Thesis (M.S.)--Virginia Polytechnic Institute and State University, 2001. / Title from PDF t.p. (viewed Dec. 17, 2005). Vita. Includes bibliographical references (p. 33-41).

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 2001. / Title from PDF t.p. (viewed on Dec. 11, 2005). Vita. Includes bibliographical references.

The work presented in this dissertation examines possible modes of action for growth inhibition by anthropogenic endocrine disrupting chemicals (EDCs) as well as endogenous hormones associated with growth in fish. Using the sheepshead minnow (SHM) (Cyprinodon variegatus) as a model, I developed methods to examine perturbations in the endocrine axis controlling fish growth, and also examined effects of EDCs on the whole fish. I used two relatively new techniques to study the endocrine growth axis, quantitative real-time PCR (TaqMan) and differential display analysis. TaqMan analysis is a highly sensitive method to measure specific sequences from a small amount of total RNA using a fluorescent probe and specific primer pairs. I optimized a TaqMan assay for SHM IGF-I to measure hepatic IGF-I mRNA concentrations. in fish injected with hormones known to influence fish growth (GH, T���, E���, insulin, or a carrier control). IGF-I mRNA levels increased in fish injected with GH, T��� and insulin, peaking at 12 h post-injection. IGF-I mRNA levels decreased significantly at 8 h and 12 h post-injection in fish injected with E���, suggesting that pharmacological levels of E��� may affect the GH/IGF-I axis and could have consequences for fish living in waters polluted by EDCs. Differences in growth were observed in fish exposed for 18 weeks to E��� or chlorpyrifos (an organophsophate). Fish exposed to the highest dose of E��� grew larger than controls only during the last week of the experiment. Fish exposed to the lower dose of E��� were not significantly different from controls. The fish exposed to all doses of chloryprifos grew significantly less than controls in a dose-dependent manner. No significant differences were found in hepatic IGF-I mRNA levels in any treatments. To establish patterns of gene up- or down-regulation, I performed differential display analysis on livers of several fish from the previous two experiments. Several genes were identified as being similar to fish including a microsatellite sequence, a choriogenin (vitelline envelope) protein mRNA sequence, a transferrin mRNA sequence and several ribosomal RNA sequences. This technique to evaluate gene expression will become more useful when more fish genes are added to the data bases. / Graduation date: 2003

The genome of the Atlantic sheepshead minnow, <u>Cyprinodon variecratus</u> (cyprinodontidae) contains at least two highly repetitive satellite DNA sequences. The major satellite sequence is present at roughly 10⁶ copies per haploid genome. The basic repeating unit averages 170 bp in length, tandemly arranged, and is bracketed by a Hind III restriction site. The Hind III satellite has little or no sequence similarity with other fish satellite DNAs reported in the 1iterature. Comparative hybridization experiments detect related sequences in the genomes of several species of pupfish of the genus <u>Cyprinodon</u> as well as in some cyprinodontid species of the genera <u>Jordanella</u> and <u>Floridichthys</u>. No significant hybridization was detected with the New World species <u>Cualac tesselatus</u> and Megupsilon aporus or within species of the Old World genera <u>Valencia</u> and <u>Aphanius</u>. / Ph. D.

Since many estuarine zones are impacted by copper contamination, there is an on-going effort to develop Biotic Ligand Models (BLMs) predicting copper toxicity in transitional environments. In the first stage of this project, a critical analysis of the BLM framework identified some aspects of the model that required further investigation. In particular, a BLM for estuaries needed (a) a better characterization of the dissolved organic matter (DOC) and its effect on copper availability, and (b) the inclusion in the model’s equation of a salinity-correction factor modulating the relationship between copper accumulation on the biotic ligand and toxicity. The first issue was addressed by modelling the data produced using a Chelex resin method to determine the labile fraction of copper in samples of mixed riverine and estuarine waters. A refined and simplified BLM equation was then presented, accounting for both the DOC characteristics and the relevance of the osmotic gradient in modulating the relationship between copper accumulation and toxicity. A critical analysis of the literature on copper toxicity and salinity led to the hypothesis that copper-exposed fish are more sensitive to osmotic stresses, as copper interferes with their osmoregulatory pathways. In particular, the cytosolic isoform-2 of the enzyme carbonic anhydrase (CA2) was identified as an osmotic effector protein targeted by copper and involved in osmotic stress response pathways, hence representing a mechanistic link between the combined effects of copper exposure and osmotic stress. To test this hypothesis, two in vivo studies were performed, using the euryhaline fish sheepshead minnow (Cyprinodon variegatus) and applying different rates of salinity changes as a way of dosing osmotic stress. The results showed a disturbance in plasma ion homeostasis after the salinity transitions, but notably the magnitude of the disturbance was greater in the copper-exposed individuals, suggesting a sensitizing effect of copper on the responses of fish to osmotic stress. Gene expression data demonstrated that CA2 is targeted by copper and confirmed the role of the enzyme in osmoregulatory pathways, as further supported by a promoter analysis of the gene coding for zebrafish CA2, which revealed the presence of osmotic-stress related elements. Overall, these results suggest that CA2 is an osmotic effector protein whose response can be activated by a medium level of osmotic stress through a combination of transcriptional and post-translational control circuits.

