DNA methylation networks underlying mammalian traits.

Using DNA methylation profiles (n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species.

The Validation of a Non-Invasive Skin Sampling Device for Detecting Cetacean Poxvirus.

Poxvirus-like lesions are widely used as a potential health indicator in cetaceans, although for this application, corroboration of Poxvirus skin disease is imperative. Aiming to address skin biopsies intrusiveness, a preliminary investigation of a non-invasive skin sampling procedure to molecularly detect CePV-1 in 12 tattoo-like-lesions from two free-ranging stranded cetaceans in the Canary Islands was performed. Skin lesions were brushed with cytology cell samplers (CCSs) and placed into 1.5 mL microcentrifuge tubes with 1 mL of RNAlaterTM Stabilization Solution. For factual comparisons, DNA extractions from sloughed skin obtained with CCS and biopsies from the same lesions were accomplished with DNA Tissue Kit STM (QuickGene, Kurabo, Japan). Moreover, a second DNA extraction from sloughed skin with DNeasyTM Blood and Tissue Kit (Qiagen, Inc., Valencia, CA, USA) was performed to ascertain kit suitability for CCS. Molecular detection of CePV-1 was performed through a real-time PCR. As a result, a 91.7% and 83.3% rates of positivity were obtained with biopsies and CCS through Quickgene, respectively, compared to the rate of 100% using CCS with Qiagen. Accordingly, CCS is a reliable non-invasive sampling device to obtain sufficient genetic material to be analyzed for CePV-1 in tattoo-skin-lesions as well as for other purposes in cetaceans under human care.

Multi-species and multi-tissue methylation clocks for age estimation in toothed whales and dolphins.

The development of a precise blood or skin tissue DNA Epigenetic Aging Clock for Odontocete (OEAC) would solve current age estimation inaccuracies for wild odontocetes. Therefore, we determined genome-wide DNA methylation profiles using a custom array (HorvathMammalMethyl40) across skin and blood samples (n = 446) from known age animals representing nine odontocete species within 4 phylogenetic families to identify age associated CG dinucleotides (CpGs). The top CpGs were used to create a cross-validated OEAC clock which was highly correlated for individuals (r = 0.94) and for unique species (median r = 0.93). Finally, we applied the OEAC for estimating the age and sex of 22 wild Norwegian killer whales. DNA methylation patterns of age associated CpGs are highly conserved across odontocetes. These similarities allowed us to develop an odontocete epigenetic aging clock (OEAC) which can be used for species conservation efforts by provide a mechanism for estimating the age of free ranging odontocetes from either blood or skin samples.

Frequency-modulated up-chirp stimuli enhance the auditory brainstem response of the killer whale (Orcinus orca).

Previous studies suggested that frequency-modulated tonal stimuli where the frequency sweeps upward (up-chirps) may enhance auditory brainstem response (ABR) amplitudes in mammals. In this study, ABRs were measured in response to up-chirps in three killer whales (Orcinus orca) and compared to ABRs evoked by broadband clicks. Chirp durations ranged from 125 - 2000 µs. Chirp spectral content was either "uncompensated," meaning the spectrum paralleled the transmitting response of the piezoelectric transducer, or "compensated," where the spectral density level was flat (+/-4 dB) across the stimulus bandwidth (10 - 130 kHz). Compensated up-chirps consistently produced higher amplitude ABRs than uncompensated clicks with the same peak equivalent sound pressure level. ABR amplitude increased with up-chirp duration up to 1400 µs, although there was considerable variability between individuals. Results suggest that compensating stimuli for the response of transducers can have a dramatic effect on broadband ABRs, and that compensated up-chirps might be useful for testing whale species where large size makes far-field recording of ABRs at the skin surface difficult.

Social interaction analysis in captive orcas (Orcinus orca).

The management of socially complex species in captivity is challenging. Research on their social behavior improves our understanding of interactions in captive animals and captive-group management. We conducted a detailed analysis of social relationships shown by the orcas kept at Loro Parque zoo and their tendency to reconcile after aggressive episodes. Affiliative interactions were the most frequent social activities compared to agonistic or sexual interactions. Within affiliative behaviors, we documented the pattern "gentle tongue bite", where an animal touches the other's tongue with his teeth but does not bite it. Affiliative interactions between a specific pair of orcas occurred significantly more often than expected by chance, and together with low levels of agonistic interactions, indicated particular affinity between some individuals. The most frequently observed low-level agonistic relationship was that of the two older males (Tekoa-Keto); however, they also showed frequent sexual and affiliative interactions. Sexual-like behaviors (pursuit, mount, and penis between males) were found in both sexes. Finally, the observed corrected conciliatory tendency (31.57%) was within the range described for other primate and cetacean species. This study provides a systematic way to assess social interactions as well as conflict management strategies in cetaceans housed in zoos and zoo-like facilities and may help to improve animal welfare and management of animals in controlled environments.

Personality in captive killer whales (Orcinus orca): A rating approach based on the five-factor model.

