This study was undertaken to elucidate the synthesis site or sites of the major yolk protein, vitellin (Vt), in the red swamp crayfish, Procambarus clarkii. Vitellin was purified from late vitellogenic ovaries (containing oocytes $\ge$456 $\mu$m in diameter). The protein was purified by ultra centrifugation, gel filtration, and finally native polyacrylamide gel electrophoresis. Once the purified form of the protein was attained it was used to create polyclonal and monoclonal antibodies by inoculating rabbits and mice, respectively. The specificity and sensitivity of the antisera were each determined by titering using sandwich enzyme-linked immunosorbent assay (ELISA). The antisera were used to design a competitive quantitative ELISA for Vt and its precursor, vitellogenin (Vg), which is immunologically identical in many crustaceans. The concentrations of Vt in ovaries and Vg in hemolymph were determined in relation to the stage of reproductive maturity as determined by oocyte diameters. The total aqueous protein concentration in developing ovaries ranged from 1% to 5% and consisted of 23% to 55% Vt. The Vt concentrations from ovaries of sixteen mid-vitellogenic females averaged 38% of the total aqueous protein. The competitive ELISA was sensitive to standard Vt concentrations as low as 62 ng/ml and was capable of distinguishing concentrations as high as 8 $\mu$g/ml. Hemolymph Vg concentrations, determined from diluted hemolymph samples (1:20), ranged from 10 $\mu$g/ml in early vitellogenic females to 2880 $\mu$g/ml in late and post-vitellogenic females. There was a correlation between ovary maturity and specific hemolymph Vg concentrations after the onset of vitellogenesis. Hemolymph Vg concentrations are attributed to leakage and reabsorption of Vg and Vt, respectively, through the thin ovarian wall. Polyclonal antibody preabsorbed to male hemolymph was used for fluorescent (FITC) and enzymatic (DAB) immunohistochemistry for Vt or Vg localization in ovary and hepatopancreas sections from females of all reproductive stages and hepatopancreata of males (controls). Immunohistochemical analysis indicated no presence of Vg in males or female hepatopancreata at any reproductive stage. Ovaries showed Vg presence in ovarian stroma associated with follicle cells or a possible subset of which we call vitellocytes. Presence of Vt in oocytes corresponded closely to timing of folliculogenesis and concentrations of Vt can be related to relative numbers of secondary follicle cells present. Finally, our results indicated that the ovary may be the sole site of Vg synthesis and that the follicle cells are responsible for the production and transport of yolk protein to developing oocytes / acase@tulane.edu

Cadmium is a heavy metal which is naturally found in soil and water. It has similar properties to copper, zinc and calcium, and it may disrupt essential metal homeostasis. The toxicity of cadmium is magnified by its extremely long biological half-life in the body, between 10 to 30 years. Although the primary body burden is found in the liver and kidneys, the testis is extremely sensitive to cadmium, and acute exposure results intesticular edema and hemorrhagic necrosis. While most animals with scrotal testes are susceptible to the testicular toxicity of cadmium, strain-related differences to this effect occur in mice. For this dissertation, the mechanism(s) of murine strain differences to cadmium-induced testicular toxicity was studied using sensitive (129/J) and resistant (A/J) mice. We found significantly decreased cadmium concentrations in the testis, epididymis and seminal vesicle 24 hr after subcutaneous cadmium exposure in the resistant A/J mice, as compared to the sensitive 129/J mice. The transport of cadmium across vascular barriers was also markedly different in the two strains. Influx rates of $\sp{109}$Cd into the brain, testis and epididymis were found to be significantly lower in A/J mice. To help identify the natural ligand for the transport system in these tissues, $\sp{65}$Zn influx rates were studied. Zinc is an essential element, and a transport system should exist to facilitate passage into tissues. Since cadmium is a nonessential and toxic metal, specific mechanisms would not be expected to be present to maintain homeostatic concentrations of this cation. Cadmium and zinc share many properties, and the two elements have been shown to compete for transport. Thus, cadmium may use the zinc transporter to enter tissues. The resistant A/J mice demonstrated a significantly reduced zinc entry into the testis and brain when compared to $\sp{129}$/J mice. Cadmium and zinc transport were significantly lower in A/J mice, suggesting that the resistant mice may have a decreased number of zinc transporters, or a mutation in the transporters that makes them less active. Therefore, the nature of murine strain resistance to the testicular effects of cadmium may be related to a relative deficiency or a mutation in the zinc transporter / acase@tulane.edu