Low oxygen tolerances of ten fishes were estimated using an original nitrogen cascade design, and reciprocally transformed to express responses as ventilated volume necessary to satisfy minimal oxygen demand (L·mg O2^-1). Values ranged from 0.52 to 5.64 L·mg^-1 and were partitioned into three statistically distinct groups. Eight stream fishes showed moderately high tolerances reflecting metabolic adaptations associated with stream intermittency. Juvenile longear sunfish and two mollies comprised the second group. High tolerance of hypoxia may allow juvenile sunfish to avoid predation, and mollies to survive harsh environmental oxygen regimens. The sheepshead minnow was the most tolerant species of low oxygen, of those examined, explaining its presence in severely hypoxic environments.

The microbiome plays a key symbiotic role in maintaining host health and aids in acquiring nutrients, supporting development and immune function, and modulating behavior. However, more research is needed to elucidate the potential impact of environmental pollutants on host microbial communities and how microbiomes can modulate the toxicity of contaminants to the host. Through a literature review of 18 studies that assessed the impacts of various anthropogenic chemicals on fish-associated microbiomes, we found that toxicants generally decrease microbial diversity, which could lead to long-term health impacts if chronically stressed, and can increase the host’s susceptibility to disease as well as the chemical resistance of certain microbes. These findings led us to explore the impacts of one of the reviewed contaminants, polycyclic aromatic hydrocarbons (PAHs), typically found in oil. The Deepwater Horizon disaster of April 2010 was the largest oil spill in U.S. history and had catastrophic effects on several ecologically important fish species in the Gulf of Mexico (GoM). This study tested the hypotheses that exposure to weathered oil would cause significant shifts in fish gut-associated microbial communities, with taxa known for hydrocarbon degradation increasing in abundance and that foraging behavior would decrease, potentially due to microbial dysbiosis via the gut-brain axis. We characterized the gut microbiome (with 16S rRNA gene sequencing) of a native GoM estuarine species, the sheepshead minnow (Cyprinodon variegatus). Fish were exposed to High Energy Water Accommodated Fractions (HEWAF; tPAH = 80.99 ± 12.5 μg/L) of oil over a 7-day period and whole gastrointestinal tracts were sampled for microbiome analyses. A foraging behavioral assay was used to determine feeding efficiency before and after oil exposure. The fish gut microbiome did not experience any significant changes in alpha or beta diversity but known hydrocarbon degrading taxa were noticeably present in oil-exposed communities and were absent in controls. We found the order Pseudomonadales, the family Paenibacillaceae, and Pseudomonas pachastrellae to be among these, with Pseudomonadales increasing in abundance. Foraging behavior was not significantly affected by oil exposure. This work highlights the need for further research to elucidate the functional metagenomic responses of the fish gut-microbiome under oil spill conditions.

No description available.

<div>Following the 2010 Deepwater Horizon oil spill, extensive research has been conducted on the toxicity of oil and polycyclic aromatic hydrocarbons (PAHs) in the aquatic environment. The location and timing of the Deepwater Horizon surface slick coincided with the spawning seasons of many important pelagic and estuarine fish species. As such, there has been particular emphasis placed on the effects of PAHs on sensitive life history stages in fish, such as the embryonic and larval periods. Additionally, the spill occurred throughout the spring and summer months which, in estuaries, are marked by regular fluctuations in abiotic environmental factors such as dissolved oxygen, salinity, and temperature. Until recently, there has been little work done to elucidate the combined effects that PAHs from oil spills and adverse environmental conditions (hypoxia, increased salinity, and elevated temperatures).</div><div>Work presented in this dissertation uses next generation sequencing technology (RNA Seq) to determine differential gene expression in larval estuarine teleosts following exposure to adverse environmental conditions and PAHs. Downstream canonical pathway and toxicological function analysis were then applied to the identified differentially expressed genes (DEGs) to predict cardiotoxic responses at the organismal level. To verify the predicted responses, a phenotypic anchoring study was conducted and identified a cardiotoxic phenotype (pericardial edema) and reduced cardiac output in embryos exposed to oil. Finally, the mechano-genetic interplay governing the morphological development of the teleost heart was investigated and correlations between developmental gene expression and blood flow forces within the cardiovascular system were identified.</div>