Molecular Detection of the Harmful Raphidophyte Chattonella subsalsa Biecheler by Whole-Cell Fluorescence in-situ Hybridisation Assay.

ABSTRACT

Species of the genus Chattonella (Raphidophyceae) are a group of marine protists that are commonly found in coastal waters. Some are known as harmful microalgae that form noxious blooms and cause massive fish mortality in finfish aquaculture. In Malaysia, blooms of Chattonella have been recorded since the 1980s in the Johor Strait. In this study, two strains of Chattonella were established from the strait, and morphological examination revealed characteristics resembling Chattonella subsalsa. The molecular characterization further confirmed the species' identity as C. subsalsa. To precisely detect the cells of C. subsalsa in the environment, a whole-cell fluorescence in-situ hybridisation (FISH) assay was developed. The species-specific oligonucleotide probes were designed in silico based on the nucleotide sequences of the large subunit (LSU) and internal transcribed spacer 2 (ITS2) of the ribosomal DNA (rDNA). The best candidate signature regions in the LSU-rRNA and ITS2-rDNA were selected based on hybridisation efficiency and probe parameters. The probes were synthesised as biotinylated probes and tested by tyramide signal amplification with FISH (FISH-TSA). The results showed the specificity of the probes toward the target cells. FISH-TSA has been proven to be a potential tool in the detection of harmful algae in the environment and could be applied to the harmful algal monitoring program.

Spesies genus Chattonella (Raphidophyceae) ialah sekumpulan protista marin yang biasa ditemui di perairan laut pantai. Sesetengahnya dikenali sebagai mikroalga berbahaya yang membentuk ledakan alga berbahaya dan menyebabkan kematian ikan secara besar-besaran dalam akuakultur ikan sirip. Di Malaysia, ledakan alga Chattonella telah direkodkan sejak tahun 1980-an di Selat Johor. Dalam kajian ini, dua strain Chattonella telah didirikan dari selat, dan pemeriksaan morfologi mendedahkan ciri-ciri yang menyerupai Chattonella subsalsa. Pencirian molekul seterusnya mengesahkan identiti spesies sebagai C. subsalsa. Untuk mengesan dengan tepat sel-sel C. subsalsa di dalam persekitaran, ujian penghibridan in-situ berpendarfluor (FISH) ke atas sel keseluruhan telah dibangunkan. Prob oligonukleotida spesies telah direka secara spesifik secara siliko berdasarkan jujukan nukleotida subunit besar (LSU) dan spacer transkripsi dalaman 2 (ITS2) gen DNA ribosom (rDNA). Calon terbaik kawasan tanda dalam LSU-rRNA dan ITS2-rDNA telah dipilih berdasarkan kecekapan penghibridan dan parameter prob. Prob telah disintesis sebagai prob biotinilasi dan diuji dengan penguatan isyarat tyramide dengan FISH (FISH-TSA). Keputusan menunjukkan kekhususan prob ke atas sel sasaran. FISH-TSA telah terbukti sebagai alat yang berpotensi dalam pengesanan alga berbahaya di alam sekitar dan boleh digunakan untuk program pemantauan alga berbahaya.