The comparative study of animal personality has received great interest in recent years. Some studies have analyzed personalities in cetaceans (exclusively in dolphins), but none have analyzed the factorial structure of personality of any species in this order. Our objective was to evaluate a sample of captive killer whales (n = 24) adapting one of the most widely used models of personality in humans and nonhuman animals: the five-factor model. A total of 38 personality descriptive adjectives were rated by 55 raters (mainly trainers and curators). Principal components analysis and regularized exploratory factor analysis revealed four statistically significant factors with acceptable standards of interrater reliability and validity, accounting for 49.85% of the variance. The first factor indicated an Extraversion factor, the second one revealed a combined factor of Conscientiousness and Agreeableness, the third one yielded in a Dominance factor, and the fourth one reflected a Careful factor very close to a combination of Conscientiousness and Agreeableness factor. The results were compared with the results obtained for humans and chimpanzees in prior studies. The similarities could be explained as a result of convergent adaptive traits despite a deep evolutionary divergence, adaptation to physically dissimilar environments, and very different neuroanatomical organization. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Effects of Polar Bear and Killer Whale Derived Contaminant Cocktails on Marine Mammal Immunity.

Most controlled toxicity studies use single chemical exposures that do not represent the real world situation of complex mixtures of known and unknown natural and anthropogenic substances. In the present study, complex contaminant cocktails derived from the blubber of polar bears (PB; Ursus maritimus) and killer whales (KW; Orcinus orca) were used for in vitro concentration-response experiments with PB, cetacean and seal spp. immune cells to evaluate the effect of realistic contaminant mixtures on various immune functions. Cytotoxic effects of the PB cocktail occurred at lower concentrations than the KW cocktail (1 vs 16 µg/mL), likely due to differences in contaminant profiles in the mixtures derived from the adipose of each species. Similarly, significant reduction of lymphocyte proliferation occurred at much lower exposures in the PB cocktail (EC50: 0.94 vs 6.06 µg/mL; P < 0.01), whereas the KW cocktail caused a much faster decline in proliferation (slope: 2.9 vs 1.7; P = 0.04). Only the KW cocktail modulated natural killer (NK) cell activity and neutrophil and monocyte phagocytosis in a concentration- and species-dependent manner. No clear sensitivity differences emerged when comparing cetaceans, seals and PB. Our results showing lower effect levels for complex mixtures relative to single compounds suggest that previous risk assessments underestimate the effects of real world contaminant exposure on immunity. Our results using blubber-derived contaminant cocktails add realism to in vitro exposure experiments and confirm the immunotoxic risk marine mammals face from exposure to complex mixtures of environmental contaminants.

Mercury and selenium status of bottlenose dolphins (Tursiops truncatus): A study in stranded animals on the Canary Islands.

The mercury (Hg) level in the marine environment has tripled in recent decades, becoming a great concern because of its high toxic potential. This study reports Hg and selenium (Se) status, and the first Se/Hg molar ratio assessment in bottlenose dolphins (Tursiops truncatus) inhabiting the waters of the Canary Islands. Total Hg and Se concentrations were determined in the blubber and liver collected from 30 specimens stranded along the coasts of the archipelago from 1997 to 2013. The median values for total Hg in the blubber and liver were 80.83 and 223.77 µg g(-1) dry weight (dw), and the median levels for Se in both tissues were 7.29 and 68.63 µg g(-1) dw, respectively. Hg concentrations in the liver were lower than 100 µg g(-1) wet weight (ww), comparable to those obtained in bottlenose dolphins from the North Sea, the Western Atlantic Ocean and several locations in the Pacific Ocean. The Mediterranean Sea and South of Australia are the most contaminated areas for both elements in this cetacean species. In addition, it must be stressed that the levels of Hg and Se in the liver showed an increasing trend with the age of the animals. As expected, a strong positive correlation between Hg and Se was observed (rs=0.960). Surprisingly, both younger and older specimens had a Se/Hg molar ratio different from 1, suggesting that these individuals may be at greater toxicological risk for high concentrations of both elements or a deficiency of Se without a protective action against Hg toxicity.

Levels and profiles of POPs (organochlorine pesticides, PCBs, and PAHs) in free-ranging common bottlenose dolphins of the Canary Islands, Spain.

