Convergent evolution of the genomes of marine mammals

Marine mammals from different mammalian orders share several phenotypic traits adapted to the aquatic environment and are therefore a classic example of convergent evolution. To investigate convergent evolution at the genomic level, we sequenced and de novo assembled 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 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 while convergent molecular evolution is relatively common, adaptive molecular convergence linked to phenotypic convergence is comparatively rare

Modelling interactions of acid–base balance and respiratory status in the toxicity of metal mixtures in the American oyster Crassostrea virginica

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology 155 (2010): 341-349, doi:10.1016/j.cbpa.2009.11.019.Heavy metals, such as copper, zinc and cadmium, represent some of the most common and serious pollutants in coastal estuaries. In the present study, we used a combination of linear and artificial neural network (ANN) modelling to detect and explore interactions among low-dose mixtures of these heavy metals and their impacts on fundamental physiological processes in tissues of the Eastern oyster, Crassostrea virginica. Animals were exposed to Cd (0.001 – 0.400 μM), Zn (0.001 – 3.059 μM) or Cu (0.002 – 0.787 μM), either alone or in combination for 1 to 27 days. We measured indicators of acid-base balance (hemolymph pH and total CO2), gas exchange (Po2), immunocompetence (total hemocyte counts, numbers of invasive bacteria), antioxidant status (glutathione, GSH), oxidative damage (lipid peroxidation; LPx), and metal accumulation in the gill and the hepatopancreas. Linear analysis showed that oxidative membrane damage from tissue accumulation of environmental metals was correlated with impaired acid-base balance in oysters. ANN analysis revealed interactions of metals with hemolymph acid-base chemistry in predicting oxidative damage that were not evident from linear analyses. These results highlight the usefulness of machine learning approaches, such as ANNs, for improving our ability to recognize and understand the effects of sub-acute exposure to contaminant mixtures.This study was supported by NOAA’s Center of Excellence in Oceans and Human Health at HML and the National Science Foundation

Marine Mammal Immunity Toward Environmental Challenges

Human activities influence contaminant concentrations in aquatic organisms. It is increasingly common to monitor the marine environment and establish geographic trends of pollution by measuring contaminant levels in animals from higher trophic levels. Marine mammals are considered an ideal model for the assessment of immunological responses to pathogens and contaminants. In fact, living their entire life (or most of it) in the sea, they act as integrators of the stressors present in the marine environment. Marine mammals may have the potential to predict contaminant effects on health, and to be indicators of the status of the marine ecosystem. The impact of infection and environmental stress is here described through the analysis of the immunological processes based on large scale genomic approaches, highly informative of the interaction of genetic, disease and environmental factors.Human activities influence contaminant concentrations in aquatic organisms. It is increasingly common to monitor the marine environment and establish geographic trends of pollution by measuring contaminant levels in animals from higher trophic levels. Marine mammals are considered to be an ideal model for the assessment of immunological responses to pathogens and contaminants. In fact, living their entire lives (or most of it) in the sea, they act as integrators of the stressors present in the marine environment. Marine mammals may have the potential to predict contaminant effects on health and to be indicators of the status of the marine ecosystem. The impact of infection and environmental stress is here described through the analysis of the immunological processes based on large-scale genomic approaches, highly informative of the interaction of genetic, disease, and environmental factors

On the revolution of cetacean evolution

The order of Cetacea with 88 species including Odontoceti (or toothed whales) and Mysticeti (or baleen whales) is the most specialized and diversified group of mammals. The blue whale with a maximum recorded length of 29.9 m for 173 t of weight is the largest animal known to have ever existed, and any dolphin's brain is most powerful and complex than any other brain in the animal kingdom, second only to primate's. Nevertheless, Cetacea are mammals that re-entered the oceans only a little over 50 million years ago, a relatively short time on the evolutionary scale. During this time cetaceans and humans have developed marked morphological and behavioral differences, yet their genomes show a high level of similarity. This present review is focused on the description and significance of newly accessible cetacean genome tools and information, and their relevance in the study of the evolution of successful phenotypic adaptations associated to mammal's marine existence, and their applicability to the unresolved disease mechanisms in humans

