The Antarctic fur seal (Arctocephalus gazella) is an important top predator and indicator of the health of the Southern Ocean ecosystem. Although abundant, this species narrowly escaped extinction due to historical sealing and is currently declining as a consequence of climate change. Genomic tools are essential for understanding these anthropogenic impacts and for predicting long-term viability. However, the current reference genome (\"arcGaz3\") shows considerable room for improvement in terms of both completeness and contiguity. We therefore combined PacBio sequencing, haplotype-aware HiRise assembly and scaffolding based on Hi-C information to generate a refined assembly of the Antarctic fur seal reference genome (\"arcGaz4_h1\"). The new assembly is 2.53Gb long, has a scaffold N50 of 55.6Mb and includes 18 chromosome-sized scaffolds, which correspond to the 18 chromosomes expected in otariids. Genome completeness is greatly improved, with 23,408 annotated genes and a Benchmarking Universal Single-Copy Orthologs (BUSCO) score raised from 84.7% to 95.2%. We furthermore included the new genome in a reference-free alignment of the genomes of eleven pinniped species to characterize evolutionary conservation across the Pinnipedia using genome-wide Genomic Evolutionary Rate Profiling (GERP). We then implemented Gene Ontology (GO) enrichment analyses to identify biological processes associated with those genes showing the highest levels of either conservation or differentiation between the two major pinniped families, the Otariidae and Phocidae. We show that processes linked to neuronal development, the circulatory system and osmoregulation are overrepresented both in conserved as well as in differentiated regions of the genome.

Pharmacokinetics studies have investigated meloxicam, a non-steroidal anti-inflammatory drug, dosing strategies in a wide variety of non-domestic animals; however, there is no prior study examining well-founded dosing for pinnipeds. To develop dosing protocols, pharmacokinetic information is needed, with an examination of differences between pinniped species. Apparently, healthy California sea lions (Zalophus californianus: CSL; n = 13) and Pacific harbor seals (Phoca vitulina richardii: PHS; n = 17) that had completed rehabilitation were enrolled into a population-based pharmacokinetic study. Each animal was administered a single oral dose of meloxicam at 0.1 mg/kg, and two blood samples were collected from each animal at varying intervals during a 96-h study period. Plasma concentrations of meloxicam were determined by high-pressure liquid chromatography. Data were analyzed with nonlinear mixed effects modeling (Phoenix® NLME™, Certara, St. Louis, MO 63105, USA). The results indicated that in PHS, peak plasma concentration (Cmax) was 0.33 μg/mL with an elimination half-life (Ke t½) of 31.53 h. In CSL, Cmax was 0.17 μg/mL with Ke t½ of 32.71 h. All animals enrolled completed the study without outward adverse clinical signs. The elimination half-life was longer than previously recommended dosing intervals for pinnipeds; however, we cannot speculate in the optimum clinical dose from these results.

Air leak syndrome (ALS) is described in human medicine as a constellation of clinical disorders including pneumomediastinum, pneumopericardium, pulmonary interstitial emphysema, pneumothorax, pneumoperitoneum, pneumoretroperitoneum and subcutaneous emphysema. The pathogenesis of ALS depends on the anatomy of the mediastinum and its associations with thoracic, abdominal and cervical connective tissues, as well as a physical phenomenon referred to as the Macklin effect. Various animal species develop diverse combinations of these lesions, although ALS has not been recognized in animals. However, this term aids pathologists in addressing this disease compilation. The aim of this retrospective study is to illustrate examples of ALS in animals by arbitrarily selecting 13 cases in dogs, cats, pinnipeds, sea otters and harbour porpoises. ALS can be classified into three groups based on aetiology: iatrogenic, secondary or spontaneous. Iatrogenic ALS was diagnosed in two cats with tracheal laceration following endotracheal intubation. Secondary ALS was identified in two dogs, one with acute respiratory distress syndrome and the other due to grass awn migration. Secondary ALS in pinnipeds was diagnosed following severe pulmonary parasitism, uraemic pneumonia and oesophageal perforation. The other marine mammals developed ALS following trauma. Spontaneous ALS was also diagnosed in one cat and one dog without any apparent predisposing causes.

