This dissertation contains three papers from two projects. The first two papers (Chapter Two and Chapter Three) are from a project entitled “Managing Flounder Openings for Maximum Revenue.” The objective of this project is to (1) estimate the monthly dockside price of summer flounder and identify seasonality in this price; and (2) set up a mathematical programming model to maximize the landing revenue by allocating the federal government quota on summer flounder across twelve months. In the first paper (Chapter Two), various forms of inverse demand equations are used to estimate the dockside price of summer flounder. These models are evaluated based on their out-of-sample forecasting performance. A structural functional form is selected. In the second paper (Chapter Three), the selected price equation for summer flounder is applied into a revenue maximization model with both the federal government quota constraint and biological constraints from twelve months. The model is solved using CONPOT Solver of GAMS 21.5. The results of the scenarios indicate that the industry should move the landing effort from the period of October – February to the period of March – August. Comparing with historical data, this method can increase $44.73 million for the industry of landing summer flounder from 1991 to 2005. The third paper (Chapter Four) investigates how China's soybean import prices and domestic prices of soybeans and soybean products affect China's soybean imports. Since 2000, soybeans have been the U.S. leading agricultural exports for bulk commodities. China is the largest importer of U.S. soybean exports. For China's soybean crushing industry, imported soybeans are inputs rather than final products and used to produce soybean meal and oil. A differential production model, which is derived from a two-stage profit maximization model in producer theory, is adopted in this research. Estimates are used to calculate conditional and unconditional price elasticities for China's soybean imports from its major source countries – the United States, Argentina, and Brazil. In addition, the Divisia index and unconditional output price elasticities are obtained for China's soybean imports. Estimation results support the hypothesis that China's soybean imports are determined by its domestic demand for soybean meal, rather than soybean oil. This implies that U.S. agribusinesses should pay attention to the dominant role of China's demand for soybean meal and animal feed. U.S. agribusinesses can also use results in this research to evaluate how China's soybean imports from different source countries will change when either international market prices or China's domestic market prices change. / Ph. D.

Nuclear hormone receptors (NHRs) are ligand-activated transcriptions factors which transduce the effects of various hormones as well as nutritional and other environmental signals. They thus function to maintain physiological homeostasis by integrating the tissue expression of specific target genes to regulate a wealth of biological processes including reproduction, development, metabolism and environmental adaptation. Mounting evidence indicates NHRs are the target of endocrine disrupting compounds (EDCs), exogenous chemicals, often of anthropogenic origin, which disrupt NHRs and thus the processes under their control. EDCs can interfere with NHR signalling by activating receptors (agonists), by inhibiting the actions of the receptor (antagonists), or by disrupting endogenous hormone synthesis, secretion, transport or metabolism. Much of the focus to date has been on the risk of EDCs to reproductive functions, via estrogen and androgen NHRs in humans, and also in aquatic organisms. However environmental pollutants also have the potential to interact with other NHRs, particularly in aquatic environments, and cause dysregulation of other critical physiological processes, including energy homeostasis, immune functions and the stress response. To address this possibility a reporter gene assay was developed, allowing the high-throughput screening of pollutants for their interactions with piscine NHRs with critical roles in energy homeostasis, stress reponse and immune functions, namely the peroxisome proliferator-activated receptors (PPARs) and corticosteroid receptors (CRs) from European plaice (Pleuronectes platessa) and European flounder (Platichthys flesus), respectively. Complementary DNA (cDNA) sequences encoding the ligand-binding domains of PPARs and CRs, critical for receptor-ligand interactions and receptor activation, were ligated to the DNA-binding domain (DBD) of the yeast Gal4 transcription activator protein to create experimental expression plasmid constructs. Co-transfection of these expression plasmids into the fathead minnow (FHM) cell line with an upstream-activating sequence (UAS)-firefly luciferase reporter gene plasmid increased luciferase expression in the presence of known PPAR and CR ligands. Several aquatic