Molecular Identification of Microbes Capable of Growing Outside Their Host Lissoclinum Patella Originated from Talise Waters, North Minahasa

Abstract

Ascidian Lissoclinum patella is known to harbor a diverse community of symbiotic microorganisms, however, information on symbiotic microbes capable of growing outside their host originated from the waters of Talise is still limited. This study aimed to molecularly identify microorganisms capable of growing outside the host L. patella collected from Talise waters, North Minahasa, Indonesia. Ascidian samples were collected by SCUBA diving and transported to the Marine Biotechnology and Marine Pharmacy Laboratory, Faculty of Fisheries and Marine Science, Sam Ratulangi University. Microbial suspensions were aseptically prepared from L. patella tissues and inoculated into sterile seawater (35 ppt salinity) enriched with NPK fertilizer (32-10-10). After approximately one month of growth outside the host, about 10 mL of the microbial culture was collected for DNA isolation. The microbe 16S rRNA gene was amplified by PCR, and the amplicons were analyzed by 1% agarose gel electrophoresis. PCR products were subsequently sequenced and compared with sequences available in the NCBI GenBank database. BLAST analysis revealed the highest sequence similarity (99.29%) to three reference sequences in the NCBI GenBank database: Oceanicella sp. strain SMB1, Oceanicella sp. strain IB204559, and Rhodovulum sp. strain SMB1. Future studies should focus on obtaining monospecies isolates on solid agar media prior to independent cultivation outside the host, thereby enabling the exploration of the molecular potential of specific species. Keywords: 16S rRNA, host, Lissoclinum patella, microbes, molecular identification   Abstrak Ascidia Lissoclinum patella diketahui berasosiasi dengan berbagai mikroba simbion, namun informasi mengenai mikroba yang mampu tumbuh di luar inangnya dari Perairan Talise masih terbatas. Penelitian ini bertujuan mengidentifikasi secara molekuler mikroba yang dapat tumbuh di luar inangnya ascidia L. patella asal Perairan Talise, Minahasa Utara. Sampel ascidia diambil menggunakan SCUBA, kemudian dibawa ke Laboratorium Bioteknologi dan Farmasetika Laut, FPIK Universitas Sam Ratulangi. Suspensi mikroba diperoleh secara aseptik dari jaringan L. patella dan diinokulasikan pada media air laut steril bersalinitas 35 ppt yang diperkaya NPK 32-10-10. Setelah tumbuh sekitar 1 bulan di luar inangnya, sekitar 10 mL medium tumbuh mikroba diambil untuk isolasi DNA, dan diamplifikasi dengan primer 16S rRNA, kemudian amplikon dielektroforesis dengan gel agarosa 1%. Produk PCR disekuens dengan penjajaran terhadap basis data NCBI GenBank. Hasil BLAST menunjukkan tingkat kemiripan tertinggi 99,29% dengan tiga sekuens referensi pada NCBI GenBank, yaitu Oceanicella sp. strain SMB1, Oceanicella sp. strain IB204559, dan Rhodovulum sp. strain SMB1. Ke depan masih perlu dilakukan isolasi monospesies pada medium agar sebelum ditumbuhkan terpisah inang, untuk eksplorasi potensi molekuler spesies spesifik.  Kata kunci: 16S rRNA, inang, Lissoclinum patella, mikroba, identifikasi molekuler