Factors that can influence the transmission of aquatic viruses in fish production facilities and natural environment are the immune defense of host species, the ability of viruses to infect host cells, and the environmental persistence of viruses. In this thesis, fish cell lines were used to study different aspects of these factors. Five viruses were used in this study: viral hemorrhagic septicemia virus (VHSV) from the Rhabdoviridae family; chum salmon reovirus (CSV) from the Reoviridae family; infectious pancreatic necrosis virus (IPNV) from the Birnaviridae family; and grouper iridovirus (GIV) and frog virus-3 (FV3) from the Iridoviridae family. The first factor affecting the transmission of fish viruses examined in this thesis is the immune defense of host species. In this work, infections of marine VHSV-IVa and freshwater VHSV-IVb were studied in two rainbow trout cell lines, RTgill-W1 from the gill epithelium, and RTS11 from spleen macrophages. RTgill-W1 produced infectious progeny of both VHSV-IVa and -IVb. However, VHSV-IVa was more infectious than IVb toward RTgill-W1: IVa caused cytopathic effects (CPE) at a lower viral titre, elicited CPE earlier, and yielded higher titres. By contrast, no CPE and no increase in viral titre were observed in RTS11 cultures infected with either genotype. Yet in RTS11 all six VHSV genes were expressed and antiviral genes, Mx2 and Mx3, were up regulated by VHSV-IVb and -IVa. However, replication appeared to terminate at the translational stage as viral N protein, presumably the most abundant of the VHSV proteins, was not detected in either infected RTS11 cultures. In RTgill-W1, Mx2 and Mx3 were up regulated to similar levels by both viral genotypes, while VHSV-IVa induced higher levels of IFN1, IFN2 and LGP2A than VHSV-IVb. The second part of the thesis examined the ability of two Ranaviruses, GIV and FV3, to infect non-host fish cells. This is referred to as cellular tropism and is one of many host-virus interaction events required to established successful infection in new organisms. Grouper iridovirus (GIV), belonging to the Ranavirus genus of the Iridoviridae family, was demonstrated to differentially express viral genes and induce apoptosis in three non-host fish cell lines rainbow trout monocyte/macrophage (RTS11), Chinook salmon embryon (CHSE-214) and fathead minnow Epithelioma papulosum cyprinii (EPC). These cells were challenged with GIV and virus entry into all three cell lines was confirmed by the expression of viral immediate early genes. The expression of the late major capsid protein gene was detected in CHSE-214 and EPC, but not in RTS11, suggesting an earlier termination in the viral replication cycle in RTS11. Approximately 12 h after infection with GIV, cell death was prominent in all three non-host cell lines. Death was later confirmed to be apoptosis by the presence of chromosomal DNA fragmentation and phosphatidylserine externalization. To determine whether apoptosis was protein related or gene expression related, the three cell lines were infected with heat-inactivated GIV and UV-treated GIV (GIVUV). The heat inactivation abolished apoptosis in all three cell lines, but each cell line responded differently to GIVUV. Relative to GIV, GIVUV caused no apoptosis in CHSE-214, decreased apoptosis in RTS11, and increased apoptosis in EPC. These results suggest that early GIV gene expression was needed for apoptosis in CHSE-214 but impeded apoptosis in EPC. At the cellular level, only EPC was a permissive host as EPC was the only cell line of the three capable of producing a moderate increase in virus titre. The three non-host cell lines present a good system for potentially identifying different components of GIV-induced apoptotic pathways in future studies. Rainbow trout are not highly susceptible to frog virus 3 (FV3) induced diseases, and had been suggested to be a potential carrier for the virus. To determine which rainbow trout cell types are permissive for FV3 and act as a potential source for virus replication in vivo, the ability of rainbow trout cell lines from gonads (RTG-2), skin (RTHDF), liver (RTL-W1), gills (RTgill-W1), intestine (RTgut-GC) and spleen (RTS11), and primary leukocyte cultures from peripheral blood (PBL) and head kidney (HKL) to support FV3 infection was examined. RTG-2 supported a moderate level of FV3 replication while viral replication in RTL-W1 was minimal. The rest of the cell lines did not support viral replication but all succumbed to the infection and were killed by FV3. Lymphocyte-like cells from PBL and HKL were not killed by FV3 while macrophage-like cells were. Most of the cell lines died by an apoptosis-independent mechanism, presumably necrosis, while the monocyte/macrophage cell line, RTS11, died by an apoptosis-dependent