Detection Moleculer of Putative 18 S rRNA Gen Protozoa Trichodina sp . Infected Larvae Gurami ( Osphronemus gouramy L ) in Balai Benih Ikan Kutasari Purbalingga Central Java

Diterima : 19/02/2021 Disetujui : 10/04/2021 Abstract Trichodina spp. are ectoparasitic pathogens of ciliata group that commonly infect both freshwater and marine fish, including gouramy fish. As a result of infection of Trichodina spp. this will lead to inhibition of fish growth and decreased fish production, resulting in low fish selling value. The rate of occurrence of Trichodina spp. that infects gurami can reach 100%. Research has been conducted to determine which one Trichodina spp. Protozoa that infects the gouramy seeds of BBI (Fish Seed Center) Kutasari Purbalingga following detection of 18S RNA gene. Gene detection method used in this research is Polymerase Chain Reaction (PCR) is a technique of DNA synthesis and amplification in vitro. This research is done following these methodes: (1) sampling of Gurami fish with purposive sampling which obtained from BBI Kutasari Purbalingga, (2) isolation of Trichodina spp., (3). Preparation of Trichodina spp. sample and its identification, and (4). Molecular character obervation following detection of 18S rRNA gene. This study obtained 10% percentage of detection of 18S rRNA genes of the species of Trichodina paraheterodentata that infect on the gouramy fish of Purbalingga. The percentage rate of detection of these genes is low when compared with the results of the detection of 18S rRNA Trichodina paraheterodentata gene that infects gouramy fish in Banjarnegara.


INTRODUCTION
Trichodina spp. is a pathogenic ectoparasite from the ciliate group that commonly attacks freshwater and marine fish (Margolis & Arthur, 1979). This ectoparasite is ± 50 nm in size, round in shape, with a bell-shaped lateral side, has a denticle ring for attachment and has cilia around its body. Ectoparasites are known to cause mortality of goldfish (Cyprinus carpio), Mujair fish (Oreochromis mossambicus) in South Africa (Van et al., 1984;Dana et al., 2002), Rainbow trout (Salmo gairdneri) and Salmon (Salmo salar) by attacking or infecting the skin and gills of fish in the UK (Mc Ardle, 1984in Dana et al., 2002. One type of fish is often infected by Trichodina spp. is gouramy. The disadvantages of ectoparasite infection are not as big as those caused by infection with other organisms such as viruses and bacteria, but ectoparasite infection can be one of the predisposing factors for infection with more dangerous pathogenic organisms (Rokhmani, 2009).
Gouramy fish (Osphronemus gouramy Lacepede) is a type of freshwater fish from the Osphronemidae family and one of the types of freshwater cultivated fish that is widely developed by farmers. Gouramy as a cultivated fish has been chosen by many farmers because it is able to reproduce naturally and is relatively easy in its cultivation. Meanwhile, as a consumption fish, gouramy is quite popular because of its delicious and savory taste. Market demand for gouramy continues to increase at a high enough price. Economically, the selling price of gouramy is relatively stable (Tim Lentera, 2003). Purbalingga is one of the districts that has gouramy production as a regional commodity. This is evidence that this district is one of the centers for gouramy cultivation in the province of Central Java.
Morphological characters are commonly used for taxonomic studies of a species because different variations between individuals often indicate different species (Darwin, 1854;Pilsbry, 1916;Permadi, 2014). (Syoc, 2010) noted that Darwin saw species that varied morphologically as variations within a species to encourage speciation. Investigations down to the genetic level can help in distinguishing 2 species that cannot be resolved morphologically. Morphological parameters of Trichodina spp. including body diameter, denticle ring diameter, adhesive disc diameter, number of denticles and membrane width of Trichodina spp. The problem that arises is the large number of biased species (cryptic species) and this problem can be solved using molecular characters, however to identify an organism using molecular techniques has not been widely used. Several molecular techniques have been developed to trace the presence of a specific DNA sequence from a particular organism, for example the use of the 18S rRNA gene sequence to determine the relationship of an organism in general morphological features. The 18S rRNA gene is often used for phylogenetic studies because it has conserved areas. The unique and conserved region can be used to characterize the organism concerned, so that it becomes the signature sequence of an organism. Likewise, the data on the nucleotide base of the 18S rRNA gene allows it to be used in constructing a phylogenetic tree that shows the ancestry and kinship of an organism (Suwanto, 2011). Molecular research can complement the morphological identification of organisms. Partial amplification of the 18S rRNA gene can be used to specifically identify Trichodina paraheterodenta (Tang et al., 2013).
Research Problems are what are the morphological characters of Trichodina spp. morphometrically isolated from gouramy at BBI Kutasari Purbalingga and What is that infected gouramy larvae at BBI Kutasari Purbalingga using 18S RNA gene detection. The objectives to detect the Trichodina spp. 18S RNA gene. which infected larvae gouramy. Benefit providing information on the results of the detection of the putative 18S RNA gene of Trichodina sp. which infected larvae gouramy
The implementation of this research was carried out from May to July 2017. Sampling of the research will be carried out at the Kutasari Purbalingga Fish Seed Center. Research observations will be carried out at the Parasitology Laboratory and the Genetics and Molecular Laboratory, Faculty of Biology, Jenderal Soedirman University.

