Abstract Recent researches have pointed out that most of marine bacteria are uncultivable. However, majority of prior researches about bacteria flora in cultivated ponds used cultivating method to researches. That means those researches ignored uncultivable bacteria in ponds and caused inaccuracy in counting bacteria. Therefore we used analyzing bacteria 16S rDNA sequences to study composition of bacteria in cultivated ponds in place of traditional bacteria taxonomy. The phylogenetic diversity of bacteria examined by analyzing the 16S rDNA sequences permits the characterization of environmental bacteria community without culturing and has been used widely. This research is to adopt both MMA medium cultivation and direct recovery of bacteria 16S rDNA sequences to investigate bacteria flora in a closed Penaeus monodon pond. We sampled from A2 (10¡Ñ8¡Ñ1.5m) test pond at the Department of Marine Resource in Sun Yat-Sen University on August 17, 1999. Then, we adopted AO (Acridine Orange) epifluorescent microscopic technique to count total direct count (TDC) and direct count of viable bacteria cell (DVC). Respective results were 2.846¡Ñ107/ml, 1.029¡Ñ107/ml, and plate count (PC) determined by MPN count method were 1.130¡Ñ105cfu/ml. In the part of cultivable bacteria, they could be separated into 9 groups by their morphologic after culturing in MMA plate at 25¢Jfor 5 days. We isolated 15 strains to analyze their 16S rDNA sequences, and separated respectively into 4 groups after comparing with the genebank. Those four groups are CFB group, low G+C Gram-positive bacteria, Alpha proteobacteria and Gamma proteobacteria. The genus Vibrio (47%) in the Gamma proteobacteria group is the dominant. In the part of uncultivable bacteria, we filtered bacteria from the water in the same pond, amplified the 16S rDNA by the polymerase chain reaction (PCR) and then cloned. After that, we randomly isolated 40 clones for sequence analysis. The bacteria belong to following groups, cyanobacteria, CFB group, Verrucomicrobia, Gram-positive eubacteria, Alpha proteobacterium, Beta proteobacterium, Gamma proteobacterium and Delta proteobacterium.

This study aims to elucidate the process and mechanism of sperm penetration through eggs of Penaeus monodon. Sperm penetration of the vitelline envelopes (VEs) of P. monodon eggs were observed with the scanning electron microscope. The characteristics of sperm proteases in the sperm extracts from seminal receptacles of females were analysized. In P. monodon, mating and sperm transfer to the thelycum of female occur soon after maturity moult. Females store the sperm in the seminal receptacles. At spawning, they release stored sperm and eggs simultaneously into the water column. The outermost investment of a newly spawned egg is the VE. Sperm bind to the VE via the tip of their anterior spike. They rapidly undergo the acrosome reaction, which composes of depolymerization of the spike and exocytosis of the acrosome vesicle, pass through the VE and become bound to the egg oolemma. The isolated sperm suspended in artificial seawater were disrupted by sonication on ice. The supernatants after microcentrifuged were collected as sperm extracts. Sperm extracts were analyzed by gelatin SDS-PAGE. Sperm extract from sperm isolated from seminal receptacles of females showed clear bands of protease activity, whereas sperm extract from vas deferens and spermatophore of males did not. This results indicated that sperm of P. monodon do proceed capacitation in the seminal receptacles, and obtain sperm protease activity after capacitation. Using fluorescent peptidyl-MCA as sperm protease substrates, high trypsin-like and aminopeptidase-like activities were observed in sperm extracts. The sperm protease activity was inhibited by trypsin inhibitors aprotinin, p-aminobenzamidine (PAB), soybean trypsin inhibitor (SBTI), N-£\-p-tosyl-L-lysinechloromethyl ketone (TLCK); but was not inhibited by chymotrypsin inhibitor N-tosyl-L-phenylalaninechloromethyl ketone (TPCK) and metalloprotease inhibitor 1,10-phenanthroline (1,10-P). The results indicated that sperm undergo the acrosome reaction and release sperm proteases including trypsin-like protease, which has been implicated in facilitating sperm passage through vitelline envelope. Sperm proteases were highly active in the weak base environment, exhibiting maximum activity at pH 8.0. The protease activities were enhanced by addition of calcium chloride and magnesium chloride in the incubation medium.

