Rachael Gray

Histology of selected tissues of the leopard seal and implications for functional adaptations to an aquatic lifestyle

The microscopic anatomy of the cardio‐respiratory system, digestive system, kidney, lymphatic system and integument was investigated in the leopard seal, Hydrurga leptonyx, by examining histological sections of tissues collected from leopard seals in Antarctica and New South Wales, Australia. The majority of the tissues had similar histological features to those described in terrestrial mammals and other pinniped species, particularly phocid seals. Differences noted included readily identifiable Purkinje cells within the endocardium, muscular rather than cartilaginous reinforcement of the smaller airways, a single capillary layer within the alveolar septa, limited and variable keratinization of the oesophageal epithelium, few lymphoid follicles within the lamina propria of the gastrointestinal tract, and an absence of a sporta perimedullaris musculosa described in the kidney of cetaceans and some pinniped species. Adaptations of the lung, spleen and integument, similar to those described in other pinnipeds, including reinforcement of the pulmonary terminal airways, prominent pulmonary interlobular septa, ample smooth muscle in the capsule and trabeculae of the spleen, increased thickness of the epidermis, well‐developed dermal sebaceous glands, and a thick blubber layer, appear to confer upon the leopard seal advantages related to its aquatic lifestyle.

Health assessment of free-ranging endangered Australian sea lion (Neophoca cinerea) pups: Effect of haematophagous parasites on haematological parameters

Evaluation of the health status of free-ranging populations is important for understanding the impact of disease on individuals and on population demography and viability. In this study, haematological reference intervals were developed for free-ranging endangered Australian sea lion (Neophoca cinerea) pups within the context of endemic hookworm (Uncinaria sanguinis) infection and the effects of pathogen, host, and environment factors on the variability of haematological parameters were investigated. Uncinaria sanguinis was identified as an important agent of disease, with infection causing regenerative anaemia, hypoproteinaemia, and a predominantly lymphocytic-eosinophilic systemic inflammatory response. Conversely, the effects of sucking lice (Antarctophthirus microchir) were less apparent and infestation in pups appears unlikely to cause clinical impact. Overall, the effects of U. sanguinis, A. microchir, host factors (standard length, body condition, pup sex, moult status, and presence of lesions), and environment factors (capture-type and year of sampling) accounted for 26-65% of the total variance observed in haematological parameters. Importantly, this study demonstrated that anaemia in neonatal Australian sea lion pups is not solely a benign physiological response to host-environment changes, but largely reflects a significant pathological process. This impact of hookworm infection on pup health has potential implications for the development of foraging and diving behaviour, which would subsequently influence the independent survival of juveniles following weaning. The haematological reference intervals developed in this study can facilitate long-term health surveillance, which is critical for the early recognition of changes in disease impact and to inform conservation management.

Unexpected absence of genetic separation of a highly diverse population of hookworms from geographically isolated hosts.

The high natal site fidelity of endangered Australian sea lions (Neophoca cinerea) along the southern Australian coast suggests that their maternally transmitted parasitic species, such as hookworms, will have restricted potential for dispersal. If this is the case, we would expect to find a hookworm haplotype structure corresponding to that of the host mtDNA haplotype structure; that is, restricted among geographically separated colonies. In this study, we used a fragment of the cytochrome c oxidase I mitochondrial DNA (mtDNA) gene to investigate the diversity of hookworms (Uncinaria sanguinis) in N. cinerea to assess the importance of host distribution and ecology on the evolutionary history of the parasite. High haplotype (h=0.986) and nucleotide diversity (π=0.013) were seen, with 45 unique hookworm mtDNA haplotypes across N. cinerea colonies; with most of the variation (78%) arising from variability within hookworms from individual colonies. This is supported by the low genetic differentiation co-efficient (GST=0.007) and a high gene flow (Nm=35.25) indicating a high migration rate between the populations of hookworms. The haplotype network demonstrated no clear distribution and delineation of haplotypes according to geographical location. Our data rejects the vicariance hypothesis; that female host natal site fidelity and the transmammary route of infection restrict hookworm gene flow between N. cinerea populations and highlights the value of studies of parasite diversity and dispersal to challenge our understanding of parasite and host ecology.