mechanism. In addition, neoplastic macrophage-like human U937 cell line, and T lymphocyte-like PEER cell line were also infected with FV3 to compare their response to that of rainbow trout immune cells. U937 cells were killed by FV3 in an apoptosis-dependent manner; however, PEER T cells did not die from FV3 infection, a result similar to the lymphocyte-like fraction of rainbow trout PBL and HKL. In summary, most rainbow trout cell lines do not support significant FV3 replication; furthermore, cells of the lymphocyte origin appeared refractory to FV3 induced cell death while those of macrophage origin underwent apoptosis as a response to FV3. The last factor affecting the transmission of aquatic viruses examined in this thesis is the persistence of viruses in the aquatic environment. Virus persistence is influenced by natural environmental factors such as temperature, pH, desiccation and salinity, but the often unexplored anthropogenic factors can play a role. Therefore, the focus of this section was on the effect of one particular anthropogenic substance, Corexit 9500, on the infectivity of aquatic viruses with different physical characteristics. The effect of Corexit 9500, a dispersant used to clean up oil spills, on invertebrates, lower vertebrates, birds and human health have been examined but there is a significant lack of study on the effect of this dispersant on aquatic viruses. In this study, the effect of Corexit 9500 on four aquatic viruses of different structural composition was examined. Corexit 9500 reduced the titre of the enveloped viral hemorrhagic septicemia virus (VHSV) at all concentrations (10% to 0.001%) examined. The titre of frog virus 3 (FV3), a virus with both enveloped and non-enveloped virions, was only reduced at the high Corexit 9500 concentrations (10% to 0.1%). Corexit 9500 was unable to reduce the titre of non-enveloped infectious pancreatic necrosis virus (IPNV), but enhanced the titre of chum salmon reovirus (CSV) by 2-4 logs. With the ability to inactivate enveloped viruses and possibly enhance some non-enveloped viruses, Corexit 9500 has the potential to alter the aquatic virosphere.

Red porgy <i>Pagrus pagrus</i> is a reef fish important to both recreational and commercial fisheries off the coast of the southeastern United States. Stock assessments performed on this species since 1985 have shown a population in decline. As a result, a number of management actions were put in place, including a harvest moratorium in 1999. Stock assessments for many marine species, including red porgy, rely on a combination of fishery-dependent and fishery-independent data. When a moratorium is in place, the flow of fishery-dependent data is interrupted, making assessments more reliant on fishery-independent information. To investigate how loss of fishery-dependent data, as during a moratorium, would affect stock assessment results for red porgy, I conducted model simulations to represent moratoria of various durations. The most recent red porgy stock assessment model developed during a 2002 workshop was used as a tool in these simulations. I found that biological reference points, such as biomass and fishing mortality, and population projections were more variable for longer simulated moratoria. When fishery-dependent data were removed, minor fluctuations in length and age frequencies resulted in larger fluctuations in estimates of biological reference points. The simulated moratoria also resulted in a slight bias toward over-estimating stock productivity. Similar simulations and analyses were conducted to determine the effects of reducing fishery-independent data from the Marine Resources Monitoring, Assessment, and Prediction (MARMAP) program. Length and age data of reduced MARMAP sample sizes were bootstrapped from original data, and used as input for the stock assessment model. Biological reference points and population projections were more variable for small MARMAP sample sizes, due to the incomplete representation of the length and age frequencies of the population. Reduced sample sizes also showed a slight bias toward predicting a more productive population. These types of simulations emphasize the benefits of investigating potential effects of data reduction on assessment results prior to implementing management strategies, such as a moratorium or sampling change, that cause data loss. Although decreasing red porgy data resulted in slight changes in assessment results, there are more data available for this species than other species in the snapper-grouper complex. For these lesser-studied species, reducing data could dramatically affect assessment capabilities. To investigate this, I compiled available data for these species and identified the stock assessment method used. I then