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[Invertebrate-Algal Symbiosis: Diversity and Evolution]Organized by Michio Hidaka1 and Toshiki Watanabe2

1Department of Chemistry, Biology, and Marine Science, Faculty of Science, University of the Ryukyus,2Ocean Research Institute, The University of Tokyo

Flexibility of Cnidarian-Zooxanthellae Symbiosis and Its Implica-tion for Stress ToleranceMamiko HiroseThe 21st Century COE Program, University of the Ryukyus

Hermatypic corals and some cnidarians harbor symbiotic dinoflagellate, Symbiodinium spp., commonly known aszooxanthellae. Zooxanthellae have been classified into seven clades based on ribosomal DNA analyses and it isbelieved that each clade consists of many species, though only several species have been described morphologi-cally. The loss of the zooxanthellae and/or algal pigments from the host results in bleaching. Zooxanthellatecnidarians show different susceptibility to bleaching when they are exposed to environmental stresses. However,it is not understood which partner of the cnidarian-zooxanthellae complex is responsible for the difference in thestress susceptibility. To answer this question, we studied the stress susceptibility of genetically identical hostsinfected with zooxanthellae of different genotypes. We infected clonal, aposymbiotic polyps (scyphistoma stage)of Cassiopea andromeda with zooxanthellae isolated from various hosts; Cassiopea sp., Aiptasia sp., Tridcnacrocea, and several hermatypic corals. We defined the genotype (clade) of isolated zooxanthellae by 18S rDNAand/or 28S rDNA RFLP. Infection was successful with zooxanthellae isolated from six out of seven hosts. Medu-sae associated with various types of zooxanthellae were formed about one month after infection. We exposed themedusae to high temperature in light or darkness for six hours, then allowed them to recover for 40 hours undernormal condition. The photochemical efficiency (Fv/Fm) and the release rate of zooxanthellae were measured dur-ing the stress treatment and recovery period. Medusae infected with zooxanthellae isolated from Aiptasia (mainlyclade B) showed the largest decline in the Fv/Fm value. The Fv/Fm did not recover and many zooxanthellae werereleased during the recovery period. Those infected with isolates from Pavona divaricata (mainly clade D)showed a small but significant decline of the Fv/Fm value, which recovered almost completely. They did not re-lease many zooxanthellae. Cassiopea jellyfish infected with zooxanthellae of different genotype showed differentsusceptibility to environmental stress. Different genotypes of zooxanthellae within the genetically identical jellyfishsuffered different degrees of damages to PSII when exposed to the same stress treatment. The host tends toexpel more zooxanthellae when the algae suffered severe PSII damage. The present results suggest thatCassiopea jellyfish can establish symbiosis with a broad range of zooxanthellae but that the symbiotic complexesshow different susceptibility to stress. The flexibility and diversity of Cassiopea–zooxanthellae symbiosis may en-able the jellyfish to adapt to various environmental conditions.

Symbiosis Between Giant Clams and Symbiotic DinoflagellatesTadashi MaruyamaResearch Program for Marine Biology and Ecology, Extremobiosphere Research Center, Japan Agencyfor Marine-Earth Science and Technology

Symbiosis between giant clams and symbiotic dinoflagellates is widely known. However, the mechanism underly-ing the symbiotic interaction still remains to be studied. In this talk, I would like to try to figure out the evolution ofthe symbiosis between giant clams and their symbiotic dinoflagellates. In giant clams, several genetically distinctsymbiotic dinoflagellates reside in the specifically differentiated organ, zooxanthellal tube, which is an extensionof stomach. Inside the clam, the symbionts secrete over 50% of their photosynthetically fixed carbon. Simulta-neously, symbionts, in the stomach, seem to be digested by the host clam. Thus, the host acquires the secretedphotosynthates and, in addition, digested symbiont as nutrients. Isolated symbiont secreted its photosynthates inthe presence of host tissue homogenate. The homogenate also induces photosynthate secretion from some freeliving dinoflagellates. These probably indicate that the symbiosis between giant clams and symbiotic algae origi-

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nated from the relationship between a filterfeeding clam that fed on microalgae and the food microalgae. Theancestor dinoflagellates were probably sensitive to some host substances in the clam tissue and were stimulatedto secrete their photosynthates when they were ingested by the clam. The algae acquired the way to resist thedigestion. The host differentiated an organ from the stomach to keep the algae inside it, and increased the effi-ciency of photosynthate acquisition.

