Annalaura Mancia

Convergent evolution of the genomes of marine mammals

Marine mammals from different mammalian orders share several phenotypic traits adapted to the aquatic environment and therefore represent a classic example of convergent evolution. To investigate convergent evolution at the genomic level, we sequenced and performed de novo assembly of the genomes of three species of marine mammals (the killer whale, walrus and manatee) from three mammalian orders that share independently evolved phenotypic adaptations to a marine existence. Our comparative genomic analyses found that convergent amino acid substitutions were widespread throughout the genome and that a subset of these substitutions were in genes evolving under positive selection and putatively associated with a marine phenotype. However, we found higher levels of convergent amino acid substitutions in a control set of terrestrial sister taxa to the marine mammals. Our results suggest that, whereas convergent molecular evolution is relatively common, adaptive molecular convergence linked to phenotypic convergence is comparatively rare.

The transcriptomic responses of the eastern oyster, Crassostrea virginica, to environmental conditions

Understanding the mechanisms by which organisms adapt to environmental conditions is a fundamental question for ecology and evolution. In this study, we evaluate changes in gene expression of a marine mollusc, the eastern oyster Crassostrea virginica, associated with the physico‐chemical conditions and the levels of metals and other contaminants in their environment. The results indicate that transcript signatures can effectively disentangle the complex interactive gene expression responses to the environment and are also capable of disentangling the complex dynamic effects of environmental factors on gene expression. In this context, the mapping of environment to gene and gene to environment is reciprocal and mutually reinforcing. In general, the response of transcripts to the environment is driven by major factors known to affect oyster physiology such as temperature, pH, salinity, and dissolved oxygen, with pollutant levels playing a relatively small role, at least within the range of concentrations found in the studied oyster habitats. Further, the two environmental factors that dominate these effects (temperature and pH) interact in a dynamic and nonlinear fashion to impact gene expression. Transcriptomic data obtained in our study provide insights into the mechanisms of physiological responses to temperature and pH in oysters that are consistent with the known effects of these factors on physiological functions of ectotherms and indicate important linkages between transcriptomics and physiological outcomes. Should these linkages hold in further studies and in other organisms, they may provide a novel integrated approach for assessing the impacts of climate change, ocean acidification and anthropogenic contaminants on aquatic organisms via relatively inexpensive microarray platforms.

A cDNA Microarray for Crassostrea virginica and C. gigas

The eastern oyster, Crassostrea virginica, and the Pacific oyster, C. gigas, are species of global economic significance as well as important components of estuarine ecosystems and models for genetic and environmental studies. To enhance the molecular tools available for oyster research, an international group of collaborators has constructed a 27,496-feature cDNA microarray containing 4460 sequences derived from C. virginica, 2320 from C. gigas, and 16 non-oyster DNAs serving as positive and negative controls. The performance of the array was assessed by gene expression profiling using gill and digestive gland RNA derived from both C. gigas and C. virginica, and digestive gland RNA from C. ariakensis. The utility of the microarray for detection of homologous genes by cross-hybridization between species was also assessed and the correlation between hybridization intensity and sequence homology for selected genes determined. The oyster cDNA microarray is publicly available to the research community on a cost-recovery basis.

A transcriptomic analysis of land-use impacts on the oyster, Crassostrea virginica, in the South Atlantic bight.

Increasing utilization and human population density in the coastal zone is widely believed to place increasing stresses on the resident biota, but confirmation of this belief is somewhat lacking. While we have solid evidence that highly disturbed estuarine systems have dramatic changes in the resident biota (black and white if you will), we lack tools that distinguish the shades of grey. In part, this lack of ability to distinguish shades of grey stems from the analytical tools that have been applied to studies of estuarine systems, and perhaps more important, is the insensitivity of the biological end points that we have used to assess these impacts. In this study, we will present data on the phenotypic adjustments as measured by transcriptomic signatures of a resilient organism (oysters) to land‐use practices in the surrounding watershed using advanced machine‐learning algorithms. We will demonstrate that such an approach can reveal subtle and meaningful shifts in oyster gene expression in response to land use. Further, the data show that gill tissues are far more responsive and provide superior discrimination of land‐use classes than hepatopancreas and that transcripts encoding proteins involved in energy production, protein synthesis and basic metabolism are more robust indicators of land use than classic biomarkers such as metallothioneins, GST and cytochrome P‐450.