Characterization and causal investigations of an alopecia syndrome in Australian fur seals (Arctocephalus pusillus doriferus)

Abstract Fur seals rely on pelage consisting of dense, fine underfur protected by guard hairs as their primary means of limiting thermoregulatory cost. A distinctive syndrome of alopecia occurs at high prevalence in 1 colony of Australian fur seals (Arctocephalus pusillus doriferus). It is characterized by bilaterally symmetrical hair loss on the dorsal body surface and a biased prevalence toward juvenile females. Light and scanning electron microscopy demonstrated that alopecia is due to fracture of the hair shaft above the skin level. No evidence of viral, bacterial, fungal, or parasite infection was found and histological examination of skin biopsies revealed no pathological variation between case and control seals. Affected animals had statistically significant lower tyrosine and zinc concentrations in hair than unaffected seals. This may increase hair brittleness and, therefore, predispose its fracture. Alopecia cases also had higher levels of heavy metals and persistent organic pollutants, which may indicate they forage in ecosystems where concentrations of pollutants are higher.

Health Assessment of the Leopard Seal, Hydrurga leptonyx , in Prydz Bay, Eastern Antarctica and NSW, Australia

The leopard seal Hydrurga leptonyx is the most widely distributed of the Antarctic pinnipeds, ranging from the Antarctic coastline to the sub-Antarctic (Bonner 1994) and less frequently, temperate and sub-tropical areas, including the Australian coast. The main population of leopard seals remains within the circumpolar pack-ice (Bonner 1994) however, there is some north–south movement of individuals, the majority of which are immature, non-breeding seals (Gwynn 1953; Brown 1957; Csordas 1963; King 1983; Rounsevell and Eberhard 1980; Rounsevell 1988; Walker et al. 1998). A periodicity in peak abundance has been observed for this northward dispersal and it is suggested that this could be related to cyclic climatic variation within the region (Harris et al. 1988; Testa et al. 1991; Croxall 1992).

Diversity of MHC DQB and DRB Genes in the Endangered Australian Sea Lion (Neophoca cinerea)

Major histocompatibility complex (MHC) class II molecules have an important role in vertebrate adaptive immunity, being responsible for recognizing, binding, and presenting specific antigenic peptides to T lymphocytes. Here, we study the MHC class II DQB and DRB exon 2 genes of the Australian sea lion (Neophoca cinerea), an endangered pinniped species that experiences high pup mortality. Following characterization of N. cinerea DQB and DRB by molecular cloning, and evaluation of diversity in pups across 2 colonies using variant screening (n = 47), 3 DQB alleles and 10 DRB variants (including 1 pseudogene allele) were identified. The higher diversity at DRB relative to DQB is consistent with other studies in marine mammals. Despite overall lower MHC class II allelic diversity relative to some other pinniped species, we observed similar levels of nucleotide diversity and selection in N. cinerea. In addition, we provide support for recent divergence of MHC class II alleles. The characterization of MHC class II diversity in the Australian sea lion establishes a baseline for further investigation of associations with disease, including endemic hookworm infection, and contributes to the conservation management of this species.

Utility of fur as a biomarker for persistent organic pollutants in Australian fur seals (Arctocephalus pusillus doriferus)

Persistent Organic Pollutants (POPs) can cause toxic effects in many species which include endocrine dysfunction, immunotoxicity, developmental defects and neoplasia. Species dominating the upper trophic level are vulnerable to these effects due to bioaccumulation. In Bass Strait, the Australian fur seal (Arctocephalus pusillus doriferus) is an important top order predator and sentinel species for ecosystem health. An alopecia syndrome is seen at high prevalence in juvenile, female Australian fur seals at Lady Julia Percy Island, Victoria, Australia. Previous investigations suggest causality could be due to an endocrine-like toxicant. The alopecia syndrome has significance for thermoregulation and is a likely risk factor for mortality. Fur collected from case (alopecic) and control (unaffected) seals sampled at Lady Julia Percy Island were analysed for POPs. To investigate the utility of fur for monitoring POPs concentrations in pinnipeds, a comparison of POPs concentrations in the fur and blubber of Australian fur seals stranded along the Victorian coast was undertaken. The concentration of selected POPs including polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), dioxin-like polychlorinated biphenyls (dl-PCBs), polybrominated diphenyl ethers (PBDEs) and perfluorooctane sulfonate/perfluorooctanoic acid (PFOS/PFOA) were determined in fur using either High Resolution Gas Chromatography-Mass Spectrometry or Liquid Chromatography-Mass Spectrometry. Results indicate detectable, and in some individuals, elevated levels of dl-PCBs, PCDD/Fs and PBDEs in juvenile fur seals sampled on Lady Julia Percy Island, with significantly higher levels of dl-PCBs in case compared to control seals. Elevated levels of dl-PCBs and PCDD/Fs were found in blubber samples collected from stranded fur seals with significant correlations between blubber and fur concentrations seen, particularly for dl-PCBs. This study discusses the significance of POPs concentrations in relation to the causality of an alopecia syndrome in the Australian fur seal, and assesses the utility of fur as a non-invasive biomarker to monitor POPs exposure in this sentinel species.