predicted assessment capabilities for each species under a moratorium and if the MARMAP survey was eliminated. A moratorium could reduce assessment capabilities for 37 of the 73 species, and 63 species would require management based on key species. Removing MARMAP data would reduce assessment capabilities of eight species, many of which are economically important. There was an overwhelming need for a reliable catch-per-effort index, information that could improve assessment capabilities of 67 species. This index could be obtained by expanding the MARMAP survey, from a fishery observer program, or from commercial logbooks. By linking the red porgy stock assessment, an evaluation of sampling regimes and data loss during a moratorium, and the expansion of this stock assessment strategy to multiple species, managers will ultimately benefit from increased ability to manage stocks experiencing varying regulations and data availability. / Master of Science

Orientador: Alpina Begossi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T01:45:12Z (GMT). No. of bitstreams: 1 Martinelli_ClaudiadeMiranda_M.pdf: 1290447 bytes, checksum: ab7d6f404f4a087cbbaea1d384d6436c (MD5) Previous issue date: 2010 / Resumo: A etnoecologia estuda o conhecimento ecológico local das comunidades humanas sobre os recursos naturais. Informações etnoecológicas analisadas e interpretadas com base na literatura ictiológica vêm contribuindo para a compreensão da ecologia dos recursos pesqueiros. Diversos estudos demonstram a importância do conhecimento tradicional, que torna mais eficiente a obtenção de informações detalhadas sobre as espécies-alvo das pescarias e sua utilização em práticas de manejo pesqueiro. Essas informações podem ser tanto utilizadas diretamente no manejo quanto como hipóteses de estudos. O objetivo desse trabalho é conhecer a etnoictiologia de Mycteroperca bonaci (sirigado), Epinephelus morio (garoupa), Cephalopholis fulva (piraúna), Pomatomus saltatrix (enchova), Centropomusundecimalis (robalo) que ocorrem na costa do Rio Grande do Norte (nordeste do Brasil), além de obter dados biológicos sobre robalo. As informações etnoictiológicas foram obtidas através de entrevistas estruturadas com 28 pescadores experientes e os dados biológicos foram obtidos através da análise das gônadas e do conteúdo estomacal de 90 indivíduos de robalo. As informações dos entrevistados sobre dieta do robalo (peixes e crustáceos), sirigado (peixes), piraúna (peixes) e enchova (peixes e crustáceos) foram concordantes com as informações científicas, já as respostas sobre a garoupa indicaram peixes, ao invés de crustáceos (como consta na literatura científica) como item alimentar mais importante da sua dieta. As informações sobre habitat do robalo (próximo à costa), do sirigado (águas profundas, dentro das pedras), da garoupa (fundos rochosos) e da piraúna (fundos rochosos)estão de acordo com a literatura científica. As respostas sobre migração e reprodução do robalo, do sirigado, da piraúna, da garoupa e da enchova foram as que mais discordaram das informações científicas, sugerindo um conhecimento dos pescadores pouco detalhado sobre esses assuntos ou uma nova informação sobre a ecologia dessas espécies na região estudada que poderia ser utilizada como hipótese em estudos futuros. As informações dos pescadores semelhantes aos dados da literatura científica podem apoiar decisões de manejo por causa da sua plausibilidade e concordância com o conhecimento científico, considerando a urgência de se fazer um plano de manejo viável para as áreas costeiras no Brasil e as dificuldades logísticas e financeiras de se fazer pesquisas ecológicas, principalmente no ambiente marinho. Para um bom manejo pesqueiro é necessário utilizar conjuntamente as informações etnoecológicas e científicas sobre a reprodução, migração, habitat e dieta das espécies. Mesmo com as limitações de tempo e esforço amostral, o presente estudo contribuiu para aumentar o conhecimento biológico sobre o robalo, o sirigado, a garoupa, a piraúna e a enchova na região nordeste do Brasil / Abstract: Ethnoecological information analyzed and interpreted based on icthiological literature contribute to the understanding of fisheries resources. Several studies demonstrate the importance of local knowledge, which optimizes the gathering of information on the target species and its utilization in fishery management practices. This information can be used directly in management or as work hypothesis. The Objective of this study is to understand the ethnoecology of Mycteroperca bonaci (sirigado), Epinephelus morio (garoupa), Cephalopholis fulva (piraúna), Pomatomus saltatrix (enchova), Centropomus undecimalis (robalo) which occur at the coast of Rio Grande do Norte (Northeast of Brazil). In addition we also aim to