Saprolegniasis pada Ikan Gurami (Osphronemus goramy Lacapede 1801) di Pulau Jawa // Saprolegniasis in Gourami Fish (Osphronemus goramy Lacapede 1801) in Java Island

Siti Mukhlishoh Setyawatisi, Promotor: Prof. Rina Sri Kasiamdari, S.Si., M.Si, ; Co-promotor 1: Prof. Dr. Bambang Retnoaji M.Sc. ; Co-promotor 2: Prof. drh. Kurniasih, M.V.Sp. Ph.D.

2024 | Disertasi | S3 Biologi

Intisari

Ikan gurami (Osphronemus goramy Lac.) adalah salah satu komoditas utama ikan indigenous Indonesia yang mudah terinfeksi saprolegniasis di setiap tahap pertumbuhannya. Penyakit ini berresiko pada kematian ikan, sehingga diperlukan kajian secara komperhensif. Penelitian ini bertujuan mengkaji saprolegniasis pada ikan gurami berdasarkan prevalensi saprolegniasis di lapangan, taksonomi dan filogeni jamur penyebab saprolegniasis, karakteristik infeksi dan respons ikan sebagai inang. Kejadian saprolegniasis dan miselium jamur dikoleksi dari ikan gurami budidaya di Jawa. Miselium diisolasi dan dikultur pada media glucose yeast-extract, air steril dan air steril-biji Cannabis. Karakterisasi dan identifikasi morfologi jamur berdasarkan struktur aseksual dan seksualnya. Identifikasi molekuler dan analisis filogentik berdasarkan sekuen Internal Transcribed Spacer Ribosomal Deoxyribonucleic Acid (ITS rDNA) jamur yang disekuen dengan metode Sanger. Konstruksi pohon filogenetik dengan UPGMA, neighbor-joining, dan maximum likelihood. Karakteristik infeksi dan respons inang dianalisis berdasarkan gejala patologis, patogenisitas (persen infeksi dan survival rate), patologi integumen, hitung leukosit, dan protein mukus kulit ikan. Sediaan integumen ikan disiapkan dengan metode Bancroft dan jaringan darah dengan apus. Protein mukus dianalisis dengan SDS-PAGE dan Liquid Chromatography High-Resolution Mass Spectrometry (LC-HRMS). Hasil pengukuran prevalensi saprolegniasis ikan gurami di pasar ikan 4%-50%, dan di kolam budidaya 0%-20%. Isolat jamur diidentifikasi sebagai Newbya (syn. Achlya) berdasarkan tipe pengeluaran spora achlyoid, dan oospora subsentrik atau sentrik. Seluruh isolat monoecious, hifa senositik, spora monomorfik, oogonium berdinding halus, multioospora, membentuk gemma. Isolat kelompok-1 oosporanya subsentrik, anteridiumnya diclinous dan diclinous-hypogynous. Isolat kelompok-2 oosporanya subsentrik dan sentrik, anteridiumnya diclinous. Sekuen ITSrDNA isolat kelompok-1 tidak identik dengan Achlya lain yang memiliki nama penunjuk spesies (identity <94>cluster terpisah. Isolat kelompok-2 teridentifikasi sebagai Newbya oblongata (syn. Achlya oblongata) (identity 99,84%) mengelompok dengan N. oblongata LC149928.1. Uji patogenisitas menunjukkan 100% ikan gurami yang dipapar zoospora Achlya sp.5 terinfeksi. Infeksi ditandai tumbuhnya miselium cottony di permukaan integumen hingga lapisan dalam hipodermis disertai lesi kemerahan, nekrosis, ulserasi, kerusakan sirip di sekitar miselium, respons peradangan (berdasar gambaran patologi anatomi-histologis integumen dan apus darah), gangguan pergerakan dan keseimbangan ikan. Gangguan mukus kulit meningkatkan luasan infeksi dan menurunkan survival rate (SR 0% untuk infeksi dengan ami-momi dan 44,44% tanpa ami momi). Hasil SDS-PAGE menunjukkan deskripsi protein mukus kulit ikan terinfeksi dan non-infeksi serupa, tetapi protein 127kDa tidak terdeteksi pada mukus ikan terinfeksi. Uji LC-HRMS mukus kulit ikan gurami, mendeteksi protein terkait respons fisiologis (?-actin, phosphotransferase, Cytochrome-C-Oxidase-subunit-2, GAPDH (phosphorylating), Sorting-nexin-33, Zic-Family-member1, dan V(D)J-recombination-activating protein-1) serta protein terkait respons imun (IL-1, TLR-3, dan TLR-22).  Kesimpulannya, prevalensi saprolegniasis gurami di pasar ikan lebih tinggi dibandingkan kolam budidaya. Jamur yang menginfeksi teridentifikasi secara morfologi, molekuler, dan filogenetik sebagai Newbya sp (Achlya sp.) yang berpotensi sebagai spesies baru dan N. oblongata (syn A. oblongata). Newbya sp (Achlya sp.) patogen pada gurami, gejala patologis berupa tumbuhnya miselium cottony di permukaan integumen hingga hipodermis, nekrosis, ulserasi, kerusakan sirip disekitar miselium, respons peradangan, gangguan pergerakan dan keseimbangan ikan. Mukus kulit penting dalam perlindungan infeksi, respons fisiologis, dan imunologis ikan gurami sebagai inang. 