Megalin and the Angiotensin type 1 receptor (AT1R) are coexpressed in several polarized epithelia including the renal proximal tubule and the yolk sac where megalin is heavily involved in receptor-mediated protein endocytosis. Because angiotensin II uptake by proximal tubule cells and yolk sac cells was only partially inhibited by AT1R specific blockers, we tested to see if megalin interacts with angiotensin peptides. Angiotensin II and Angiotensin-(1-7) were chosen on the basis of their physiological relevance and their dissimilar affinity for the AT1R. Cell experiments were conducted on several proximal tubule cell lines (HRCEs, IRPPT cells and NRK-52E cells) and BN/MSV cells. Uptake experiments were performed in cell monolayers exposed to fluorescent-labeled angiotensin II or angiotensin-(1-7) +/- different competitors, and the amount of cell-associated fluorescence (an indication of internalized angiotensin) was determined by flow cytometry. Anti-megalin antisera and AT1R blockers (olmesartan or candesartan) were used to interfere with uptake via megalin and the AT1R respectively. Anti-megalin antisera universally interfered with angiotensin II uptake by proximal tubule and BN/MSV cells, but the degree of reduction was cell type-dependent (ranging from 80 to 30% in reduction, p < 0.05 in all cases vs. positive control). Angiotensin-(1-7) uptake by BN/MSV cells was also prevented by anti-megalin antisera (63%, p < 0.001). Angiotensin II interfered with uptake of metallothionein (a known ligand for megalin) by BN/MSV cells. Flow cytometry analysis of binding experiments performed in brush border membrane vesicles freshly isolated from kidney cortices of CD-1 mice showed that anti-megalin antisera interfered with angiotensin II and Angiotensin-(1-7) binding (27% and 30% respectively, p < 0.05 vs. positive control). Molecular interactions of megalin with angiotensin II and angiotensin-(1-7) were studied by surface plasmon resonance. Angiotensin II and angiotensin-(1-7) bind megalin dose and time-dependently and with a similar affinity (&sim;3 mM). Collectively, the data from these studies demonstrate that megalin binds and internalizes angiotensin II and angiotensin-(1-7). These results also indicate that angiotensin II or angiotensin-(1-7) internalization in the proximal tubule might be megalin-dependent and that the scavenger receptor megalin may play a role in regulating proximal tubule and therefore intrarenal angiotensin peptide levels / acase@tulane.edu

acase@tulane.edu

Technologically advanced countries use more than 10,000 chemicals routinely and approximately 1,500 new chemicals are introduced annually (Connell and Miller 1984). While many of these chemicals are harmless, some have catastrophic effects on aquatic ecosystems and show the most severe effects on higher trophic level consumers, including wading birds through bioaccumulation and biomagnification (Ohlendorf et al. 1979, Spalding et al. 1994). The present study is the first to address three primary questions: (1) Can non-sacrificial sampling of wading birds reflect food chain contamination reliably? (2) Are nestling growth rates or other reproductive indices of the birds affected by food chain contamination? (3) Are reproductive indices related to tissue concentrations of contaminants in these birds? Tissues of White Ibis (WI) and Little Blue Herons (LBH) were sampled from a colony of wading birds near Devil's Swamp, an area known to be polluted with organochlorines and heavy metals, and from four other colonies as controls. Eggs, blood, guano, and food samples were analyzed for hexachlorobenzene, hexachlorobutadiene, lead, and cadmium contamination. Eggshells and feathers were also tested for metal content. LBH chicks were measured twice weekly to ascertain growth rates The data indicate for the first time that food chain contamination by heavy metals can be readily assessed from guano samples collected from chicks of both WI and LBH, and demonstrate differences in metal contamination between polluted and control colonies. This was not true for organochlorine contaminants, which were distributed independently of the metals in the environment. These data are also original in establishing a relationship between the presence of cadmium in feathers and reduced growth rates in LBH, and in demonstrating that lead-exposed chicks had increased nestling mortality. The findings of the present study expand our understanding of wading birds as indicators of environmental contamination in several ways: (1) non-sacrificial sampling can reflect food chain contamination by heavy metals; (2) nestling survival and growth rates are affected by exposure to heavy metals in the food chain; and (3) wading birds bioaccumulate metals and their tissues can thus serve as reliable bioindicators of contamination / acase@tulane.edu

No description available.

No description available.

