Os filamentos branquiais da lagosta Americana, Homarus americanus, foram dissociados em solução salina fisiológica e os diversos tipos celulares separados em gradiente de 30, 40, 50 e 80% de sacarose através de centrifugação de alta velocidade. As células provenientes de cada solução de sacarose foram incubadas em 65Zn2+ visando caracterizar a tomada de zinco para cada tipo celular. A caracterização do acúmulo de zinco em cada célula foi investigada na presença e ausência de 10mM de cálcio (CaCl2), variadas concentrações de NaCl e pH, presença de 100 µM de verapamil, nifedipina (inibidores de canais de Ca2+) e ionóforo de cálcio, A23187. O influxo de 65Zn2+ foi descrito pela cinética de Michaelis-Menten nas concentrações de zinco variando de 1-1000 µM.. O cálcio externo não afetou o transporte de Zn para as células presentes no gradiente de 30% a 50% de sacarose, mas atuou como estimulador para as células em gradiente de 80% de sacarose. O cálcio reduziu o Km e a velocidade máxima de transporte (Vmax) para as células de 30% de sacarose, enquanto duplicou aparentemente a velocidade máxima de transporte para as células provenientes do meio em 80% de sacarose. Os resultados sugerem que o cálcio, sódio e prótons entram nas células branquiais através de um canal para cátion com ampla especificidade. Diferenças observadas no transporte de zinco em relação aos diferentes tipos de células aparentemente estão relacionadas com as diferentes taxas de afinidade de cada transportador em cada tipo celular. O transporte de 65Zn2+ também foi estudado em filamentos branquiais isolados e intactos, demonstrando propriedades de transporte muito parecidas com as observadas pelas células em gradiente de 80% de sacarose. Os resultados sugerem que a tomada de Zn se dá por processo de transporte na membrana apical das brânquias. Um modelo experimental para o transporte de Zn em células de brânquias de lagostas é apresentado. / Gill filaments of the American lobster, Homarus americanus, were dissociated in a physiological saline and separated into several cell types on a 30, 40, 50, and 80% sucrose gradient. Cells from each sucrose solution were separately resuspended in physiological saline and incubated in 65Zn2+ in order to assess the nature of metal uptake by each cell type. Characteristics of zinc accumulation by each kind of cell was investigated in the presence and absence of 10 mM calcium (CaCl2), variable NaCl concentrations and pH values, and 100 µM verapamil, nifedipine (calcium channel inhibitors), and the calcium ionophore, A23187. 65Zn2+ influxes were hyperbolic functions of zinc concentration (1-1000 µM) and followed the Michaelis-Menten kinetics. External calcium was neutral to cells from 30% to 50% sucrose, but stimulatory for cells from 80% sucrose. However, calcium reduced both apparent zinc binding affinity (Km) and maximal transport velocity (Jmax) for 30% sucrose cells, while doubling the apparent maximal transport velocity for 80% sucrose cells. Results suggest that calcium, sodium, and protons enter gill epithelial cells by way of an endogenous broad-specificity cation channel and trans-stimulate metal uptake by a plasma membrane carrier system. Differences in zinc transport observed between gill epithelial cell types appear related to apparent affinity differences of the transporters in each kind of cell. Low affinity cells from 30% sucrose were partially inhibited by calcium, while high affinity cells from 80% sucrose were stimulated. 65Zn2+ transport was also studied by isolated, intact, gill filament tips. These intact gill fragments generally displayed the same transport properties as did cells from 80% sucrose and provided support for metal uptake processes being an apical phenomena. A working model for zinc transport by lobster gill cells is presented.