Kata kunci: saprolegniasis, Achlya, Newbya, molekuler, filogeni, ikan gurami, patologi

Abstract

Gourami (Osphronemus goramy Lac.) was one of the main commodities of indigenous freshwater fish in Indonesia, which were easily infected by saprolegniasis at all stages of their life, so a comprehensive study is needed for infection control. This research aims to examine saprolegniasis in gourami based on the prevalence of the field’s saprolegniasis, the taxonomy of saprolegniasis fungal agent based on morphological, molecular, and phylogenetic characteristics, characteristics of infection, and response of gourami as a host. The occurrence of saprolegniasis and fungal mycelium were collected from three main areas of gourami cultivation in Java. Mycelium was isolated and cultured on glucose yeast extract, sterile water, and sterile water-cannabis seeds. Characterization and identification of fungal morphology based on its asexual and sexual structures. Molecular identification and phylogenetic analysis based on sequences of Internal Transcribed Spacer Ribosomal Deoxyribonucleic Acid (ITS rDNA) of fungi sequenced by the Sanger method. Phylogenetic tree construction with neighbor-joining, and maximum likelihood. Characteristics of infection and host response were analyzed based on pathological symptoms, percent infection, survival rate, integument pathology, leukocyte count, and fish skin mucus protein. Fish were infected with zoospores through the immersion method. Fish integument preparation was prepared by Bancroft’s method and blood tissue by smear. Mucus protein was analyzed in SDS-PAGE and Liquid Chromatography High-Resolution Mass-Spectrometry (LC-HRMS). 

The results showed the prevalence of gourami’s saprolegniasis 4%-50% in fish markets, and 0%-20% in cultivation ponds. Fungal isolates were identified as Newbya (syn. Achlya) based on the type of achlyoid spore release, and subcentric or centric oospores. All isolates were monoecious, coenocytic hyphae, monomorphic spores, smooth-walled oogonium, multi-oospores, and formed gemma. Group-1 isolates have subcentric oospores, diclinous and diclinous-hypogynous antheridia (this character has never been reported). Group-2 isolates have subcentric and centric oospores, and diclinous antheridia only. The ITS rDNA sequences of group-1 isolates were not identical to other Achlya with specific epithet names (identity <94>Newbya oblongata (syn. Achlya oblongata) (identity 99.84%) and clustered with N. oblongata LC149928.1. 100% of gourami fish exposed to Achlya sp.5 zoospores were infected on the second day. Infected fish characterized by the growth of cottony mycelium on the surface of the integument to the inner layer of the hypodermis accompanied by reddish lesions, necrosis, ulceration, damage to the fins around the mycelium, inflammatory response (based on the pathological anatomy-histology of the integument and blood smears), impaired movement and balance of the fish. Disruption of skin mucus increases the extent of infection and decreases the survival rate (SR 0% for infection with ami-momi and 44.44% without ami-momi). The SDS-PAGE results showed that the description of the skin mucus protein of infected and non-infected fish was similar, but a protein with 127kDa of molecular weight was not detected in the infected fish. LC-HRMS test of gourami skin mucus, detecting proteins related to physiological responses (?-actin, phosphotransferase, Cytochrome-C-Oxidase-subunit-2, GAPDH (phosphorylating), Sorting-nexin-33, Zic-Family member1, and V(D)J-recombination-activating protein-1) and proteins related to immune responses (IL-1, TLR-3, and TLR-22). In conclusion, the prevalence of gourami saprolegniasis in fish markets was higher than in aquaculture ponds. The infecting fungi were identified morphologically, molecularly, and phylogenetically as Newbya sp (Achlya sp.), which can potentially be a new species, and N. oblongata (syn A. oblongata). Newbya sp (Achlya sp.) was a pathogen in gourami, pathological symptoms include the growth of cottony mycelium on the surface of the integument to the hypodermis, necrosis, ulceration, damage to the fins around the mycelium, inflammatory response, impaired movement and balance of the fish. Skin mucus is important in protecting against infection, physiological, and immunological responses of gourami as a host. 

Keywords: saprolegniasis, Achlya, Newbya, molecular, phylogeny, gourami fish, pathology

Kata Kunci : Kata kunci: saprolegniasis, Achlya, Newbya, molekuler, filogeni, ikan gurami, patologi // Keywords: saprolegniasis, Achlya, Newbya, molecular, phylogeny, gourami fish, pathology