<p></p><p>In salmonids, the decision to migrate or remain resident is influenced by the status, and hence condition, of individuals. Status has been suggested to arise from the temperament of fish. In this study the links between standard metabolic rate and the levels of aggressiveness and shy/boldness were examined for 0+, hatchery-raised brown trout (<em>Salmo trutta</em>). I hypothesized, from the results of earlier studies (Cutts <em>et al</em>., 1998; Yamamoto <em>et al</em>., 1998), that high metabolic rates (MR) would be positively correlated to levels of aggression and boldness. The study was conducted in 200 L aquaria in which aggressiveness was measured by allowing each fish to interact with a mirror image of itself, and shy/boldness was tested by measuring the amount of time a fish used before exploring a new area. Standard metabolic rate was measured in a flow-through respirometer. In contrast to my expectations, there was no correlation between the different behavioural measures and the metabolic rate of fish. Also, no correlation between boldness and aggressiveness of fish was found. In additional testing aggressiveness correlated positively with the condition of fish (in coherence with Harwood <em>et al</em>., 2003) but, contrary to earlier studies (Överli <em>et al.,</em> 2004; Schjolden & Winberg, 2007), not with the speed of acclimatization. The difference in results between this test and earlier studies, concerning the degree of correlation between MR and aggressiveness, suggests that the strength of this link differs between species of salmonids. Also, it may suggest changeability in the MR – behaviour link in different environments. Last, the status and condition of individuals cannot be unambiguously explained by temperament alone, but arise from a wider array of physiological and environmental factors.</p><p> </p>

This dissertation investigated larval release and larval behavior of the Caribbean spiny lobster Panulirus argus and the spotted spiny lobster P. guttatus. These species were examined under laboratory conditions to determine the phase relationship between larval release and natural environmental cycles. P. argus displayed a nocturnal tidal rhythm, while P. guttatus displayed a circadian rhythm in larval release. P. argus releases larvae near the time of nocturnal high slack water, while P. guttatus released larvae near the time of sunrise. The role of 'pumping pheromones' in controlling larval release behaviors was tested by measuring the pumping response of ovigerous P. argus to (1) hatch water, (2) homogenized-embryo water, (3) embryo-conditioned water, and (4) water containing homogenized-egg cases. Lobsters with late-stage embryos displayed increased pleopod pumping with increased concentration of hatch water. Water individually conditioned with homogenized late-stage embryos, intact late-stage embryos, and homogenized egg-cases induced pumping activity in females with late-stage embryos, indicating the presence of a chemical cue. I quantified pumping responses upon exposure to synthetic peptides to determine if they mimicked pheromones that induce larval release behaviors. Pumping behavior was evoked by oligopeptides with a basic amino acid at the carboxy-terminus, preceded by several neutral amino acids. Carboxyl-terminal arginine peptides serve as pheromone mimics. I investigated whether these peptides originate from the action of trypsin-like enzymes by conducting a bioassay measuring pumping activity of ovigerous P. argus subjected to increasing concentrations of trypsin, trypsin inhibitor, and a combination of the two. Pumping activity increased with increasing concentrations of trypsin and trypsin inhibitor, while behaviors ceased when ovigerous females were subjected to a complex of the two. Pheromones are generated by trypsin-like enzymes assisting in the degradation of the egg membranes at the time of hatching. Vertical swimming behaviors of stage-I phyllosoma larvae of P. argus and P. guttatus were observed under laboratory conditions. P. argus larvae displayed a pattern of twilight vertical migration, while P. guttatus larvae displayed nocturnal diel vertical migration (DVM). Rhythms persisted for 5-6 cycles under constant conditions, indicating that an endogenous rhythm in activity plays a proximate role in DVM for both species. / Dissertation

<p>Scallops, a family of swimming bivalve mollusks, have dozens of eyes arrayed along the edges of their valves. Relatively little is known about the form and function of these unusual eyes. To learn more about them, we studied the visually influenced behavior of scallops, as well as the morphology and spectral sensitivity of their eyes. Of particular interest was whether or not the simple neural architecture of these animals constrains the number of visually-influenced behaviors they can perform. We were also interested to learn whether scallop eyes, despite providing relatively poor visual acuity, show optical refinements, such as corrections for spherical and chromatic aberration, that are known from the eyes of animals with better vision. In the following dissertation, Chapter 2 discusses the visually-influenced behaviors of scallops. It has been argued that bivalve mantle eyes only act as predator-detectors, but the behavioral trials described in this chapter suggest that vision may serve additional purposes in scallops. For example, it was found that visual cues relating to flow conditions may influence scallop feeding behavior. Chapter 3 presents a comparative study of scallop eye morphology. Here, it is found that eye morphology varies considerably between scallop species and that highly mobile scallops have better vision than less mobile or immobile species. Evidence is also presented that one of the two scallop retinas may perform tasks of similar importance to all species, such as predator detection, while the other retina may perform tasks more important to mobile species, such as those associated with the visual detection of preferred habitats. Chapter 4 investigates the spectral sensitivity of the two retinas in the mantle eyes of two scallop species. It is found that there is both inter- and intra-specific variation in scallop spectral sensitivity and that color perception in scallops may be influenced by both environmental light conditions and chromatic aberration caused by their lens. The research in this dissertation provides insight into how vision functions in animals that, like scallops, have a vast number of eyes, but a limited capacity for neural processing. Despite such limitations, it is evident that scallops display a wide range of visual behaviors and have eyes with highly-refined optics.</p> / Dissertation