Multiomix Mikrobiota, Ekspresi Gen, dan Metabolit Usus Udang Vaname (Litopenaeus vannamei) Terkait P{ertumbuhan dan Imunitas

Abstract

Udang vaname (Litopenaeus vannamei) merupakan komoditas ekspor unggulan akuakultur. Keberlanjutan budidaya udang yang ramah lingkungan dengan produksi yang tinggi dapat diwujudkan melalui upaya peningkatan imunitas dan performa pertumbuhan udang vaname dengan pemanfaatan probiotik yang berbasis inang atau host-associated probiotic. Ekplorasi, skrining, dan uji aplikasi probiotik yang terintegrasi dengan pemanfaatan teknologi multiomik merupakan metode terbaru untuk mendapatkan probiotik yang lebih cepat dan akurat. Tujuan umum penelitian ini adalah untuk mengembangkan probiotik berbasis inang udang vaname sehat melalui pendekatan multiomik. Ruang lingkup penelitian ini terdiri dari lima tahap. Penelitian tahap pertama bertujuan untuk mengevaluasi profil metagenomik mikrobiota dan skrining kandidat probiotik baru dari sampel usus udang vaname sehat pada berbagai stadia. Metagenomik mikrobiota di usus udang pada stadia naupli, pascalarva-10 (PL-10), udang umur 40 hari, dan udang umur 80 hari dilakukan menggunakan penanda sekuen gen 16S rRNA daerah hipervariabel V3-V4. Jumlah sekuen yang terdeteksi menunjukkan total 54 filum dan sejumlah 34 dari 557 genus teridentifikasi sebagai “core intestine bacteria”. Metadata tersebut digunakan sebagai dasar skrining kandidat probiotik dan dilanjutkan sesuai analisis pengujian probiotik potensial yang meliputi uji patogenisitas, resistansi antibiotik, antibakteri Vibrio parahaemolyticus Vp5, aktivitas enzim amilase, lipase, protease, dan chitinase, serta morfologi bakteri menggunakan SEM (scanning electron microscope). Tiga isolat berhasil diidentifikasi sebagai novel kandidat indigenus probiotik yaitu Shewanella algae A3 (nomor aksesi GenBank OR915453), Shewanella algae A1 (nomor aksesi GenBank OR915455), dan Serratia marcescens Van80 UB3 (nomor aksesi GenBank PP411195). Penelitian tahap kedua bertujuan untuk mengevaluasi dosis lethal dose-50 (LD-50) dan efek perbedaan konsentrasi infeksi V. parahaemolyticus Vp5 pada udang vaname menggunakan pendekatan metabolomik usus udang. Udang vaname berukuran 2,00 ± 0,50 g diinfeksi dengan V. parahaemolyticus Vp5 dosis 103, 104, 105, dan 106 CFU/ml yang kemudian dianalisis LD-50, survival rate (SR), histologi usus dan hepatopankreas, dan profil metabolomik usus udang berdasarkan metode GC-MS (gas chromatography mass spectrometry). Bakteri V. parahaemolyticus Vp5 digunakan sebagai bakteri patogen dalam analisis performa imunitas dan konsentrasi yang digunakan adalah sesuai LD-50 yaitu 105 CFU/ml.Hasil analisis metabolomik menunjukkan bahwa V. parahaemolyticus Vp5 mengubah profil metabolit dimana udang sehat memiliki konsentrasi pentacosane dan octosane-1-iodo lebih tinggi dibandingkan udang sakit. Hepatopankreas dan usus mengalami kerusakan berdasarkan analisis histologi. Defferential metabolites (DMs) menunjukkan bahwa V. parahaemolyticus Vp5 menghambat sistem imun, metabolisme steroid, dan metabolisme kolesterol. Biomarker analisis menunjukkan bahwa eicosane dan tetracosane meningkat pada usus udang vaname yang terinfeksi akut. Pentacosane, octacosane-1-iodo, eicosane, dan tetracosane merupakan biomarker penting di usus udang vaname yang berasosiasi dengan infeksi V. parahaemolyticus Vp5 berdasarkan pendekatan metabolomik. Penelitian tahap ketiga bertujuan mengevaluasi efek pemberian pakan dengan suplementasi kandidat indigenus probiotik pada respons imun, ekspresi gen, histologi, dan ketahanan udang vaname terhadap bakteri patogen V. parahaemolyticus Vp5. Udang sehat (2,00 ± 0,50 g) digunakan dalam penelitian ini dan metode pemberian probiotik melalui pakan yang diberikan selama 30 hari. Lima