32 research outputs found
ANALISIS RASIO RNA/DNA UDANG WINDU Penaeus monodon HASIL SELEKSI TUMBUH CEPAT Andi
No full textSeleksi udang windu Penaeus monodon tumbuh cepat menggunakan marker DNA telah berhasil dilakukan dalam upaya perakitan strain unggul udang windu. Udang windu hasil seleksi menunjukkan adanya peningkatan pertumbuhan dibandingkan dengan tanpa seleksi (kontrol). Rasio RNA/DNA merupakan salah satu parameter yang telah banyak digunakan dalam menentukan kualitas ikan/udang di antaranya adalah pertumbuhan. Penelitian ini bertujuan untuk mengetahui rasio RNA/DNA pada udang windu hasil seleksi tumbuh cepat dan kontrol (tanpa seleksi). Sampel udang windu tumbuh cepat yang digunakan berukuran bobot 50,66±16,51 g dan panjang 17,55±1,93 cm; sedangkan udang kontrol berukuran bobot 29,64±11,93 g dan panjang 14,78±2,53 cm. Metode isolasi total RNA dilakukan dengan menggunakan kit isogen, sedangkan genom DNA menggunakan metode konvensional fenol kloroform. Konsentrasi RNA dan DNA hasil isolasi diukur menggunakan GeneQuant. T-test dari Statistix Versi 3,0 digunakan untuk membedakan rasio RNA/DNA antara kedua perlakuan yang dianalisis. Hasil penelitian menunjukkan bahwa genom DNA dan total RNA mempunyai tingkat kemurnian yang tinggi. Hasil analisis t-test menunjukkanbahwa rasio RNA/DNA udang windu tumbuh cepat (4,51) berbeda secara nyata (P<0,05) dengan udang windu kontrol (3,19). Kecenderungan rasio RNA/DNA semakin tinggi dengan semakin beratnya bobot badan, di mana rasio RNA/DNA udang betina (4,96) lebih tinggi (P<0,05) dari udang jantan (2,93). Analisis regresi menunjukkan bahwa rasio RNA/DNA udang windu memiliki hubungan erat dengan panjang (R=0,5628) dan bobot (R=0,6539). Hasil penelitian ini berimplikasi bahwa parameter rasio RNA/DNA dapat dijadikan sebagai indikator pertumbuhan udang windu
GENETIC VARIABILITY AND POPULATION STRUCTURE OF GROUPER (Epinephelus suillus) FROM MAKASSAR STRAIT AND BONE BAY, SOUTH SULAWESI, INDONESIA
No full textRandom Amplified Polymorphic DNA (RAPD) was employed to determine the genetic variability and population structure of grouper (Epinephelus suillus) from Makassar Strait and Bone Bay, South Sulawesi, Indonesia. Genomic DNA was isolated from preserved muscle tissue using Phenol-Chloroform technique. Among 24-screened arbitrary primers, ten primers (OPA-02, OPA-06, OPA-08, OPA-10, OPA-15, OPA-16, OPA-17, OPA-18, OPA-19 and CA-05) were selected to generate RAPD fingerprinting of grouper populations. The ten primers generated a total of 212 fragments (loci) and 120 polymorphic fragments in their size ranging from 250 to 2,500bp. The high polymorphism (60%) was obtained from Makassar population followed by Bone (59%) and Pare-Pare populations (50%). Similarity index of individuals was 0.86±0.07 for Pare-Pare, 0.80±0.11 for Makassar and 0.82±0.07 for Bone population. Fifteen fragments from ten primes were identified as species-specific markers of E. suillus. The UPGMA cluster analysis showed that the dendrogram seemed to be clustered according to their geographical location, where Pare-Pare population was genetically closer to Makassar population (D=0.20) than to Bone population (D=0.24)
CHARACTERISTICS OF VIRAL PROTEIN, VP-15, OF WHITESPOT SYNDROME VIRUS ISOLATED FROM INFECTED TIGER SHRIMP Penaeus monodon (Fabricius, 1798)
No full textWhite spot syndrome virus (WSSV) has caused mass mortality on tiger shrimp (Penaeus monodon) culture and adversely affects prawn industry worldwide including Indonesia. It is well known that the protein structure of WSSV plays an important role in the virus infection and morphogenesis process. A viral protein structure called VP-15 is located in the nucleocapsid of virion virus. The protein structure involves in the life cycle of WSSV in host cells. A gene encoding VP-15 could be involved in constructing the RNA interference (RNAi), so it is needed to isolate and characterize for RNAi technology purpose. The study was aimed to isolate and characterize the VP-15 from the infected WSSV tiger shrimp. The characterization of VP-15 was undertaken through assessment of nucleotide sequence, amino acid deduction, alignment nucleotide/protein searches using Genetyx and BLAST