gather biological data about the robalo. The ethnoictiological information was gathered through structured interviews with 28 fishermen and the biological data was gathered through analysis of gonads and stomach content of 90 robalo individuals. The information of the fishermen about diet of robalo (fish and crustaceans), sirigado (fish), piraúna (fish) and enchova (fish and crustaceans) agreed with the scientific information. The answers about the garoupa diet indicated fish, instead of crustaceans (as wrote in scientific literature), as a primary food item. The information about the habitat of robalo (next to the coast), sirigado (deep water, inside the rocks), garoupa ( rocky bottoms) and piraúna (rocky bottoms) agreed with the scientific information. The mostly of answersabout migration and spawning of robalo, sirigado, garoupa, piraúna e enchova disagreed with the information in scientific literature, what suggests that the fisher?s knowledge is less accurate about this issues or a new information about the ecology of these species can be use as hypothesis in future studies in the region. The fishermen information that agreed with the scientific information can support coastal management decisions because of its plausibility and agreement with the scientific knowledge, considering the urgency to make a coast management plan viable to Brazilian coastal areas and the logistic and financial difficulties to do marine ecological research / Mestrado / Ecologia / Mestre em Ecologia

O presente projeto analisou o perfil dos ácidos graxos, embriogênese e ontogenia do sistema digestório das larvas da garoupa verdadeira Epinephelus marginatus durante os primeiros dias de desenvolvimento. Além disso, descrevemos como os embriões se dividem, eclodem e desenvolvem suas principais características, e como se desenvolve seu sistema digestório ao longo dos primeiros dias de desenvolvimento, e como utilizam os AG durante os primeiros 8 dias de desenvolvimento. Observamos que as larvas da garoupa apresentam desenvolvimento de suas estruturas digestórias relativamente lento, no entanto, estas são capazes de capturar, ingerir e digerir presas já a partir do 4º DAE. Os ovos da garoupa verdadeira são compostos por elevados percentuais de PUFA nos fosfolipídios e para o processo de eclosão utilizam preferencialmente os SFA dos fosfolipídios. Além disso, os PUFA da série n3 sobrepõe-se aos da série n6, principalmente o DHA, que apresentaram valores elevados em comparação com outras espécies marinhas tanto nos fosfolipídios quanto nos triglicérides nos três primeiros dias de desenvolvimento. As larvas apresentam uma elevada necessidade dos HUFAs DHA/EPA, e durante essa fase é importante a utilização de alimento vivo de tamanho reduzido (copépodes ou &ldquo;SS strain&rdquo; Brachionus rotundiformes) enriquecidos com valores da relação entre DHA/EPA acima de 2,0. Com isso, todo o conhecimento gerado durante esses dias de desenvolvimento já permitem a aplicação deste conhecimento no processo de embriogênese e larvicultura da garoupa verdadeira contribuindo para alavancar sua domesticação e produção em cativeiro e elaborar futuramente um programa de repovoamento desta espécie contribuindo para sua conservação. / This project analyzed the fatty acids profile, embryogenesis and ontogeny of the digestive system of dusky grouper Epinephelus marginatus larvae during the first days of development. Furthermore, we described how the embryos are divided, hatch and develop its main features, and how develops its digestive system during the first days of development, and how they use the FA during the first 8 days of development. We observed that the grouper larvae present relative slow development of their digestive structures, however, the larvae are able to capture, ingest and digest preys already from 4º DAE. The dusky grouper eggs are composed by high percentages of PUFA in phospholipids and for the hatching process it uses preferably the SFA of phospholipids. Additionally, the n3 series PUFAs overlaps the n6 series, especially DHA, which exhibited high values compared to other marine species both on phospholipids as in the triglycerides during the first three days of development. The larvae exhibit a high requirement of HUFAs DHA/EPA, and during this phase is important to use live food of small size (copepods or &ldquo;SS strain&rdquo; Brachionus rotundiformes) enriched with the ratio of DHA/EPA levels above 2.0. Thus, all the knowledge generated during these days of development allow us the application of this knowledge in the embryogenesis and hatchery process of the dusky grouper contributing to leverage its domestication and production in captivity and eventually draw up a restocking program of this species contributing to their conservation.