Genome-Wide DNA Methylation Protocol for Epigenetics Studies

Epigenetic modification are heritable changes in gene expression not encoded by the DNA sequence therefore playing a significant role in a broad range of biological processes and diseases. A key player of the epigenetic modifications is the DNA methylation, a process involving the covalent transfer of a methyl group to the C-5 position of the cytosine ring of DNA forming 5-methylcytosine (5mC), catalyzed by DNA methyltransferases. Altering the structure of the chromatin, DNA methylation has the potential to down-regulate gene expression. The here presented protocol shows a method to obtain DNA samples ready for NGS sequencing for genome-wide methylation analysis

The chronicles of the contaminated Mediterranean seas: a story told by the cetaceans' skin genes

Wild animals in their natural environment could provide a big source of information, but sampling can be very challenging, above all for protected species, like marine mammals. Nevertheless, significant data can be obtained sampling stranded animals right after their death, taking into account proper sampling time and methodology. RNA samples from the skin of 12 individuals including the species Stenella coeruleoalba, Tursiops truncatus, and Grampus griseus were used to test 4 potential gene markers of anthropogenic contaminants exposure. The individuals were sampled in 3 geographic areas: the Adriatic, Ionian and Tyrrhenian seas. Three out of the 4 genes tested showed higher expression in the samples collected from the Adriatic Sea. Minute skin samples tell the story of the specific geographic location where the marine mammal spent its life, thanks to the different impact on gene expression exerted by different contamination levels

Functional Genomics and Cell Biology of the Dolphin (Tursiops runcatus): Establishment of Novel Molecular Tools to Study Marine Mammals in Changing Environments

The dolphin (Tursiops truncatus) is a mammal that is adapted to life in a totally aquatic environment. Despite the popularity and even iconic status of the dolphin, our knowledge of its physiology, its unique adaptations and the effects on it of environmental stressors are limited. One approach to improve this limited understanding is the implementation of established cellular and molecular methods to provide sensitive and insightful information for dolphin biology. We initiated our studies with the analysis of wild dolphin peripheral blood leukocytes, which have the potential to be informative of the animal’s global immune status. Transcriptomic profiles from almost 200 individual samples were analyzed using a newly developed species-specific microarray to assess its value as a prognostic and diagnostic tool. Functional genomics analyses were informative of stress-induced gene expression profiles and also of geographical location specific transcriptomic signatures, determined by the interaction of genetic, disease and environmental factors. We have developed quantitative metrics to unambiguously characterize the phenotypic properties of dolphin cells in culture. These quantitative metrics can provide identifiable characteristics and baseline data which will enable identification of changes in the cells due to time in culture. We have also developed a novel protocol to isolate primary cultures from cryopreserved tissue of stranded marine mammals, establishing a tissue (and cell) biorepository, a new approach that can provide a solution to the limited availability of samples. The work presented represents the development and application of tools for the study of the biology, health and physiology of the dolphin, and establishes their relevance for future studies of the impact on the dolphin of environmental infection and stress

Induced Pluripotent Stem Cells Produced From Cryopreserved Pygmy Sperm Whale (Kogia Breviceps) Lung Tissue

Concern over marine mammal health has risen in the last decade due to increases in mortality, unusual mortality events and previously undetected pathologies. Developing environmental trends, such as global warming and acute environmental insults, such as large-scale oil spills have been implicated. Unfortunately, the limited availability of biological samples hinders basic biological/toxicological studies. To compensate for the limited availability of samples and to make the most of rare samples that do become available, we have pursued methods to establish and expand cultures of primary cell types and reconstituted tissues from marine mammals for long-term use as surrogates for freshly isolated samples. To this end, we first developed a method to cryopreserve tissues from deceased/stranded individuals and thereby initiate establishment of a tissue bank biorepository. We were able to establish conditions and perform this successfully on lung tissue from a Pygmy Sperm Whale (PSW; Kogia breviceps). Using these conditions, we were able to establish cultures of viable primary lung cell types from tissue fragments that had been cryopreserved several months earlier (immediately after the stranding event). We then applied genetic or chemical means for generating induced pluripotent stem (iPS) cells to one of these primary cultures (lung fibroblasts). We observed that the genetic means, involving the forced expression of Klf4, Oct3/4, Sox2 and Myc, did produce PSW cells with long-term expansion and differentiative capability, while the chemical means using valproic acid (driving histone deacetylase inhibition and chromatin decondensation) did not. Finally, we employed specialized culture conditions to differentiate in bulk PSW cells with vascular endothelial and airway epithelial-like properties. The cryopreservation of viable tissue samples and the generation of iPS-like cells with unlimited expansion & pluripotent differentiative capacities from marine mammals is anticipated to have far-reaching impacts on levels ranging from basic biology to environmental policy related to stressors of marine and land mammalian health

