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.

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.

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.

Cetacean morbillivirus: current knowledge and future directions.

We review the molecular and epidemiological characteristics of cetacean morbillivirus (CeMV) and the diagnosis and pathogenesis of associated disease, with six different strains detected in cetaceans worldwide. CeMV has caused epidemics with high mortality in odontocetes in Europe, the USA and Australia. It represents a distinct species within the Morbillivirus genus. Although most CeMV strains are phylogenetically closely related, recent data indicate that morbilliviruses recovered from Indo-Pacific bottlenose dolphins (Tursiops aduncus), from Western Australia, and a Guiana dolphin (Sotalia guianensis), from Brazil, are divergent. The signaling lymphocyte activation molecule (SLAM) cell receptor for CeMV has been characterized in cetaceans. It shares higher amino acid identity with the ruminant SLAM than with the receptors of carnivores or humans, reflecting the evolutionary history of these mammalian taxa. In Delphinidae, three amino acid substitutions may result in a higher affinity for the virus. Infection is diagnosed by histology, immunohistochemistry, virus isolation, RT-PCR, and serology. Classical CeMV-associated lesions include bronchointerstitial pneumonia, encephalitis, syncytia, and lymphoid depletion associated with immunosuppression. Cetaceans that survive the acute disease may develop fatal secondary infections and chronic encephalitis. Endemically infected, gregarious odontocetes probably serve as reservoirs and vectors. Transmission likely occurs through the inhalation of aerosolized virus but mother to fetus transmission was also reported.

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.

Masticatory muscles of the great-gray kangaroo (Macropus giganteus).

The great-gray kangaroo (Macropus giganteus) belongs to the Diprotodontia suborder (herbivorous marsupials of Australia) of the order of marsupials. We dissected the masticatory muscles in the great-gray kangaroo and classified them based on their innervation. Three (two male and one female) adult great-gray kangaroos (M. giganteus), fixed with 10% formalin, were examined. The masseter muscle of the great-gray kangaroo was classified into four layers (superficial layers 1, 2, 3, and a deep layer), all innervated by masseteric nerves. Layer 1 of the masseter muscle was well developed and the deep layer inserted into the masseteric canal. The zygomaticomandibular muscle, which belongs to both the masseter and temporalis muscles, was innervated by both the masseteric nerve and posterior deep temporal nerve, and the temporalis muscle was innervated by the anterior and posterior deep temporal nerves. The medial pterygoid muscle, which was innervated by the medial pterygoid nerve, was divided into superficial and deep portions. The lateral pterygoid muscle was divided into superior and inferior heads by the buccal nerve. We propose that the relationship of the masticatory muscles in the kangaroo has evolved by passive anterior invasion of the deep layer of the masseter by the medial pterygoid muscle via the masseteric canal, associated with the development of an anteroposterior mode of mastication.

Comparative anatomy of the buccinator muscle in cat (Felis domestica).

Published descriptions of the buccinator muscle of the cat (Felis domestica) differ from those for the same muscle in other mammals. Only an oral component of the muscle has been described in cats, not a buccal part. The purpose of this study was to identify the buccinator muscle in the cat and report on its anatomical features in detail. Dissections of the facial muscles were carried out on 12 specimens of adult cats (6 males and 6 females) that had been fixed with 10% formalin. We then observed the facial muscles and traced their innervations, arteries, and veins under a binocular microscope. The buccinator muscle in the cat was identified underneath an orbicularis oris, arising from the lower buccal membrane and from the molar region of the alveolar border of the mandible. It was about 3 mm wide at its origin, 4 mm wide at its insertion, and about 11 mm in length from origin to insertion. This contrasts with humans, in whom the muscle arises not only from the mandible, but also from the maxilla. Apart from this difference, this muscle in cats displays the following similarities to the buccinator muscle of other mammals: 1) it is innervated by the facial nerve; 2) it supports the buccal membrane; 3) it seems to insert into the modiolus; 4) its bundles run antero-posteriorly; 5) the posterior part of the muscle is located medially to the masseter muscle; 6) the parotid duct, facial nerve, artery, and vein run lateral to the muscle; 7) it is located deeper than other facial muscles; and 8) the buccal nerve runs on its surface. These relationships are spatially similar to those of the buccinator muscle in mammals. This muscle may aid in mastication, including suckling, and in expelling air forcibly, like the buccinator in humans.