title: Direct visualization of the novel pathogen, Spiroplasma eriocheiris, in the freshwater crayfish Procambarus clarkii (Girard) using fluorescence in situ hybridization.
abstract: Spiroplasma eriocheiris is the first spiroplasma strain known to be pathogenic to freshwater crustaceans. It has caused considerable economic losses both in the freshwater crayfish Procambarus clarkii (Girard) and in some other crustaceans. The monitoring of the pathogen in crustacean populations and study of its behaviour in the laboratory require the development of reliable diagnostic tools. In this article, we improved microscopic identification of S. eriocheiris by combining in situ hybridization with specific fluorescently labelled oligonucleotide probes. The established fluorescence in situ hybridization (FISH) allowed simultaneous visualization, identification and localization of S. eriocheiris in the tissues of diseased crayfish P. clarkii and exhibited low background autofluorescence and ideal signal-to-noise ratio. With the advantages of better tissue penetration, potentially more specific and stable, we designed three species-specific oligonucleotide probes utilizing the sequences of 16S-23S rRNA intergenic spacer regions (ISRs) of S. eriocheiris. Positive hybridization signals were visualized in haemocytes and connective tissues of hepatopancreas, cardiac muscle and gill from diseased crayfish. This unique distribution pattern matched the pathological changes when diagnosed by H&E staining and indicated that S. eriocheiris probably spread throughout the tissues in P. clarkii by hemokinesis. This assay will facilitate our understanding of the pathogenesis of S. eriocheiris and enhance the early diagnosis of the novel pathogen.
authors.affiliation: Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing, China.|Freshwater Fisheries Research Institute of Jiangsu Province, Nanjing, China.
authors.forename: Z F
authors.initials: ZF
authors.lastname: Ding
authors.affiliation: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China.
authors.forename: S Y
authors.initials: SY
authors.lastname: Xia
authors.affiliation: Freshwater Fisheries Research Institute of Jiangsu Province, Nanjing, China.
authors.forename: H
authors.initials: H
authors.lastname: Xue
authors.affiliation: Freshwater Fisheries Research Institute of Jiangsu Province, Nanjing, China.
authors.forename: J Q
authors.initials: JQ
authors.lastname: Tang
authors.affiliation: Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing, China.
authors.forename: Q
authors.initials: Q
authors.lastname: Ren
authors.affiliation: Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing, China.
authors.forename: W
authors.initials: W
authors.lastname: Gu
authors.affiliation: Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing, China.
authors.forename: Q G
authors.initials: QG
authors.lastname: Meng
authors.affiliation: Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing, China.
authors.forename: W
authors.initials: W
authors.lastname: Wang
mesh_terms: D000818:Animals; D017756:Aquaculture / Q000379:methods*; D003400:Astacoidea / Q000382:microbiology*; D005618:Fresh Water; D043143:Hepatopancreas / Q000382:microbiology / Q000473:pathology; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D013149:Spiroplasma / Q000166:cytology* / Q000235:genetics
keywords: Procambarus clarkii; Spiroplasma eriocheiris; fluorescence in situ hybridization; pathogenesis