perlakuan menggunakan 150 ekor udang yang meliputi grup K (+) diinfeksi dan tanpa probiotik, grup K (-) tidak diinfeksi dan tanpa probiotik, grup A diinfeksi dan pakan probiotik S. algae A3, grup B diinfeksi dan pakan probiotik Serratia marcescens Van80 UB3, dan grup C diinfeksi dan pakan probiotik campuran S. algae A3 dan Serratia marcescens Van80 UB3 masing-masing dengan dosis 106 CFU/g. Udang vaname yang diberi campuran pakan indigenus probiotik S. algae A3 dan Serratia marcescens Van80 UB3 menunjukkan nilai sintasan tertinggi, level ekspresi gen proPO dan serine protease pada organ usus, otot, dan hepatopankreas meningkat signifikan, level ekspresi gen cathepsin-L terkait pertumbuhan otot menurun, respons imun meningkat, dan histologi hepatopankreas dan usus menunjukkan bahwa suplementasi probiotik tidak merusak jaringan. Suplementasi indigenus probiotik dengan campuran S. algae A3 dan Serratia marcescens Van80 UB3 dosis 106 CFU/g mampu meningkatkan proteksi terhadap V. parahaemolyticus Vp5. Penelitian tahap keempat bertujuan untuk mengevaluasi performa pertumbuhan, imunitas, ekspresi gen, dan aktivitas enzim pencernaan udang vaname yang diberikan pakan dengan suplementasi kandidat indigenus probiotik. Sebanyak 600 ekor udang pascalarva-10 (PL-10) digunakan dalam empat perlakuan yaitu grup A (S. algae A3), grup B (Serratia marcescens Van80 UB3), grup C (campuran bakteri S. algae A3 dan Serratia marcescens Van80 UB3) masing-masing dengan dosis 106 CFU/g pakan dan grup kontrol. Hasil penelitian menunjukkan bahwa performa pertumbuhan, aktivitas enzim pencernaan, level ekspresi gen IGF-1, ecdysteroid, dan myosin di usus, hepatopankreas, dan otot di grup C lebih tinggi dibandingkan kontrol. Penelitian ini merekomendasikan suplementasi campuran S. algae A3 dan Serratia marcescens Van80 UB3 dengan dosis 106 CFU/g pakan dapat meningkatkan performa pertumbuhan dan imunitas skala laboratorium. Penelitian tahap kelima bertujuan untuk mengevaluasi parameter produksi dan pendekatan metabolomik pada pemberian pakan dengan suplementasi kandidat indigenus probiotik S. algae A3 dan Serratia marcescens Van80 UB3 selama 30 hari di udang vaname skala lapangan. Sebanyak 200 ekor udang (bobot: 3 ± 0,33 g) digunakan pada grup P (probiotik dengan perbandingan setara 106 CFU/g S. algae A3 dan Serratia marcescens Van80 UB3) dan grup K (tanpa pakan probiotik) dengan lima ulangan dan dipelihara selama 90 hari yang kemudian dilakukan analisis metabolomik usus udang dengan metode LC-HRMS (liquid chromatography high resolution mass spectrometry), performa pertumbuhan, dan respon imun. Udang vaname dengan perlakuan probiotik memiliki nilai sintasan lebih tinggi yaitu 78,67 ± 3,81 % secara signifikan dibandingkan kontrol 23,50 ± 4,82 %. Parameter respons imun dan total biomassa panen lebih tinggi di grup P dibandingkan dengan grup K. Sebanyak 52 metabolit berhasil diidentifikasi dari sampel usus udang vaname. Berdasarkan biomarker analisis diperoleh metabolit spinganine, DL-stachydrine, dan DL-ß-Leucine mengalami peningkatan kelimpahan dan metabolit linoleamide turun pada grup P. Perlakuan pakan probiotik mempengaruhi tiga jalur yaitu metabolisme glycine, serine, dan threonine, metabolisme pirimidin, dan biosintesis unsaturated fatty acids. Hasil penelitian menunjukkan bahwa pemberian pakan indigenus probiotik di udang vaname dapat meningkatkan produktivitas udang. Hasil penelitian ini menunjukkan bahwa indigenus probiotik Shewanella algae A3 dan Serratia marcescens Van80 UB3 yang diperoleh dari tahapan ekplorasi, skrining, dan uji aplikasi probiotik yang terintegrasi dengan pemanfaatan teknologi multiomik yaitu metagenomik dam metabolomik terbukti mampu meningkatkan respons imun, performa pertumbuhan, dan produktivitas udang vaname.