Dissertation (Ph.D.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references.

Thesis (Ph.D.)--Memorial University of Newfoundland, 1999. / Accompanying floppy disk consists of Appendix H: Presentation of models in the STELLA II environment. Bibliography: leaves 217-249.

Thesis (M.S.)--Corpus Christi State University, 1980. / "Conducted in cooperation with the U.S. Department of Commerce, NOAA, National Marine Fisheries Service under P.L. 88-309 (Project no. 2-202-R)." Includes bibliographical references (p. 24-26).

This study embraces two aspects of closed system culture: (a) a comparative study of four different biological filtration treatments, and (b) a quantitative determination of the nitrogen flow in a particular biological filtration system. Twenty 0,5 g juvenile Penaeus indicus Milne Edwards were cultured for approximately two months in four different kinds of sea water recirculatory systems with bacterial filtration, algal filtration, algar plus bacterial filtration and water exchange (25% every 3 days). Growth was comparable to that found in the literature for P. indicus. Nitrification occurred in all systems irrespective of the type of biological filtration employed. Ammonia, nitrite and nitrate levels were reduced by the algae, but nitrate accumulated to approximately 30 mg NO¯³ -Nℓ¯¹ in those systems without algae. Depletion of trace elements (Cu, Fe, Zn, Hn and Co) by the algae did not occur due to trace metal replenishment from the addition of food. No marked changes occurred in the concentrations of the major cations (Ca⁺⁺, Mg⁺⁺, Na⁺ and K⁺), but a small increase in Ca⁺⁺ levels was detectable in those systems with oyster shell chips in the filtration medium. Only minor changes occurred in oxygen or pH values in the various systems tested. Inorganic carbon levels showed major increases in the algal filtration systems, while a slight drop occurred in the bacterial filtration systems. The increase in the algal filtration systems was thought to be due to the formation of carbonates and bicarbonates by the reaction of respiratory CO₂ with unionized ammonia and water. Suggestions are made as to how closed systems can be improved upon. By determining the rate of excretion of P.indicus as related to size and temperature, its faecal production, and the nitrogen content of P.indicus, its food and faeces, the nitrogen flow within a system could be calculated. P.indicus was found to have an assimilation efficiency of 88,93% and a food conversion ratio of 3,49:1. Of the total dietary nitrogen input into the system, P.indicus ingested 69,02% - 19,80% of which was used for growth, 41,58% excreted and 7,64% lost as faeces. The remaining 30,98% of the total nitrogen input can be regarded as excess food nitrogen. The total nitrogen loading on the biological filtration system was found to be 80,20% of the total input of dietary nitrogen. The ability of various types of filamentous algae to remove these nitrogen compounds was determined. Suggestions are made as to the application of this information on nitrogen flow to the culture of P.indicus

A series of studies were conducted on an emerging disease in farmed penaeid shrimp. This disease was first named as Early Mortality Syndrome (EMS) or more descriptively as Acute Hepatopancreatic Necrosis Syndrome (AHPNS). As part of the outcome of this research, the etiology of EMS/AHPNS was demonstrated. EMS was first classified as an idiopathic disease because no causative agent had been identified. Preliminary studies conducted in Vietnam in 2012 by the University of Arizona Aquaculture Pathology Laboratory (UAZ-APL) indicated that EMS is infectious (Tran et al., 2013). The agent was identified as a unique strain of Vibrio parahaemolyticus. Hence, EMS has a bacterial etiology confirmed by satisfying Koch's Postulates. Further studies focusing on the bacterial isolate causing EMS revealed that the agent could produce toxin(s), which is responsible for the primary pathology in affected shrimp. Since the causative agent has been identified, we propose a new name for EMS as Acute Hepatopancreatic Necrosis Disease (AHPND). Characterizations of the AHPND-causing Vibrio parahaemolyticus: Biochemical methods and molecular methods were used. Based on these results, various diagnostic methods were developed including polymerase chain reaction (PCR) test and biochemical tests. Other aspects of the AHPND causing V. parahaemolyticus were also run to determine such as antibiogram and the development of resistance mechanism of the bacteria exposed to farm conditions with antibiotic medications, pathogenicity, and infection dose of the bacteria, mode of infection, mechanisms governing the toxin production, and effects of environmental parameters on the invasion of the agent. Some proposed control measures for AHPND: Several antibiotic-free approaches were tested to determine viable control methods for AHPND. The principle proposed control method is to increase biosecurity. With the PCR method that has been developed, potential sources of the pathogen such as post-larvae and broodstock can be tested. As more and more insights of the pathogen were explored, the behavior of the pathogen was further elucidated. Based on this, control methods such as using polyculture with tilapia, probiotics, and bioflocs system were also tested. Several improvements in shrimp farming practices that may reduce the outbreak of the disease were also proposed.

