Genetic mechanism of body size variation in groupers:Insights from phylotranscriptomics

Animal body size variation is of particular interest in evolutionary biology,but the genetic basis remains largely unknown.Previous studies have shown the presence of two parallel evolutionary genetic clusters within the fish genus Epinephelus with evident divergence in body size,providing an excellent opportunity to investigate the genetic basis of body size variation in vertebrates.Herein,we performed phylotranscriptomic analysis and reconstructed the phylogeny of 13 epinephelids originating from the South China Sea.Two genetic clades with an estimated divergence time of approximately 15.4 million years ago were correlated with large and small body size,respectively.A total of 180 rapidly evolving genes and two positively selected genes were identified between the two groups.Functional enrichment analyses of these candidate genes revealed distinct enrichment categories between the two groups.These pathways and genes may play important roles in body size variation in groupers through complex regulatory networks.Based on our results,we speculate that the ancestors of the two divergent groups of groupers may have adapted to different environments through habitat selection,leading to genetic variations in metabolic patterns,organ development,and lifespan,resulting in body size divergence between the two locally adapted populations.These findings provide important insights into the genetic mechanisms underlying body size variation in groupers and species differentiation.

Assessment of Genetic Variability and Inter-specific Relationships among Four Species of Groupers (Genus Epinephelus) from South China Sea Using Microsatellite Markers

[Objective] The study aimed to evaluate the genetic variability and interspecific relationships among four species of groupers from South China Sea, including E. fario, E. merra, E. malabaricus and E. coioides. [Method] Twenty one mircosatellite loci of groupers were selected from GenBank and eight high polymorphic loci were used to further genetic analysis. [Result] The mean number of alleles per locus （A）, effective number of alleles （Ne）, mean polymorphism information content （PIC）, observed heterozygosity （Ho） and expected heterozygosity （He） were 4.38±1.60, 3.69±0.86, 0.69±0.08, 0.67±0.08, 0.72±0.06 in E. malabaricus; 3.88±1.13, 3.55±1.04, 0.66±0.10, 0.68±0.21, 0.70±0.08 in E.coioides; 6.00±1.07, 4.68±0.65, 0.78±0.03, 0.73±0.25, 0.79±0.03 in E. fario; 5.50±1.07, 4.58±0.80, 0.76±0.05, 0.75±0.18, 0.78±0.04 in E. merra, respectively. [Conclusion] We compared the values above, the order of the genetic variability among these grouper species was E. fario E. merra E. malabaricus E. coioides. We found that the level of genetic variability of these groupers species was relatively higher than that of other marine fish, so their genetic status was good. In addition, the analyisis of genetic relationship showed that E. malabaricus and E. coioides was the closest and it was the farthest between E. merra and E. coioides.

饲料磷水平对虎龙杂交斑生长、骨矿化及肝脏、血液相关生理生化指标的影响

本文旨在探究饲料不同磷(P)水平对虎龙杂交斑生长、骨矿化及肝脏、血液相关生理生化指标的影响。实验设计了7组以干物质计,其等能(以每100 g计)、等蛋白、等脂分别为1421.2 kJ、47%、8%的实验饲料,饲料中磷的实测值分别为1.05%、1.10%、1.20%、1.37%、1.52%、1.59%和1.70%,相应缩写为P1.05、P1.10、P1.20、P1.37、P1.52、P1.59及P1.70,每组3个平行。实验鱼[初始体质量(12.14±0.05)g]每日表观饱食投喂两次(8:00和16:30),养殖周期为6周。分析结果显示,P1.05组体质量增长率(WG)、特定生长率(SGR)和肥满度(CF)均极显著高于其他实验组(P<0.01),蛋白质增长值(PPV)在P1.05和P1.10组高于其他实验组,但各组间无显著差异(P>0.05)。随着饲料磷水平升高,P1.05组血浆及肝脏碱性磷酸酶(ALP)、甘油三酯(TG)含量高于其他实验组,而全鱼灰分以及血浆、肝脏总胆固醇(TC)含量呈现相反趋势。P1.05组血浆钙(Ca)含量在各实验组中最高,而磷含量最低,脊椎骨Ca、P含量在P1.05组均低于其他实验组。P1.05组肝脏胰岛素样生长因子1(igf-1)基因表达水平显著高于其他实验组(P<0.05)。综上所述,在本实验条件下,w=1.05%饲料磷水平已满足虎龙杂交斑幼鱼生长的需要,过高磷水平则会导致实验鱼生长下降。