program, and dendrogram construction analysis. The results showed that VP-15 was successfully isolated in form of ORFDNA with a fragment size of 243 bp. The phylogenetic tree analysis revealed three clusters corresponding to the time (year) of isolates collection. The VP-15 consisted of 80 amino acids, two start codons (ATG), one stop codon (TAA), and one Kozak context (AAAATGG). Hydrophilic amino acid was the highest composition (44.2%), followed by neutral (31.2%) and hydrophobic (24.6%) amino acid groups. The VP-15 was rich in amino acid of lysine (21.3%), arginine (22.9%) and serine (24.6%). The successful isolation of VP-15 is a very important step in providing a basic yet suitable material in constructing the dsRNA vaccine to control shrimp diseases in aquaculture.</jats:p
PERFORMA REPRODUKSI UDANG WINDU, Penaeus monodon TRANSGENIK PASCA INSEMINASI BUATAN MENGGUNAKAN SUMBER SPERMATOFOR YANG BERBEDA
No full textUdang windu transgenik merupakan udang hasil rekayasa dengan mengintroduksikan gen antivirus yang diisolasi dari udang windu untuk menghasilkan fenotipe yang lebih baik. Domestikasi udang transgenik telah dilakukan dan berhasil memijah/bertelur, tetapi umumnya telurnya infertil yang disebabkan tidak terjadinya pembuahan di tambak pemeliharaan. Udang betina tidak kawin ditandai tidak membawa spermatofor di telikumnya. Upaya untuk mendapatkan telur fertil udang dengan inseminasi buatan (IB) perlu dilakukan. Tujuan penelitian untuk mengevaluasi performa reproduksi udang betina transgenik dan mutu larva yang dihasilkan pasca IB menggunakan sumber spermatofor yang berbeda. Penelitian ini dirancang dengan tiga perlakuan yaitu: IB menggunakan spermatofor udang windu jantan transgenik (SJT), spermatofor udang windu jantan alam Sulawesi Selatan (SulSel) (SJS) dan spermatofor udang windu jantan alam Aceh (SJA). IB dilakukan pada udang windu betina transgenik setelah dua hari moulting. Hasil penelitian menunjukkan bahwa udang windu betina transgenik pasca IB perlakuan SJT menghasilkan total telur fertil sebanyak 766.949 butir, perlakuan SJS 535.644 butir dan perlakuan SJA 678.016 butir dengan daya tetas telur fertil yaitu: pada SJT, SJS, dan SJA masing-masing adalah 53,5%; 53,7%; dan 55,0%. Uji vitalitas larva dengan perendaman dalam larutan formalin 150-200 mg/L, perendaman air tawar: 5-15 menit, dan pengeringan 3-9 menit menghasilkan sintasan larva udang yang relatif sama pada ketiga perlakuan. Nilai morfologi larva perlakuan SJT, SJA, dan SJS adalah masing-masing 85,0; 84,5; dan 75,0. Dari hasil penelitian ini mengindikasikan bahwa performa reproduksi udang windu betina transgenik dan mutu larva yang dihasilkan pasca IB tidak dipengaruhi oleh sumber spermatofor induk udang windu jantan Penaeus monodon.Transgenic tiger shrimp, Penaeus monodon has been developed in the last decade to equip shrimp with immunity against viral diseases. However, the effort to produce large quantities of specific pathogen resistance (SPR) tiger shrimp seed is hampered by several constraints in the domestication process. The successfulness of domesticated broodstock in producing larvae is very low due to low fertilization rate. An artificial insemination (AI) offers a solution to increase fertilization rate in crustacean. This study was aimed to evaluate the reproductive performance of female transgenic tiger shrimp broodstock and their larval quality after artificially inseminated with males from different sources. The spermatophores of male from different sources i.e. transgenic male spermatophore (SJT), wild male from South Sulawesi (SJS), and wild male from Aceh (SJA) were collected through electric shock and inseminated to female transgenic broodstock two days after moulting. The results showed that the total numbers of fertile eggs produced from SJT, SJS, and SJA treatment were 766,949 pcs; 535,644 pcs; and 678,016 pcs, respectively and not significantly different (P>0.05). Similar to the number of fertile eggs, the hatching rate of eggs of SJT (53.5%), SJS (53.7%), and SJA (55.0%) also did not indicate any significant differences (P>0.05). On the larval vitality test by soaking the larvae in formalin and freshwater as well as by air exposure at different duration showed no significant difference on the survival rate (P>0.05) as indicated by score value at each treatment of 85.0, 84.5, and 75.0 for SJT, SJS, and SJA, respectively. In conclusion, the reproductive performance of female transgenic tiger shrimp and their larval quality were not affected by the different sources of spermatophores inseminated artificially during the spawning cycle
