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 sheepshead has two readily isolatable humoral immunoglobulins, a 16S tetrameric form and a 6S monomeric form. The 16S tetrameric form is composed of two subpopulations, one being a disulfide linked form (~700,000 daltons) and the other a noncovalently linked population of predominantly disulfide linked dimers (~350,000 daltons). The 6s immunoglobulin (~140,000 daltons) is composed of two noncovalently linked units (~70,000 daltons) each having one heavy and one light chain. The 6S immunoglobulin is antigenically deficient to the 16S immunoglobulin, this deficiency may be due to the heavy chain of the 6s protein lacking a~25,000 dalton segment present in the heavy chain of the 16S molecule. Cutaneous mucus and bile also contain immunoglobulins. The mucus contains three proteins that can be considered immunoglobulins: a 6S form which is antigenically indistinguishable from the serum 6S immunoglobulin: a ~700,000 dalton form which does not have a "dimeric" subpopulation as observed with the serum 16S protein; and a dimeric form of ~350,000 daltons. The dimeric form may have a secretory piece since the reduced mucus dimeric protein shows an additional polypeptide chain at ~95,000 daltons. All of the cutaneous mucus high molecular weight immunoglobulins have heavy and light chains identical to the serum high molecular weight immunoglobulins (~70,000 and ~25,000 daltons). Bile immunoglobulin is dimeric and composed of two noncovalently linked monomers of ~l60,000 daltons. The bile heavy chains are ~55,000 daltons; the light chains are ~25,000 daltons. The bile immunoglobulin does not appear to be a different class of protein from that of the serum or mucus immunoglobulins. In vivo administration of radiolabeled 16S and 6S serum immunoglobulins indicates that the 6S protein is not a degradation product of the 16S form. The half lives of the 16S and 6s forms are both ~16 days. Furthermore, the secretory immunoglobulins of the bile and mucus are not due to simple transudation or active transport of the predominant serum immunoglobulins. This result suggests that the secretory immunoglobulins of the sheepshead may be locally synthesized.

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.

Phenotypic plasticity, the capacity of a single genotype to exhibit variable phenotypes in different environments, is common in many species. A sample of wild caught Archosargus probatocephalus, also known as sheepshead, from Florida was randomly divided into two treatment groups: one group was fed soft prey, Mercenaria sp. muscle tissue, and the other group was fed hard prey, Mercenaria sp. in the shell, for 365 days. It was hypothesized that the sheepshead fed hard prey would have a thicker tooth enamel layer containing more calcium, and therefore be stronger than the tooth enamel layer of those fed soft prey items. Additionally, the mean functional jaw surface area, the percentage of tooth coverage of functional jaw surface, number of teeth per jaw, correlation between standard length and mean total tooth height, and the combined surface area of the teeth, when compared between the two treatments, should be greater in the hard prey treatment. The seventeen jaws of two prey groups were acquired postmortem and each jaw was divided into four quadrants. The largest tooth in each quadrant was removed from the jaw, longitudinally sectioned, and examined using scanning electron microscopy (SEM) to measure the enamel and dentin layers. Using the SEM backscatter electron detector the elemental composition of the different layers was determined at multiple locations. Finally, data was analyzed using analyses of variance (ANOVA’s) to compare mean tooth height, calcium content in enamel and dentin layers, mean functional jaw number of teeth per jaw, and upper to lower jaw overall enamel and dentin thickness between each treatment. Phenotypic plasticity was identified in three areas: percentage of jaw surface covered by teeth, a positive correlation between total tooth height and enamel height in hard prey treatment, and a positive correlation between total tooth height and soft prey treatment dentin height; but not in the other areas studied. It is apparent that phenotypic plasticity can increase an individual’s ability to survive in a variable food resource environment by changing some aspects of tooth morphology, but the ability to change in response to stimuli was not found in all areas of tooth structure. i

Microbial life on Mars is not visible from orbit or by rover cameras, but the fracture networks and scarp morphologies associated with fractures they could live in are measurable. We conducted a field analogue study of 92 normal fault scarps in the Sheepshead Mountains, Oregon to examine the correlation between scarp morphology and vegetation growth in the Steens Basalt. While vegetation is not expected on Mars, the fracture networks that sustain vegetation offer the same micro-environment that would support and protect endoliths. Structural variables were measured in the field and infrared spectra of fault scarps were measured using a handheld multispectral camera and vegetation indices were calculated from these images. Statistical analysis of the scarp morphologic parameters indicate that interconnectedness of fractures is key for elevated vegetation and is represented by a range of parameters. Results support a model for ideal slopes to investigate for preserved biological activity on Mars.

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.