As aquaculture has become a significant provider of the human diet, the interest to have better quality of sea and fresh products has been increasing. However the potential hazards associated with pathogenic agents resulting in losses to the industry are major concerns that provided the motivation for this study.The use of ultraviolet irradiation is an alternative to disinfect water in inlet and outlet water sources. However the ultraviolet disinfection method has some drawbacks including no disinfectant residuals and high cost of lamp fouling and replacement. The ultraviolet system needs to be calibrated according with the life time of the ultraviolet lamps.The MS-2 coliphage in this study is an approach to determine a good indicator for determining if an ultraviolet system can be effective in an aquaculture recirculation system. The susceptibility of this system can provide an indication if WSSV can be inactivated and possible other pathogenic agents.The WSSV experiment was successful in reducing mortality. Further studies have to be completed and analyzed before recommending for control of other pathogens.

As principais enfermidades virais detectadas na carcinicultura mundial sÃo a SÃndrome da Mancha Branca (WSSV), InfecÃÃo Hipodermal e Necrose HematopoiÃtica (IHHNV), SÃndrome de Taura (TSV) e a SÃndrome da CabeÃa Amarela (YHV), esta Ãltima sem registro nos cultivos brasileiros. As doenÃas virais acima relacionadas nÃo estÃo restritas ao ambiente de cultivo. O WSSV foi detectado em vÃrias espÃcies de peneÃdeos e em outros grupos de crustÃceos silvestres. O IHHNV parece ser restrito aos peneÃdeos, tendo tambÃm sido detectado em populaÃÃes silvestres. O presente estudo teve como Ãrea de investigaÃÃo uma gamboa do EstuÃrio do Rio Juaguaribe, localizada na comunidade de Canavieiras, Aracati-CE, que recebe efluentes de fazendas de carcinicultura. A pesquisa objetivou verificar possÃveis escapes da espÃcie Litopenaeus vannamei para o ambiente de entorno e a ocorrÃncia do WSSV e IHHNV em populaÃÃes de camarÃes silvestres. Foram capturados 450 camarÃes por meio de arrastos e tarrafeamentos em trÃs estaÃÃes durante trÃs coletas realizadas nos meses de marÃo, abril e maio de 2006. Para a pesquisa viral foram utilizadas a ferramenta diagnÃstica de biologia molecular, reaÃÃo em cadeia da polimerase (PCR) e histologia clÃssica. A identificaÃÃo dos animais demonstrou o escape da espÃcie exÃtica. NÃo foi detectada a presenÃa do WSSV nas amostras analisadas. Contudo, foi detectado o IHHNV em 17 indivÃduos, sendo seis L. vannamei, oito Farfantepenaeus subtilis, um Farfantepenaeus brasiliensis e dois Litopenaeus schmitti, ficando demonstrada a suscetibilidade dessas espÃcies nativas ao IHHNV.

The immunostimulatory property of glucan isolates from three marine yeasts (Debaryomyces hansenii S8, Debaryomyces hansenii S169 and Candida tropicalis S186) and one Baker's yeast (Saccharomyces cerevisiae S36) as examined for potential application as immunostimulants in Penaeus monodon postlarvae against White Spot Syndrome Virus (WSSV) infection. Structural characterization of the glucan component in the isolates by proton nuclear magnetic resonance (NMR) indicated similar structures containing (1-3)-linked anhydroglucose repeat units (AGRUs) in the backbone with (1-6)-linked AGRUs in side chains that are (1-6)-linked to the backbone AGRUs. Glucan from C. tropicalis (S186) with the highest molecular weight and the lowest level of branching supported maximum survival (69%) followed by the other two marine yeast (S169 and S8) glucans of 27% and 23% respectively while glucan from Baker's yeast, S. cerevisiae S36 with the lowest molecular weight and the highest level of branching exhibited poor survival (4%) in P. monodon post challenge WSSV. The present study showed that the glucan isolate from marine yeast with a higher molecular weight and a lower degree of branching acts as better immunostimulants in P. monodon postlarvae than did the glucan isolate from S. cerevisiae.

