Analysis of DNA Fragment Obtained from Groupers (Epinephelus fuscoguttatus) Challenged by Vibrio alginolyticus

The objective of this study was to analyse the size of DNA fragment from both resistant and susceptible groupers (Epinephelus fuscoguttatus) after infection by Vibrio alginolyticus. This study was conducted by two steps, firstly, determination of Lethal Concentration (LC50) of Vibrio alginolyticus to obtain fish which resistant and susceptible against Vibrio alginolyticus infection. Secondly, analysis of DNA fragment from both resistant and susceptible fish against Vibrio alginolyticus infection by PCR-RAPD method. In order to analyse the fragment of DNA from the fish, DNA was extracted and the concentration was counted using Kit. Sixteen primers were used, which six primers (RAPD 1-6) were from RAPD Kit, other primers were OPA-14, -A, -B, -C, and -D, YNZ 22, UBC-122, -158, -456, and -457. The results demonstrated that the concentration of bacteria that caused 50 % of fish mortality (LC50)was 7.4 × 105 CFU/L. Based on this concentration, the number of fish which were resistant and susceptible against Vibrio alginolyticus infection were obtained. For the DNA fragment analysis, the concentration of DNA after extraction ranges from 448 μg/ml to 3320 μg/ml with the purity ranges from 86-95 %. From 16 primers used for PCR-RAPD, only 6 primers showed DNA fragments in the gel electrophoresis. Those primers are OPA-14, YNZ 22, UBC-122, -158, -456, and -457. The number of DNA fragments was higher in the group of resistant fish (average 6.4 fragments) than in the group of susceptible fish (average 4.9 fragments). Fifty seven percent (57%) from resistant fish showed specific DNA fragments with size 2.0 kb, indicate that these fragment sizes have a potential role to be used as a marker for obtaining the resistant fish against bacterial infection.

ANALISIS KERAGAMAN GENETIK PADA IKAN KERAPU MACAN (Epinephelus fuscoguttatus) YANG TAHAN TERHADAP PENYAKIT YANG DISEBABKAN BAKTERI Vibrio alginolyticus

The research was purposed to analyse genetic similarity of tiger grouper (Epinephelus fuscoguttatus) inorder to select grouper that was resistant to Vibrio alginolyticus. In this study, fish were challenged with Vibrioalginolyticus and genetic similarity was examined by a PCR-RAPD method. Eight RAPD primers were used forPCR-RAPD analysis. The results showed that only three primers of RAPD primer (YNZ-22, UBC-456, danUBC-457) generated high number of RAPD fragments. The resistant group of tiger groupers generated higherpolymorph RAPD fragments than the susceptible groups. RAPD primer YNZ-22 and UBC-457 generated 82 %and 71 % of RAPD polymorph fragments from resistant group of fish and 71% and 60 % from susceptiblegroups, respectively. Primer YNZ-22 is a best genetic marker to analyse genetic similarity of tiger groupersproduced specific marker ranging from 1,2-2,0 kb. Genetic distances between the population of resistant fishand population of susceptible fish was 0.5091 and between individual of the resistant fish and susceptible fishwas 0.7032. Keywords: Genetik diversity, Epinephelus fuscoguttatus, V. alginolyticus, PCR, RAPD

Penggunaan Marka DNA dalam Seleksi Induk Ikan Kerapu Macan (Epinephelus fuscoguttatus) yang Tahan Terhadap Bakteri Vibrio alginolyticus

The objective of the present study was to use selected RAPD markers (Primer UBC-122 and YNZ-22) in parental selection to the bacterial Vibrio alginolyticus resistance in Tiger groupers (Epinephelus fuscoguttatus).  Eighteen fish parent samples were challenged with Vibrio alginolyticus and parental selection was examined by a PCR-RAPD method.   The results showed that from 18 fish samples, primer YNZ-22 generated 18 fragments while primer UBC-122 generated 8 fragments only (KHP, KB, HB, PB, MKH, MH, K and MK).  In addition, the average number of fragments from each samples generated from primer-YNZ was higher (3,2) than from UBC-122 (2,3). This indicates that primer YNZ-22 is better than primer UBC-122 to use in parental selection to the Vibrio alginolyticus resistance in Tiger groupers.  From the parental selection, five fish parents (KHP, PB, MH, K and  MK) were selected for their high resistance to the bacterial Vibrio alginolyticus