pollutants including pharmaceuticals, industrial by-products and biocides were tested for their potential to disrupt PPAR and CR functions by interacting with these receptors in an agonistic or antagonistic manner. Several fibrates, a group of pharmaceutical compounds used to treat dyslipidemia in humans by targeting the PPARs, were able to activate plaice Gal4-PPARα and Gal4-PPARβ in the reporter gene assay, indicative of an interaction with PPAR receptors in non-target species. Fibrates which did not activate Gal4-PPARα were able to inhibit the activation of Gal4-PPARα by the PPARα-specific agonist, Wy14643, suggesting differential effects of fibrates on human and flounder PPARs. In addition some metabolites of widespread phthalate ester pollutants were also agonists of the Gal4-PPARα and Gal4-PPARβ constructs. The Gal4-PPARγ construct was unresponsive to almost all the compounds tested, including the mammalian PPARγ agonist, rosiglitazone. The exception to this was the phthalate metabolite monobenzylphthalate, which induced a small increase in firefly luciferase in Gal4-PPARγ transfected cells. All of the above effects required concentrations of at least 10 µM, which are unlikely to be encountered in the aquatic environment. In contrast bis(tributyltin) oxide (TBTO), a notorious environmental pollutant, inhibited Gal4-PPARα and Gal4-CR constructs at concentrations as low as 1 nM and 100 nM, respectively. These concentrations are lower than those reported in aquatic environments, or in fish tissues, making TBTO a candidate endocrine disruptor in fish by inhibiting PPARα and CR signalling. A European flounder cDNA microarray was used to investigate the trasnscriptional responses of flounder hepatocytes to TBTO (10 nM) exposure. Exposure to TBTO and Wy14643, both alone and in combination, indicated a TBTO-driven downregulation of several potential PPARα-target genes with functions in the immune system, the proteasome, and lipid metabolism, although, based on mammalian comparisons, some potential PPARα-target genes were also upregulated, indicating differences in mammalian and fish PPAR-target genes or reflecting the complexity of organisms at a higher organisational level than cell-based assay systems. However, the microarray-based approach was useful in formulating further hypotheses about the effects of TBTO on PPARα signalling. Overall, these results indicate that exogenous chemicals entering the aquatic environment can interfere with NHRs with functions in energy homeostasis, immune functions and stress, in non-target organisms. The cell-based reporter gene assay is a useful tool for identifying potential endocrine disruptors which target PPARs and CRs and would be a useful method in a first tier testing approach, limiting the use of live animal models and enabling investigation into specific receptors which are targets of endocrine disrupting compounds. Although more work is required to confirm the physiological consequences of TBTO inhibition of PPARα, the results presented here indicate that organisms inhabiting TBTO-polluted environments may experience suppression of the immune system, an increase in non-functional or misfolded proteins through suppression of genes involved in the ubiquitin/proteasome system and a disruption in lipid homeostasis.

Made available in DSpace on 2014-08-20T13:32:53Z (GMT). No. of bitstreams: 1 dissertacao_vinicius_campos.pdf: 483644 bytes, checksum: 9caf292597102dea88bfa2da68dc9158 (MD5) Previous issue date: 2009-03-03 / Food intake in vertebrates is a complex process involving several endocrine and neural pathways. Among the orexigenic factors, the most notably are the neuropetides as the neuropeptide Y (NPY) that is a 36 amino acid peptide which plays a key role in food intake. Studies evaluating fish NPY expression showed that this peptide is involved in appetite stimulation. However, despite the recent advances, our present knowledge of the regulation of feeding behavior in fish is limited and based in a few fish species, and there is increasing evidence of speciesspecific differences. The Brazilian flounder Paralichthys orbignyanus is being considered for aquaculture, and it is important to understand the mechanisms regulating feeding in order to improve its performance in captivity. The objectives of this study were to clone NPY cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain NPY expression during 24 h by real-time RT-PCR. A 597 bp NPY cDNA was cloned from Brazilian flounder brain by 3´RACE method. NPY expression was detected in all peripheral tissues analyzed, but with predominant expression in the brain. No significant differences were observed in brain NPY gene expression over a 24 h evaluation period. No correlation was observed among plasma glucose, total protein, cholesterol, triglycerides and NPY expression levels during 24 hours. These results indicated that NPY may play roles in flounder peripheral tissues and may be not involved short-term