Submitted by Socorro Pontes (socorrop@ufersa.edu.br) on 2017-03-23T14:59:59Z No. of bitstreams: 1 EmanuellyCRP_DISSERT.pdf: 744501 bytes, checksum: c98036be80df6f22f868dcbb58ac0ac7 (MD5) / Made available in DSpace on 2017-03-23T14:59:59Z (GMT). No. of bitstreams: 1 EmanuellyCRP_DISSERT.pdf: 744501 bytes, checksum: c98036be80df6f22f868dcbb58ac0ac7 (MD5) Previous issue date: 2016-08-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / White spot syndrome is an acute conatgious disease whose etiologic agent is the White Spot Syndrome Virus (WSSV) responsible for serious economic losses in world acquaculture. Since 2012, it has been notified WSSV occurrence in the state of Rio Grande do Norte - RN, one of most important shrimp production region. Despite its importance, there is a lack of information about the origins and biodiversity of circulating virus. The aim of investigation was to detect WSSV in Litopenaeus vannamei from farms in the RN as well as perform genotyping of the isolates. A total of five farms were surveyed, from which 10 samples were collected each one. We selected two ponds of each of the farms, chosen because clinical suspicion of WSSV. Genomic DNA was extracted to detect the virus by polymerase chain reaction (PCR). Subsequently, was performed by PCR genotyping of the positive DNA samples analysing variable number of tandem repeats - VNTR in three molecular markers (ORFs 75, 94 and 125). The results revealed the presence of virus in only one of the 5 analyzed farms. The loci ORF 75 and ORF 94 were identified in isolates from positive farm and no amplification failure for ORF 125. The number of repeating units (RUs) was calculated, and found to 9 repetitions ORF 75 and 10 RUs to ORF 94, a pattern that had not been described, stressing the need for more strain typing for screening and surveillance of the disease / A síndrome da mancha branca é uma doença notificável cujo agente etiológico é o Vírus da Síndrome da Mancha Branca (White Spot Syndrome Virus - WSSV) responsável por graves perdas econômicas na carcinicultura mundial. Desde 2012, têm sido verificados registros oficiais da presença do vírus no estado do Rio Grande do Norte - RN , região de extensa produção camaroneira. Apesar da importância, pouca informação tem sido produzida quanto às origens e biodiversidade dos virus circulantes. Diante disso, objetivou-se investigar a presença do vírus em criações de Litopenaues vannamei oriundos de fazendas do RN, bem como realizar a genotipagem dos isolados. Um total de cinco fazendas foram submetidas ao estudo, das quais foram coletadas 10 amostras de cada. Foram selecionados dois viveiros de cada uma das fazendas, escolhidos mediante suspeita clínica de WSSV. O DNA genômico foi extraído para investigação da presença ou ausência do vírus através da reação em cadeia da polimerase (PCR). Posteriormente, foi realizada por PCR a genotipagem dos isolados utilizando as repetições em tandem de número variável – VNTR (ORFs 75, 94 e 125). Os resultados revelaram a presença do vírus em apenas uma das 5 fazendas analisadas. As regiões (ORF 75 e ORF 94) foram identificadas nos isolados da fazenda positiva e não houve amplificação para o ORF 125. O número de unidades repetidas (RUs) foi calculado, sendo verificado 9 repetições para o ORF 75 e 10 para o ORF 94, um padrão que ainda não havia sido descrito, alertando para a necessidade de mais isolados caracterizados geneticamente para o rastreamento e vigilância epidemiológica da doença / 2017-03-23

Viruses are very abundant in water and hence diseases caused by viruses are common in marine organisms. These diseases create great problems for the commercial farming of crustaceans and mussels. One of the most common and most disastrous diseases for shrimp is caused by the white spot syndrome virus (WSSV), which is spread all around the world and also is infecting many different species of crustaceans including freshwater crayfish. Although during recent years knowledge has been gathered on the ways in which invertebrates defend themselves against bacteria and fungi virtually nothing is known about the defence processes elicited by virus. The aim of this work was to develop a model to use for studies of virus-host interactions in vivo and in vitro. Temperature was found to be important for the virus infectivity and at lower temperature the virus apparently did not replicate, but if animals kept at low temperature for more than 40 days were transferred to higher temperatures they died quickly due to an increased virus replication. In crayfish infected with the virus it was found that hemocytes did not degranulate and the melanization reaction was also inhibited in the hemocyes. Thus it is apparent that this virus interacts with the immune system and hemocytes in particular and to be able to study this in some greater detail it was necessary to develop a cell culture to study virus-host interactions at the molecular level. Hence, we have developed a stem cell culture from the hematopoietic tissue (hpt) that will differentiate and mature into hemocytes and which can be used to replicate the WSSV in the presence of an endogenous cytokine, astakine. Astakine is the first cytokine like-factor described which is directly involved in hematopoiesis in an invertebrate.