MOREIRA, Renato Teixeira. Cultivo de Arthrospira platensis em meio orgânico: extração de polissacarídeos sulfatados e sua administração a camarões Penaeus vannamei. 2015. 67 f. : Tese (doutorado) - Universidade Federal do Ceará. Centro de Ciências Agrárias, Departamento de Engenharia de Pesca, Fortaleza-CE, 2015 / Submitted by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-27T14:53:18Z No. of bitstreams: 1 2015_tese_rtmoreira.pdf: 1566320 bytes, checksum: 67faa730cbea3b35ee8201d967647744 (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-07-27T14:53:30Z (GMT) No. of bitstreams: 1 2015_tese_rtmoreira.pdf: 1566320 bytes, checksum: 67faa730cbea3b35ee8201d967647744 (MD5) / Made available in DSpace on 2016-07-27T14:53:30Z (GMT). No. of bitstreams: 1 2015_tese_rtmoreira.pdf: 1566320 bytes, checksum: 67faa730cbea3b35ee8201d967647744 (MD5) Previous issue date: 2015 / A aquicultura ocupa cada vez mais espaço no cenário mundial, em virtude do avanço tecnológico e do aumento da demanda por proteína animal, consolidando-se como a atividade que mais cresce no agronegócio. Por outro lado, existe a possibilidade do desenvolvimento de enfermidades ao longo do cultivo, bem como, causar impactos no meio ambiente, com o lançamento de compostos orgânicos na água, eutrofizando o ecossistema aquático. Diante deste cenário, as cianobactérias como a Arthrospira platensis, surgem como um forte aliado dos produtores, seja para absorção de compostos orgânicos e tratamento de efluentes, seja pela produção de compostos que apresentam atividade biológica, promovendo o bem-estar dos animais cultivados. Assim, o objetivo desse estudo foi produzir A. platensis em efluente de cultivo da tilápia do Nilo (Oreochromis niloticus), extrair e avaliar o efeito imunomodulador dos polissacarídeos sulfatados (PS) em juvenis de Penaeus vannamei. Os PS foram obtidos a partir de um extrato aquoso quente e administrados por meio de banhos de imersão em juvenis de P. vannamei, nas concentrações de 0, 60, 120 e 240 mg L-1. Após a imersão, foi realizada a contagem total de hemócitos (THC) e verificada a concentração de proteínas totais da hemolinfa (CP) e atividade das enzimas fenoloxidase (PO) e α2-macroglobulina no soro. Após a avaliação dos imunoparâmetros, verificou-se que na concentração de 120 mg L-1 ocorreu a melhor atividade imunoestimulante. Esta concentração foi utilizada para se observar a reação dos parâmetros imunológicos quando expostos ao tempo 0 (sem exposição), 45, 90, 135 e 180 minutos, bem como a atividade antimicrobiana contra Vibrio alginolyticus. A microalga mostrou um bom desenvolvimento dentro do sistema de produção orgânico. Foram extraídos 19,85 g de PS, com um rendimento de 8,5% da biomassa utilizada. Durante o fracionamento dos PS foram observados duas frações majoritárias com 0,5 e 1,2 M de NaCl. Após três horas de imersão em diferentes concentrações de PS, os imunoparâmetros apresentaram um aumento gradativo à medida que se elevava a concentração dos PS. Ainda, a estimulação do sistema imunológico dos camarões ocorreu com 90 e 135 minutos de exposição aos PS. A enzima fenoloxidase atingiu sua atividade máxima (72,33 Umin-1mg-1) com 135 minutos de exposição, seguida de um aumento da atividade da enzima α2-macroglobulina com 180 minutos. O soro dos animais coletados com 90 e 135 minutos foi mais efetivo em inibir o desenvolvimento de V. alginolyticus, sendo necessários apenas 1,51 ± 0,08 e 1,59 ± 0,02 mg mL-1 de proteínas para combatê-lo. Os resultados obtidos nos demais tempos não diferiram do tempo zero. Assim, conclui-se que os PS de A. platensis foram capazes de ativar o sistema