Whole genomes reveal multiple candidate genes and pathways involved in the immune response of dolphins to a highly infectious virus.

Wildlife species are challenged by various infectious diseases that act as important demographic drivers of populations and have become a great conservation concern particularly under growing environmental changes. The new era of whole genome sequencing provides new opportunities and avenues to explore the role of genetic variants in the plasticity of immune responses, particularly in non-model systems. Cetacean morbillivirus (CeMV) has emerged as a major viral threat to cetacean populations worldwide, contributing to the death of thousands of individuals of multiple dolphin and whale species. To understand the genomic basis of immune responses to CeMV, we generated and analysed whole genomes of 53 Indo-Pacific bottlenose dolphins (Tursiops aduncus) exposed to Australia's largest known CeMV-related mortality event that killed at least 50 dolphins from three different species. The genomic data set consisted of 10,168,981 SNPs anchored onto 23 chromosome-length scaffolds and 77 short scaffolds. Whole genome analysis indicated that levels of inbreeding in the dolphin population did not influence the outcome of an individual. Allele frequency estimates between survivors and nonsurvivors of the outbreak revealed 15,769 candidate SNPs, of which 689 were annotated to 295 protein coding genes. These included 50 genes with functions related to innate and adaptive immune responses, and cytokine signalling pathways and genes thought to be involved in immune responses to other morbilliviruses. Our study characterised genomic regions and pathways that may contribute to CeMV immune responses in dolphins. This represents a stride towards clarifying the complex interactions of the cetacean immune system and emphasises the value of whole genome data sets in understanding genetic elements that are essential for species conservation, including disease susceptibility and adaptation.

Concentrations of some legacy pollutants have increased in South Australian bottlenose dolphins from 1989 to 2014.

Information about pollution and its potential impact in Australian marine wildlife is scarce. To fill this knowledge gap, our study investigated concentrations of legacy pollutants as well as naturally produced methoxylated polybrominated diphenyl ethers (MeO-PBDEs) in blubber, liver, kidney and muscle of Indo-Pacific bottlenose dolphins (Tursiops aduncus) from two large inverse estuaries in South Australia from 1989 to 1995 and 2009-2014. Our results show that concentrations of most pollutant classes are relatively low compared to the literature but at the higher end of the ranges reported for marine mammals in Australia. Results for some individuals exceed toxicity thresholds indicative of immunotoxicity in marine mammals. It is important to note that concentrations of some compound classes, particularly PBDEs and polychlorinated biphenyls (PCBs), increased over a time interval of 20 years thereby placing more individuals at risk in recent years. Some of the highest concentrations of persistent organic pollutants (POPs) were measured in juveniles, which may jeopardize their development and the success of future generations. These results indicate that legacy pollutants may play a role in the long-term health of T. aduncus and should be included in biomonitoring efforts.

Pathology of the skeleton of Indo-Pacific bottlenose dolphins Tursiops aduncus: a comparison of adjacent gulfs in South Australia.

Studies of skeletal pathology of marine mammals can contribute to conservation measures, yet few have focused on causative factors. Museums hold vast collections of skeletons relevant to this knowledge gap. Indo-Pacific bottlenose dolphin Tursiops aduncus carcasses (n = 162) were collected from Gulf St Vincent (GSV) and Spencer Gulf (SG), South Australia (SA), between 1988 and 2013, and post-mortem examinations were carried out. After preparing skeletons, their gross pathology was classified into 4 categories: lytic lesions, degenerative lesions, fractures and malformations. Comparisons were made between gulfs based on pathology type, relative age, period of collection and cause of death (anthropogenic vs. non-anthropogenic). Pathology prevalence (76%) was higher than reported in other studies but observed pathologies were similar. More than one pathology was observed in 88% of dolphins that had pathology. Vertebrae were often the site of pathology. Prevalence of lesions were: fractures (82%), lytic lesions (75%) including spondylo-osteomyelitis and osteomyelitis, degenerative lesions (57%) including spondylosis deformans, and malformations (17%). Prevalence of pathology increased with dolphin relative age, and GSV dolphins had more pathology than those in SG. In SG, anthropogenic cases had more pathology than non-anthropogenic cases, and dolphins collected after 2000 had more degenerative lesions than those collected before 2000. There were more malformations in SG dolphins than those from GSV, although this was not statistically significant. In one anomalous case, an individual was found with 5 detached transverse processes on 4 lumbar vertebrae. Heavy metal toxicity reported for T. aduncus from SG may be related to the malformations reported in this study.

Whale fall chemosymbiotic communities in a southwest Australian submarine canyon fill a distributional gap.

