RNA-Seq analysis of seasonal and individual variation in blood transcriptomes of healthy managed bottlenose dolphins.

BACKGROUND: The blood transcriptome can reflect both systemic exposures and pathological changes in other organs of the body because immune cells recirculate through the blood, lymphoid tissues, and affected sites. In human and veterinary medicine, blood transcriptome analysis has been used successfully to identify markers of disease or pathological conditions, but can be confounded by large seasonal changes in expression. In comparison, the use of transcriptomic based analyses in wildlife has been limited. Here we report a longitudinal study of four managed bottlenose dolphins located in Waikoloa, Hawaii, serially sampled (approximately monthly) over the course of 1 year to establish baseline information on the content and variation of the dolphin blood transcriptome. RESULTS: Illumina based RNA-seq analyses were carried out using both the Ensembl dolphin genome and a de novo blood transcriptome as guides. Overall, the blood transcriptome encompassed a wide array of cellular functions and processes and was relatively stable within and between animals over the course of 1 year. Principal components analysis revealed moderate clustering by sex associated with the variation among global gene expression profiles (PC1, 22 % of variance). Limited seasonal change was observed, with < 2.5 % of genes differentially expressed between winter and summer months (FDR < 0.05). Among the differentially expressed genes, cosinor analysis identified seasonal rhythmicity for the observed changes in blood gene expression, consistent with studies in humans. While the proportion of seasonally variant genes in these dolphins is much smaller than that reported in humans, the majority of those identified in dolphins were also shown to vary with season in humans. Gene co-expression network analysis identified several gene modules with significant correlation to age, sex, or hematological parameters. CONCLUSIONS: This longitudinal analysis of healthy managed dolphins establishes a preliminary baseline for blood transcriptome analysis in this species. Correlations with hematological parameters, distinct from muted seasonal effects, suggest that the otherwise relatively stable blood transcriptome may be a useful indicator of health and exposure. A robust database of gene expression in free-ranging and managed dolphins across seasons with known adverse health conditions or contaminant exposures will be needed to establish predictive gene expression profiles suitable for biomonitoring.

Isolation and phenotyping of potential stem cells from the umbilical cord of the bottlenose dolphin(Tursiops truncatus).

We have successfully isolated cells with stem-like properties from bottlenose dolphin (Tursiops truncatus) umbilical cord. Our results show that this cetacean species has embryonic fetal and adult stem cells as do humans and other studied mammals. This accomplishment allows to eventually investigate whether dolphins, due to their unique adaptations to aquatic environments, have special stem cell lineages or distinctive mechanisms of cell programming. Further characterization of their potency to differentiate into multiple cell lineages would fulfill numerous applicative purposes. We characterized, developed and refined a new protocol for obtaining potential stem cells from umbilical cord tissues of the bottlenose dolphin. Tissue samples were taken from umbilical cords of successful deliveries immediately after placenta ejection and collection from the water. Umbilical cord samples (2-3 cm3) were excised and subjected to enzymatic digestion and mechanical dissociation. Viable cells from specimens resident in the Oceanografic Valencia were cultured and subsequently isolated and tested for pluripotent characteristics (cell morphology, phenotype and expression of surface markers). Cell viability was confirmed also after freezing/thawing. The established protocol is suitable for collection/isolation/culture of dolphin potential mesenchymal stem cells from dolphin umbilical cord, which can be deposited in cell banks for future research needs.

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.

De novo transcriptome assembly and RNA-Seq expression analysis in blood from beluga whales of Bristol Bay, AK.

Assessing the health of marine mammal sentinel species is crucial to understanding the impacts of environmental perturbations on marine ecosystems and human health. In Arctic regions, beluga whales, Delphinapterus leucas, are upper level predators that may serve as a sentinel species, potentially forecasting impacts on human health. While gene expression profiling from blood transcriptomes has widely been used to assess health status and environmental exposures in human and veterinary medicine, its use in wildlife has been limited due to the lack of available genomes and baseline data. To this end we constructed the first beluga whale blood transcriptome de novo from samples collected during annual health assessments of the healthy Bristol Bay, AK stock during 2012-2014 to establish baseline information on the content and variation of the beluga whale blood transcriptome. The Trinity transcriptome assembly from beluga was comprised of 91,325 transcripts that represented a wide array of cellular functions and processes and was extremely similar in content to the blood transcriptome of another cetacean, the bottlenose dolphin. Expression of hemoglobin transcripts was much lower in beluga (25.6% of TPM, transcripts per million) than has been observed in many other mammals. A T12A amino acid substitution in the HBB sequence of beluga whales, but not bottlenose dolphins, was identified and may play a role in low temperature adaptation. The beluga blood transcriptome was extremely stable between sex and year, with no apparent clustering of samples by principle components analysis and <4% of genes differentially expressed (EBseq, FDR<0.05). While the impacts of season, sexual maturity, disease, and geography on the beluga blood transcriptome must be established, the presence of transcripts involved in stress, detoxification, and immune functions indicate that blood gene expression analyses may provide information on health status and exposure. This study provides a wealth of transcriptomic data on beluga whales and provides a sizeable pool of preliminary data for comparison with other studies in beluga whale.

Transcriptomic analysis of bottlenose dolphin (Tursiops truncatus) skin biopsies to assess the effects of emerging contaminants.

Chemicals discovered in water at levels that may be significantly different than expected are referred to as contaminants of emerging concern (CECs) because the risk to environmental health posed by their occurrence/frequency is still unknown. The worldwide distributed compounds perfluorooctanoic acid (PFOA) and bisphenol A (BPA) may fall into this category due to effects on endocrine receptors. We applied an ex vivo assay using small slices of bioptic skin from the bottlenose dolphin, Tursiops truncatus, cultured and treated for 24 h with different PFOA or BPA concentrations to analyze global gene expression. RNA was labeled and hybridized to a species-specific oligomicroarray. The skin transcriptome held information on the contaminant exposure, potentially predictive about long-term effects on health, being the genes affected involved in immunity modulation, response to stress, lipid homeostasis, and development. The transcriptomic signature of dolphin skin could be therefore relevant as classifier for a specific contaminant.