A transcriptomic analysis of the stress induced by capture-release health assessment studies in wild dolphins (Tursiops truncatus).

The health of wild bottlenose dolphins (Tursiops truncatus) is typically evaluated by the study of animals that are captured and released back into the wild after examination. The impact of such studies on gene expression in peripheral blood cells was investigated using microarray and quantitative polymerase chain reaction methods. Significantly increased expression was observed in two major classes of genes: (i) energy metabolism, and (ii) responsiveness to stress and trauma, the latter effect suggesting the initiation of an acute‐phase response. The value of data obtained in capture/release studies may need to be weighed against the potential physiological impacts of such studies.

Tollip Is a Mediator of Protein Sumoylation

Tollip is an interactor of the interleukin-1 receptor involved in its activation. The endosomal turnover of ubiquitylated IL-1RI is also controlled by Tollip. Furthermore, together with Tom1, Tollip has a general role in endosomal protein traffic. This work shows that Tollip is involved in the sumoylation process. Using the yeast two-hybrid technique, we have isolated new Tollip partners including two sumoylation enzymes, SUMO-1 and the transcriptional repressor Daxx. The interactions were confirmed by GST-pull down experiments and immunoprecipitation of the co-expressed recombinants. More specifically, we show that the TIR domain of the cytoplasmic region of IL-1RI is a sumoylation target of Tollip. The sumoylated and unsumoylated RanGAP-1 protein also interacts with Tollip, suggesting a possible role in RanGAP-1 modification and nuclear-cytoplasmic protein translocation. In fact, Tollip is found in the nuclear bodies of SAOS-2/IL-1RI cells where it colocalizes with SUMO-1 and the Daxx repressor. We conclude that Tollip is involved in the control of both nuclear and cytoplasmic protein traffic, through two different and often contrasting processes: ubiquitylation and sumoylation.

Health status, infection and disease in California sea lions (Zalophus californianus) studied using a canine microarray platform and machine-learning approaches

Conservation biologists face many challenges in assessing health, immune status and infectious diseases in protected species. These challenges include unpredictable sample populations, diverse genetic and environmental backgrounds of the animals, as well as the practical, legal and ethical issues involved in experimentation. The use of whole genome scale transcriptomics with animal samples obtained in a minimally invasive manner is an approach that shows promise for health assessment. In this study we assessed the utility of a microarray to identify changes in gene expression predictive of health status by interrogating blood samples from California sea lions (Zalophus californianus) in rehabilitation. A custom microarray was developed from the commercially available dog microarray (Canis familiaris) by selecting probes that demonstrated reliable cross-hybridization with RNA in sea lion blood. This custom microarray was used for the analysis of RNA from 73 sea lion blood samples, from animals with a broad spectrum of health changes. Both traditional classifying techniques and newer artificial neural network approaches correctly classified sea lions with respect to health status, primarily distinguishing between leptospirosis infection and domoic acid exposure. Real time PCR validation for a small set of genes, followed by sequencing, showed good correlation with array results and high identity (96-98%) between the dog and sea lion sequences. This approach to health status classification shows promise for disease identification in a clinical setting, and assessment of health status of wildlife.

The vitamin D3 transcriptomic response in skin cells derived from the Atlantic bottlenose dolphin.

