title: In situ identification of symbiotic dinoflagellates, the genus Symbiodinium with fluorescence-labeled rRNA-targeted oligonucleotide probes.
abstract: Fluorescence in situ hybridization has been used for the identification and analysis of populations of the dinoflagellate Symbiodinium that lives symbiotically in marine invertebrates. Conditions for in situ hybridization of Symbiodinium were optimized and used to identify the clade to which the isolate belongs using specific probes. The optimized in situ hybridization procedure used a combination of chlorophyll removal and permeabilization with hot ethanol. Incubation of the cells in 50% ethanol at 80 degrees C for 20 min rendered the cell wall permeable to Cy3-labeled probes. Symbiodinium clade-specific probes were designed based on 18S rRNA sequences. Symbiodinium A, B and C were distinguished by in situ hybridization with the specific probes SymA, SymB and SymC, respectively. The hybridization results using clade-specific probes corresponded with results obtained using restriction fragment length polymorphism (RFLP) analysis. Symbiodinium isolated from jellyfish Cassiopea sp. and sea anemone Aiptasia sp. were classified as belonging to clades A and B using the FISH procedure established in this study.
authors.affiliation: Department of Biotechnology, Tokyo University of Agriculture and Technology 2-24-16, Naka-cho, Koganei, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Yokouchi
authors.forename: Haruko
authors.initials: H
authors.lastname: Takeyama
authors.forename: Hideaki
authors.initials: H
authors.lastname: Miyashita
authors.forename: Tadashi
authors.initials: T
authors.lastname: Maruyama
authors.forename: Tadashi
authors.initials: T
authors.lastname: Matsunaga
mesh_terms: D000818:Animals; D002463:Cell Membrane Permeability; D004141:Dinoflagellida / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D015345:Oligonucleotide Probes; D012337:RNA, Ribosomal, 18S / Q000032:analysis*; D013559:Symbiosis
chemical_list: D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012337:RNA, Ribosomal, 18S