The effect of anthropogenic pollution in marine mammals worldwide has become an important issue due to the high concentrations found in many areas. The present study represents the first report of pollutants in free-ranging cetaceans from the Canary Islands, where there are 12 marine Special Areas of Conservation (SACs), because of the presence of bottlenose dolphins (Tursiops truncatus). We selected this resident population of dolphins as a bioindicator to gain knowledge concerning the toxicological status of the cetaceans of this protected area. In 64 biopsy samples of live free-ranging animals sampled from 2003 to 2011, we determined the concentrations of 18 polychlorinated biphenyls (PCBs), 23 organochlorine pesticides (OCPs) and 16 polycyclic aromatic hydrocarbons (PAHs). We found high levels of many of these pollutants, and some of them were detectable in 100% of the samples. The median value for ∑OCPs was 57,104 ng g(-1) lipid weight (lw), and the dichlorodiphenyldichloroethylene (p,p'-DDE) accounted for 70% of this amount. Among PCBs, congeners 180, 153 and 138 were predominant (82% of ∑PCBs; median = 30,783 ng g(-1) lw). Concerning the analyzed PAHs, the total median burden was 13,598 ng g(-1) lw, and phenanthrene was the compound measured at the highest concentration followed by pyrene and by naphthalene. Surprisingly, we have found that organohalogen pollutants exhibit an upward trend in recent years of sampling. Thus, according to the guidelines outlined in the EU's Marine Strategy Framework Directive, further monitoring studies in Canary Islands are required to contribute to the conservation of the resident populations of marine mammals in this region.

Assessment of the levels of polycyclic aromatic hydrocarbons and organochlorine contaminants in bottlenose dolphins (Tursiops truncatus) from the Eastern Atlantic Ocean.

The concentrations of 18 polychlorinated biphenyls (PCBs), 23 organochlorine pesticides (OCPs), and 16 polycyclic aromatic hydrocarbons (PAHs) were determined in the blubber and liver of 27 bottlenose dolphins (Tursiops truncatus) stranded along the Canary Islands coasts from 1997 to 2011. DDTs (mean of 60,960 and 445 ng/g lw., respectively) and PCBs (mean of 47,168 and 628 ng/g lw., respectively) were the predominant compounds in both tissues. Among PCBs the highly chlorinated PCB 180, 153 and 138 were the predominant congeners. We found a p,p'-DDE/∑DDTs ratio of 0.87 in blubber and 0.88 in liver, which is indicative of DDT ageing. All the samples showed detectable values of any of the 16 PAH studied. Phenanthrene was the most frequently detected and at the highest concentration. According to our results, concentrations of OCPs, and especially PCBs, are still at toxicologically relevant levels in blubber of bottlenose dolphins of this geographical area.

Development of an indirect immunofluorescence technique for the diagnosis of toxoplasmosis in bottlenose dolphins.

The diagnosis of toxoplasmosis is often complicated by the lack of specific clinical symptoms or postmortem features, in humans and other animals. The only diagnostic test described so far for the serological diagnosis of Toxoplasma gondii in marine mammals is the modified agglutination test (Dubey et al., Am J Vet Res 48(8):1239-1243, 1987). The development of more sensible and specific immunological techniques requires specific antibodies, which are currently unavailable in the scientific market. Indirect immunofluorescence (IIF) is one of the most widely used methods for the diagnosis of toxoplasmosis in humans (Auer et al., Parasitol Res 12:965-970, 2000). In order to develop and apply this technique to the bottlenose dolphin (Tursiops truncatus), immunoglobulins were firstly purified using ion-exchange chromatography. The purified immunoglobulins were then injected in New Zealand rabbits in order to obtain polyclonal antibodies. These antisera were validated by the IIF technique, using as controls serum samples of dolphins infected by Toxoplasma. The results were visualized using antirabbit IgG labeled with fluorescein. This newly developed and specific serological assay was then tested with the dolphin collection of Loro Parque, Tenerife, Spain (group I), and L'Oceanogràfic of Valencia, Spain (group II). The obtained results in this study showed that none of the dolphins from group 1 were infected by T. gondii and two animals were positive in group 2. Furthermore, we conclude that this study has produced antibodies with high specificity against dolphin immunoglobulins and an IIF method which may be used as immunological diagnostic tools, especially for the serological diagnosis of toxoplasmosis.

Vocal sharing and individual acoustic distinctiveness within a group of captive orcas (Orcinus orca).

Among vocal learners, some animal species are known to develop individually distinctive vocalizations, and others clearly learn to produce group signatures. The optimal vocal sharing hypothesis suggests that vocal divergence and convergence are not compulsorily exclusive and both can be found at different levels in a given species. Being individually recognizable is socially important even in species sharing vocal badges. Acoustic divergence is not systematically controlled as it can simply be due to interindividual morphological differences. We tested that hypothesis in a species known to learn their family vocal dialect socially: the orca (Orcinus orca). We identified 13 different call types, including some shared by all group members, some shared only by 2 or 3 individuals, and others particular to 1 individual. Sharing was higher between males than between females. Three of our 4 orcas each produced a unique call type, which was preferably emitted. The call types shared by all orcas still presented individual acoustic distinctiveness that could, to some degree, be explained by morphological differences. We found evidence for strong similarities between some of the call types of our captive orcas and the call types of their ancestors, which are Canadian and Icelandic free-ranging orcas. Our findings suggest that captive orcas use a complex vocal repertoire enabling each individual to produce sounds that are similar to some of their partners', which might be used as social badges to advertise their preferential bonds, as well as individual-specific calls. Our findings open new lines of research concerning the functional value of a balanced "diverging-converging" vocal system.