EXPRESSION OF ANTIVIRAL GENE ON TIGER SHRIMP Penaeus monodon AT DIFFERENT TISSUE AND BODY SIZE
No full textThe role of tiger shrimp defense against invading pathogen on molecular level such antiviral gene expression is limited to be reported. Gene expression is a process which codes information of genes that is converted to the protein as a phenotype. Distribution of PmAV antivirus gene, that has been reported as an important gene on non-specific response immune, is needed to be observed to several organs/tissues and size of tiger shrimp. The aim of this study is to determine the distribution of gene antiviral expression at several organ/tissue and size of shrimp. The organs/tissues observed in this study were: gill, hepatopancres, muscle tissue, eyes, heart, stomach, gonad, and intestine. While the size of shrimp consisted of three groups, those are: (A) 10-20 g/ind., (B) 30-40 g/ind., and (C) 60-70 g/ind. Analysis of antiviral gene expression was performed by RNA extraction, followed by the cDNA syntesis, and amplification of gene expression by semi-quantitative PCR. The result of PCR optimation showed the optimal concentration of cDNA and primer was 1 μL and 50 mol, respectively for PCR final volume of 25 μL. Antiviral gene was expressed on the hepatopancreas and stomach in percentage of 50.0% and 16.7%, respectively. While the highest percentage of individual expressing the antiviral gene was observed in the shrimp size of C (66.7%), followed by B (50.0%) and A (16.7%). The result of study implied that the hepatopancreas has importantly involed in tiger shrimp defense mechanism on viral infection
ANALYSIS OF IMMUNE RESPONSES ON TRANSGENIC TIGER SHRIMP (Penaeus monodon) AGAINST PATHOGENIC BACTERIUM Vibrio harveyi
No full textVibriosis is one of main diseases of the black tiger shrimp Penaeus monodon infected by pathogenic bioluminous bacterium Vibrio harveyi that can cause mass mortalities in shrimp culture. The bacteria can also trigger the disease white spot syndrome virus (WSSV). An effort to produce shrimp disease-resistant strains has been done through transgenesis technology with antiviral gene transfection. By this technology, it is expected an increase in the immune response of shrimp in a variety of diseasecausing pathogens. This study aimed to determine the immune responses (total haemocytes, haemocyte differentiation, and phenoloxydase activity) of transgenic tiger shrimp against pathogenic bacterium V. harveyi. Research using completely randomized design, which consists of two treatments and three replications. Test animals being used were transgenic and non-transgenic shrimp with size, weight 3.93±1.25 g and a total length of 7.59±0.87 cm. Treatments being tested were the injection of bacterium V. harveyi (density of 5x106 cfu/mL) of 0.1 mL/individual on transgenic (A) and non-transgenic shrimp (B). Immune response parameters such as total haemocytes, haemocyte differentiation, and phenoloxydase activity were observed on day 1, 3, and 6 days after challenging. Data were analyzed using t-test by SPSS software. The results showed that the total haemocyte of transgenic shrimp was not significantly different (P>0.05) from non-transgenic shrimp, but haemocyte differentiation and phenoloxydase activity were significantly different (P<0.05) especially on sixth days after being exposed to the bioluminescent bacteria. The study results implied that transgenic shrimp has a better immune response compared than non-transgenic shrimp
GENETIC VARIABILITY OF THREE POPULATIONS OF FLYING FISH, Hirundichthy oxycephalus FROM MAKASSAR STRAIT