The freshwater crayfish, Pacifastacus leniusculus, mounts a strong innate immune response against microbes such as viruses and bacteria. In this thesis, a novel RNA interference (RNAi) method mediated with histone H2A was developed and applied in crayfish hematopoietic tissue cell cultures for gene functional studies. Further, the interactions between host (crayfish) and pathogens (white spot syndrome virus and Aeromonas hydrophila, respectively) were studied using RNAi technology in live animals. An antilipopolysaccharide factor isolated from viral challenged crayfish by suppression subtractive hybridization was shown to interfere with the propagation of white spot syndrome virus both in vivo and in vitro in crayfish, suggesting an important role of this factor in antiviral defense. Besides, RNAi of phenoloxidase, a critical immune effector involved in melanization, revealed that phenoloxidase activity is necessary for crayfish immune defense against a highly pathogenic bacterial infection in crayfish. In addition, RNAi was also employed to study a marker protein gene involved in hemocyte maturation in crayfish. Taken together, these studies may provide more insights into the immune responses against pathogen invasion as well as hemocyte ontogenesis in crustaceans.

White shrimp (Litopenaeus vannamei) were cultured in isolated environment using zero-water exchange to investigate optimum cultivation method. Except density experiment, culture density was 100 shrimps/m2 in all other experiments. The results indicated that simple quarantine facility could prevent virus infection of pond shrimps. After 83 days of cultivation, survival rate, final weight, yield and growth rate (90.7%, 16.8 g, 1.52 kg/m2, 1.36 g/week) of zero-water exchange group was not significantly different from those of water exchange group, but FCR was lower (p<0.05) instead. Addition of brown sugar increased final weight and growth rate of shrimp and lowered FCR (18.6 g, 1.60 kg/m2, 1.64 ¡Ó 0.04) (p<0.05), but nitrification was inhibited. Application of two mats per tank gave highest increases in final weight and yield and lowest FCR (p<0.05). Bottom sand increased final weight and yield and lowered FCR (p<0.05), stabilized pH and increased de-nitrification efficiency. Salinity of 25 ppt had highest final weight and yield and lowest FCR (p<0.05). Density of 200 shrimps/m2 had highest final weight and lowest FCR (p<0.05) and yield twice as that of density of 100 shrimps/m2. At the end of cultivation, water quality condition between 37% and 32% protein feed were not significantly different. But, the shrimps of 37% protein feed had higher final weight, lower FCR (p<0.05) and 26% more yield. The above results indicated that the risk of shrimps infected by virus could be prevented using zero-water exchange culture method. As long as solid was held suspending by sufficient agitating, good water quality could be maintained. Hence, zero-water exchange culture could not only decrease electricity of water pump and quantity of water use, but also increase the value of product and incorporation efficiency of feeds.