A whale fall community of chemosymbiotic invertebrates living on cetacean bones has been identified off southwestern Australia during a Remotely Operated Vehicle (ROV) survey at bathyal depths within the Bremer Marine Park, which is part of important marine mammal areas (IMMA) of the Albany Canyon Region. Cetacean bones on the seafloor of the Hood Canyon, consisted of isolated skulls of three species of beaked whales (family Ziphiidae): Mesoplodon cf. layardii, M. grayi, and M. hectori, a few vertebrae, and lower jaws. One of the beaked whale skulls (Mesoplodon cf. layardii) was sampled and found to be intensely colonised by hundreds of specimens of a bathymodilinae mussel ("Adipicola" s.l.). Live polychaetes (Phyllochaetopterus?), skeneimorph gastropods, and amphipods (Seba, Leptamphopus) colonised the skull bone, which represent a later stage (sulfophilic) of carcass decomposition. The reducing sediment below the skull was inhabited by lucinid (Lucinoma) and vesicomyid (Calyptogena) chemosymbiotic bivalves. Additionally, the sediment thanatocoenosis comprised shells of various other chemosymbiotic bivalves, such as Acharax, thyasirids, lucinids, vesicomyids, and limpets, representing the complex ecological turnover phases through time in this whale fall chemosynthetic habitat. With one exception, all bones recovered were colonized by bathymodiolin mussels. This is the first documented case of a chemosynthetic community and associated chemosymbiotic fauna relating to beaked whales, and the first fully documented record of a whale fall community within the Australian Southern Ocean region.

Exploring lipid affinities of persistent organic pollutants and MeO-PBDEs in blubber of marine mammals.

Although lipophilic compounds have been the focus of numerous studies in marine mammals, their association with lipids is widely accepted, but rarely scrutinized. This pilot study aimed to investigate potential relationships between individual lipids from different lipid classes identified through a non-targeted Nuclear Magnetic Resonance (NMR) based lipidomics approach and legacy POPs in the blubber of long-finned pilot whales, sperm whales, common bottlenose dolphins, and Indo-Pacific bottlenose dolphins. Concentrations of selected POPs such as HCB and HCHs in sperm whales from Tasmania were found to differ from those in long-finned pilot whales and common bottlenose dolphins from the same location. Profiles of NMR spectra measured in blubber of sperm whales were also distinctly different compared to the pilot whales and common bottlenose dolphins. Two groups of Indo-Pacific bottlenose dolphins from South Australia that were 20 years apart showed highly comparable profiles of NMR signals despite having higher concentrations of several POP classes in the more recent group. More specific correlations were investigated between selected POPs (n = 12) and all detected NMR signals (n = 63) in all species. Outcomes were species-specific, but difficult to interpret due to the lack of available literature for marine mammals and the small sample sizes per species. Because of the key role of lipids in the bioaccumulation of POPs and in the incidence of diseases, more attention should be given to the identification and characterization of lipid species in future toxicological studies. However, future studies should focus on one marine mammal species to increase sample sizes and limit the number of confounding factors, such as diet, that can influence POP and lipid levels and profiles.

Unusual causes of fatal upper aerodigestive tract obstruction in wild Bottlenose Dolphins (Tursiops aduncus).

Necropsy examination of dolphins living in Gulf St Vincent, Australia is routinely undertaken to enable the evaluation of disease processes and to provide rapid medicolegal assessment of any inflicted and/or accidental injuries. Two Indo-Pacific Bottlenose Dolphins (Tursiops aduncus) are reported to demonstrate conditions that may result in unexpected death involving upper airway compromise by quite unusual mechanisms. In the first case an adult male was found with extensive soft tissue trauma suggesting human interaction. At necropsy, death was due instead to upper airway obstruction from an impacted Slender-spined Porcupine Fish (Diodon nichthemerus) in the posterior pharynx and upper esophagus. In the second case, an adult male dolphin was found to have died, following several weeks' illness, from upper airway obstruction due to extensive respiratory tract papillomatosis within the blowhole. Given the infectious etiology of this condition the local population will be monitored for similar lesions. These cases demonstrate rare causes of upper airway obstruction in wild dolphins that were identifiable only after detailed necropsy examination. The possibility of human involvement in the deaths could be excluded.

Structure and properties of baleen in the Southern right (Eubalaena australis) and Pygmy right whales (Caperea marginata).