Os filamentos branquiais da lagosta Americana, Homarus americanus, foram dissociados em solução salina fisiológica e os diversos tipos celulares separados em gradiente de 30, 40, 50 e 80% de sacarose através de centrifugação de alta velocidade. As células provenientes de cada solução de sacarose foram incubadas em 65Zn2+ visando caracterizar a tomada de zinco para cada tipo celular. A caracterização do acúmulo de zinco em cada célula foi investigada na presença e ausência de 10mM de cálcio (CaCl2), variadas concentrações de NaCl e pH, presença de 100 µM de verapamil, nifedipina (inibidores de canais de Ca2+) e ionóforo de cálcio, A23187. O influxo de 65Zn2+ foi descrito pela cinética de Michaelis-Menten nas concentrações de zinco variando de 1-1000 µM.. O cálcio externo não afetou o transporte de Zn para as células presentes no gradiente de 30% a 50% de sacarose, mas atuou como estimulador para as células em gradiente de 80% de sacarose. O cálcio reduziu o Km e a velocidade máxima de transporte (Vmax) para as células de 30% de sacarose, enquanto duplicou aparentemente a velocidade máxima de transporte para as células provenientes do meio em 80% de sacarose. Os resultados sugerem que o cálcio, sódio e prótons entram nas células branquiais através de um canal para cátion com ampla especificidade. Diferenças observadas no transporte de zinco em relação aos diferentes tipos de células aparentemente estão relacionadas com as diferentes taxas de afinidade de cada transportador em cada tipo celular. O transporte de 65Zn2+ também foi estudado em filamentos branquiais isolados e intactos, demonstrando propriedades de transporte muito parecidas com as observadas pelas células em gradiente de 80% de sacarose. Os resultados sugerem que a tomada de Zn se dá por processo de transporte na membrana apical das brânquias. Um modelo experimental para o transporte de Zn em células de brânquias de lagostas é apresentado. / Gill filaments of the American lobster, Homarus americanus, were dissociated in a physiological saline and separated into several cell types on a 30, 40, 50, and 80% sucrose gradient. Cells from each sucrose solution were separately resuspended in physiological saline and incubated in 65Zn2+ in order to assess the nature of metal uptake by each cell type. Characteristics of zinc accumulation by each kind of cell was investigated in the presence and absence of 10 mM calcium (CaCl2), variable NaCl concentrations and pH values, and 100 µM verapamil, nifedipine (calcium channel inhibitors), and the calcium ionophore, A23187. 65Zn2+ influxes were hyperbolic functions of zinc concentration (1-1000 µM) and followed the Michaelis-Menten kinetics. External calcium was neutral to cells from 30% to 50% sucrose, but stimulatory for cells from 80% sucrose. However, calcium reduced both apparent zinc binding affinity (Km) and maximal transport velocity (Jmax) for 30% sucrose cells, while doubling the apparent maximal transport velocity for 80% sucrose cells. Results suggest that calcium, sodium, and protons enter gill epithelial cells by way of an endogenous broad-specificity cation channel and trans-stimulate metal uptake by a plasma membrane carrier system. Differences in zinc transport observed between gill epithelial cell types appear related to apparent affinity differences of the transporters in each kind of cell. Low affinity cells from 30% sucrose were partially inhibited by calcium, while high affinity cells from 80% sucrose were stimulated. 65Zn2+ transport was also studied by isolated, intact, gill filament tips. These intact gill fragments generally displayed the same transport properties as did cells from 80% sucrose and provided support for metal uptake processes being an apical phenomena. A working model for zinc transport by lobster gill cells is presented.

Many management strategies are available to fishery managers to improve the sustainability of a fishery; however, it is not always clear how implemented strategies interact with the demographics of the exploited species. Management decisions are often made in order to maximize egg production and recruitment or to preserve specific size classes to increase reproductive output. Consequences of these strategies could include variation from a natural size structure of the population, exaggerated sexual dimorphism, skewed sex ratios, and suboptimal mating conditions. To examine the possibility of these consequences, I ran a series of deterministic models to simulate the fished and unfished population dynamics of two clawed lobster species, American lobster (Homarus americanus) and European lobster (Homarus gammarus), under a variety of management strategies. Protection of ovigerous females takes two forms in this model. The American fishery in the Gulf of Maine requires fishers to v-notch ovigerous females for up to four years of protection from fishing. The European fishery in southern Norway bans the harvest of ovigerous females, which translates to one year of protection. I compared the relative impact of the two levels of ovigerous female protections on factors that may be important for reproductive success: size structure, dimorphism, sex ratio, and egg production for both species. I then considered a case study on European lobster to evaluate the interaction of a no-take marine protected area with a slot limit to compare relative impacts to egg production, overall size structure and dimorphism. Results showed that American lobster females benefitted greatly from strict protections such as v-notching in terms of mean size increase and egg production, but mean size dimorphism and the overall proportion of females also increased with higher fishing pressure. European lobster females also benefitted from protections, but less dramatically than American lobsters, and with lower size dimorphism. In the case study, European lobsters benefited most from the implementation of a no-take marine protected area in combination with a slot limit to preserve the largest individuals of both sexes which improved overall egg production. By taking a simulation approach to evaluate these different management strategies on two closely related species of