regulation of food intake at low-temperatures in Brazilian flounder. / O apetite e a ingestão de alimentos em vertebrados é um processo complexo que envolve várias rotas neurais e endócrinas. Dentre os fatores orexigênicos, destacamse os neuropeptídeos, como o neuropeptídeo Y (NPY), o qual é constituído de 36 aminoácidos e desempenha papel chave na regulação do apetite. Estudos avaliando a expressão do NPY em peixes demonstraram que este peptídeo está envolvido no estímulo da ingestão. Entretanto, apesar dos avanços recentes, o atual conhecimento sobre a regulação fisiológica do apetite é limitado e baseado em poucas espécies de peixes com evidencias de diferenças interespecíficas. O linguado Paralichthys orbignyanus tem sido considerado para a aqüicultura e com isso, torna-se importante compreender os mecanismos de regulação do apetite para incrementar o seu desempenho em cativeiro. Os objetivos deste trabalho compreenderam clonar o gene do NPY, avaliar os níveis de RNA mensageiro nos diferentes tecidos do linguado e também avaliar a expressão do NPY no cérebro durante 24 h por RT-PCR em tempo real. Um fragmento de 597 pb do cDNA do NPY foi clonado a partir do RNAm do cérebro do linguado através do método RACE 3 . A expressão do NPY foi detectada em todos os tecidos analisados (cérebro, baço, coração, intestino, estômago, brânquia, fígado, rim, músculo e testículo), mas com predominante expressão no cérebro. A expressão do NPY não cérebro demonstrou variações durante 24 horas de avaliação. Nenhuma correlação foi observada entre glicose, proteínas totais, colesterol e triglicerídeos plasmáticos. Estes resultados indicam que o NPY pode desenvolver funções em outros tecidos além do cérebro e também pode não estar associado na regulação da alimentação em curto prazo em baixas temperaturas.

Many of the habitats occupied by fish are highly dynamic, naturally demonstrating substantial abiotic fluctuations over diurnal, tidal or seasonal cycles. It is also the case that throughout their 545 million year evolutionary history, fish have existed in aquatic environments very different to those of the present day. However, the past several decades have seen unprecedented rates of environmental change, at local and global scales, arising from human activities. The two major themes of the present thesis are: 1) Respiratory responses of fish to changes in environmental oxygen and temperature in the context of exploring intra- and inter-specific trait variation and its ecological implications 2) The effects of environmental factors (oxygen, carbon dioxide, temperature and seawater chemistry) on the intestinal precipitation and excretion of calcium carbonate by marine teleosts. In the first study (chapter two) a comprehensive database of fish critical PO2 (Pcrit) data compiled from the published literature is presented. The systematic review of this literature provided the opportunity to critically examine methodologies for determining Pcrit as well as its usefulness as an indicator of hypoxia tolerance in fish. The second study (chapter three) examines whether inter- and intra-specific variation in thermal and hypoxia tolerance in two reef snapper species (Lutjanus carponotatus and Lutjanus adetii) reflects their distributions across the contrasting biophysical environments of the reef flat and reef slope surrounding Heron Island on the Great Barrier Reef. L. carponotatus was clearly the most thermally and hypoxia tolerant of the two species, demonstrating a ~3.5 °C wider thermal tolerance zone (higher CTmax, lower CTmin) and ~26% lower Pcrit than L. adetii. These results suggest that the contrasting distribution of these species between flat and slope reef zones is reflected in their physiological tolerances. However, there was no evidence of intra-species variation in tolerance between flat and slope caught L. carponotatus individuals, indicating that this species does not form physiologically distinct subpopulations between these reef zones. The third study (chapter four) experimentally quantified the effect of hypercarbia (3000 μatm) and hypoxia (50% air saturation) on gut carbonate production by the European flounder (Platichthys flesus). Both hypercarbia and hypoxia resulted in a significant increase in carbonate excretion rate (1.5-fold and 2.4-fold, respectively) and acted synergistically when combined. In the final study (chapter five), gut carbonate production was measured in the European flounder undergoing conditions simulating the ‘calcite seas’ of the Cretaceous. The results of this study support the hypothesis that ocean conditions prevalent during the Cretaceous period resulted in piscine carbonate production rates substantially higher (~14-fold) than the present day. Ultimately, this thesis directly links the environmental physiology of fish at the individual level to wider scale implications (past, present and future), ranging from local ecological patterns all the way up to global carbon cycles.