Over the last decades the crustacean aquaculture sector has been steadily growing, in order to meet global demands for its products. A major hurdle for further growth of the industry is the prevalence of viral disease epidemics that are facilitated by the intense culture conditions. A devastating virus impacting on the sector is the White Spot Syndrome Virus (WSSV), responsible for over US $10 billion in losses in shrimp production and trade. The Pathogenicity of WSSV is high, reaching 100 % mortality within 3-10 days in penaeid shrimps. In contrast, the European shore crab Carcinus maenas has been shown to be relatively resistant to WSSV. Uncovering the basis of this resistance could help inform on the development of strategies to mitigate the WSSV threat. C. maenas has been used widely in studies on ecotoxicology and host-pathogen interactions. However, like most aquatic crustaceans, the genomic resources available for this species are limited, impairing experimentation. Therefore, to facilitate interpretations of the exposure studies, we first produced a C. maenas transcriptome and genome scaffold assembly. We also produced a transcriptome for the European lobster (Homarus gammarus), an ecologically and commercially important crustacean species in United Kingdom waters, for use in comparing WSSV responses in this, a susceptible species, and C. maenas. For the C. maenas transcriptome assembly we isolated and pooled RNA from twelve different tissues and sequenced RNA on an Illumina HiSeq 2500 platform. After de novo assembly a transcriptome encompassing 212,427 transcripts was produced. Similar, the H. gammarus transcriptome was based on RNA from nine tissues and contained 106,498 transcripts. The transcripts were filtered and annotated using a variety of tools (including BLAST, MEGAN and RSEM) and databases (including GenBank, Gene Ontology and KEGG). The annotation rate for transcripts in both transcriptomes was around 20-25 % which appears to be common for aquatic crustacean species, as a result of the lack of well annotated gene sequences for this clade. Since it is likely that the host immune system would play an important role in WSSV infection we characterized the IMD, JAK/STAT, Toll-like receptor and other innate immune system pathways. We found a strong overlap between the immune system pathways in C. maenas and H. gammarus. In addition we investigated the sequence diversity of known WSSV interacting proteins amongst susceptible penaeid shrimp/lobster and the more resistant C. maenas. There were differences in viral receptor sequences, like Rab7, that correlate with a less efficient infection by WSSV. To produce the genome scaffold assembly for C. maenas we isolated DNA from muscle tissue and produced both paired-end and mate pair libraries for processing on the Illumina HiSeq 2500 platform. A de novo draft genome assembly consisting of 338,980 scaffolds and covering 362 Mb (36 % of estimated genome size) was produced, using SOAP-denovo2 coupled with the BESST scaffolding system. The generated assembly was highly fragmented due to the presence of repetitive areas in the C. maenas genome. Using a combination of ab initio predictors, RNA-sequencing data from the transcriptome datasets and curated C. maenas sequences we produced a model encompassing 10,355 genes. The gene model for C. maenas Dscam, a gene potentially involved in (pan)crustacean immune memory, was investigated in greater detail as manual curation can improve on the results of ab initio predictors. The scaffold containing C. maenas Dscam was fragmented, thus only contained the latter exons of the gene. The assembled draft genome and transcriptomes for C. maenas and H. gammarus are valuable molecular resources for studies involving these and other aquatic crustacean species. To uncover the basis of their resistance to WSSV, we infected C. maenas with WSSV and measured mRNA and miRNA expression for 7 time points spread over a period of 28 days, using RNA-Seq and miRNA-Seq. The resistance of C. maenas to WSSV infection was confirmed by the fact that no mortalities occurred. In these animals replicating WSSV was latent and detected only after 7 days, and this occurred in five of out 28 infected crabs only. Differential expression of transcripts and miRNAs were identified for each time point. In the first 12 hours post exposure we observed decreased expression of important regulators in endocytosis. Since it is established that WSSV enters the host cells through endocytosis and that interactions between the viral protein VP28 and Rab7 are important in successful infection, it is likely that changes in this process could impact WSSV infection success. Additionally we observed an increased expression of transcripts involved in RNA interference pathways across many time points, indicating a longer term response to initial viral exposure. miRNA sequencing showed several miRNAs that were differentially expressed. The most striking finding was a novel C. maenas miRNA that we found to be significantly downregulated in every WSSV infected individual, suggesting that it may play an important role in mediating the response of the host to the virus. In silico target prediction pointed to the involvement of this miRNA in endocytosis regulation. Taken together we hypothesize that C. maenas resistance to WSSV involves obstruction of viral entry by endocytosis, a process probably regulated through miRNAs, resulting in inefficient uptake of virions.

