STUDI PERBANDINGAN MORFOLOGI DAN MOLEKULER Clinostomum complanatum (DIGENEA: CLINOSTOMIDAE) PADA IKAN AIR TAWAR DI RIAU DAN YOGYAKARTA

Ir.Morina RIauwaty S, Dipl.Biol.M.P, Prof. drh. Kurniasih, MV.Sc., Ph.D.

2011 | Disertasi | S3 Sain Veteriner

Clinostomiasis adalah salah satu penyakit yang disebabkan oleh trematoda digenea Clinostomum complanatum atau lebih dikenal dengan “yellow grub”. Parasit ini bersifat zoonotik yang menyebabkan penyakit “laryngo-pharyngitis” dan dapat mengakibatkan kematian karena terjadi Asphyxia pada manusia. Infeksi Clinostomum complanatum pada ikan air tawar di Indonesia mengakibatkan kematian ikan dan kerugian ekonomi. Penelitian ini bertujuan untuk mengetahui perbedaan morfologi dan molekuler Clinostomum complanatum (Digenea: Clinostomidae) pada ikan air tawar di Riau dan Yogyakarta. Sampel ikan sepat (Trichogaster trichopterus) diperoleh dari sungai Sail, Riau, sedangkan ikan betok (Anabas testudineus) ditangkap di Kali Progo, Yogyakarta. Metaserkaria Clinostomum sp. yang ditemukan di rongga dada dan rongga perut, diambil dengan menggunakan jarum, diawetkan dalam formalin 10% dan etanol absolut. Pemeriksaan morfologi dengan pewarnaan Semichon’s acetocarmine, Scanning Electron Microscope (SEM). Pemeriksaan molekuler dengan metode Polymerase Chain Reaction (PCR) yang terdiri dari ekstraksi, amplifikasi, PCR-RFLP, elektroforesis dan sekuensing. Data yang diperoleh dari hasil sekuensing dianalisis dengan metode maximum parsimony dan neighbour-joining. Jarak genetik dihitung menggunakan model parameter Kimura. Data yang diperoleh dari hasil PCR-RFLP dianalisis secara deskriptif. Hasil identifikasi morfologi dan Scanning Electron Microscope (SEM) diketahui bahwa metaserkaria Clinostomum sp. asal Riau merupakan spesies Clinostomum baru, sedangkan metaserkaria Clinostomum sp. asal Yogyakarta adalah Clinostomum complanatum. Hasil PCR rDNA metaserkaria Clinostomum sp. pada ITS region mengandung band yang jelas pada 1300 bp. Hasil PCR-RFLP dengan enzim restriksi PstI dan AluI menunjukkan restriksisitas yang berbeda dari kelima Clinostomum sp, namun dengan enzim RsaI dan HaeIII tidak dapat mendigesti dengan baik. Penelitian menyimpulkan bahwa terdapat variasi intraspesifik antara metaserkaria Clinostomum sp. dari Riau dan Yogyakarta. Hasil analisis filogenetik diketahui metaserkaria Clinostomum sp. asal Yogyakarta identik secara molekuler dan satu klaster dengan metaserkaria Clinostomum complanatum. Clinostomum sp. asal Riau diduga spesies baru yang satu klaster dengan Clinostomum phalacrocorasis (perbedaan > 2%).

Clinostomiasis is one of diseases caused by digenea trematoda Clinostomum complanatum, known well as \"yellow grubs\". This is a zoonotic parasite and causes the \"laryngo-pharyngitis\", and death caused by asphyxia in humans. Clinostomum complanatum infection of freshwater fish in Indonesia causes high fish mortality and economic losses. The objectives of research were a) to identify and to compare the morphological and molecular characteristics of C. complanatum (Digenea: Clinostomidae) infecting freshwater fishes in Riau and Yogyakarta, and b) to find out their strain variation. Climbing perch (Trichogaster trichopterus) infected by Clinostomum sp. were caught from Sail River of Riau, while climbing gouramy (Anabas testudineus) caught from Kali Progo, Yogyakarta. Excysted Clinostomum sp. was found in the body cavity and and visceral organ, were aseptically taken using needle, preserved in 10% formalin and absolute ethanol solution. Morphological examination was done with Semichon’s acetocarmine staining, Scanning Electron Microscope (SEM). Molecular examination performed by Polymerase Chain Reaction (PCR) method consisting of extraction, amplification in the ITS region, PCR-RFLP, electrophoresis and sequencing. Data obtained from sequencing results were analysed by maximum parsimony and neighbour-joining method. Genetic distances were calculated using Kimura parameter model. Based on morphological observation and Scanning Electron Microscope (SEM) the worm from Riau was categorized as a new species, whereas worm from Yogyakarta was identified as Clinostomum complanatum. The PCR rDNA product of Clinostomum sp. at ITS region had a clear band of 1300 bp. Product of RFLP using PstI and AluI restriction enzyme showed different site among the five worms, however, the RsaI and HaeIII enzyme could not digest well. There was intraspecific variation in Clinostomum sp. metacercariae in Riau and Yogyakarta. Phylogenetic analysis showed that Clinostomum sp. from Yogyakarta identic to Clinostomum complanatum, whereas Clinostomum sp. from Riau were suspected as a new species (difference > 2%) which is included in one cluster with Clinostomum phalacrocorasis.

Kata Kunci : Clinostomum complanatum, PCR, RFLP, Filogenetik, Riau, Yogyakarta