Diagnosis of nervous necrosis virus in orange-spotted grouper,Epinephelus coioides, by a rapid and convenient RT-PCR method

Viral nervous necrosis （VNN） causes high mortality in marine fish, especially in the grouper, worldwide and in China. Since there is no effective vaccine or drug to deal with VNN, early detection and prevention is important to block its outbreak. In this study, a reverse transcription-polymerase chain reaction （RT-PCR） was developed for the rapid, convenient, and sensitive detection of the VNN pathogen, nervous necro- sis virus （NNV）, in the grouper. The whole process was completed within 3.5 h from the RNA extraction to PCR product visualization. The detection limit of this method was 200 copies of NNV RNA standard, which corresponded to 200 copies of virus particles. This RT-PCR method was specific to the NNV detection with no cross-reactivity to other fish viral disease pathogens, such as infectious pancreatic necrosis virus （IPNV）, infectious hematopoietic necrosis virus （IHNV）, spring viraemia of carp virus （SVCV）, epizootic haematopoietic necrosis virus （EHNV）, and large yellow croaker iridovirus （LYC1V）. With this method, the orange-spotted grouper （Epinephelus coioides） fry from hatcheries with or without incidence of the VN- N epidemic in Fujian Province were detected. The results showed that all or 93% of the fry from the two hatcheries with incidence of the epidemic were diagnosed as positive, while 40% or 25% of fry from the t- wo hatcheries without the VNN epidemic were also detected as NNV positive, indicating that this RT-PCR method can be used for rapid, sensitive detection of NNV infection and applied in the VNN epidemic alert.

Whole-genome sequencing of leopard coral grouper(Plectropomus leopardus) and exploration of regulation mechanism of skin color and adaptive evolution

Leopard coral groupers belong to the Plectropomus genus of the Epinephelidae family and are important fish for coral reef ecosystems and the marine aquaculture industry. To promote future research of this species, a high-quality chromosome-level genome was assembled using PacBio sequencing and Hi-C technology. A 787.06 Mb genome was assembled, with 99.7%(784.57 Mb) of bases anchored to 24 chromosomes. The leopard coral grouper genome size was smaller than that of other groupers, which may be related to its ancient status among grouper species. A total of 22 317 proteincoding genes were predicted. This high-quality genome of the leopard coral grouper is the first genomic resource for Plectropomus and should provide a pivotal genetic foundation for further research. Phylogenetic analysis of the leopard coral grouper and 12 other fish species showed that this fish is closely related to the brown-marbled grouper.Expanded genes in the leopard coral grouper genome were mainly associated with immune response and movement ability, which may be related to the adaptive evolution of this species to its habitat. In addition, we also identified differentially expressed genes(DEGs) associated with carotenoid metabolism between red and brown-colored leopard coral groupers. These genes may play roles in skin color decision by regulating carotenoid content in these groupers.