Three series of studies were conducted to quantify the effectiveness of dietary incorporation of b-1,3-glucan (BG) from Schizophyllum commune in enhancing the immunity and resistance of grass prawn Penaeus monodon to vibriosis and white spot syndrome virus (WSSV) infections. In the first series of studies, three experiments were conducted to evaluate the effectiveness of dietary b-1,3-glucan on shrimp growth and resistance to vibriosis. Weight gain, survival and feed efficiency of juvenile shrimp (0.5 ¡Ó 0.1 g) were not significantly different (P>0.05) after being fed the diets containing BG 0, 0.2, 2, 10 g/kg diet for 18 weeks. Subadult shrimp (20.4 ¡Ó 2.1 g) fed the diet containing of BG 2 g/kg diet for 10 days showed a significantly (P<0.001) enhanced resistance against vibriosis. Postlarvae fed with the BG diet (2 g/kg diet) were more resistant (P<0.001) against starvation and V. harveyi challenges than the postlarvae fed non-BG diet. Additive disease resistance was observed when polyphosphorylated L-ascorbic acid (PAA) was used together with BG. In challenge tests with V. damsela, shrimp fed with PAA (0.2 g/kg diet) + BG (2 g/kg diet) diet for 20 days had a survival rate up to 60%. In the second series of studies, two experiments were conducted to evaluate the effectiveness of dietary b-1,3-glucan on wound healing and immunity in spawners. Dietary supplement of BG reduced the chance of infections, but did not help wound healing as did the supplement of PAA. And regardless of indoor or outdoor rearing, the survival rate of brooder (135 ¡Ó 25 g) fed the BG (2 g/kg diet) diet was higher (P<0.001) than that of the non-BG group. Fed the BG brooders showed enhanced haemocyte phagocytic activity, cell adhesion and superoxide anion production then the control group. Third series of studies evaluated the effectiveness against white spot syndrome virus (WSSV). Six days after being challenged with WSSV, 12.2 % of the BG-treated (2 g/kg diet for 15 days) postlarvae (PL15) and 20 % BG-treated (2 g/kg diet for 20 days) juveniles (5.5 ¡Ó 0.5 g) were still alive; while all non-BG-treated shrimp died. In order to quantify the effectiveness of BG to WSSV, juveniles (6.5 ¡Ó 0.4 g) were fed diets containing graded levels of BG. The results showed that shrimp fed the diet containing BG 10 g/kg for 20 days had the highest (P<0.001) survival rate (42 %) among all groups. Shrimp that received diets supplemented with BG at a dosage >2 g/kg recuperated 9 ~ 12 days after WSSV challenge; while the group fed diets with no or 1 g/kg BG suffered from rapid decrease in total haemocyte count, phagocytic activity, phenoloxidase, O2-, superoxide dismutase (SOD) production and subsequent high mortality. The results in this study showed that b-1,3-glucan is effective in enhancing the phagocytic activity, phenoloxidase, O2- and SOD productions and consequently the resistance of postlarval, juvenile, subadult and brooder P. monodon against vibriosis and viral infections. Since prolonged use of BG, even at optimal dietary levels, decreased the immunity of the shrimp, care therefore must be taken to maximize its effectiveness. A cycle of dietary BG supplement of 2 ~ 10 g/kg diet for 20 days with an intermission of 10 days may serve the purposes.