Gastropoda

Cone snails are carnivorous marine mollusks, utilizing their neuropeptide-rich venom for prey capture. The venom of Conus brunneus, a wide-spread Eastern Pacific vermivore, has not been extensively studied. In the current work, peptides from the dissected venom were characterized and tested using preliminary bioassays. Six peptides (A-F) were isolated and tested. Three peptide identities were determined by comparison with previously reported data: bru9a (A), bru3a (F), and an a-conotoxin (E). Preliminary screening in a stroke-related model of induced glutamate excitotoxicity in primary neuronal cells and PC12 cell cultures indicated potential neuroprotective activity of peptide fractions A, D, and F. Further testing is necessary to determine and verify structure, activity, target, and mechanism of action of the promising peptides from C. brunneus, which may prove effective neuropharmacological agents to treat stroke.

The presence of heavy metals and other pollutants is detrimental to marine ecosystems. The queen conch, once an important fisheries species in the Florida Keys, has not sufficiently recovered after a 25-year fishery closure. Research has shown high levels of copper and zinc in the gonads and digestive glands of adult conch found in the nearshore waters. Four sites relevant to queen conch larval recruitment were tested in 2010 for the presence of copper and zinc in the water, phytoplankton, sediment, and seagrass epiphytes over seven months. Both metals were detected in all sample types and no seasonal or geographical differences were detected. Surface water concentrations from the field were used to conduct acute and chronic toxicity tests on various ages of queen conch larvae and their phytoplankton food source. When zinc concentrations (0-40 (So(Bg/L) similar to those measured in situ were used, there was no significant impact on conch larval survival although some velar lobe de velopment was impaired. However, field concentrations of copper (0-15 (So(Bg/L), which often surpassed water quality standards, negatively impacted growth, survival, and development of the larvae. Chronic exposure to copper, through the water and food, disrupted the metamorphic success of competent larvae and decreased post-metamorphosis survival. Exposure to copper at later life stages increased mortality, suggesting that heavy metals have a negative effect on larval recruitment in localized areas of the Florida Keys. Structural equation modeling revealed that copper and zinc are moving through the systems differently and are best represented by two different models.

Cone snails are predatory marine gastropods that use venom for means of predation and defense. This venom is a complex mixture of conopeptides that selectivity binds to ion channels and receptors, giving them a wide range of potential pharmaceutical applications. Conus brunneus is a wide spread Eastern Pacific cone snail species that preys upon worms (vermivorous). Vermivorous cone snails have developed very specific biochemical strategies for the immobilization of their prey and their venom has not been extensively studied to date. The main objective of this dissertation is the characterization of novel conopeptides isolated from Conus brunneus. Chapter 1 is an introduction and background on cone snails and conopeptides. Chapter 2 details the isolation and characterization of a novel P-superfamily conotoxin. Chapter 3 presents the 3D solution structure of the novel P-superfamily conotoxin. Chapter 4 details the isolation and characterization of two novel M-superfamily conotoxins. Chapter 5 covers the use of nano-NMR to characterize a novel P-superfamily conotoxin using nanomole quantities of sample. Chapter 6 is a reprint of a paper published in the Journal of the American Chemical Society in which we combined and implemented techniques developed in the previous chapters to report the presence of D-(Sd(B-Hydroxyvaline in a polypeptide chain. This dissertation contains the first reported work of a P-superfamily structure obtained directly from the crude venom therefore accurately representing native post-translational modifications. In this paper, crude cone snail venom was characterized by: high performance liquid chromatography, nuclear magnetic resonance spectroscopy, nanonuclear magnetic resonance spectroscopy, mass spectrometry, amino acid analysis, Edman degradation sequencing, and preliminary bioassays.