lobster, this thesis provides a basis for understanding how fishery decisions can achieve their sustainability goals in addition to quantifying some of the unintended impacts of management on parameters that may be important to overall reproductive success. / Master of Science / Fishery managers make decisions about a fishery based on the species' biology to optimize catch while also ensuring that enough individuals remain to keep the population alive for years to come. However, it is not always clear what types of unintended consequences these decisions may have. Management decisions are often made in order to maximize egg production, to keep small individuals alive until they can reproduce, or to preserve the large individuals that have high reproductive potential. Some consequences of these strategies could include a reduction in the number of large individuals with high fishing pressure, increased female body size compared to males, unequal numbers of females and males, and less optimal conditions for mating. To examine the possibility of these consequences, I ran a series of models to simulate the fished and unfished populations of two clawed lobster species, American lobster (Homarus americanus) and European lobster (Homarus gammarus), under a variety of management strategies. The American lobster fishery in the Gulf of Maine requires a practice known as v-notching, which protects egg-bearing females for up to four years. The Norwegian fishery for European lobster bans the harvest of egg-bearing females, which is protection that lasts for one year. I compared the impact of the two types of egg-bearing female protections on factors that may be important for reproductive success: abundance of large lobsters, unequal numbers of females and males, difference in size between females and males, and overall production of eggs for both species. I then considered a case study on European lobster to test how a no-take marine protected area and a slot limit (minimum and maximum harvest size limits) affected those same factors. Results showed that American lobster females benefited greatly from longer lasting protections by growing to larger sizes and producing more eggs, but the average size difference between females and males and the overall proportion of females also increased as fishing became more intense. European lobster females also benefitted from protections, but less dramatically than American lobsters, and with lower mean differences in size between females and males. In the case study, European lobsters benefitted most from the no-take marine protected area in combination with a slot limit to preserve the largest individuals of both sexes which improved overall egg production. Using a simulation makes it easier to tease apart the effects of these different management strategies on two closely related species of lobster. This thesis helps managers see how fishery decisions affect lobsters in both desired and unintended ways. With this information, managers can better improve the sustainability of the fishery through considering what a specific species needs to improve reproductive success.

The series of experiments that comprises this thesis consider the use of prebiotics and probiotics to improve success in European lobster Homarus gammarus culture. Research was conducted at the National Lobster Hatchery and The University of Plymouth, UK between April 2007 and March 2010. Great potential was revealed for the application of biotics as dietary supplements in the culture of larval and juvenile H. gammarus. Specific biotics, namely; prebiotic mannon oligosaccharide (MOS) and probiotic Sanolife® (Bacillus spp.) were revealed to increase growth and survival during the culture of early lobster life stages. Lobsters that showed enhanced development and/or survival were found to possess improved feed conversion, immune status and/or stress tolerance. Furthermore it was found that biotic fed lobsters displayed shifts in gastrointestinal (GI) microbiology and enhanced physical GI structures, which may account for improved feed conversion and consequently growth. The work presented thus demonstrates that by making positive changes in GI structure and bacterial community composition combined with influencing immune status culture success can be positively enhanced through the biotic supplementation of diets. However, it must be considered that the outcome of dietary biotic supplementation is heavily influenced by the situation in question.

Previous nutritional physiology research using L-histidine and zinc in American lobster intestine (Homarus americanus) has suggested that these solutes can be co-transported as complexes (Histidine-Zinc-Histidine) across the intestine using a peptide transporter. Furthermore, transport of L-leucine was shown to be inhibited by high calcium concentrations. Dipeptide and bis-complex transport and the role of calcium were investigated in the perfused intestines of lobster and Atlantic white shrimp (Litopenaeus setiferus). Following trans-intestinal transport, serosal medium was analyzed for amino acid composition by gas chromatography. In lobster, the transport of glycylsarcosine (Gly-Sar) from mucosa to serosa was stimulated two-fold with luminal pH 8.5, compared to the pH 5.5 control. Mucosa to serosa and serosa to mucosa fluxes of Gly-Sar were measured; the dipeptide was transported intact in both directions, but the net flux was from mucosa to serosa. The use of 0.5mM calcium chloride stimulated Gly-Sar transport two-fold, compared to 25 mM. In shrimp, the addition of 50 µM zinc chloride increased the rate of L-histidine transport, while Gly-Sar inhibited histidine transport in the presence of zinc. The rate of histidine transport was significantly higher with 1mM calcium chloride than with 25mM. These results suggest that shrimp transport bis-complexes in a manner similar to lobster. High calcium concentration had an inhibitory effect on both amino acid and dipeptide transport. Proposed mechanisms accounting for the effects of metals and calcium on trans-intestinal transports of both amino acids and dipeptides by lobster and shrimp digestive tracts are discussed.