imunológico dos camarões logo após 90 minutos de exposição, como também contribuíram para aumentar a atividade antibacteriana do soro dos animais. / Aquaculture takes up more and more space on the world stage, as a result of technological advances and increased demand for animal protein, consolidating itself as the fastest growing activity in agribusiness. On the other hand, there is the possibility of the development of illnesses over the cultivation, as well as impact on the environment, with the release of organic compounds in water, eutrophicating aquatic ecosystem. Given this scenario, microalgae Arthrospira platensis, as to appear as a strong ally of the producers, whether for absorption of organic compounds and treatment of effluents, is for the production of compounds that exhibit biological activity, promoting the welfare of farmed animals. Thus, the objective of this study was to produce. platensis cultivation sewage Nile tilapia (Oreochromis niloticus), extract and evaluate the immunomodulatory effect of sulfated polysaccharide (PS) in juvenile Penaeus vannamei. The PS were obtained from a hot aqueous extract and administered through immersion baths in juveniles of P. vannamei, at concentrations of 0, 60, 120 and 240 mg L-1. After soaking, the total count of hemocytes (THC) and checked the concentration of total proteins (CP) and activity of enzymes fenoloxidase (PO) and α2-macroglobulina. After the evaluation of the imunoparâmetros, it was found that the concentration of 120 mg L-1 the best immunostimulating activity. This concentration was used to observe the reaction of immunological parameters when exposed to the weather (no exposure) 0, 45, 90, 135 and 180 minutes, as well as the antimicrobial activity against Vibrio alginolyticus. The microalgae showed a good development within the organic production system. Were extracted 19,85 g PS, with a yield of 8,5% of the biomass used. During the fractionation of PS were observed two fractions with 0,5 and 1,2 M majority of NaCl. After three hours of immersion in different concentrations of PS, the imunoparâmetros showed an increase gradually as amounting to concentration of PS. Still, stimulation of the immune system of shrimp took place with 90 and 135 minutes of exposure to the PS. The fenoloxidase enzyme reached its maximum activity (72,33 Umin-1 mg-1) with 135 minutes of exposure, followed by an increase of the α2-macroglobulina enzyme activity with 180 minutes. The serum of animals collected with 90 and 135 minutes was more effective in inhibiting the development of V. alginolyticus, and needed only 1,51 ± 0,08 and 1,59 ± 0,02 mg mL-1 of proteins to fight it. The results obtained in other times did not differ from time zero. Thus, it is concluded that the PS of A. platensis were able to activate the immune system in shrimp shortly after 90 minutes of exposure, but also contributed to increase the antibacterial activity of the serum of animals.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Aquaculture takes up more and more space on the world stage, as a result of technological advances and increased demand for animal protein, consolidating itself as the fastest growing activity in agribusiness. On the other hand, there is the possibility of the development of illnesses over the cultivation, as well as impact on the environment, with the release of organic compounds in water, eutrophicating aquatic ecosystem. Given this scenario, microalgae Arthrospira platensis, as to appear as a strong ally of the producers, whether for absorption of organic compounds and treatment of effluents, is for the production of