Pacific white shrimp (Litopenaeus vannamei) is Indonesia’s famous aquaculture export commodity. The high production, sustainability, and eco-friendly shrimp culture can be rised by increasing the immune response and growth performance using a host-associated probiotics approach. The exploration, screening, and application of probiotics based on multi-omic technology is a new signature for finding novel probiotics more accurately and faster. This study aims to develop host-associated probiotics from healthy shrimp using multiomics approaches. This study was conducted in five stages. The first stage aims to evaluate metagenomic microbiota profiling and screening approach of potential indigenous probiotics from the healthy shrimp intestine in different stages. Metagenomics microbiota in the shrimp intestine is conducted using nauplii, post larval-10, juvenile (40 days), and adult (80 days) using 16S rRNA gene V3-V4 hypervariable region. Fifty-four phylum was indentified, 34 genera from 557 genera were detected as core intestine bacteria. The metadata was used as a database for screening probiotics candidates and continued for probiotics assessment methods such as patogenicity test, antibiotic resistance, anti bacterial against Vibrio parahaemolyticus Vp5, amylase, lipase, protease, and chitinase enzyme activity, and bacterial morphology using SEM (scanning electron microscope). Three isolates was identified as novel indigenous probiotics candidates such as Shewanella algae A3 (accession number of GenBank OR915453), Shewanella algae A1 (accession number of GenBank OR915455), and Serratia marcescens Van80 UB3 (accession number of GenBank PP411195). The second stage of this study aimed to evaluate the metabolomics profile of shrimp intestine after different concentrations of V. parahaemolyticus Vp5 challenges. Healthy cultured shrimp weighing 2 ± 0,50 g were challenged using the intramuscular method with four different concentrations (103, 104, 105, and 106 CFU/ml) of V. parahaemolyticus Vp5 and then subjected to non-targeted volatile metabolite analysis via gas chromatography-mass spectrometry (GC-MS). Among the five groups tested, the three exposed to higher concentrations of V. parahaemolyticus Vp5 (104, 105, and 106 CFU/ml) showed significant mortality rates (P<0,05). Sixty-two metabolite compounds were identified from the intestine samples. Metabolomic analysis revealed that V. parahaemolyticus Vp5 altered the metabolite profile. The healthy shrimp had a significantly greater concentration of pentacosane and octacosane-1-iodo than the infected shrimp. Pentacosane and octacosane-1-iodo were lost in abundance in acute infection with V. parahemolyticus Vp5. The hepatopancreas and intestine were damaged based on histopathological examination. The differential metabolites (DMs) exhibit that this patogen bacteria disrupt the immune system, steroid metabolism, and cholesterol metabolism. Our findings showed that based on biomarker analysis, metabolites eicosane and tetracosane were increased in acute infection of V. parahaemolyticus Vp5. Pentacosane, octacosane-1-iodo, eicosane and tetracosane were represented as putative important intestine biomarkers associated with V. parahaemolyticus Vp5 infection in shrimp. The third stage aimed to evaluate the effect of indigenous probiotics S. algae A3 and Serratia marcescens Van80 UB3 on immunity, histopathology, gene expression, and its protection against patogen bacteria AHPND-caused strain V. parahaemolyticus Vp5 of Pacific white shrimp (2 ± 0,50 g) by a 30-day feeding experiment. Five treatment groups using 150 shrimp of post larvae-10 (PL-10) including 106 CFU/g single indigenous probiotic