Sampling Technique
This research will be conducted using a survey method with purposive sampling technique. The gouramy which was taken from BBI Kutasari Purbalingga was put into plastic container then taken to the Laboratory of Parasitology, isolated and taken ectoparasite species Trichodina spp. only, whose characteristics are known by reference. Sampling was done once. Samples were taken in genomic DNA isolation at the Molecular Laboratory of the Faculty of Biology, Jenderal Soedirman University and carried out DNA extraction and PCR. Trichodina was isolated from the caudal fin of Gurami (Osphronemus goramy) to obtain its DNA through the extraction method, DNA amplification using PCR (Polymerase Chain Reaction) techniques using universal primers. Then, DNA Visualization uses electrophoresis techniques.

Molecular Identification
Genomic DNA Isolation of Trichodina sp.DNA Trichodina sp. extracted from the gouramy fin tissue. DNA extraction method with working steps: 1. Pellets (Trichodina sp.) are added 600 µl of Nuclei lysis Solution into tube 1.5 (homogeneous for 10 seconds) 2. 3 µl of RNAse was added to the tissue which had been given Nuclei Lysis solution after that it was homogenized 3. Incubate in a water bath at 37 ° C for 5-10 minutes 4. Added 200 µl of Protein Prespitation Solution 5. Vortex for 10 seconds 6. Incubate in the refrigerator at -20 ° C for 5 minutes 7. Centrifugation at 4 ° C at 13,000 rpm for 4 minutes 8. The supernata at the top was taken and then transferred into a new 1.5 tube 9. Add 600µl of isopropanol to the tube that has been moved (homogeneous by means of back and forth) 10. Centrifugation 13,000 for 1 minute at 4 ° C 11. Supernata at the top are removed 12. The supernata below are added with 600 µl of 70% Ethanol (homogenized) 13. Centrifuge at 4 ° C 13,000 rpm for 1 minute 14. Ethanol removed and dried for 30 minutes 15. Add 100 µl of DNA Rehydration solution then incubate at 20 ° C (over night)

Amplification of the 18S rRNA gene using PCR technique
The amplification of the 18S rRNA gene from Trichodina paraheterodentata was carried out using PCR techniques. The primers used were Forward primers (5 '-AAC CTG GTT GAT CCT GCC ATG-3') and Reverse primers (5 '-TGA TCC TTC TGC AGG TTC ACC TAC-3') which produced 600 bp DNA amplicons (Tang et al., 2013). The gene amplification procedure uses the PCR technique with the following steps: 1. Preparation of PCR mixture (PCR mix) in a volume of 15 µl in an eppendorf tube (1.5 ml) with the composition in Table 1.