Phylogeny of Ascidian-Prochloron SymbiosisEuichi HiroseDepartment of Chemistry, Biology, and Marine Science, Faculty of Science, University of the Ryukyus

In the tropics, certain colonial ascidians harbor the prokaryotic photosymbiont Prochloron. Stable symbiosis ofProchloron has been exclusively found in about 30 ascidian species belonging to the family Didemnidae. Free-living Prochloron has never been recorded and host species always harbor Prochloron cells. In the host colonies,Prochloron cells usually inhabit the peribranchial and common cloacal cavity or, exceptionally, are intracellularlydistributed in the tunic, as in the case of Lissoclinum punctatum. While Prochloron cells are not associated withhost gametes or embryos, the larvae usually possess them that are acquired from their mother colony during (orjust before) the hatch from the colony. The vertical transmission of the symbionts strongly suggests the impor-tance of Prochloron to the host for survival and the occurrence of co-evolution between host ascidians and sym-bionts. On the other hand, the mode of vertical transmission is various among the host species, suggesting mul-tiple origins of the ascidian-Prochloron symbiosis in didemnids. To date, host ascidians bearing Prochloron arefound in four genera of the family Didemnidae: Didemnum, Trididemnum, Lissoclinum, and Diplosoma. Sincethere also exist non-symbiotic species in each genus, the symbiosis may have established independently at leastonce in these four lineages.In order to elucidate the evolutionary history of the ascidian-Prochloron symbiosis, we investigated the molecularphylogeny of symbiotic/non-symbiotic didemnids and Prochloron based on 18SrDNA and 16SrDNA sequences,respectively. Our phylogenetic trees of didemnid ascidians (4 non-didemnid species and 48 photosymbioticsamples) indicated a monophyletic origin of the family Didemnidae, as well as each of the didemnid genera, ex-cept the genus Lissoclinum. The results strongly support the hypothesis that establishment of the ascidian-Prochloron symbiosis occurred independently in the Didemnidae lineage at least once in each of the genera thatpossess symbiotic species. On the contrary, our phylogenetic trees of Prochloron (15 samples from 9 host spe-cies) indicated that neither host species nor sampling sites restrain the genetic variations of the symbiotic algae.These results suggest that the combination of host and Prochloron is opportunistic and the vertical transmissionis not the only source of the photosymbionts. If horizontal transmission of symbionts occurs, new questionsshould arise: 1) free-living Prochloron does exist? 2) why symbiosis with Prochloron have never been establishedin non-didemnid ascidians and other metazoans? It is possible that there are undiscovered animals having sym-biotic relationship with Prochloron.The present study was supported by Japan-Australia Research Cooperative Program and Grants-in Aid for Sci-entific Research (#14740473, #16570081) from JSPS, Nihon University Multidisciplinary Global Research Grant,and the 21st Century COE program of University of the Ryukyus.

Cloning and Characterization of Novel Fluorescent Proteinsfrom Anthozoan Animals and Their Applications to Cell Biologi-cal ResearchSatoshi Karasawa1,2 and Atsushi Miyawaki11Laboratory for Cell Function Dynamics, Brain Science Institute (BSI), The Institute of Physical andChemical Research (RIKEN), 2Amalgaam Co. Ltd.,

The Green Fluorescent Protein (GFP), which was cloned from the jellyfish Aequorea victoria, and its homologsfrom the species Anthozoa have been useful tools for cell biological research. They have been used not only forlabeling proteins, but also as reporters for gene expression and intracellular conditions. We report here the mo-lecular cloning of novel fluorescent proteins (FPs) from Anthozoa, as well as their characteristics and applicationsto cell biological research.