Effect of Laminarin on Growth and Immunity of Pearl Gentian Grouper

[ Objective] The paper was to study the effects of laminarin on growth performance and immunity of pearl gentian groupers. [ Method] Four separate 30 m2 concrete pools were used as a closed circulating water aquaculture system for the experiment. Simultaneously, approximately 1 800 groupers were placed in each of the two pools for treatment or control. The initial weight of the individual fish was （275.55 ＋29.79）g. They were fed with either a control diet or one con- raining 0.6% laminarin in the pools for 66 d. [ Result] At the end of the feeding trial, the weight gain of the groupers in treatment group was increased by 29.03% （ P 〈 0.05 ）, the specific growth ratio was increased by 23.88% （ P 〈 0.05 ）, and the feed conversion rate was decreased by 15.20% （ P 〈 0.05 ） compared to control group, while there was no significant difference in muscle nutritional components between treatment group and control group （ P 〉 0.05 ）. For each kilogram weight gain of an individual fish, the cost of the treatment diet was 1.96 yuan less than that of control diet, i. e. , a reduction of 12.54%. On the other hand, the activities of alkaline phosphatase （AKP）, lysozyme （LZM） and total superoxide dismutase （T-SOD） of the groupers in treatment group were significantly higher than those in control group （P 〈 0.05 ）. [ Conclusion ] Addition of 0.6% laminarin in the regular feed would improve the growth performance and immunity of pearl gentian groupers in aquaculture.

DISEASE RESISTANCE AND HUMORAL IMMUNOMODULATORY EFFECTS OF VITAMIN C ON GROUPER, EPINEPHELUS AWOARA

The disease resistance and humoral immunomodulatory effects of vitamin C administered orally to grouper, Epinephelus awoara maintained on a frozen fish diet supplemented with vitamin C at 500, 1000, 1500 and 2000 mg/kg were investigated. After 20 weeks, the growth rates of the groups with high level of vitamin C apparently increased. The untreated fish had symptoms of vitamin C deficiency. The endogenous liver tissue vitamin C levels were found to reflect well the dietary treatments. After intraperitoneal injection or bath challenge with a virulent strain of Vibrio vulnificus , fish fed with high level vitamin C showed significantly higher survival rate compared with the normal control group. Vaccination with formalin inactivated V. vulnificus significantly enhanced the specific antibody production in fish treated with vitamin C, and completely protected from strong bacterial challenge the groups fed on fish with vitamin C 1500 and 2000 mg/kg diet.

Protective immunity of orange-spotted grouper(Epinephelus coioids) against a nervous necrosis virus isolated from China,and determination of the complete sequences of the virus

On the basis of the sequence and analysis of genome from the orange-spotted nervous necrosis virus（ OGNNV）, China strain, a pair of special primers were designed according to the nucleotide sequences of RNA2 from OGNNV. The major capsid protein （ MCP）gene of OGNNV was cloned by means of reverse-transcriptase polymerase chain reaction （RT-PCR） and ligated into the pET32a expression plasmid. The MCP gene of OGNNV was 1 017 bases, encoded a protein of 338 amino acid with a molecular mass of 37.1 kDa. Recombinant protein with a molecular mass of 57.4 kDa was expressed in E. coli BL21 （DE3）. Vaccine was prepared from the recombinant protein expressed in recombinant cells. The juvenile orange-spotted groupers （8 cm in average length） were immunized by intraperitoneal injection. Group A was challenged with infected tissue filtrates 25 d post-vaccination. The mortality in the vaccined group （ A1,30% ） was a little higher than the unvaccined group （ B2, 27.8% ）. Group B was challenged after three vaccine injections. The mortality in the vaccined group （B1, 16.7% ） was lower than the unvaccined group （132, 27.8% ）, And the relative percentage survival （RPS） value of vaccined group, compared with the unvaccined group, was 40%. The anti-recombinant protein sera with a 1 ： 100 dilution were mixed with double volume of infected tissue filtrates and incubated at 4 ℃ for 12 h and then intramuscularly injected into the juvenile orange-spotted grouper. Treatment of infected tissue filtrates with anti-recombinant protein serum resulted in a significantly lower mortality of fish （ Group C1, mortality of 18.18% ）, compared with the fish （ Group C2, mortality of 40% ） which received infected tissue filtrates treated with control serum. Results implied the potential use of the capsid protein in immunization against OGNNV.

