PENENTUAN SPESIES Myxobolus PADA IKAN AIR TAWAR DI BERBAGAI PULAU DI INDONESIA SECARA KONVENSIONAL DAN MOLEKULER

Agus Priyono, M.Si., Prof. drh. Kurniasih, MVSc, Ph.D.

2012 | Disertasi | S3 Sain Veteriner

Penyakit akibat infestasi Myxobolus sering menyerang budidaya ikan air tawar di Indonesia dan menyebabkan wabah kematian yang tinggi. Penelitian bertujuan untuk (1) mengetahui berbagai spesies Myxobolus menyerang ikan air tawar di Indonesia berdasarkan pemeriksaan morfologi; (2) Untuk mengidentifikasi perbedaan spesies Myxobolus secara morfologi permukaan spora (Scanning Electron Microscope); (3) Untuk mengidentifikasi perbedaan spesies Myxobolus berdasarkan analisa secara molekuler dengan metode PCR-RFLP dan sequencing di daerah 28S dan 18S rDNA dan (4) Untuk mengetahui kekerabatan spesies Myxobolus yang ditemukan di Indonesia secara phylogeny. Sampel ikan diambil dari berbagai wilayah di Indonesia seperti Yogyakarta (Jawa Tengah), Kediri dan Blitar (Jawa Timur), Tabanan (Bali), Merauke (Papua), Pekanbaru (Sumatera) dan Entikong (Kalimantan). Ikan yang menunjukkan gejala klinis berupa operkulum tidak dapat menutup sempurna dan kejadian kematian tinggi pada ikan muda. Spora Myxobolus membentuk kapsula/nodul, biasanya menyerang insang, otak, mata dan kulit. Nodul dibuka untuk diperiksa morfologi dan diwarnai dengan Lugol’s Iodin. Nodul difiksasi dengan larutan formalin 10% untuk pemeriksaan histopatologi dan difiksasi dengan larutan glutaraldehyde untuk pemeriksaan Scanning Electron Microscope (SEM), difiksasi dengan larutan etanol absolute untuk pemeriksaan molekuler. Myxobolus diekstraksi DNA dan dilakukan amplifikasi pada daerah 18S dan 28S rDNA, kemudian dilakukan PCR-RFLP dan sequencing. Hasil pemeriksaan morfologi, SEM dan histopatologi dianalisa secara deskriptif. Hasil sequencing rDNA dianalisa secara Maximum Parsimony dan Neighbor Joining untuk memperoleh phylogeny antar spesies.

Disease caused by Myxobolus infestation was often occurred in fish aquaculture in Indonesia, they caused a high mortality. The aim of study is 1) to identify the Myxobolus sp. obtained of fresh water fish in Indonesia based on morphology, 2) to identify species based on Scanning Electron Microscopy (SEM), 3) to identify species Myxobolus sp. based on molecular study with PCR-RFLP method, and 4) phylogenetic tree based on sequencing in 28S and 18S rDNA region. Sampel was collected from fresh water fish of Central Java, East Java, Bali, Papua, Riau and West Kalimantan. Fresh water fish with unclosed operculum and high mortality in the juvenile was used as a sample. The spores of Myxobolus sp. from the cyst, usually attacked gill, brain, eye, and skin. The cyst were opened from morphological examination and stained with Lugol’s solution. The spores were examined by wet mounts preparation, fixed with 10% of formaline solution for histopathological examination, fixed with the glutaraldehyde for SEM, and with ethanol absolute solution for moleculer analyses. The DNA of Myxobolus sp. was ectracted and amplified in 28S and 18S rDNA region then analized with PCR-RFLP and rDNA sequencing. The morphological, SEM, and histopathological result were analized with descriptive method. The sequencing result was analized with Maximum Parsimony and Neighbour Joining Methods to get a phylogenic tre. Based on morphology, samples from 6 regions showed a small differences in shape and size of spores. The ultrastructure of spores showed the lining ridges with specific patterns (from Java, Pekanbaru and Merauke) and smooth ciliated surface (from Entikong and Bali) by using SEM. Restriction product with HaeIII, RsaI, and Bam showed a clearly differences among the samples except the samples from Bali and the Central Java. Based on the phylogenetic tree showed that samples from Merauke and East Java. Based on the phylogenetic tree showed that samples from Merauke and East Java were identical with M.koi, sample from Kediri (East Java) was identical with M.ampullicapsulatus, samples from Entikong and Bali were Myxobolus sp. Myxosoma cerebralis have not been found from the fresh water fish in Indonesia.

Kata Kunci : Myxobolus, morfologi, molekuler, phylogeny.