The American horseshoe crab (HSC), Limulus polyphemus, is one of four species of horseshoe crabs found throughout the world, and the only one found in North America. It is an economically and ecologically important species throughout its native range from Maine to the Yucatan Peninsula. Harvested for fertilizer and livestock feed in the 19th century, the species is now harvested as bait for whelk and eel fisheries, and for their blood by the biomedical industry. The Atlantic States Marine Fisheries Commission (ASMFC) started to formally manage HSC in 1998 with its Interstate Fisheries Management Plan (IFMP). Unique emphasis and harvest limits have been placed on the Delaware Bay stock, as it is commercially exploited and a critical food source for the threatened red knot, Calidris canutus rufa. Previously, estimates of relative and total abundance of HSC in the Delaware Bay area were based on a design-based approach using a stratified random sampling design. In Chapter 1 of this work, I developed hurdle models for each of the six HSC demographic groups to standardize catch-per-unit-effort (CPUE) and estimate relative abundance using a model-based approach. It was determined that while the two approaches resulted in mostly convergent estimates of relative abundance, external factors such as month, time-of-day, and average depth have major effects on the observed CPUE of all demographic groups. Chapter 2 involved the development of hurdle models for the three species of bycatch frequently caught in our trawls, channeled whelk (Busysotypus canaliculatus), knobbed whelk (Busycon carica), and summer flounder (Paralichthys dentatus). It was found that channeled whelk relative abundance has been at a historical low since 2016, while summer flounder has been at a consistent high. Recent estimates of knobbed whelk relative abundance have been less variable than previously seen, with estimates since 2016 being similar to those seen before 2012. These results provide the first estimates for whelk population trends in the mid-Atlantic region and add to the growing knowledge of summer flounder relative abundance in the area. In Chapter 3, I applied the hurdle models developed in Chapter 1 to estimate the total abundance of HSC in the Delaware Bay area. For this work, I developed two spatio-temporal variograms to estimate bottom temperature and bottom salinity at unmeasured cells per month in the time series. The results showed that night estimates of total abundance were consistently higher than daytime estimates, and estimates from September or November resulted in the highest estimated catch for all demographic groups. The results suggest that when comparing September model-based estimates at night to those of the design-based approach, nearly a third of all previous design-based estimates significantly underestimated the total abundance of HSC in the Delaware Bay area. This result suggests that the ASMFC can recommend increased harvest limits for mature individuals if that action aligns with the goals of their adaptive resource management (ARM) framework. / Master of Science / The American horseshoe crab (HSC), Limulus polyphemus, is one of four species of horseshoe crabs found throughout the world, and the only one found in North America. It is an economically and ecologically important species throughout its native range from Maine to the Yucatan Peninsula. Harvested by the millions in the 19th century, the species is still harvested as bait for whelk and eel fisheries, and for their blood by the biomedical industry, on the order of hundreds of thousands. Formal management of HSC by the Atlantic States Marine Fisheries Commission (ASMFC) began in 1998, and allowable catch and landings have decreased since 1999. A strong focus has been placed on the Delaware Bay population, as it is the center of HSC abundance and provides a critical food source for the threatened red knot, Calidris canutus rufa, while also being commercially exploited. To effectively manage the species, it is important to accurately estimate relative and total abundance so that proper harvest limits can be set. Previously, estimates of relative and total abundance of HSC in the Delaware Bay area were based on a design-based approach using a stratified random sampling design. In Chapter 1 of this work, I developed hurdle models (a generalized linear model that models the probability of observations and the observed positive counts using two separate regression models that are then combined) for each of the six HSC demographic groups to remove the effect of external factors (year, latitude, longitude, depth [inshore/offshore], topography, average trawl depth, time-of-day, month, bottom temperature, bottom salinity, and distance from shore) on our observed catch-per-unit-effort (CPUE) and estimate relative abundance using a model-based approach. It was determined that while the two approaches resulted in mostly convergent estimates of relative abundance, factors like month, time-of-day, and average depth had major effects on the observed CPUE of all demographic groups. Chapter 2 involved developing similar hurdle models for three species of bycatch frequently caught in our trawls, i.e., channeled whelk (Busysotypus canaliculatus), knobbed