Karenia brevis dinoflagellate blooms off the west coast of Florida can create devastating effects on marine communities when they release a neurotoxin known as a brevetoxin. These blooms, informally referred to as red tides, can cause massive fish kills, necessitate closures of shellfish fisheries, and can even leave lingering toxins that impact shelf communities long after the bloom has dissipated. As a result, much effort has been put into studying K. brevis bloom initiation and dynamics. However, how K. brevis blooms impact Florida's fisheries is not fully understood because the relationship between K. brevis cell counts and fish mortality is poorly described. To study this relationship and the ecosystem response to K. brevis blooms, Ecopath with Ecosim (EwE) modeling is used to force K. brevis bloom mortality on the shelf ecosystems by using a recently developed time series that indexes K. brevis bloom severity. This index dynamically drives K. brevis bloom mortality in EwE in a historical reconstruction scenario from 1980 to 2009. Three hypotheses on ecosystem response are explored using Gag grouper as a case study. We postulate a) that K. brevis blooms impose bottom-up and top-down effects on the food web, b) that episodic perturbations by these blooms shape the community structure and c) that fishing pressure exacerbates those effects. Results support the hypothesis that K. brevis blooms pose top-down food web pressures, which is seen by evidence of trophic cascading. Changes in community structure with bloom mortality are also evidenced by changes seen in biodiversity and richness. An exacerbation of those effects as a result of heavy fishing pressure is evident, however, is only seen during severe bloom events. Little to no changes were found in the mortality from K.brevis blooms during blooms of average severity, and less mortality was imposed on the system during blooms of particularly low severity. However, this may be an artefact of the mode of action of K. brevis in EwE. Investigation of bloom effects on Gag showed that natural mortality rates of Gag appear to be largely influenced by mortality incurred during K. brevis blooms relative to the low rate of predation on Gag. Moreover, consumption rates of Gag on its prey were found to increase under a realistic schedule of these blooms. This may be due to a combination of effects, including increased mortality on competitors (making more prey available for Gag) and a lowering of the mean age of the Gag stock, which increases population productivity.

Movement patterns and space use by mature fishes are critical in determining the effectiveness of marine reserves in conserving spawning stock biomass and/or providing biomass to adjacent fisheries through 'spillover'. Home range sizes, activity patterns, site fidelity and habitat preferences were determined for acoustically-tagged snappers and groupers using a rigorously-calibrated array of omnidirectional hydroacoustic receivers deployed in the diverse coral reef environments of a no-take marine reserve (NTMR) network in the Dry Tortugas, Florida. An individual-based localizing tendency model of reef fish movement was parameterized from fine-scale acoustic telemetry data and integrated into a Spatial Management Performance Assessment (SMPA) simulation model for reef fish populations developed to quantitatively evaluate performance of no-take marine reserves in the Dry Tortugas, Florida. Spatially-explicit SMPA models were parameterized for three overfished stocks in the lucrative snapper-grouper fishery: red grouper (Epinephelus morio), black grouper (Mycteroperca bonaci), and mutton snapper (Lutjanus analis). SMPA models were used to evaluate the impacts of a variety of life histories, movement strategies and speeds, and management regulations upon long-term stock sustainability, as measured by annual changes in spawning potential ratio (SPR), and long-term stock productivity, as measured by annual changes in fisheries yield-in-weight per recruit (Yw/R). Under assumptions of constant regional fishing pressure, constant recruitment, and 'realistic' fish movement, SMPA simulation runs from initial conditions in 2000 suggested that by 2014, the Tortugas NTMR network should function to restore red grouper populations to 30% SPR, a Federal management benchmark for sustainability. Mutton snapper were the most mobile of the species investigated; if mutton snapper movements are ignored, their population is predicted to attain 30% SPR by 2014, but given 'realistic' mobility, they may not attain this target by 2021 without additional protections. Black grouper are currently fished at over 9 times sustainable levels. SMPA simulations suggest coupling an increase in minimum size at capture of 20 - 25 cm with NTMR implementation would result in substantial short term losses in yield, but would restore both black grouper and mutton snapper populations to 30% SPR by 2021 and lead to increased long-term yields. Although marine reserve sites are often chosen opportunistically, these findings strongly suggest that reserve designs (e.g. proper sizes and configurations) must take into account the scales and patterns of movement exhibited by the exploited stocks they are intended to protect. These modeling efforts also suggested reserves are not a panacea; in order to promote sustainability for severely depleted stocks, they must be accompanied by an overall reduction in fishing capacity. Although important questions remain concerning the movements of reef fish in response to habitat and density dependent processes, our analyses of realistic reef fish behaviors suggest that the NTMRs of the Dry Tortugas promote substantial gains in SPR, promoting long-term stock sustainability and enhanced egg production. Increased rates of movement diminish these benefits, but may also mitigate short-term losses in yield associated with NTMR establishment.

