Skin Transcriptomes of common bottlenose dolphins (Tursiops truncatus) from the northern Gulf of Mexico and southeastern U.S. Atlantic coasts.

Common bottlenose dolphins serve as sentinels for the health of their coastal environments as they are susceptible to health impacts from anthropogenic inputs through both direct exposure and food web magnification. Remote biopsy samples have been widely used to reveal contaminant burdens in free-ranging bottlenose dolphins, but do not address the health consequences of this exposure. To gain insight into whether remote biopsies can also identify health impacts associated with contaminant burdens, we employed RNA sequencing (RNA-seq) to interrogate the transcriptomes of remote skin biopsies from 116 bottlenose dolphins from the northern Gulf of Mexico and southeastern U.S. Atlantic coasts. Gene expression was analyzed using principal component analysis, differential expression testing, and gene co-expression networks, and the results correlated to season, location, and contaminant burden. Season had a significant impact, with over 60% of genes differentially expressed between spring/summer and winter months. Geographic location exhibited lesser effects on the transcriptome, with 23.5% of genes differentially expressed between the northern Gulf of Mexico and the southeastern U.S. Atlantic locations. Despite a large overlap between the seasonal and geographical gene sets, the pathways altered in the observed gene expression profiles were somewhat distinct. Co-regulated gene modules and differential expression analysis both identified epidermal development and cellular architecture pathways to be expressed at lower levels in animals from the northern Gulf of Mexico. Although contaminant burdens measured were not significantly different between regions, some correlation with contaminant loads in individuals was observed among co-expressed gene modules, but these did not include classical detoxification pathways. Instead, this study identified other, possibly downstream pathways, including those involved in cellular architecture, immune response, and oxidative stress, that may prove to be contaminant responsive markers in bottlenose dolphin skin.

Alterations in eicosanoid composition during embryonic development in the chorioallantoic membrane of the American alligator (Alligator mississippiensis) and domestic chicken (Gallus gallus).

Eicosanoids are signaling lipids known to regulate several physiological processes in the mammalian placenta, including the initiation of parturition. Though all amniotes construct similar extraembryonic membranes during development, the composition and function of eicosanoids in extraembryonic membranes of oviparous reptiles is largely unknown. The majority of effort placed in eicosanoid investigations is typically targeted toward defining the role of specific compounds in disease etiology; however, comprehensive characterization of several pathways in eicosanoid synthesis during development is also needed to better understand the complex role of these lipids in comparative species. To this end, we have examined the chorioallantoic membrane (CAM) of the American alligator (Alligator mississippiensis) and domestic chicken (Gallus gallus) during development. Previously, our lab has demonstrated that the CAM of several oviparous species shared conserved steroidogenic activity, a feature originally attributed to mammalian amniotes. To further explore this, we have developed a liquid chromatography/tandem mass spectrometry method that is used here to quantify multiple eicosanoids in the CAM of two oviparous species at different stages of development. We identified 18 eicosanoids in the alligator CAM; the cyclooxygenase (COX) pathway showed the largest increase from early development to later development in the alligator CAM. Similarly, the chicken CAM had an increase in COX products and COX activity, which supports the LC-MS/MS analyses. Jointly, our findings indicate that the CAM tissue of an oviparous species is capable of eicosanoid synthesis, which expands our knowledge of placental evolution and introduces the possibility of future comparative models of placental function.

Persistent organic pollutants in blood samples of Southern Giant Petrels (Macronectes giganteus) from the South Shetland Islands, Antarctica.

Seabirds play an important role as top consumers in the food web and can be used as biomonitors of exposure to pollutants. Contamination studies involving non-destructive sampling methods are of considerable importance, allowing better evaluation of the levels of pollutants and their toxic effects. In the present study, organohalogen contaminants were analyzed in 113 blood samples from Southern Giant Petrel (Macronectes giganteus) adults and chicks collected in the austral summer of 2011/2012 and 2012/2013 from colonies on Elephant and Livingston Islands, South Shetland, Antarctica. Polychlorinated biphenyls (PCBs), hexachlorobenzene (HCB), pentachlorobenzene (PeCB), mirex, dichlorodiphenyltrichloroetane and derivatives (DDTs) and chlordanes were detected in all birds, whereas polybrominated diphenyl ethers (PBDEs) were not detected in any blood samples. No significant differences were found in organochlorine levels between sampling events. Adults exhibited significantly higher levels than chicks, except for PeCB. PCBs, HCB, mirex and DDTs were statistically similar in males and females from Elephant Island. Females on Livingston Island exhibited higher HCB values than males, but no sex differences were found regarding other organochlorines. The similarity in organochlorine levels between sexes in birds with very marked sexual segregation in feeding habits during the breeding season may indicate that significant amounts of contaminants are acquired during migration to lower latitudes, when the diets of males and females are similar. Birds sampled on Livingston Island exhibited significantly lower levels of PCBs, HCB, DDTs, mirex and chlordanes in comparison to those on Elephant Island, which could be the result of distinct foraging patterns between the two colonies. Organochlorine levels were similar between years in birds captured in two consecutive breeding seasons. Blood samples from Southern Giant Petrels adults and chicks proved to be useful for the comparison of intraspecific contamination levels and appear to be adequate for the long-term assessment of organohalogen contaminants in antarctic top predators. Organochlorine contaminants in blood samples of Southern Giant Petrels reflected intra-specific differences and suggested distinct foraging patterns between colonies.