Macroparasites in Antarctic Penguins

Parasitism is a highly common mode of living in animals being parasite species very abundant. Parasites affect in a different ways the host life through subtle effects to more dramatic effects causing population crashes and then regulating host populations. Antarctica and the Southern Ocean wildlife show also parasites although the published information is very scarce. This is even in the case of the most studied group of Antarctic seabirds, the penguins. In this chapter, we analyze the published information about the presence, epidemiology, life cycles, and effects of macroparasites, helminths, and ectoparasites in Antarctic penguins. Most of the publications only give information about the presence/absence of parasites, and very few give data about epidemiology such as prevalence or intensity of parasitization. The information about intermediate host is almost absent, and parasite effects have been addressed very few times. Moreover, the information is based on few areas, and there is not any long-term data set which makes difficult a broad understanding of the impact of parasites in the ecology of penguins. Nevertheless, the little information allows extracting some conclusions. First, the diversity of parasite species is very low which can be explained by the narrow diet spectrum and the harsh conditions. Second, helminths occur at higher prevalence than ectoparasites. In general, a trend of decreased macroparasite prevalence towards more southerly locations can be identified, although the small number of studies precludes a robust conclusion. Third, general parasite effects have been reported causing tissue damage, changes in immune parameters, reduction in body mass, reduction of breeding success, and transmission of diseases, this later in the case of ticks. Finally, it is expected that climate change will affect host-parasite interaction in penguins due to changes in the parasite distribution, host exposure, or resistance, but a higher number of studies with good quality data at long term are needed to confirm the expectations and a deeper understanding of the ecological aspects of parasites such as life cycle, epidemiology, and health impacts in the penguins.

Primers for amplifying major histocompatibility complex class II DQB and DRB exon 2 in the Australian sea lion (Neophoca cinerea)

Abstract Major histocompatibility complex (MHC) molecules have an essential role in the vertebrate adaptive immune response. These genes are widely studied to investigate evolutionary mechanisms, including pathogen-driven positive selection. The current gap in knowledge of the MHC in the Australian sea lion (Neophoca cinerea) is attributed to the absence of immunogenetic resources in the species, and is critical to understanding the consequences of small population sizes and association with hookworm infection, identified at high prevalence in pups. Here, we use conserved primers in exons 1 and 3 to obtain sequences for development of N. cinerea-specific primers to amplify multiple MHC class II DQB and DRB exon 2 loci, to facilitate study of MHC class II diversity and disease association.

Comparative ecology of Escherichia coli in endangered Australian sea lion (Neophoca cinerea) pups

The dissemination of human-associated bacteria into the marine environment has the potential to expose wildlife populations to atypical microbes that can alter the composition of the gut microbiome or act as pathogens. The objective of the study was to determine whether endangered Australian sea lion (Neophoca cinerea) pups from two South Australian colonies, Seal Bay, Kangaroo Island and Dangerous Reef, Spencer Gulf, have been colonised by human-associated Escherichia coli. Faecal samples (n = 111) were collected to isolate E. coli, and molecular screening was applied to assign E. coli isolates (n = 94) to phylotypes and detect class 1 integrons; mobile genetic elements that confer resistance to antimicrobial agents. E. coli phylotype distribution and frequency differed significantly between colonies with phylotypes B2 and D being the most abundant at Seal Bay, Kangaroo Island (55% and 7%) and Dangerous Reef, Spencer Gulf (36% and 49%), respectively. This study reports the first case of antimicrobial resistant E. coli in free-ranging Australian sea lions through the identification of class 1 integrons from an individual pup at Seal Bay. A significant relationship between phylotype and total white cell count (WCC) was identified, with significantly higher WCC seen in pups with human-associated phylotypes at Dangerous Reef. The difference in phylotype distribution and presence of human-associated E. coli suggests that proximity to human populations can influence sea lion gut microbiota. The identification of antimicrobial resistance in a free-ranging pinniped population provides crucial information concerning anthropogenic influences in the marine environment.