Grouper nervous necrosis viruses belong to the Betanodavirus genus in the Nodaviridae family that is a group of small, non-enveloped icosahedron viruses. More than 30 species of fish are infected by the betanodaviruses, which cause massive mortality in hatchery-reared larvae and juveniles. The infection causes great economic losses to aquaculture and sea-ranching. To study the effects of N-terminus and disulfide bonds of capsid protein on particle formation and thermal stability of grouper nervous necrosis virus, virus-like particles (VLPs) of dragon grouper nervous necrosis virus (DGNNV) were used. Deletion of 35 residues at the N-terminus completely ruined the VLP assembly. When deletions were restricted to 4, 16, or 25 N-terminal residues, the assembly of VLPs remained. Site-directed mutagenesis was used to investigate the effects of N-terminus of capsid protein on particle formation and thermal stability of grouper nervous necrosis virus. Althought all arginine mutants could produce VLPs, the relative amounts and thermal stabilities of arginine-mutated VLPs were decrease. The VLPs from £GN25-R29A and £GN25 mutants have similar structural properties on particle formation and thermal stability. Therefore, the effects of Arg29 mutations are negligible. The relative amounts and thermal stabilities of VLPs from £GN25-R30A and £GN25-R31A mutants are lower than £GN25-R29A VLP. When 25 amino acids at N-terminus of DGNNV capsid protein were removed, Arg30 and Arg31 are important for particle formation and particel stability. Although particle could form as 12 positively charged amino acids were lost (¡µN25-R293031A), the efficiency of particles assembly were decrease to 1.2 ¡Ó 0.9% as compare to wild-type VLPs (WT-VLPs). Site-directed mutagenesis and chemical reducing reagents were used to investigate the roles of disulfide bonds in particle formation and thermal stability of grouper nervous necrosis virus. The homogeneous particles from C187A, C331A and C187A/C331A mutants are indistinguishable from the native virus and WT-VLPs in their sizes and shapes. C115A and C201A mutants could not produce VLPs. The dissociated capsomers from arginine- or cysteine-mutant VLPs all can be reassembled to icosahedrons with efficiencies as high as 100%. When VLP particles are pre-fabricated, the reducing agent cannot disrupt the VLP icosahedron structure. The thiol reduction only caused effects on the disulfide linkages inside the icosahedrons. £]-mercaptoethanol-treated WT-VLPs could not tolerate the thermal effects at a temperature higher than 70¢XC. Once the disulfide linkages in dissociated capsomers were entirely disrupted by £]-mercaptoethanol treatment, the resulting capsomers could not reassemble back to icosahedron particles.These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles. In the observation of Cryo-EM, the shapes and sizes of the N-terminus truncated particle (£GN25-VLP) are indistinct from the full-length particle (WT-VLP). The maximum diameter of DGNNV is approximately 380 Å. Like that of the insect nodaviruses, the surface morphologies of £GN25-VLP and WT-VLP are consistent with a T = 3 quasi-equivalent lattice. The protrusions (~154 to 192 Å), the inner shell of the capsid (~112 to 154 Å), and the RNA (¡Õ112 Å) were observed in the DGNNV structure. The protrusion domain is consisting of three capsid subunits, and the interactions between these subunits are different. Deletion of 25 residues at the N-terminus did not affect VLPs formation and the structure of £GN25-VLP is similar to WT-VLPs. Resolutions was calculated by Fourier shell correlation, and the resolution of WT-VLPs and £GN25-VLPs is 6.5Å and 11.8Å, respectively.