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The green FP isolated from Galaxea fascicularis (“Azami-sango” in Japanese) was named “Azami-green (AG).”With a high extinction coefficient, fluorescence quantum yield, and acid stability, AG emits brighter green fluores-cence than EGFP in cultured mammalian cells. Kusabira-Orange (KO) isolated from “Kusabira-ishi (Fungiaconcinna)” emits orange fluorescence, filling the gap between yellow (YFP) and red (RFP). Midoriishi-Cyan(MiCy) is a cyan FP from “Midori-ishi (Acropara sp.)” and has a high fluorescence quantum yield. We utilized theKO/MiCy combination as a novel donor/acceptor pair for fluorescence resonance energy transfer (FRET) mea-surements of caspase-3 activity during apoptosis and succeeded in clearly “visualizing the enzymatic activity” inliving mammalian cells. Kaede, which was cloned from Trachyphyllia geoffroyi, is a “photoconvertible” fluorescentprotein: It originally emits green fluorescence. After a short exposure to UV or violet light, Kaede becomes to emitlonger wavelengths (i.e. red) of fluorescence. By restricting the UV irradiation to a single cell, for example, wecan label the cell red in the background of green fluorescent cells.These newly-identified Anthozoa-derived FPs have a variety of interesting characteristics and will make usefultools for cell biological research.

Host-Symbiont Interactions in Paramecium-Chlorella SymbiosisMasahiro Fujishima and Yuuki KodamaBiological Institute, Faculty of Science, Yamaguchi University

Paramecium bursaria harbors several hundred symbiotic algae in their cytoplasm. The algae-free P. bursaria caneasily be produced from the algae-bearing cell by growing the cell in dark condition, and the cell still has theability to grow even without the symbiotic algae. The association of P. bursaria with Chlorella sp. has long beenregarded as mutualistic symbiosis. For example, the endosymbiotic algae can be supplied with nitrogen by theirhost. The algae-bearing P. bursaria can grow well compared with the algae-non-bearing cell. The algae within thesymbiotic unit show a higher rate of photosynthetic oxygen production than in the isolated state and thus guaran-tee oxygen supply for the host. The algae-free P. bursaria shows negative phototaxis, but the algae-bearing cellshows positive phototaxis for algal photosynthesis. When the algae-free P. bursaria are mixed with the symbioticalgae isolated from the symbiotic P. bursaria, most of the algae ingested by the host are digested but someescape from the host’s digestion and succeed to be maintained in the host cytoplasm.Symbiotic associations between these eukaryotic cells are excellent models for studying cell-to-cell interactionand evolution of eukaryotic cell by endosymbiosis between different eukaryotic cells. However, the mechanismand timing used by the algae to escape from digestive vacuole (DV) of the host have not yet been revealed. In P.bursaria, each symbiotic alga is enclosed in perialgal vacuole (PV) derived from the host DV, so that the alga isprotected from lysosomal fusion. We elucidated the timing of acidification and lysosomal fusion and the appear-ance of the PV membrane around the each alga during early infection by using pulse label of 1.5-min and chasemethod with pH indicator dye-stained yeasts and isolated algae from P. bursaria. Acidification of the vacuolesand digestion of Chlorella sp. began at 0.5 and 2 min, respectively. All vacuoles containing single green Chlorellasp. that had been present in the host cytoplasm before 0.5 h after mixing were digested till 0.5 h. At 1 h aftermixing, however, the single green alga appeared again in the host cytoplasm from the digestive vacuoles con-taining both the non-digested and the digested algae, and the ratio of such cells increased to about 40% at 3 h.At 48 h, the single green alga began to grow by cell division, indicating that these algae had succeeded to estab-lish endosymbiosis. In the contrast to previously published studies, our data show that the alga can escape fromthe host’s digestive vacuole considerably after the acidosomal and the lysosomal fusion of the vacuole, and suc-ceed to establish endosymbiosis.