whelk (Busycon carica), and summer flounder (Paralichthys dentatus). It was found that channeled whelk relative abundance has been at a historical low since 2016, while summer flounder has been at a consistent high. Recent estimates of knobbed whelk relative abundance have been less variable than previously seen, with estimates since 2016 being similar to those seen before 2012. These results provide the first estimates for whelk population trends in the mid-Atlantic region and add to the growing knowledge of summer flounder relative abundance in the area. In Chapter 3, I applied the hurdle models developed in Chapter 1 to estimate the total abundance of HSC in the Delaware Bay area. To do this, the bottom temperature and salinity had to be estimated for each geographic cell. This was accomplished by developing two spatio-temporal variograms which allowed me to estimate either variable at an unmeasured point and time based on its spatial and temporal distance from a measured value in a process known as spatio-temporal kriging. The results showed that night estimates of total abundance were consistently higher than daytime estimates and that estimates from September or November resulted in the highest estimated total abundance for all demographic groups. The results suggest that when using September model-based estimates at night to compare against the design-based approach, nearly a third of all previous design-based estimates significantly underestimated the total abundance of HSC in the Delaware Bay area. This outcome could justify ASMFC increasing recommended harvest limits for mature individuals if that action aligns with the goals of their adaptive resource management (ARM) framework.

Effects of man made pollutants on an ecosystem are initiated at the cellular level where a prime determinant for survival of an organism is its ability to metabolise and excrete toxic chemicals or their metabolites, thereby preventing cellular toxicity or damage to germ cell DNA. Cytochrome P450 (CYP) enzymes are responsible (in concert with the remainder of the Ah battery enzymes) for the metabolism of numerous xenobiotics and endogenous compounds, including the metabolic activation of most environmental toxic chemicals and carcinogens. Genetic polymorphisms which affect performance of these enzymatic detoxification systems may alter tolerance to pollutants and thus survival in polluted environments. Alterations in the susceptibility of individuals and the development of resistant populations has arisen by forced selection of populations with variant genes, resulting in increased detoxification capacity. There is evidence for such scenarios of variations in activities of pollutant biotransforming enzymes of fish contributing to survival in polluted estuarine environments and several chemically resistant populations have been identified in the USA and Europe. In fish it has been demonstrated that CYP1A enzyme activity is required to activate some carcinogenic xenobiotics to a metabolic state in which they can form DNA adducts. The mechanism of reduced CYP1A expression in highly contaminated populations may therefore represent resistance to chemical stressors. European flounder (Platichthys flesus) from some waterways which have a long history of severe sedimentary contamination do not show elevated levels of CYP1A. The aim of the current study was to investigate whether any heritable differences were apparent between offspring from parents inhabiting long-term polluted and pristine areas. Flounder were obtained from a highly polluted estuary in the UK and crossed with fish from a relatively pristine environment. Offspring were raised in communal tanks in order to standardise environmental conditions, and allow investigations into the genetic variation of CYP1A. To allow identification of offspring to parental fish, polymorphic microsatellite loci were isolated and characterised for the flounder. Novel cDNA probes to transcription factors in the detoxification pathway (AhR2 and ARNT2) were cloned for flounder, and RT-PCR / Southern blot methods were developed for quantitation of gene transcript levels. A novel method of CYP1A quantification using real-time PCR was developed. PAH and PCB exposure trials were carried out on mixed batch offspring, and CYP1A gene transcript levels assessed using Northern blot and real-time PCR techniques. Offspring were genotyped to their parents using the microsatellites obtained, and CYP1A transcript levels were correlated with clean and polluted areas. CYP1A was further correlated to transcription factor expression, and data are presented. Following exposure to the commercial PCB mixture, Aroclor 1254, CYP1A transcript levels were found to be significantly lower in families whose parents originated from a polluted area. This observation indicates that there is a possible genetic component to variation in CYP1A levels, and that these fish may have acquired a heritable tolerance to polluted areas. The lack of induction, or correlation with CYP1A levels, of AhR2 and ARNT2 expression indicates a possible AhR independent pathway for the metabolism of PCBs in the flounder. © Tom Dixon 2003 http://www.tomdixon.org

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