compounds that exhibit biological activity, promoting the welfare of farmed animals. Thus, the objective of this study was to produce. platensis cultivation sewage Nile tilapia (Oreochromis niloticus), extract and evaluate the immunomodulatory effect of sulfated polysaccharide (PS) in juvenile Penaeus vannamei. The PS were obtained from a hot aqueous extract and administered through immersion baths in juveniles of P. vannamei, at concentrations of 0, 60, 120 and 240 mg L-1. After soaking, the total count of hemocytes (THC) and checked the concentration of total proteins (CP) and activity of enzymes fenoloxidase (PO) and α2-macroglobulina. After the evaluation of the imunoparÃmetros, it was found that the concentration of 120 mg L-1 the best immunostimulating activity. This concentration was used to observe the reaction of immunological parameters when exposed to the weather (no exposure) 0, 45, 90, 135 and 180 minutes, as well as the antimicrobial activity against Vibrio alginolyticus. The microalgae showed a good development within the organic production system. Were extracted 19,85 g PS, with a yield of 8,5% of the biomass used. During the fractionation of PS were observed two fractions with 0,5 and 1,2 M majority of NaCl. After three hours of immersion in different concentrations of PS, the imunoparÃmetros showed an increase gradually as amounting to concentration of PS. Still, stimulation of the immune system of shrimp took place with 90 and 135 minutes of exposure to the PS. The fenoloxidase enzyme reached its maximum activity (72,33 Umin-1 mg-1) with 135 minutes of exposure, followed by an increase of the α2-macroglobulina enzyme activity with 180 minutes. The serum of animals collected with 90 and 135 minutes was more effective in inhibiting the development of V. alginolyticus, and needed only 1,51  0,08 and 1,59  0,02 mg mL-1 of proteins to fight it. The results obtained in other times did not differ from time zero. Thus, it is concluded that the PS of A. platensis were able to activate the immune system in shrimp shortly after 90 minutes of exposure, but also contributed to increase the antibacterial activity of the serum of animals. / A aquicultura ocupa cada vez mais espaÃo no cenÃrio mundial, em virtude do avanÃo tecnolÃgico e do aumento da demanda por proteÃna animal, consolidando-se como a atividade que mais cresce no agronegÃcio. Por outro lado, existe a possibilidade do desenvolvimento de enfermidades ao longo do cultivo, bem como, causar impactos no meio ambiente, com o lanÃamento de compostos orgÃnicos na Ãgua, eutrofizando o ecossistema aquÃtico. Diante deste cenÃrio, as cianobactÃrias como a Arthrospira platensis, surgem como um forte aliado dos produtores, seja para absorÃÃo de compostos orgÃnicos e tratamento de efluentes, seja pela produÃÃo de compostos que apresentam atividade biolÃgica, promovendo o bem-estar dos animais cultivados. Assim, o objetivo desse estudo foi produzir A. platensis em efluente de cultivo da tilÃpia do Nilo (Oreochromis niloticus), extrair e avaliar o efeito imunomodulador dos polissacarÃdeos sulfatados (PS) em juvenis de Penaeus vannamei. Os PS foram obtidos a partir de um extrato aquoso quente e administrados por meio de banhos de imersÃo em juvenis de P. vannamei, nas concentraÃÃes de 0, 60, 120 e 240 mg L-1. ApÃs a imersÃo, foi realizada a contagem total de hemÃcitos (THC) e verificada a concentraÃÃo de proteÃnas totais da hemolinfa (CP) e atividade das enzimas fenoloxidase (PO) e α2-macroglobulina no soro. ApÃs a avaliaÃÃo dos imunoparÃmetros, verificou-se que na concentraÃÃo de 120 mg L-1 ocorreu a melhor atividade