ARTICLE IN PRESS Organisms, Diversity & Evolution 8 (2008) 66–76 www.elsevier.de/ode Symbiosis between Symbiodinium (Dinophyceae) and various taxa of Nudibranchia (Mollusca: Gastropoda), with analyses of long-term retention Ingo Burghardta,Ã, Kristina Stemmera, Heike Wa¨geleb aLehrstuhl fu¨r Evolutionso¨kologie und Biodiversita¨t der Tiere, Ruhr-Universita¨t Bochum, 44780 Bochum, Germany bInstitut fu¨r Evolutionsbiologie und O¨kologie, Rheinische Friedrich-Wilhelms-Universita¨t Bonn, and Zoologisches Forschungsmuseum A. Koenig, Bonn, Germany Received 25 July 2006; accepted 16 January 2007 Abstract Long-term retention of zooxanthellae in five different species belonging to two different nudibranch groups (Aeolidoidea and Dendronotoidea) was investigated. Specimens belonging to the species Phyllodesmium briareum, Phyllodesmium colemani, Phyllodesmium longicirrum, Pteraeolidia ianthina and Melibe engeli were cultivated for 70–270 days under various feeding conditions, and photosynthetic activity was analysed by taking pulse amplitude modulated (PAM) fluorometer measurements. All five species showed stable symbiosis and long-term retention of zooxanthellae. Interspecific differences are discussed. Morphological adaptations for housing zooxanthellae in the digestive glandular system were investigated and documented by histological means. r 2007 Gesellschaft fu¨r Biologische Systematik. Published by Elsevier GmbH. All rights reserved. Keywords: Nudibranchia; Zooxanthellae; Symbiosis; Phyllodesmium; Evolution; Pulse amplitude modulated fluorescence Introduction (‘zoochlorellae’, e.g. Carteria Diesling, 1866, Prochloron (Lewin, 1975)) living in symbiotic relationships with The mutualistic symbiosis of members of various marine invertebrates (e.g. didemnid tunicates) are known clades in Nudibranchia with zooxanthellae (unicellular from only one nudibranch, the temperate species Aeolidia dinoflagellates of the genus Symbiodinium Freudenthal, papillosa (Linne´, 1761), although this symbiosis does not 1962) was already described by Rousseau (1934, 1935). seem to be stable (McFarland and Muller-Parker 1993). Assignment to the genus Symbiodinium with its known The zooxanthellae are housed inside the cells of the eight clades (see LaJeunesse 2001; Rodriguez-Lanetty digestive gland. In general, the nudibranchs take up 2003; Ulstrup and van Oppen 2003; Pochon et al. 2006) Symbiodinium through their prey, mostly by feeding on was verified by investigating zooxanthellae from several soft corals, in some cases also on hard corals (Rudman nudibranchs belonging to the family Facelinidae using 1981b, 1991; Kempf 1984; Wa¨gele and Johnsen 2001; molecular means (Melkonian, pers. comm.). Other taxa Burghardt and Wa¨gele 2004; Burghardt et al. 2005). Some nudibranchs, e.g. Melibe Rang, 1829, do not feed on ÃCorresponding author. cnidarians, and the source of their zooxanthellae remains E-mail address: [email protected] (I. Burghardt). unverified. The general advantages of a symbiosis with 1439-6092/$ - see front matter r 2007 Gesellschaft fu¨r Biologische Systematik. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.ode.2007.01.001 ARTICLE IN PRESS I. Burghardt et al. / Organisms, Diversity & Evolution 8 (2008) 66–76 67 zooxanthellae have already been discussed by several Phyllodesmium briareum (specimen #1), P. longicirrum authors. The nudibranchs’ incorporation of zooxanthellae (specimens #1 and #2), and Pteraeolidia ianthina (specimen results in a cryptic appearance (Rudman 1987; Wa¨gele and #1) were kept under natural moderate light conditions Johnsen 2001; Gosliner 1987; Burghardt and Wa¨gele 2004, (uptoamaximumof350 mmol quanta mÀ2 sÀ1 at solar 2006; Burghardt et al. 2005; Burghardt and Gosliner 2006) noon) for the whole time of the experiments at the Lizard and photosynthetic products (Hoegh-Guldberg and Hinde Island Research Station (LIRS; for details see Burghardt 1986; Hoegh-Guldberg et al. 1986; Rudman 1991; Wa¨gele et al. 2005). Natural light conditions mean natural light and Johnsen 2001; Burghardt and Wa¨gele 2004, 2006; climate conditions in which irradiance, spectral irradiance Burghardt et al. 2005; Burghardt and Gosliner 2006). The and photoperiod are driven by the sun’s position (sun additional nutrients produced by the symbionts allow the angle), clouds, and the extinction coefficient of water and slugs to survive food shortages lasting from weeks to depth. Melibe engeli and Pteraeolidia ianthina (specimen (several) months (Burghardt and Wa¨gele 2004, 2006; #2) were kept in natural light conditions (shade) during Burghardt et al. 2005; Burghardt and Gosliner 2006). The the first 10–14 days, and after transfer to Germany symbiosis with zooxanthellae might be a key character for under controlled artificial light conditions (plant lamp increased radiation in certain taxa (Wa¨gele 2004). Osram Lumilux 11, Germany; up to a maximum of In the past, zooxanthellae inside nudibranchs were 70 mmol quanta mÀ2 sÀ1) in the laboratory at the Ruhr- mainly detected by histological and ultrastructural University Bochum. Phyllodesmium colemani was kept investigations (Rudman 1981a, b, 1982a, b, 1991; Kempf under the same conditions in Bochum. 