High-throughput simple sequence repeat (SSR) markers development for the kelp grouper (<i>Epinephelus bruneus</i>) and cross-species amplifications for Epinephelinae species

The kelp grouper (Epinephelus bruneus), belonging to one of the largest genera among the subfamily Epinephelinae, is a commercially important fish in Japan. There are limited data about the genomics of this species. To provide tools for addressing both population genetics studies and gene mapping, dito pentanucleotide simple sequence repeat (SSR) markers were developed using 454 pyrosequencing. Among the 1466 SSR markers developed, 1244 primer sets produced strong PCR products, of which 905 (72.7%) were polymorphic in kelp grouper. Cross-species utility of the 905 polymorphic SSR markers was tested in four additional Epinephelinae species of Hyporthodus septemfasciatus, Plectropomus leopardus, Epinephelus lanceolatus and Epinephelus coioides. Results revealed that, respectively, 401 (44.3%), 136 (15.0%), 434 (49.0%) and 538 (59.4%) SSRs showed specific polymorphic products. Of these, 40 SSR markers (33 di-, 1 tri- and 6 tetra-nucleotides) showed polymorphism in all species tested. Additionally, three AGAT SSR motifs which accounted for 42.9% of the nondi-nucleotide markers were found in the 40 SSR markers. This indicates that the AGAT SSR motif has a high potential as a highly versatile SSR marker in grouper Epinephelinae. The SSR markers developed in this study can be employed to obtain reliable genetic variability estimates for groupers (Epinephelinae).

Establishment of Real-time Fluorescent RT-LAMP Detection Method for Grouper Nervous Necrosis Virus

Nervous necrosis virus （NNV） is the causative agent of fulminant infectious diseases in marine fishes such as grouper. Specific primers were designed based on the conserved sequence of capsid protein （CP） gene of red-spotted grouper nervous necrosis virus （NNV）. By optimizing the reaction conditions, a rapid and simple reverse transcription loop-mediated isothermal amplification （RT-LAMP） method was established for NNV detection. After adding SYTO-9 fluorescent dye in the reaction system, the amplification curve was monitored in real time using a fluorescence detector, and the result was obviously easy to assess. Moreover, the specificity and sensitivity of the established method was analyzed. The results showed that the established RT-LAMP method has good specificity with a detection limit of 1.3 pg/μl. The detection sensitivity of the established RT-LAMP method is 100 times that of the conventional RT-PCR method, and the detection duration is only 40 min. The established RT-LAMP method is suitable for quarantine and rapid detection of grouper nervous necrosis virus.

Advances in the Study on Nutrient Requirements of Grouper (Epinephelus sp.): a Review

The paper reviews the recent advances in studying grouper nutrition requirement for the development of cost-effective and environmentally friendly artificial diets. It consists of seven parts: protein and amino acid, lipid and essential fatty acid, carbohydrate, vitamin, mineral, alternative protein source, broodstock and larval nutrition. The review provides some basic information for further investigation of nutrient requirements of groupers.

Study on the Release of Grouper Epinephelus for Stock Enhancement

Two methods, tagging and ink injection, are used in the grouper releasing experiment. The studied fishes are the juvenile of Epinephelus akaara and Epinephelus awoara both artificially cultivated and caught from natural waters, and their wild adults. Experimental results show that the inhabiting behaviours for both juvenile and adult fishes show distinct regionality, which move within an area of 2 n mile diameter 651 days and 48 days after being released, respectively. With the tagging method, the tagged fishes could only be recaptured in the year of release while, with the injection method, they could be caught in the 1st, 2nd and 3rd years. It is confirmed that in the cement tank, the tagged fishes lose their 1/3 tags. That means that the tagging method is not fit for the release research while the injection method is. Generally, the recaptured rates of injected fishes are 1.4 ~ 4.5 %, 3.1 ~ 13.4 % and 2.7 % for the 1st, 2nd and 3rd years, respectively.

