestilla starvation experiments including PAM data. lugubris, all specimens were exposed to different irradiances of natural sunlight between 0 and 1500 flmol quanta m·2S·l for measuring the operational MATERIAL AND METHODS quantum yield (<PIle) and obtaining photosynthesis For this study, nudibranchs from different Versus irradiance curves (P-E curves). The P-E­ localities in the Indopacific and the Red Sea were curves and the resulting values (Pmax' Ek, et) were sampled. Phyllodesmium briareum was collected at analysed by the statistics software Kaleidagraph 3.6. Cobia Hole, Lizard Island (Great Barrier Reef, For the calculations the following equation was Australia) at a depth of 16m on Briareum violacea used (for further details see Wiigele and Johnsen in July 2002. Phestilla lugubris was collected at 2001): Loomis Reef, Lizard Island under a Porites coral in P = Pmax * (1- exp (-et * E/Pma)) 0.5m depth in August 2004. Melibe cf. bucephala P = photosynthetic rate at a given actinic irradiance, Bergh, 1902, Aeolidiella alba Risbec, 1928, Spurilla P = <Pu; E where: australis Rudman, 1982 and Limenandra nodosa P = <P Ue [mol charge separation . mol quanta Haefelfinger and Stamm, 1958 were collected on a absorbed'l] .E [flmol quanta m 2S·l] field trip to Dahab (Gulf of Aquaba, Red Sea, Egypt) <PUe = operational quantum yield in May 2003. All Red Sea specimens were collected Pmax = maximum photosynthetic rate (same units as at Dahab lagoon, in shallow water from 0.5 to 1.5m P) depth between coral rubble. Dermatobranchus sp. et maximum light utilization coefficient (et = (<pu; E) was collected near Pulau Talise, north of Sulawesi . RI) (Indonesia) in July 2003. This specimen was found E = Irradiance (PAR, 400-700nm; [flmol quanta m·2 under coral rubble in about 1m depth. S·l]) Phyllodesmium briareum and Phestilla lugubris were kept under natural moderate light conditions Ek= Pmax I et = light saturation index in [f.illlol qum;tta 2 1 (up to a maximum of ~350 f.illlol quanta m 2 S·l at m· s· ] solar noon) for the whole time of the experiments at For curve linear regression the Webb equation the Lizard Island Research Station (LIRS; for details was used (Webb et al. 1974). For methodological see Burghardt et al., in press). Natural light details see Wiigele and Johnsen (2001) and conditions means natural light climate conditions in Burghardt et al. (in press). After the long term which irradiance, spectral irradiance and experiments (and before their natural death) all photoperiod are driven by the sun's position (sun specimens were preserved in formalinlseawater for angle), clouds, the extinction coefficient of water and later histological investigations. depth. All Red Sea specimens were kept in natural light conditions (shade) during the first 10-14 days and after transfer to Germany kept under controlled RESULTS artificial light conditions (up to a maximum of 70 Comparisons of the maximum yield of Photosynthetic characteristics of "solarpowered" Nudibranchia 3 "Solarpowere d" Nudib ranchi a 0.7 0.6 • o 0.5 ..~... ... 0.4 0.3 • 0.2 0.1 O-t-----,~--.___--.--~---r---__.__-~_e.....--...__-__.__-____,--....._-__I A o 5 10 15 20 25 30 35 40 45 50 55 60 Phestilla lugubris Spurila australis 0.8 0.8 ~---"7iIJ!::'--'-'-"--'---'---'------, 0.7 0.7 0.6 0.6 0.5 0.5