No full textFlying fish, Hirundichthy oxycephalus is one of economically important marine species to Indonesia, particularly in Makassar Strait and Flores Sea. However, there is a limited published data on genetic variation in molecular marker level of this species. Random Amplified Polymorphic DNA (RAPD) was employed in this study to determine the genetic variability of three populations of flying fish collected from Takalar, Pare-Pare, and Majene in Makassar Strait. Genomic DNA was isolated from preserved muscle tissue using phenol-chloroform technique. Two selected arbitrary primers (CA-01 and P-40) were performed to generate RAPD finger printing of flying fish populations. The two primers generated a total of 81 fragments (loci) and 50 polymorphic fragments with size ranging from 125 to 1,250 bp. There were no significant differences in number of fragment and number of polymorphic fragment among populations. The high polymorphism (63.5±7.4%) was obtained from Takalar population followed by Pare-Pare (58.3±19.6%) and Majene population (57.7±0.8%). Similarity index of individuals was 0.60±0.17 for Takalar, 0.63±0.17 for Majene and 0.75±0.21 for Pare-Pare population. Seven fragments were identified as species-specific markers of H. oxycephalus. The UPGMA cluster analysis showed that the Takalar population was genetically closer to Pare-Pare population (D= 0.0812) than to Majene population (D= 0.1873)
PENGKLONAN GEN PENYANDI VIRAL PROTEIN 15 (VP-15) WSSV DAN APLIKASINYA SEBAGAI VAKSIN REKOMBINAN PADA UDANG WINDU
No full textInfeksi white spot syndrome virus (WSSV) dapat menyebabkan kematian massal pada budidaya udang windu Penaeus monodon di Indonesia. Infeksi yang terjadi secara sistematis tersebut disebabkan oleh peran gen nucleocapsid viral protein (VP-15). Upaya pengembangan gen VP-15 WSSV untuk menginduksi respons imun dan menetralisasi terhadap infeksi WSSV pada udang windu perlu dilakukan. Penelitian ini bertujuan untuk mengisolasi dan merekombinasikan gen penyandi VP-15 WSSV sebagai vaksin dsRNA, serta menganalisis aplikasinya pada udang windu. Gen VP-15 diisolasi dari udang windu yang terinfeksi WSSV, dikloning ke dalam suatu vektor dan ditransformasikan ke sel kompeten (bakteri Escheria coli DH5a). Plasmid diisolasi untuk mengonfirmasi insert region gen VP-15 melalui sekuensing nukleotida. Pembuatan vaksin rekombinan dilakukan secara in-vitro menggunakan kit MEGAscript RNAi dan diaplikasikan ke udang windu melalui metode injeksi dengan dosis tunggal 0,2 µg dan kontrol (tanpa injeksi vaksin). Hewan uji yang digunakan berukuran panjang 14,75±3,17 g dan bobot 11,64±0,76 cm; serta dipelihara pada wadah bak fiber volume 250 L dengan kepadatan 10 ekor/bak. Hasil penelitian menunjukkan bahwa gen penyandi VP-15 telah diisolasi dari udang windu dan vaksin rekombinan telah dihasilkan secara in-vitro. Analisis sekuens nukleotida memperlihatkan bahwa sisipan gen DNA VP-15 sebesar 253 bp dan menunjukkan kemiripan yang tinggi (99%) pada GenBank. Penggunaan vaksin rekombinan dsRNA dengan dosis 0,2 µg memperlihatkan sintasan udang windu yang dapat mencapai 40,0% dibandingkan dengan kontrol hanya 3,3% (peningkatan 36,7%). Gambaran histopatologi pada jaringan hepatopankreas udang windu pada perlakuan kontrol menunjukkan adanya kerusakan inti sel, akibat infeksi WSSV. Gene VP-15 berpotensi sebagai bahan vaksin rekombinan dsRNA dalam mencegah infeksi WSSV.Infection of white spot syndrome virus (WSSV) causes bulk mortalities of tiger shrimp Penaeus monodon cultured in Indonesia. The nucleocapsid viral protein-15 (VP-15) is strongly suspected to be responsible for the systemic infection of WSSV. The development of VP-15 WSSV gene for inducing the immune response to and neutralize WSSV infection of tiger shrimp is vitally needed. The aim of this study was to isolate and clone the gene encoding VP-15 WSSV as dsRNA vaccine and assess the vaccine application to tiger shrimp. VP-15 gene was isolated from the genomic DNA of infected tiger shrimps, cloned into the vector, and transformed into competent cells (Escheria coli DH5a). The plasmid was isolated to confirm the insert region gene of VP-15 by the nucleotide sequence. Production of dsRNA vaccine was performed by in-vitro using MEGAscript RNAi kit and applied to tiger shrimp through muscular injection at a single dosage of 0.2 µg and without dsRNA as a control treatment. The average