Development and application of quantitative detection method for nervous necrosis virus(NNV) isolated from sevenband grouper Hyporthodus septemfasciatus

Objective:To develop the rapid and efficient quantitative detection tool for nervous necrosis virus isolated from sevenband grouper Hyporhodus septemfasciatus.Methods:The viral genes of the NNV(SGYeosu08) isolated from sevenband grouper were phylogenetically analyzed.In addition,novel quantitative PCR primers based on the genomic sequence of SGYeosu08 isolate were designed and compared it with the conventional bio-assay method(TCID_(50)) using in vitro and in vivo samples.Results:The phylogenetic analysis of viral genes demonstrated the relationship of SGYeosu08 with members of red-spotted grouper nervous necrosis virus(RGNNV).The qNNV_Rl primer set(R1_F and R1_R) and the qNNV_R2 primer set(R2_F and R2_R) revealed 93%primer efficiency(regression:y=-0.2861 x + 9.9401,R^2= 0.9976)and the revealed 108%primer efficiency(regression:y=-0.3172 x + 10.0611,R^2= 0.9982),respectively.Its comparison with viral infectivity calculated by TCID_(50) method showed similar kinetic pattern at in vitro and NNV challenged fish(in vivo) samples.Conclusions:Result show that this method is rapid and efficient to diagnose NNV infection compare to traditional bioassay method(TCID_(50)).

Soybean meal as a source of protein in formulated diets for tiger grouper, <i>Epinephelus fuscoguttatus</i>juvenile. Part I: Effects on growth, survival, feed utilization and body compositions

The effects of fish meal (FM) replacement with graded level of soybean meal (SBM) on growth performance, feed utilization, survival rate and body composition were investigated in juvenile tiger grouper, Epinephelus fuscoguttatus (initial body weight 13.9 ±0.65 g). Six experimental diets were formulated to contain 50% crude protein, 16% crude lipid and 365.8 kcal/100g feed with SBM replacing FM protein at 0% (SM 0), 10% (SM 0), 20% (SM 20), 30% (SM 30), 40% (SM 40) and 20% with phytase (SM 20P) replacement levels. At the end of the ten-week feeding trial, there were no significant differences detected in terms of growth performances (weight gain and specific growth rate), feed conversion ratio (FCR) and survival rates of fish fed with the control diet (SM 0), SM 20, SM 30 and SM 20P. Net protein utilization of fish fed SM 20P was higher than those fed with other diets suggesting an improved utilization of nutrients with phytase addition in the diet. Replacements of FM protein with SBM at 10% and 40% have resulted in significantly lower growth and poorer FCR than other replacement levels. Survival rates remain high (≥90%) throughout the trial. Whole-body proximate composition of the fish was significantly affected by the inclusion of SBM in the diets. It can be concluded that 20%-30% of FM protein replacement with SBM is recommended and addition of phytase in the SBM-based diet should be considered to improve nutrient utilization of tiger grouper juvenile.

Generation and Characterization of Monoclonal Antibodies Against Red-Spotted Grouper Nervous Necrosis Virus(RGNNV)

Nervous necrosis virus(NNV)can infect more than 120 fish species worldwide and has caused high mortality and sig-nificant economic losses to the aquaculture industry.Among different genotypes of NNV,the red-grouper nervous necrosis virus(RGNNV)is the most widely distributed one with the highest number of susceptible fish species.In this study,the capsid protein(Cp)gene of RGNNV was recombined and expressed in Escherichia coli strain BL21(DE3)and the recombinant Cp(rCp)was used as an immunogen to produce monoclonal antibodies(MAbs)through hybridoma cell fusion technology.Three MAbs were produced and characterized by indirect enzyme-linked immunosorbent assay(ELISA),western blotting,and immunofluorescence assay(IFA).Wes-tern blotting result showed that the MAbs could specifically react with the capsid protein of RGNNV.The result of IFA showed that the MAbs could recognize virions in RGNNV-infected grouper spleen(GS)cells.These results indicate that the MAbs can specifi-cally recognize RGNNV virions and can be used to produce a rapid detection method.This study provides a foundation for further studies on the rapid diagnosis of RGNNV and its infection mechanisms.