IMPROVEMENT METHOD OF GENE TRANSFER IN Kappaphycus alvarezii

Method of foreign gene transfer in red seaweed Kappaphycus alvarezii has been reported, however, li-mited number of transgenic F0 (broodstock) was obtained. This study was conducted to improve the method of gene transfer mediated by Agrobacterium tumefaciens in order to obtain high percentage of K. alvarezii transgenic. Superoxide dismutase gene from Melastoma malabatrichum (MmCu/Zn-SOD) was used as model towards increasing adaptability of K. alvarezii to environmental stress. The treat-ments were the culture media and recovery duration, and each treatment consisted of three replica-tions. The best method was co-cultivation using liquid media, then recovery was conducted in liquid media for 10 days. That treatment allowed higher transformation percentage (90%), regeneration effi-ciency (90%), putative bud efficiency (100%), number of buds and explants sprouted (100%) and transgenic explants (100%). The transgenic explants showed an amplification PCR product of Mm-Cu/Zn-SOD gene fragment, whereas the non-transgenic explants showed no amplification product.  All results revealed that suitable method of transgenesis for K. alvarezii has been developed. Keywords:       Agrobacterium tumefaciens, culture media, Kappaphycus alvarezii, recovery duration, transformatio

IMPROVEMENT METHOD OF GENE TRANSFER IN Kappaphycus alvarezii

<p><em>Method of foreign gene transfer in red seaweed </em><em><span style="text-decoration: underline;">Kappaphycus</span></em><em> <span style="text-decoration: underline;">alvarezii</span> has been reported, however, li-mited number of transgenic F</em><em>0 (broodstock) </em><em>was obtained. This study</em><em> </em><em>was conducted to improve the method of gene transfer mediated by </em><em><span style="text-decoration: underline;">Agrobacterium</span></em><em> <span style="text-decoration: underline;">tumefaciens</span> in order to obtain high percentage of <span style="text-decoration: underline;">K.</span> <span style="text-decoration: underline;">alvarezii </span>transgenic. Superoxide dismutase gene from <span style="text-decoration: underline;">Melastoma</span></em> <em> <span style="text-decoration: underline;">malabatrichum</span> (MmCu/Zn-SOD) was used as model towards increasing adaptability of <span style="text-decoration: underline;">K.</span> <span style="text-decoration: underline;">alvarezii</span> to environmental stress. The treat-ments were the culture media and recovery duration, and each treatment consisted of three replica-tions. The best method was co-cultivation using liquid media, then recovery was conducted in liquid</em><em> </em><em>media for</em><em> </em><em>10</em><em> </em><em>days. That treatment allowed higher transformation percentage </em><em>(</em><em>90%</em><em>)</em><em>, regeneration effi-ciency </em><em>(</em><em>90%</em><em>)</em><em>, putative bud efficiency</em><em> (100%)</em><em>, number of buds and explants sprouted </em><em>(100%) </em><em>and transgenic </em><em>explants (</em><em>100%</em><em>)</em><em>. The </em><em>transgenic explants showed an amplification PCR product of </em><em>Mm-Cu/Zn-SOD </em><em>gene fragment</em><em>, whereas the non-transgenic explants showed no amplification product.  All results revealed that suitable method </em><em>of transgenesis </em><em>for <span style="text-decoration: underline;">K.</span> <span style="text-decoration: underline;">alvarezii</span> has been developed. </em></p><p><strong><em>Keywords</em></strong><em>:       <span style="text-decoration: underline;">Agrobacterium</span> <span style="text-decoration: underline;">tumefaciens</span>, culture media, <span style="text-decoration: underline;">Kappaphycus</span> <span style="text-decoration: underline;">alvarezii</span>, recovery duration, transformation </em></p

THE EFFECT OF VITAMIN C AND AEROMONAS VACCINE ON THE IMMUNE RESPONSE AND DISEASE RESISTANCE OF GROUPER (Epinephelus fuscoguttatus)

We evaluated the effectiveness of vitamin C and Aeromonas salmonicida vaccine in grouper (Epinephelus fuscoguttatus) for increasing immune responses and protection against A .salmonicida. The vitamin C used was polyethoxylated ascorbic and tocopherol. The vaccine was prepared from formalin-killed cells and concentrated extracellular products of a single isolate A. salmonicida. Bath immersion vitamin C and vaccine trials were conducted for 60 min. Fish used had a mean weight 25 g. Control groupers were injected with tryptic soy broth. The results showed that vitamin C enhanced phagocytic activity in head kidney leucocytes of grouper 7, 14, 28 and 36 days after treatments. A significant different of the antibody titre was found between control fish and the treated fish at 42 days after treatments. In addition, at day 42, Relative Percent Survival (RPS) for control group was 53.3 %, vitamin C-treated group was 80.0 % and vaccinated group was 90.0 %. The results of this study suggest that bath immersion of vitamin C provided an increasing of phagocytic activity (non-specific immune responses), titre antibody (specific immune responses) and protection against A. salmonicida infection in grouper. A. salmonicida vaccine also en-hanced titre antibody and protection against A. salmonicida infection in grouper