size of tiger shrimps used was 14.75±3.17 g in weight and 11.64±0.76 cm in length and stocked in 250 L fiber tank at 10 ind./tank. The results of the study showed the VP-15 gene was successfully isolated from the tiger shrimps and the recombinant vaccine was produced by in-vitro. The analysis of nucleotide sequence showed that the inserted DNA was 253 bp and showed a high similarity (99%) with VP-15 gene deposited in the GenBank. The application of dsRNA vaccine showed that the dosage of 0.2 ¼g resulted in the survival rate of 40.0% compared with without dsRNA (control) of 3.3% (36.7% increment). Hepatopancreas histology indicated obvious damages to cell nucleus in the un-vaccinated tiger shrimp caused by the virus infection. We suggest that the VP-15 gene is a very promising dsRNA recombinant vaccine against WSSV infection
RECONFIRMING THE SPECIES OF MUD CRAB GENUS SCYLLA (DE HAAN, 1833) IN BALIKPAPAN, EAST KALIMANTAN PROVINCE, INDONESIA BASED ON MITOCHONDRIAL 16S rRNA
No full textTaxonomy of mud crab species under the genus Scylla has been misidentified for several years due to their high morphological similarity. In Indonesia, some reports concerning mud crab have been published with misleading identification results where the species under the genus Scylla all named as Scylla serrata. The study was conducted to reconfirm the validity of species in the mud crab genus Scylla collected from Balikpapan mangrove, East Kalimantan, Indonesia and to analyze the genetic variation of the first generation (G-1) offspring, based on mitochondrial 16S rRNA sequence. The animal test used for species identification was a representative sample of mud crab. Ten of the G-1 crablet were randomly sampled for genetic variation analysis. Fragment of the 16S rRNA gene was isolated by PCR technique and purified for sequencing purpose. The mtDNA sequences were analyzed using Genetyx, BLAST-N, and DnaSP to get a consensus sequence, similarity index, haplotype and sequence diversity, and the number of haplotypes. The results showed that the 16S rRNA gene was successfully isolated with a single band in size of approximately 600 bp. The mud crab morphologically identified as Scylla tranquebarica was genetically confirmed as a species of S. tranquebarica. High haplotype diversity (0.9254) and low nucleotide diversity (0.1256) were revealed in the G-1 mud crab population, while the number of haplotypes was 7.5
SURVIVAL AND RESPONSE MOLTING OF MUD CRAB (Scylla olivacea) INJECTED WITH MURBEY (Morus spp.) LEAVE EXTRACT
No full textThe soft shell crab productivity has been hampered due to the long rearing time and unsimultaneous molting of the crab. This study aimed to determine the effect of murbey (Morus spp.) leave extract as molting stimulant on Scylla olivacea and its best extract dosage to be applied in soft shell crabs production technology. Application of murbey extract was conducted by using injection method with 5 treatments such as (a) 0 ppm (as control); (b) 100 ppm; (c) 125 ppm; and (d) 150 ppm for 12 individual per treatment. The results showed that the highest molting percentage (50%) was obtained at the concentration of 100 ppm. Meanwhile, the control (0 ppm), 125 ppm, and 150 ppm treatments displayed the same molting response (33.3%). The fastest latent molting time (29 days) was found at the treatment of 125 ppm and the slowest one of 44 days at 100 and 150 ppm treatments. The best growth of crabs injected with murbey leaves extract was at the concentration of 100 ppm with the carapace width of 6.0 mm and the body weight of 32.98 g, while the lowest was obtained at the concentration of 150 ppm with the carapace width of 3.8 mm and the body weight of 25.43 g. Crabs treated with murbey extract at the concentrations of 100, 125, and 150 ppm exhibited survival rate of 91.7 % vs. the control of 83.3%. Murbey leaves extract have been proven to be effective in stimulating molting mud crab (Scylla olivacea). The 100 ppm exhibited the best response for growth and molting percentage, while the 125 ppm showed the best performance for latent period molting of the crab. Keywords: molting response, survival rate, Scylla olivacea, murbei leaves, Morus spp