imunoestimulante. Esta concentraÃÃo foi utilizada para se observar a reaÃÃo dos parÃmetros imunolÃgicos quando expostos ao tempo 0 (sem exposiÃÃo), 45, 90, 135 e 180 minutos, bem como a atividade antimicrobiana contra Vibrio alginolyticus. A microalga mostrou um bom desenvolvimento dentro do sistema de produÃÃo orgÃnico. Foram extraÃdos 19,85 g de PS, com um rendimento de 8,5% da biomassa utilizada. Durante o fracionamento dos PS foram observados duas fraÃÃes majoritÃrias com 0,5 e 1,2 M de NaCl. ApÃs trÃs horas de imersÃo em diferentes concentraÃÃes de PS, os imunoparÃmetros apresentaram um aumento gradativo à medida que se elevava a concentraÃÃo dos PS. Ainda, a estimulaÃÃo do sistema imunolÃgico dos camarÃes ocorreu com 90 e 135 minutos de exposiÃÃo aos PS. A enzima fenoloxidase atingiu sua atividade mÃxima (72,33 Umin-1mg-1) com 135 minutos de exposiÃÃo, seguida de um aumento da atividade da enzima α2-macroglobulina com 180 minutos. O soro dos animais coletados com 90 e 135 minutos foi mais efetivo em inibir o desenvolvimento de V. alginolyticus, sendo necessÃrios apenas 1,51  0,08 e 1,59  0,02 mg mL-1 de proteÃnas para combatÃ-lo. Os resultados obtidos nos demais tempos nÃo diferiram do tempo zero. Assim, conclui-se que os PS de A. platensis foram capazes de ativar o sistema imunolÃgico dos camarÃes logo apÃs 90 minutos de exposiÃÃo, como tambÃm contribuÃram para aumentar a atividade antibacteriana do soro dos animais.

Biological and molecular characteristics of the pathogenic mollicute, Spiroplasma penaei, isolated from the hemolymph of infected Pacific white shrimp, Penaeus vannamei, were investigated. The doubling times of a S. penaei were 6.13 h (2% NaCl) and 3.43 h (no salt) under aerobic conditions, and 6.63 h (2% NaCl) and 3.22 h (no salt) under anaerobic conditions. Small diffuse white colonies with granular centers, surrounded by small satellite colonies that appeared embedded in the agar matrix, were detected on solid M1D medium (2% Noble agar) under aerobic conditions at 28°C. The genome size of the S. penaei was 1778 Kb, as determined by pulsed-field gel electrophoresis using undigested DNA. Reduction of virulence of S. penaei was not detected in serial passage 24 and 76 isolates but passage 131 isolate was attenuated as indicated by the number of surviving shrimp and histological findings of challenged P. vannamei. Toxicity was not detected in supernatant fractions of M1D medium cultures of S. penaei isolates. The most predominant host responses to the S. penaei reference isolate and to serial passage isolates were hemocytic nodules and hemocytic infiltration observed in hematoxylin and eosin-stained histological sections. Transmission electron microscopy of the lymphoid organ of experimentally infected P. vannamei depicted S. penaei without cell wall and free in the cytoplasm of lymphoid organ cells. The lesions observed in histological sections were verified by in situ hybridization using a digoxigenin (DIG)-labeled probe specific to the spiralin gene of Spiroplasma spp. Evolutionary relationship trees, based on five partial DNA sequences of 16S rDNA, 23S rDNA, 5S rDNA, gyrB, rpoB genes and two complete DNA sequences of 16S-23S and 23S-5S ISR, were constructed using the distance-based Neighboring-Joining method with Kimura-2-parameter substitution model. The NJ trees based on all DNA sequences investigated in this study positioned S. penaei in the Citri-Poulsonii clade and corroborates the observations by other investigators using the 16S gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to S. insolitum and distantly related to Spiroplasma sp. SHRIMP from China.