A (S. algae A3), 106 CFU/g single indigenous probiotic B (Serratia marcescens Van80 UB3), 106 CFU/g multi indigenous probiotic C (mix equal portion of S. algae A3 and Serratia marcescens Van80 UB3), common feeding without infection (positive control), and common feeding (negative control) were designed. After 30 days of feeding trial, the shrimp were challenge with V. parahaemolyticus Vp5 and survival percentage was recorded for 14 days. As a result, Pacific white shrimp showed a significant higher survival rate, enhance gene expression-related immunity in the intestine, muscle, and hepatopancreas including proPO and serine protease, and increased gene expression of cathepsine L- related muscle in C group. Immune response parameters after 14-day challenge was higher in multi indigenous probiotic C group compared to the other groups. Histological analysis of the intestine and hepatopancreas indicated that probiotic supplement treatment has better conditions of normal tissue than the negative control. Finally, the results strongly recommended enriched Pacific white shrimp diets with 106 living bacteria in mix equal to the portion of S. algae A3 and Serratia marcescens Van80 UB3 per gram of diet. The fourth stage aimed to evaluate the effect of probiotic fed on growth performance. Four treatment groups using 600 shrimp of post larvae-10 (PL-10) including 106 CFU/g single indigenous probiotic A (S. algae A3), 106 CFU/g single indigenous probiotic B (Serratia marcescens Van80 UB3), 106 CFU/g multi indigenous probiotic C (mix of S. algae A3 and Serratia marcescens Van80 UB3), and common feeding (control) were designed. The Pacific white shrimp exhibited improved growth, enhance digestive capacity, normal histology, increased expression of growth-related genes, and stronger immunity when fed indigenous probiotics. Relative gene expression of IGF-1, ecdysteroid, and myosin in the intestines, hepatopancreas, and muscles in group C was higher than in the control group. Finally, the results strongly recommended enriched Pacific white shrimp diets with 106 living bacteria mix indigenous probiotics per gram of diet to enhance shrimp growth performance and immunity. The fifth stage evaluated the effect of indigenous probiotics S. algae A3 and Serratia marcescens Van80 UB3 on metabolomics and parameters production of Pacific white shrimp by a 30-day feeding experiment and 30-day maintenance in the semi-field trial. Five treatment groups using 200 shrimp (BW: 3 ± 0,33 g) including 106 CFU/g indigenous probiotic (mix equal portion of S. algae A3 and Serratia marcescens Van80 UB3) (P group) and common feeding without probiotics diet as control (K group) were designed. As a result, Pacific white shrimp showed a significant higher survival rate, enhance immune response, and increase total biomass of harvest in P group compare to the K group. Fifty-two metabolites compounds were identified from the intestine samples. Based on biomarker analysis, metabolites spinganine, DL-Stachydrine, and DL-ß-Leucine were increased and metabolites linoleamide was decreased in diets probiotics group. The probiotics diet are interfere the three pathways including glycine, serine, and threonine metabolism, pyrimidine metabolism, and biosynthesis of unsaturated fatty acids. Finally, the results proved that indigenous probiotics feeding enriched Pacific white shrimp diets could raise shrimp productivity. The study concluded that indigenous probiotics Shewanella algae A3 and Serratia marcescens Van80 UB3, resulting from exploration, screening, and application with multi-omics approach, can enhance immune response, growth performance, and shrimp culture productivity.