Hemotropic mycoplasmas are bacteria that attaches to erythrocytes surface, which some species presents zoonotic concerns. In the suborder Pinnipedia, genera Otaria and Arctocephalus are prominent in Brazil. This study investigated the occurrence of hemoplasmas in Arctocephalus sp. and Otaria flavescens found dead along the coast of a Southern Brazilian State. DNA from 135 spleen samples were extracted and subjected to conventional PCR protocols, targeting the 16 S rRNA and 23 S rRNA gene. Three (2.22 %) Arctocephalus australis were positive in the 16 S rRNA gene, and no samples amplified in the 23 S rRNA gene. Samples from this study clustered with Zalophus californianus and Arctocephalus tropicalis mycoplasmas on a Bayesian phylogenetic analysis. Genetic diversity analysis suggested distinct genotypes, indicating A. australis as a new host for hemoplasma, and also a potential putative novel hemoplasma genotype. These findings raises future awareness for pinnipeds conservation, and adds Mycoplasma spp. to be taken into consideration when clinically evaluating rescued animals.

The molecular evolution of the mammalian heater protein UCP1 is a powerful biomarker to understand thermoregulatory strategies during species radiation into extreme climates, such as aquatic life with high thermal conductivity. While fully aquatic mammals lost UCP1, most semiaquatic seals display intact UCP1 genes, apart from large elephant seals. Here, we show that UCP1 thermogenic activity of the small-bodied harbor seal is equally potent compared to terrestrial orthologs, emphasizing its importance for neonatal survival on land. In contrast, elephant seal UCP1 does not display thermogenic activity, not even when translating a repaired or a recently highlighted truncated version. Thus, the thermogenic benefits for neonatal survival during terrestrial birth in semiaquatic pinnipeds maintained evolutionary selection pressure on UCP1 function and were only outweighed by extreme body sizes among elephant seals, fully eliminating UCP1-dependent thermogenesis.

Despite decades of research, surprisingly little is known about the mechanism(s) by which an individual\'s genotype is encoded in odour. Many studies have focused on the role of the major histocompatibility complex (MHC) owing to its importance for survival and mate choice. However, the salience of MHC-mediated odours compared to chemicals influenced by the rest of the genome remains unclear, especially in wild populations where it is challenging to quantify and control for the effects of the genomic background. We addressed this issue in Antarctic fur seals by analysing skin swabs together with full-length MHC DQB II exon 2 sequences and data from 41 genome-wide distributed microsatellites. We did not find any effects of MHC relatedness on chemical similarity and there was also no relationship between MHC heterozygosity and chemical diversity. However, multilocus heterozygosity showed a significant positive association with chemical diversity, even after controlling for MHC heterozygosity. Our results appear to rule out a dominant role of the MHC in the chemical encoding of genetic information in a wild vertebrate population and highlight the need for genome-wide approaches to elucidate the mechanism(s) and specific genes underlying genotype-odour associations.

Regulation of mitochondrial oxidative phosphorylation is essential to match energy supply to changing cellular energy demands, and to cope with periods of hypoxia. Recent work implicates the circadian molecular clock in control of mitochondrial function and hypoxia sensing. Because diving mammals experience intermittent episodes of severe hypoxia, with diel patterning in dive depth and duration, it is interesting to consider circadian-mitochondrial interaction in this group. Here, we demonstrate that the hooded seal (Cystophora cristata), a deep-diving Arctic pinniped, shows strong daily patterning of diving behaviour in the wild. Cultures of hooded seal skin fibroblasts exhibit robust circadian oscillation of the core clock genes per2 and arntl. In liver tissue collected from captive hooded seals, expression of arntl was some 4-fold higher in the middle of the night than in the middle of the day. To explore the clock-mitochondria relationship, we measured the mitochondrial oxygen consumption in synchronized hooded seal skin fibroblasts and found a circadian variation in mitochondrial activity, with higher coupling efficiency of complex I coinciding with the trough of arntl expression. These results open the way for further studies of circadian-hypoxia interactions in pinnipeds during diving.