Skin distress associated with xenobiotics exposure: An epigenetic study in the Mediterranean fin whale (Balaenoptera physalus)

The phenotypic plasticity of many organisms is mediated in part by epigenetics, the heritable changes in gene activity that occur without any alterations to DNA sequence. A major mechanism in epigenetics is the DNA methylation (DNAm). Hypo- and hyper-methylation are generalized responses to control gene expression however recent studies have demonstrated that classes of contaminants could mark specific DNAm signatures, that could usefully signal prior environmental exposure. We collected skin and blubber from 6 free-ranging fin whale (Balaenoptera physalus) individuals sampled as a part of a previous published study in the northern Mediterranean Sea. Genomic DNA extracted from the skin of the fin whales and levels of contaminants measured in the blubber of the same individuals were used for DNAm profiling through reduced representation bisulfite sequencing (RRBS). We tested the hypothesis that differences in the methylation patterns could be related to environmental exposure to contaminants and load in the whale tissues. The aims of this study were to determine the DNAm profiles of the methylation contexts (CpGs and non-CpGs) of differently contaminated groups using the RRBS, and to identify potential contaminant exposure related genes. Amount and proportion of methylcytosines in CpG and non-CpG regions (CHH and CHG) was very similar across the 6 samples. The proportion of methylcytosines sites in CpG was n = 32,682, the highest among all the sequence contexts (n = 3216 in CHH; n = 1743 in CHG). The majority of the methylcytosine occurred in the intron regions, followed by exon and promoter regions in CpG, CHH and CHG. Gene Ontology results indicated that DNAm affected genes that take place in cell differentiation and function in cutaneous, vascular and nervous systems. The identification of cellular response pathways allows a better understanding of the organism biological reaction to a specific environmental challenge and the development of sensitive tools based on the predictive responses. Eco-epigenetics analyses have an extraordinary potential to address growing issues on pollution biomonitoring, ecotoxicity assessment, conservation and management planning

Inflammation Induced Chronic Fatiguing Illnesses: A steady march towards understanding mechanisms and identifying new biomarkers and therapies.

Illnesses characterized by chronic fatigue are often defined by symptoms and not by objective biomarkers that support both diagnosis and treatment. Without readily obta ined b io marke rs, c linica l ma na ge me nt ca n be compromised by lack of certainty. This uncertainty creates a wide spectrum of possible therapies that in many cases is reduced to trial and error medicine, resulting in patient frustration and resource exhaustion, with little improvement in health status. Modern medicine must leverage modern science to bring common research tools into the clinic for patient diagnostics. Using biomarkers previously confirmed as useful in diagnosis and treatment of chronic inflammatory response syndrome (CIRS), including transcriptomics, the authors present evidence of benefit in assessment of a “symptoms-only illness.” These immune biomarkers, such as transforming growth factor beta (TGFb), vasoactive intestinal peptide (VIP), melanocyte stimulating hormone (MSH), split produc ts o f co mp le me nt act ivat io n, and ma ny ot hers disc ussed he re, are now a va ilab le for use a s c linica l diagnostics, but rarely ordered in cases of chronic illness. In cases of cognitive decline, new technology for brain MRI analysis, NeuroQuant, can pick up small changes in brain structures that are frequently missed by radiologists, but consistently shown in CIRS. By focusing on persistent symptoms seen in antibiotic-treated Lyme disease (Post-Lyme Syndrome, PLS), CIRS-biomarkers have utility to define both an initial infectious process and a subsequent inflammatory illness. Genomic testing can determine predisposition to chronic stages of Lyme after acute illness and use of Next Generation Sequencing now brings transcriptomics to the Lyme community, to assess remaining abnormalities at any given treatment stage of PLS. Application of these new, object ive tes t ing o ffe r ings w ill re vea l t he mo le c ular pathophysiology of illness, avoiding over-reliance on symptoms and antibody testing alone. This will help providers direct highly targeted therapies on an individual basis, in this era of personalized medicine