1984; Marin and Ros 1991; Wa¨gele and Johnsen 2001). All starving specimens were kept without their Wa¨gele and Johnsen (2001) and Burghardt et al. (2005) documented food in aquaria to prevent the uptake of have already discussed that the existence of Symbiodinium fresh zooxanthellae. All aquaria were cleaned regularly inside the digestive gland cells does not necessarily prove to remove faeces and prevent algal growth. The animals a symbiotic relationship with the host. Wa¨gele and on Lizard Island were kept in a flow-through water Johnsen (2001) were the first who introduced the now system, in the other cases seawater was replaced by established method of using a pulse amplitude modulated sterilised fresh seawater every 1 or 2 weeks. (PAM) fluorometer to distinguish digested from photo- A PAM fluorometer (Diving-PAM, Walz, Germany) synthetically active zooxanthellae in situ,i.e.insidethe was used to detect in vivo photosynthetic activity of seaslugs. Burghardt and Wa¨gele (2004) and Burghardt zooxanthellae in the investigated specimens by measur- et al. (2005) performed long-term experiments with ‘solar- ing the fluorescence emitted by photosystem II (PSII) of powered’ nudibranchs of the genus Phyllodesmium chlorophyll a. This allows distinction between active Ehrenberg, 1831 and of Pteraeolidia ianthina (Angas, photosynthetic zooxanthellae and digested ones inside 1864), and showed intra- and interspecific differences in the nudibranch, and interspecific differences could also the efficiency of the symbiosis by means of PAM data. be detected (Wa¨gele and Johnsen 2001; Burghardt and In this study, we compare data from various nudi- Wa¨gele 2004, 2006; Burghardt et al. 2005; Burghardt branch species belonging to two subtaxa in Cladobran- and Gosliner 2006). chia. These are Phyllodesmium briareum (Bergh, 1896), The PAM consists of a main instrument, a cosinus- P. longicirrum (Bergh, 1905), P. colemani Rudman, 1991, corrected light collector, and an optic fibre. The fibre and Pteraeolidia ianthina (Angas, 1864) as representatives detects the fluorescence of the zooxanthellae inside the of the Aeolidoidea, and Melibe engeli Risbec, 1937 as a nudibranch by measuring the ground fluorescence (F0) representative of the Dendronotoidea. All species have using a very low light source. F0 is defined as the shown long-term retention of zooxanthellae and seem to fluorescence measured in dark-acclimated tissues. have evolved different morphological adaptations and A flash of approximately 10,000 mmol quanta mÀ2 sÀ1 behaviour for this symbiosis. We discuss the respective for 0.8 s is applied via the optic fibre to shut down the efficiency of their symbiosis with Symbiodinium by reaction centres of PSII and therefore obtain maximum presenting results of long-term starvation experiments fluorescence (Fm). including PAM data, and examine the role of this The maximum quantum yield of fluorescence for PSII symbiosis in evolution. Histological data are compared (FIIe-max) is defined as with PAM data to highlight the morphological adapta- tions of each species for cultivating zooxanthellae. FIIeÀmax ¼ðF m À F 0Þ=F m. Measurements of the maximum fluorescence yield Material and methods (FIIe-max) were taken in darkness. Between measure- ments, breaks of at least 10 min allowed the reaction For this study, nudibranchs from various localities in centres of PSII to recover after light saturation. FIIe-max the Indopacific and the Red Sea were sampled. The was plotted versus time in order to show long-term specimens investigated are listed in Table 1. photosynthetic activity as a function of time (Wa¨gele ARTICLE IN PRESS 68 I. Burghardt et al. / Organisms, Diversity & Evolution 8 (2008) 66–76 Table 1. Synopsis of investigated specimens, collecting data, and environmental conditions during experiments Species # Locality Coll. date Depth (m)/substrate Cultivation Cult. (days) conditions (d ¼ day) Phyllodesmium 1 Cobia Hole, July 2002 16/Briareum violacea Starvation: d0–70 70 briareum Lizard Island, (Roule, 1908) GBR, Australia 2 Orpheus Island, July 1995 –/Briareum violacea Directly preserved À GBR, Australia Phyllodesmium 1 Moalboal, Cebu Nov. 2003 –/Tubipora musica Starvation: d0–74 74 colemani Island, Linnaeus, 1758 Philippines Phyllodesmium 1 Bird Island, Feb. 2005 7/Sarcophyton sp. Starvation: d0–116; 160 longicirrum Lizard Island, fed with GBR,