Data Analyses
Positivity of the DNA amplicon of 18S rRNA gene from the study sample

RESULT AND DISCUS
The morphology of the body parts of Trichodina paraheterodentata shows several features and varying shapes of denticles, namely having a blunt blade apex tip and a straight tapered but thicker denticle ray shape. Trichodina paraheterodentata research results were found in gouramy seeds on the tail fin. According to Basson & Van As (2006), Trichodina paraheterodentata is found mostly on the skin surface and is rarely found in gills. Meanwhile, research by Rokhmani et al. (2015) (Rokhmani et al., 2015).
Trichodina spp. are spherical and supported by a rigid ring of interconnected discs called chitinoids (Durborow, 2003). The ability to infect Trichodina spp., In fish seeds is related to the structure and morphology of the denticle ring of Trichodina spp. (Kabata, 1985). The structure and morphology of the denticle rings are the morphometric characters of Trichodina spp. which can be used to determine the type of Trichodina spp. One of the morphometric size characters of Trichodina spp. in the form of a denticle tip. The denticle tips of Trichodina spp. There are two types, namely sharp and blunt (Windarto et al., 2013).
The results Amplification of the 18S RNA gene in Trichodina sp. from Purbalingga was done using the PCR method. The interpretation can be seen in Figures 1 to 4. Each band on the gel indicates the presence of Trichodina paraheterodentata.    The results of DNA isolation visualized on agarose gels in Figures 1 and 4 produced 19 DNA bands measuring 250 -500 bp, all DNA bands showed the presence of Trichodina paraheterodentata in gouramy taken from BBI Kutasari Purbalingga. The entire DNA band is contaminated (smear), appears due to the presence of RNA contaminants or nuclease. The intact DNA is characterized by the absence of an electrophoretic DNA smear. This is important because in the PCR process, the intact DNA will provide relatively more accurate results (Syafaruddin & Tri, 2011).
Detection of the 18S rRNA gene in Trichodina spp. 10% Trichodina paraheterodentata is found in gouramy seeds belonging to BBI Strongasari Purbalingga. This result is low, when compared to the research of Rokhmani (2017) which detected the 18S rRNA gene in gouramy seeds belonging to Banjarnegara Rakit fish farmers, which was 45%. Based on the environmental conditions between the two sampling locations, BBI Kutasari Purbalingga is quite good, while in the Rakit Banjarnegara fish farmer the environment is less or bad enough. Observations on the maintenance of gouramy seeds at BBI Kutasari Purbalingga show that the aquatic environment is good enough so that it does not support parasites to breed. According to Syafaruddin & Tri (2011), Trichodina spp. can reproduce rapidly, has a wide spread, and is a parasite commonly found in freshwater fish and can infect various types of fish. Incidence of infection with Trichodina sp. The host is influenced by water quality and poor pond maintenance techniques, namely high stocking density and calm, inundated and non-flowing ponds. The high stocking density will cause the fish to come into contact with each other so that parasites will be easily transmitted to other fish.

CONCLUSION
Based on the results and discussion that has been described, it can be concluded that, The morphological description of Trichodina spp. identified was Trichodina paraheterodentata and the detection percentage of the 18S rRNA gene for the protozoan species Trichodina paraheterodentata that infected the gouramy seeds from Purbalingga was 10%. The percentage of detection of this gene was low when compared to the results of the detection of the 18S rRNA Trichodina paraheterodentata gene that infected Banjarnegara gouramy seeds (45%).

ACKNOWLEDGEMENT
We would like to deliver our greatest gratitude, especiallyfor the Dean of Biology Faculty, as well as the head of entomology and parasitology including my staffs and students who provide assistance and contributions in this study. We would also like to thank the Head of Unsoed Reseach Centre for providing funds through the scheme of Competence Research BLU Unsoed No : 1248/UN23.14/PN.01.00/ 2017, February 3, 2017.