Transcriptomic and metabolomic insights into the role of the flgK gene in the pathogenicity of Pseudomonas plecoglossicida to orange-spotted grouper(Epinephelus coioides)

Pseudomonas plecoglossicida is the pathogen responsible for visceral white spot disease in large yellow croaker(Larimichthys crocea)and orangespotted grouper(Epinephelus coioides).Previously,RNA sequencing showed that P.plecoglossicida flgK gene expression was significantly up-regulated in orange-spotted grouper spleens during infection.To explore the role of flgK in P.plecoglossicida pathogenicity,RNA interference(RNAi)was performed to silence the P.plecoglossicida flgK gene,and the mutant(flgK-RNAi strain)with the best silencing efficiency(89.40%)was chosen for further study.Results showed that flgK gene silencing significantly attenuated P.plecoglossicida motility,adhesion,and biofilm formation.Compared to those fish infected with the wild-type strain of P.plecoglossicida,orange-spotted grouper infected with the flgK-RNAi strain showed a 55%increase in the survival rate and a one-day delay in time of first death,with fewer pathogens in the spleen and fewer white spots on the spleen surface.RNAi of flgK significantly affected the transcriptome and metabolome of the spleen in infected orange-spotted grouper.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis showed that the C-type lectin receptor signaling pathway was the most significantly changed immune-related pathway and the mitogen-activated protein kinase(MAPK)signaling pathway was related to multiple immunerelated pathways.Furthermore,arginine biosynthesis and glycerophospholipid metabolism were the most significantly changed metabolism-related pathways.These findings suggest that flgK is a virulence gene of P.plecoglossicida.Furthermore,flgK appears to be involved in the regulation of motility,adhesion,and biofilm formation in P.plecoglossicida,as well as in the regulation of inflammatory and immune responses of orange-spotted grouper to P.plecoglossicida infection.

Optimum temperature for the growth performance of juvenile orange-spotted grouper (Epinephelus coioides H.)

Effects of water temperature （17, 21, 25, 30 and 35℃） and body size （14.75-281.41 g initial body weight） on food consumption, growth, feed conversion, and dry matter content in orange-spotted grouper fed to satiation were investigated. The combined effect of temperature （T, ℃） and body weight （W, g） on maximum food consumption （Cmax, g/day） was described as： InCmax=-7.411＋0.828 InW＋0.317T4）.004 7T2, and the optimum feeding temperature was 33.9℃. The combined effect of temperature and body weight on growth （G） was described as： lnG=-4.461-0.2081nW＋0.394T-0.006 3T^2. The optimum growth temperature was 31.4℃, whereas overall growth rates were high at 25, 30 and 35 ℃. Feed conversion efficiencies （FCE, %）, increasing first and then decreasing with increasing temperature, averaged from 1.8 to 2.1 in terms of dry weight of food fish. The optimum temperature for FCE tended to be lower than that for growth or feeding. Dry matter content increased with both increasing water temperature （17, 25, 30 and 35℃） and body weight, and the combined effect of temperature and body weight on dry matter content （DM, %） was described as： lnDM =3.232＋0.01 4 lnW-0.004 4T＋0.001 2TInW.