Baleen is a resilient and keratinised filter-feeding structure attached to the maxilla of mysticete whales. It is strong and tough, yet a pliant and resilient material, that withstands extreme pressures in the oral cavity during feeding. We investigated the structure, water content, wettability and mechanical properties of baleen of the Southern right (SRW) and Pygmy right whales (PRW), to understand the effects of hydration on the physical and mechanical properties of baleen. Sixty 25 × 15mm baleen subsamples were prepared from one individual of SRW and PRW. Half were hydrated in circulated natural seawater for 21 days and half were dry. Water content analysis showed that SRW baleen was 21.2% water weight and PRW was 26.1%. Wettability testing indicated that surfaces of both hydrated and dried SRW and PRW baleen were hydrophilic, with hydrated samples of both species having lower contact angle values. For the SRW, the average contact angle of hydrated baleen was 40° ± 13.2 and 73° ± 6 for dried samples. Hydrated PRW baleen had an average contact angle of 44° ± 15.3, which was lower than in dried samples (74° ± 2.9). Three-point bending mechanical tests showed that the average maximum flexural stress of dried SRW (134.1 ± 34.3 MPa) and PRW samples (117.8 ± 22.3 MPa) were significantly higher than those of hydrated SRW (25.7 ± 6.3 MPa) and PRW (19.7 ± 4.8 MPa) baleen. Scanning electron microscope images showed the stratification of the outer cortical layer, with cross-linked keratin fibres observed within and between baleen keratin sheets. Hydrated baleen, as in its natural and functional behaviour, has greater flexibility and strength, attributes necessary for the complex filter feeding mechanism characteristic of whales. Hydration must be considered when addressing the physical and mechanical properties of baleen, especially when using dried museum specimens.

Heavy metal toxicity of kidney and bone tissues in South Australian adult bottlenose dolphins (Tursiops aduncus).

Metallothioneins (MT) concentration, renal damage, and bone malformations were investigated in 38 adult Tursiops aduncus carcasses to determine any associations with cadmium, copper, zinc, mercury, lead and selenium. Significantly higher concentrations of cadmium, copper, and zinc in the liver were observed in dolphins showing evidence of more advanced renal damage. No significant differences in metal or selenium concentrations in the liver were observed between groups differing in level of bone malformations. Some dolphins displayed evidence of toxicity and knowledge of metal toxicity pathways were used to elucidate the cause of these abnormalities. Two dolphins had high metal burdens, high MT concentrations, renal damage, and evidence of bone malformations, indicating possible severe and prolonged metal toxicity. One dolphin showed evidence of renal damage, but the lack of any other symptoms suggests that this was unlikely to be caused by metal toxicity. We recommend examining a range of metal toxicity symptoms simultaneously to aid in distinguishing metal toxicity from unrelated aetiologies.

Genome-wide association study of an unusual dolphin mortality event reveals candidate genes for susceptibility and resistance to cetacean morbillivirus.

Infectious diseases are significant demographic and evolutionary drivers of populations, but studies about the genetic basis of disease resistance and susceptibility are scarce in wildlife populations. Cetacean morbillivirus (CeMV) is a highly contagious disease that is increasing in both geographic distribution and incidence, causing unusual mortality events (UME) and killing tens of thousands of individuals across multiple cetacean species worldwide since the late 1980s. The largest CeMV outbreak in the Southern Hemisphere reported to date occurred in Australia in 2013, where it was a major factor in a UME, killing mainly young Indo-Pacific bottlenose dolphins (Tursiops aduncus). Using cases (nonsurvivors) and controls (putative survivors) from the most affected population, we carried out a genome-wide association study to identify candidate genes for resistance and susceptibility to CeMV. The genomic data set consisted of 278,147,988 sequence reads and 35,493 high-quality SNPs genotyped across 38 individuals. Association analyses found highly significant differences in allele and genotype frequencies among cases and controls at 65 SNPs, and Random Forests conservatively identified eight as candidates. Annotation of these SNPs identified five candidate genes (MAPK8, FBXW11, INADL, ANK3 and ACOX3) with functions associated with stress, pain and immune responses. Our findings provide the first insights into the genetic basis of host defence to this highly contagious disease, enabling the development of an applied evolutionary framework to monitor CeMV resistance across cetacean species. Biomarkers could now be established to assess potential risk factors associated with these genes in other CeMV-affected cetacean populations and species. These results could also possibly aid in the advancement of vaccines against morbilliviruses.

Metals and selenium in the liver and bone of three dolphin species from South Australia, 1988-2004.

Metal and selenium concentrations (wet weight) were determined in the liver (Cd, Hg, Pb, Zn, Cu and Se) and bone (Pb and Cd) of common dolphins (Delphinus delphis, N=71) and bottlenose dolphins (Tursiops truncatus, N=12, and Tursiops aduncus, N=71) stranded or by-caught in South Australia from 1988 to 2004. Differences in metal burdens existed between species, stranding location, and relative age. T. aduncus had the greatest mean tissue burdens of liver Pb (0.45 mg/kg), Cd (6.45 mg/kg), Hg (475.78 mg/kg), Se (178.85 mg/kg) and Zn (93.88 mg/kg) and bone Pb (2.78 mg/kg), probably reflecting their coastal habitat and benthic prey. Mean Cu was highest in T. truncatus (21.18 mg/kg). Bone Cd was measured only in T. aduncus and averaged 0.05 mg/kg. Stranding location impacted metal burdens. Dolphins from Spencer Gulf had higher mean levels of liver Pb (0.39 mg/kg) while Gulf St Vincent dolphins had greater liver Hg (444.64 mg/kg), liver Se (163.12 mg/kg), and bone Pb (2.85 mg/kg). This may be due to high anthropogenic inputs of Pb and Hg into Spencer Gulf and Gulf St Vincent respectively. Liver Cd, Hg, Se and Pb increased with age in all species while Cu decreased with age, in keeping with previous studies. Se and Hg were positively correlated. The possibility that metallothioneins are driving observed correlations between Zn, Cd, Hg and Cu are discussed. Future research must investigate the toxicological consequences of the metal concentrations reported.

