Thesis (M. Agr. Sc.)--University of Queensland, 2002. / Includes bibliographical references.

Thesis (Ph.D.) - University of Queensland, 2006. / Includes bibliography.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 196-222). Also available in print.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

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.

Presently, most bacterial diseases of eel (Anguilla japonica) are controlled by antibiotics. However, antibiotics not only kill the bacterial pathogens but also kill those bacteria which might be beneficial to eels. In the meantime, application of antibiotics may result in spreading and accumulation of the resistance genes which may in turn lower the efficacy the antibiotics in the future and may threat public health. The recent trend to such problems is to screen non-pathogenic bacteria which are competitive to the pathogenic bacteria in the same environments. The eel pathogen, Edwardsiella tarda, was chosen as the target in this study. Bacterial strains were isolated from different eel ponds and tested for the ability to inhibit the growth of E. tarda. Of 2,412 strains tested, eight of them showed the inhibition capability. The molecular weights of the bioactive ingredients are all smaller than 12,000 daltons indicating they are not protein in nature. One of the strains is Bacillus cereus, four of the strains are Bacillus sphaericus, two of the strains are Bacillus laterosporus, and one of the strains of identified as Pseudomonas areuginosa competed extremely well with E. tarda. These antagonistic bacteria may have the potential of becoming as bio-control agents.Tiger shrimp (Penaeus monodon) is an important agricultural product in Taiwan. The over all production peaked in 1988, since then the outbreak of viral infection has caused the shrimp aquaculture a heavy damage. The current production is merely 1/10 of the peak. Many solutions were proposed to solve the problem, such as: increase the immunity of the shrimp, study pumping of the underground water has caused serious land subsidence in the coastal areas. Therefore, conservation of water is the trend of current aquaculture. In this study, culturable bacteria were isolated from a closed tiger shrimp pond. The taxonomy of the bacteria was based on 16S rDNA sequence phylogeny. Roughly 8 groups (genera) of bacteria were identified, including: Vibrio, Pseudoalteromonas, Porphyrobacter, Flavobacterium, Rhodthermus and three uncertain genera.

Three experiments were conducted to evaluate the effects of dietary chitin and chitosan on growth, immune responses and resistance of grass prawn Penaeus monodon against white spot syndrome virus (WSSV) and Vibrio infections. In the first experiment, two levels (0.5¡B1 g/100g diet) of chitin and three levels (0.5¡B1¡B5 g/100g diet) of chitosan were evaluated. The results show that weight gain of the shrimp fed on diet containing no chitosan or the lowest level of chitosan (0.5 %) was higher than other groups. In the second experiment, four levels of chitosan (0¡B0.5¡B1¡B5 g/100g diet) were tested. Weight gains of the control (0 %) and 0.5 % chitosan groups were significantly (P<0.05) higher than the 0.1 and 1 % chitosan groups. Shrimp survival rate was not influenced by chitosan inclusion. The test shrimp of the first experiment were evaluated for their immune responses after dietary exposures. The results show that phenoloxidase activity and superoxide dismutase were not significantly different (P>0.05) among treatments. The production of superoxide anion in the 0.5 % chitin group was significantly (P<0.05) lower than the other groups at day 3 and 12. The last experiment evaluated the effectiveness of dietary chitosan against infection of WSSV and Vibrio damsela. Shrimp were fed for 20 days on test diets containing four levels of chitosan (0¡B0.5¡B1¡B5 g/100g diet) and then challenged by injection of WSSV or Vibrio solution. In the WSSV challenge, except at day 7, shrimp survivals were not different among treatments. At day 7, however, the survival rates of the shrimp fed the diet containing 0.1 or 1 % chitosan were significantly (P<0.05) higher than those of the other groups. When challenged with Vibrio damsela, there was no difference in shrimp survival among dietary treatments. The present study shows that dietary chitin and chitosan do not significantly enhance immune responses and disease resistance of juvenile P. monodon. Dietary incorporation of chitin or chitosan negatively affects shrimp growth.

Clinical signs and lesions of Taura syndrome virus (TSV) infection in Penaeus monodon were investigated by histological and in situ hybridization (ISH) analyses. Mortality among P. monodon inoculated with 2 genotypic variants of TSV (Th04Pm and Th04Lv) appeared on Day 3, with 2 out of 10 shrimp dying. Severe necrosis of cuticular epithelial cells and lymphoid organ spheroids, indicative of acute and chronic phase lesions of TSV infection, respectively, were detected in the samples. Both Th04Pm and Th04Lv belonged to a phylogenetic family of Asian TSV isolates. The results demonstrate that both mortality and histological lesions are associated with TSV infection in P. monodon.Infection with 4 genotypic variants of TSV (Bz01, Th04, UsHi94, and Ve05) in TSV-resistant (TSR) and TSV-susceptible (Kona) Litopenaeus vannamei was investigated. Survival probabilities of TSR shrimp were higher than those for Kona shrimp with all 4 variants. Th04, UsHi94, and Ve05 gave no Taura syndrome lesions with TSR shrimp. In contrast, TSR shrimp challenged with Bz01 and Kona shrimp with all 4 TSV variants exhibited severe necrosis of cuticular epithelial cells and lymphoid organ spheroids. Real-time reverse transcription polymerase chain reaction (RT-PCR) revealed that mean TSV copy numbers in TSR shrimp infected with Bz01, Th04, and UsHi94 were significantly (p &lt; 0.0005) lower than those in Kona shrimp. In contrast, mean TSV copy numbers in TSR and Kona shrimp infected with Ve05 were not significantly different (p &gt; 0.4). The results show that TSR L. vannamei are susceptible to infection but give high survival rates following challenge by all 4 variants of TSV.To identify the viral replication site within shrimp infected cells, the viral RNA was located in association with virus-induced membrane rearrangement by electron microscopic ISH. Ultrastructure in the infected cells, analyzed by transmission electron microscopy, included the induction and proliferation of intracellular vesicle-like membranes, while the intracytoplasmic inclusion bodies and pyknotic nuclei were frequently seen. TSV RNA and TSV particles were found to be associated with the membranous structures. The results suggest that the proliferating membranes carry the RNA replication complex and that they are the site of nascent viral RNA synthesis.