Development of Co-agglutination Method for Viral Nervous Necrosis from Grouper （Epinephelus sp,） in Batam

Viral nervous necrosis （VNN） has been reported to infect larvae and juvenile of humpback grouper, and until now, this virus is one of main problem for humpback grouper. Co-agglutination test proved to be a simple, rapid and reliable diagnostic test suitable for use in the field or laboratory without any special apparatus. The study aimed to study application of a co-agglutination test using Staphylococci sensitized with specific antibody for the diagnostic of VNN in grouper. First, brain and eye organ samples from diseased fish are homogenized with phosphate buffer saline （PBS） of pH 7.2. Then, the supernatant is collected after centrifugation at 8,000 rpm for 20 min, and finally one drop of the supematant and one drop of anti VNN antibody sensitized Staphylococci suspension are mixed on a glass slid and observation of the agglutination is performed after 5-10 min. The result shows that co-agglutination technique detects positive VNN in the brain and eye samples. The co-agglutination technique may provide valid result in a very short time as compared with complex method, such as enzyme-linked immunosorbant essay （ELISA） and fluorescient antibody technique （FAT） that require a high cost. Thus, this test can detect VNN faster, simple and more economic.

Sequence and Bioinformatics Analysis on MSTN Gene of the Hybrid Grouper Derived from(Epinephelus fuscoguttatus×Epinephelus polyphekadion)

[Objectives]This study aimed to investigate the sequence structure and function of Myostatin(MSTN)gene in the hybrid grouper(Epinephelus fuscoguttatus,♀×Epinephelus polyphekadion,♂).[Methods]Genetic DNA samples were extracted from the caudal fins of the hybrid grouper and its parents to amplify their MSTN genes.Then,MSTN gene sequences were analyzed using bioinformatics tools to predict their protein structures and functions.[Results]The hybrid grouper and its parents shared the same MSTN gene structure,consisting of three exons and two introns.Nucleotide sequence of the gene could be translated into 376 amino acids,including an N-terminal signal peptide,a proteolytic processing site(RXXR motif),and nine conserved cysteine residues at C-terminal,which were the typical features of transforming growth factor beta(TGF-β)superfamily proteins.Alignment of protein sequence showed that MSTN was highly conserved between the hybrid grouper and its parents.Especially,exon 3,an important functional domain,exhibited a sequence similarity of 100%among them.In addition,four variable amino acid residues were detected in exon 2 at positions 141,153,185 and 186 in the hybrid grouper,but they did not affect the secondary structure of the protein.[Conclusion]These results will provide molecular information for future investigation on the growth and heterosis of hybrid grouper species,and on the roles of MSTN gene in regulating the growth traits of the hybrid grouper.

Detection of Fish Disease Caused by Iridovirus on Grouper （Epinephelus sp.） and Pomfret Star （Trachinotus blochii） with Co-agglutination Method in Tanjungpinang, Indonesia

Iridovirus infection often causes death and considerable economic losses in the aquaculture industry. This research applies the co-agglutination method that is fast, cheap and accurate in confirming the diagnosis of the cause of an outbreak of illness caused by iridovirus in the field, so that remedial action can be taken quickly and appropriately to minimize the impact of wider losses. Samples were taken from the grouper and pomfret star farms that are experiencing outbreaks of infectious diseases in the months from May to August 2015, Tanjungpinang, Indonesia. The sick and allegedly attacked by iridovirus samples showed abnormal swimming clinical symptoms, weakness and the swollen spleen. The swollen spleen of sick fish created suspension in phosphate buffered saline （PBS） with pH 7.2, and then centrifuged at 8,000 rpm for I0 rain. The supernatant after centrifuge was used as the test sample. On a clean object glass, 50 μL of the supematant was treated with 50 μL kit co-agglutination pre-prepared. The positive results were shown by the agglutination reaction after 10-15 rain, while as a negative control, PBS was reacted with co-agglutination kit that looked homogeneous （no agglutination）. It was showed that the grouper （Epinepkelus sp.） on four farms and pomfret star （Thracinotus blochii） on one farm that experienced an outbreak of infectious disease in Tanjungpinang showed positively infected iridovirus. The same positive iridovirus result was also demonstrated by examination using polymerase chain reaction （PCR） at 570 bp. So, the causative agent of plague on grouper and pomfret star was iridovirus. In addition, the co-agglutination test based on serology is more quick, cheap and accurate.