Animal vocal communication often relies on call sequences. The temporal patterns of such sequences can be adjusted to other callers, follow complex rhythmic structures or exhibit a metronome-like pattern (i.e., isochronous). How regular are the temporal patterns in animal signals, and what influences their precision? If present, are rhythms already there early in ontogeny? Here, we describe an exploratory study of Cape fur seal (Arctocephalus pusillus pusillus) barks-a vocalisation type produced across many pinniped species in rhythmic, percussive bouts. This study is the first quantitative description of barking in Cape fur seal pups. We analysed the rhythmic structures of spontaneous barking bouts of pups and adult females from the breeding colony in Cape Cross, Namibia. Barks of adult females exhibited isochrony, that is they were produced at fairly regular points in time. Instead, intervals between pup barks were more variable, that is skipping a bark in the isochronous series occasionally. In both age classes, beat precision, that is how well the barks followed a perfect template, was worse when barking at higher rates. Differences could be explained by physiological factors, such as respiration or arousal. Whether, and how, isochrony develops in this species remains an open question. This study provides evidence towards a rhythmic production of barks in Cape fur seal pups and lays the groundwork for future studies to investigate the development of rhythm using multidimensional metrics.

The increased size and enhanced compliance of the aortic bulb-the enlargement of the ascending aorta-are believed to maintain blood flow in pinnipeds during extended periods of diastole induced by diving bradycardia. The aortic bulb has been described ex vivo in several species of pinnipeds, but in vivo measurements are needed to investigate the relationship between structure and function. We obtained ultrasound images using electrocardiogram-gated transesophageal echocardiography during anesthesia and after atropine administration to assess the relationship between aortic bulb anatomy and cardiac function (heart rate, stroke volume, cardiac output) in northern fur seals (Callorhinus ursinus) and Steller sea lions (Eumetopias jubatus). We observed that the aortic bulb in northern fur seals and Steller sea lions expands during systole and recoils over the entire diastolic period indicating that blood flow is maintained throughout the entire cardiac cycle as expected. The stroke volumes we measured in the fur seals and sea lions fit the values predicted based on body size in mammals and did not change with increased heart rates, suggesting that greater stroke volumes are not needed for aortic bulb function. Overall, our results suggest that peripheral vasoconstriction during diving is sufficient to modulate the volume of blood in the aortic bulb to ensure that flow lasts over the entire diastolic period. These results indicate that the shift of blood into the aortic bulb of pinnipeds is a fundamental mechanism caused by vasoconstriction while diving, highlighting the importance of this unique anatomical adaptation.

We evaluated the Precision Xtra™ ketometer as part of a larger study categorizing fasting status of free-ranging Steller sea lion (Eumetopias jubatus; SSL) pups which necessitated the identification of plasma β-hydroxybutyrate concentrations ([β-HBA]) around a threshold of <0.3 and ≥0.3 mmol/l. Whole blood samples mixed with sodium heparin (NaHep) or ethylenediaminetetraacetic acid liquid anticoagulants were tested <10 minutes after collection (n = 14; triplicate technical replicates). Plasma (stored at -80°C, NaHep, Thaw1) measured via our laboratory\'s Reference Assay (Sigma Aldrich, St. Louis, MO, Kit #MAK041) served as the standard [β-HBA] for ketometer comparisons. Our observed β-HBA range (0.0-1.6 mmol/l), consistent with published [β-HBA] of free-ranging Otariid pups, represented the lower 20% of the ketometer\'s range (0.0-8.0 mmol/l). The maximal coefficient of variation (%CV) of ketometer technical replicates was 9.1% (NaHep, whole blood). The majority of ketometer technical replicate sets (84%, including all matrices, anticoagulants and thawings) were identical (CV = 0%). We found linear relationships and agreement of ketometer [β-HBA] between whole blood preserved with different anticoagulants and between whole blood and plasma (Thaw1) measurements. The ketometer produced results with linearity to the Reference Assay for both whole blood and plasma (Thaw1). We identified a non-linear relationship between plasma at Thaw1 and Thaw2 (tested four months apart, NaHep), as only samples with higher SSL [β-HBA] decreased in concentration, and all others remained the same. With respect to categorizing SSL pup fasting in our larger study, the ketometer\'s %Accuracy, %Sensitivity and %Specificity for samples with Reference Assay β-HBA <0.2 and >0.4 mmol/l were 100%. We adopted a modified procedure: plasma samples with mean ketometer concentrations ±0.1 mmol/l of 0.3 mmol/l β-HBA were re-evaluated using the Reference Assay, improving measurement precision from tenths (ketometer) to thousandths (assay) mmol/l. The Precision Xtra™ ketometer was valuable to our application over the range of [β-HBA] observed in SSL pup plasma and whole blood samples.