In order to investigate the effects of C-terminus modification of Dragon Grouper Nervous Necrosis Virus capsid protein on the virus particle formation, we used E. coli expression system to express DGNNV capsid protain with different truncations at C-teminus fused with six or three histidines (His-Tag). These poly-His tagged clones, including ¡µC334-C6H, ¡µC335-C6H, ¡µC336-C6H, ¡µC337-C6H, C3H and C6H (His6 tagged at the C-teminus of wild-type capsid protein)¡Awere expressed and VLPs formation ability were examined. Wild-type and N-terminal recombination (N6H, His6 tagged at the N-teminus of wild-type capsid protein) were also used for comparison. These His-tagged VLPs can be further purified by Ni-NTA agarose, and their thermal stability of mutant VLPs were analyzed by Circular Dichroism. The Western blotting and ELISA assay were utilized to analyzed N-teminus or C-terminus was located at the surface of virus icosahedron. Once the four amino acids at the C-terminus of capsid protein were truncated (¡µC334-C6H), the mutated cpasid protein cannot assemble into VLPs. The same phenomenon was also observed in C6H. The related productions of wild-type, ¡µC335-C6H, ¡µC336-C6H, ¡µC337-C6H, C3H VLPs were about 100%, 56%, 116%, 141%, and 193%, respectively. Using Circular Dichroism to observe the thermal stability of mutant VLPs, the results revealed that the Tm of mutant VLPs were about 3oC lower than wild-type VLPs (61oC). The results of Western blotting and ELISA assay suggest that the C-termius of DGNNV capisid protein was exposed to the surface of virus structure.

Grouper in Taiwan is of high value, but nervous necrosis virus infection causes 100% mortality. Our laboratory had developed a good expression system to produce virus-like particles that induced immune functions. In this study, cells producing monoclonal antibody against the virus-like particles were used to induce BALB/c mice production of high titer ascites. The ascite from the H1 cells generated 6000-fold of antibodies higher than cell culture, using enzyme-linked immune-sorbent assay, although relatively less than the previous monoclonal cells of mX, 8000-fold. In the electron microscopy, the ascite antibody bound to various mutants of virus-like particles. Using the polymerase chain reaction to amplify fragments of IgG cDNAs, we will clone and express such cDNAs to efficiently produce the desired monoclonal antibodies.

Overfishing of stock and decreasing biodiversity are grave concerns for the U.S. and the rest of the world. Understanding and applying spatial and temporal information of marine species’ reproductive ecology and critical life habitat is vital to the development of effective strategies for marine resource management. In the Caribbean, one of the critical science gaps hindering effective management is the lack of information on how environmental factors may make fish spawning aggregation (FSA) sites optimal for spawning. Understanding the patterns of seafloor characteristics of spawning aggregation sites is of great interest to managers who need a means to efficiently design marine protected areas to help rebuild regional fish stocks. The specific goals of the study were: (1) to map the seafloor at historically known grouper and snapper spawning aggregation sites in three different countries, and (2) to characterize quantitatively the geomorphology of the sites including horizontal and vertical curvature profiles of the reefs, bottom depth at spawning sites, distance between spawning sites and shelf-edges/reef promontory tips, and the shortest distance between the spawning sites and 100 m water depth. These data were field-collected with a GPS and single-beam eco-sounder that provided latitude/longitude and depth. The point data were interpolated to surfaces in GIS to determine slope, aspect, curvature, and distance from spawning sites and three-dimensional reef structures. This study revealed that all 12 known Nassau grouper spawning aggregation sites in Belize and 5 known sites in the Cayman Islands were located at convex-shaped seaward extending reefs (reef promontories) jutting into deep water, within 1 km of reef promontory tips. However, spawning aggregations did not always occur at the tips of reef promontories, though all were found along the shelf edges within 1 km of promontory tips. Sixteen sites were multi-species spawning sites. These general characteristics were used to predict an undiscovered multi-species spawning aggregation in Belize. A successful prediction in Belize, together with the compiled data from multiple sites indicate: 1) reef promontories are vital locations for transient reef fish spawning aggregations, and 2) this study provides a potential tool for prediction of unknown spawning sites in the Caribbean.