The Atlantic bottlenose dolphin has attracted attention due to the evident impact that environmental stressors have taken on its health. In order to better understand the mechanisms linking environmental health with dolphin health, we have established cell cultures from dolphin skin as in vitro tools for molecular evaluations. The vitamin D3 pathway is one mechanism of interest because of its well established chemopreventative and immunomodulatory properties in terrestrial mammals. On the other hand, little is known of the physiological role of this molecule in aquatic animals. 1,25-dihydroxyvitamin D3 (1,25D3), the bioactive and hormonal form of vitamin D3, exerts its biological function by binding to the vitamin D receptor (VDR), a ligand-activated regulator of gene transcription. Therefore, we investigated the transcriptomic changes induced by 1,25D3 administration in dolphin skin cells. Identification of specific genes activated by 1,25D3 has provided clues to the physiological function of the vitamin D3 pathway in the dolphin. We found that exposure of the cells to 1,25D3 upregulated transactivation of a vitamin D-sensitive promoter. cDNA microarray analysis, using a novel dolphin array, identified specific gene targets within this pathway, and real-time PCR (qPCR) confirmed the enhanced expression of select genes of interest. These transcriptional changes correlated with an increase in VDR levels. This is the first report of the presence and activation of the vitamin D3 pathway in a marine mammal, and our experimental results demonstrate a number of similarities to terrestrial animals. Conservation of this pathway in the Atlantic bottlenose dolphin is consistent with the importance of nonclassic functions of vitamin D3, such as its role in innate immunity, similar to what has been demonstrated in other mammals.

Microarray applications to understand the impact of exposure to environmental contaminants in wild dolphins (Tursiops truncatus)

It is increasingly common to monitor the marine environment and establish geographic trends of environmental contamination by measuring contaminant levels in animals from higher trophic levels. The health of an ecosystem is largely reflected in the health of its inhabitants. As an apex predator, the common bottlenose dolphin (Tursiops truncatus) can reflect the health of near shore marine ecosystems, and reflect coastal threats that pose risk to human health, such as legacy contaminants or marine toxins, e.g. polychlorinated biphenyls (PCBs) and brevetoxins. Major advances in the understanding of dolphin biology and the unique adaptations of these animals in response to the marine environment are being made as a result of the development of cell-lines for use in in vitro experiments, the production of monoclonal antibodies to recognize dolphin proteins, the development of dolphin DNA microarrays to measure global gene expression and the sequencing of the dolphin genome. These advances may play a central role in understanding the complex and specialized biology of the dolphin with regard to how this species responds to an array of environmental insults. This work presents the creation, characterization and application of a new molecular tool to better understand the complex and unique biology of the common bottlenose dolphin and its response to environmental stress and infection. A dolphin oligo microarray representing 24,418 unigene sequences was developed and used to analyze blood samples collected from 69 dolphins during capture-release health assessments at five geographic locations (Beaufort, NC, Sarasota Bay, FL, Saint Joseph Bay, FL, Sapelo Island, GA and Brunswick, GA). The microarray was validated and tested for its ability to: 1) distinguish male from female dolphins; 2) differentiate dolphins inhabiting different geographic locations (Atlantic coasts vs the Gulf of Mexico); and 3) study in detail dolphins resident in one site, the Georgia coast, known to be heavily contaminated by Aroclor 1268, an uncommon polychlorinated (PCB) mixture. The microarray was able to distinguish dolphins by sex, geographic location, and corroborate previously published health irregularities for the Georgia dolphins. Genes involved in xenobiotic metabolism, development/differentiation and oncogenic pathways were found to be differentially expressed in GA dolphins. The report bridges the advancements in dolphin genome sequencing to the first step towards providing a cost-effective means to screen for indicators of chemical toxin exposure as well as disease status in top level predators.

Quantitative methods to characterize morphological properties of cell lines

Descriptive terms are often used to characterize cells in culture, but the use of nonquantitative and poorly defined terms can lead to ambiguities when comparing data from different laboratories. Although recently there has been a good deal of interest in unambiguous identification of cell lines via their genetic markers, it is also critical to have definitive, quantitative metrics to describe cell phenotypic characteristics. Quantitative metrics of cell phenotype will aid the comparison of data from experiments performed at different times and in different laboratories where influences such as the age of the population and differences in culture conditions or protocols can potentially affect cellular metabolic state and gene expression in the absence of changes in the genetic profile. Here, we present examples of robust methodologies for quantitatively assessing characteristics of cell morphology and cell–cell interactions, and of growth rates of cells within the population. We performed these analyses with endothelial cell lines derived from dolphin, bovine and human, and with a mouse fibroblast cell line. These metrics quantify some characteristics of these cells lines that clearly distinguish them from one another, and provide quantitative information on phenotypic changes in one of the cell lines over large number of passages. Published 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1069–1078, 2012