Cetacean café coronary.

The carcass of a young adult male Indian Ocean Bottlenose dolphin (Tursiops cf aduncus) was found floating in an estuary near Adelaide, South Australia. An autopsy revealed that death had been caused by obstruction of the upper aerodigestive tract by a 660 mm Cobbler Carpetshark (Sutorectus tentaculatus). Similar airway obstruction in humans while feeding has been termed café coronary syndrome. Although death may have merely resulted from over-enthusiastic feeding, the possibility of neurological impairment was considered, and limited toxicological analyses of tissues was undertaken. No increase in organochlorine pesticides was found, however the possibility of heavy metal poisoning was not excluded. Formal neuropathology was unable to be undertaken. When sudden death in other mammal species mimics cases that are found in humans, similar underlying mechanisms may be present.

The use of carcasses for the analysis of cetacean population genetic structure: a comparative study in two dolphin species.

Advances in molecular techniques have enabled the study of genetic diversity and population structure in many different contexts. Studies that assess the genetic structure of cetacean populations often use biopsy samples from free-ranging individuals and tissue samples from stranded animals or individuals that became entangled in fishery or aquaculture equipment. This leads to the question of how representative the location of a stranded or entangled animal is with respect to its natural range, and whether similar results would be obtained when comparing carcass samples with samples from free-ranging individuals in studies of population structure. Here we use tissue samples from carcasses of dolphins that stranded or died as a result of bycatch in South Australia to investigate spatial population structure in two species: coastal bottlenose (Tursiops sp.) and short-beaked common dolphins (Delphinus delphis). We compare these results with those previously obtained from biopsy sampled free-ranging dolphins in the same area to test whether carcass samples yield similar patterns of genetic variability and population structure. Data from dolphin carcasses were gathered using seven microsatellite markers and a fragment of the mitochondrial DNA control region. Analyses based on carcass samples alone failed to detect genetic structure in Tursiops sp., a species previously shown to exhibit restricted dispersal and moderate genetic differentiation across a small spatial scale in this region. However, genetic structure was correctly inferred in D. delphis, a species previously shown to have reduced genetic structure over a similar geographic area. We propose that in the absence of corroborating data, and when population structure is assessed over relatively small spatial scales, the sole use of carcasses may lead to an underestimate of genetic differentiation. This can lead to a failure in identifying management units for conservation. Therefore, this risk should be carefully assessed when planning population genetic studies of cetaceans.

Eighteen-year study of South Australian dolphins shows variation in lung nematodes by season, year, age class, and location.

Between 1990 and 2007, carcasses of opportunistically collected short-beaked common dolphins (Delphinus delphis; n=238), Indo-Pacific bottlenose dolphins (Tursiops aduncus; n=167), and common bottlenose dolphins (Tursiops truncatus; n=15) were examined for parasites and life history data. Three species of lung nematodes (Halocercus lagenorhynchi, Stenurus ovatus, Pharurus alatus) were identified in surface nodules, subsurface lesions, or airways. Nematode burdens were light to heavy and, in many cases, would have compromised the dolphins' health. The number of dolphins infected was related to species, year, season, age class, and geographic region. Nematodes were found in all three species but were more prevalent in short-beaked common dolphins (mean annual prevalence=26%) than in bottlenose dolphins (12%). There was a significant increase in prevalence of nematodes in short-beaked common dolphins in 2005-06 (63%) compared to 1990-2004 (14%), with a peak in April-June. More young short-beaked common dolphins were infected than subadults and adults and, during the unusual infection event, there were more dependent calves (<130 cm) than juveniles. There were also more infections in dependent bottlenose dolphin (Tursiops spp.) calves but no increase in overall prevalence was detected during 2005-06. Because neonates of both short-beaked common dolphins and bottlenose dolphins were infected, mother-to-calf transmission is suspected for these species in South Australia. Numbers of infections in short-beaked common dolphins were higher in Gulf St Vincent than elsewhere in South Australia, particularly in 2005-06. The cause of the unusual infection event in short-beaked common dolphins is unknown. We discuss the influence of dolphin diet, life history, and external factors.