Identification of trichotoxin, a novel chlorinated compound associated with the bloom forming Cyanobacterium, Trichodesmium thiebautii.

Trichodesmium is a suspected toxin-producing nonheterocystous cyanobacteria ubiquitous in tropical, subtropical, and temperate seas. The genus is known for its ability to fix nitrogen and form massive blooms. In oligotrophic seas, it can dominate the biomass and be a major component of oceanic primary production and global nitrogen cycling. Numerous reports suggest Trichodesmium-derived toxins are a cause of death of fish, crabs, and bivalves. Laboratory studies have demonstrated neurotoxic effects in T. thiebautii cell extracts and field reports suggest respiratory distress and contact dermatitis of humans at collection sites. However, Trichodesmium toxins have not been identified and characterized. Here, we report the extraction of a lipophilic toxin from field-collected T. thiebautii using a purification method of several chromatographic techniques, nuclear magnetic resonance (NMR), mass spectroscopy (MS), and Fourier transformed-infrared spectroscopy (FT-IR). Trichotoxin has a molecular formula of C(20)H(27)ClO and a mass of 318 m/z and possesses cytotoxic activity against GH(4)C(1) rat pituitary and Neuro-2a mouse neuroblastoma cells. A detection method using liquid chromatography/mass spectrometry (LC/MS) was developed. This compound is the first reported cytotoxic natural product isolated and fully characterized from a Trichodesmium species.

Induced thyme product prevents VEGF-induced migration in human umbilical vein endothelial cells.

Compounds with anti-angiogenic properties are useful in combating cancer by preventing new blood vessel formation to support the tumor. In this report we introduce a rapid method for screening potential anti-angiogenic compounds in a model system that stimulates the production of secondary defense chemicals in plants. This methodology identified an inducible vascular factor (IVF3), which was found to be inhibitory in all of the model systems tested. Thyme plants were exposed to highly vascular mint plants and the methanol extracts were analyzed by reverse phase HPLC. The thyme compounds induced by the invading mint tissue, and not present in the thyme plants grown alone, were tested in a vertical plate assay measuring root length as a quantitative assay for drug sensitivity. The HPLC-purified extract, referred to as IVF3, reduced the growth of root vascular tissue compared to the control and vehicle control, and 50% as well as known angiogenesis inhibitors, VEGF receptor tyrosine kinase inhibitor and amiloride hydrochloride. Extracted compounds that were effective inhibitors of plant roots were assayed in Madin Darby canine kidney epithelial cells (MDCK) for toxicity, and in human umbilical vein endothelial cells (HUVEC) for their effect on migration. IVF3 was effective at limiting HUVEC migration in VEGF-stimulated cultures. In vivo video capture of intersegmental vessel circulation between 48 and 72 h post fertilization in the developing vasculature of zebrafish embryos showed IVF3 also significantly reduced ISV functional circulation. This report demonstrates the anti-angiogenic effects of IVF3 extract in endothelial cells and in an intact vertebrate model for angiogenesis.

Metal complexes and free radical toxins produced by Pfiesteria piscicida.

Metal-containing organic toxins produced by Pfiesteria piscicida were characterized, for the first time, by corroborating data obtained from five distinct instrumental methods: nuclear magnetic resonance spectroscopy (NMR), inductively coupled plasma mass spectrometry (ICP-MS), liquid chromatography particle beam glow discharge mass spectrometry (LC/PB-G DMS), electron paramagnetic resonance spectroscopy (EPR), and X-ray absorption spectroscopy (XAS). The high toxicity of the metal-containing toxins is due to metal-mediated free radical production. This mode of activity explains the toxicity of Pfiesteria, as well as previously reported difficulty in observing the molecular target, due to the ephemeral nature of radical species. The toxins are highly labile in purified form, maintaining activity for only 2-5 days before all activity is lost. The multiple toxin congeners in active extracts are also susceptible to decomposition in the presence of white light, pH variations, and prolonged heat. These findings represent the first formal isolation and characterization of a radical forming toxic organic-ligated metal complex isolated from estuarine/marine dinoflagellates. These findings add to an increased understanding regarding the active role of metals interacting with biological systems in the estuarine environment, as well as their links and implications to human health.