A novel method for identifying fine-scale bottom-use in a benthic-foraging pinniped.

BACKGROUND: For diving, marine predators, accelerometer and magnetometer data provides critical information on sub-surface foraging behaviours that cannot be identified from location or time-depth data. By measuring head movement and body orientation, accelerometers and magnetometers can help identify broad shifts in foraging movements, fine-scale habitat use and energy expenditure of terrestrial and marine species. Here, we use accelerometer and magnetometer data from tagged Australian sea lions and provide a new method to identify key benthic foraging areas. As Australian sea lions are listed as endangered by the IUCN and Australian legislation, identifying key areas for the species is vital to support targeted management of populations. METHODS: Firstly, tri-axial magnetometer and accelerometer data from adult female Australian sea lions is used in conjunction with GPS and dive data to dead-reckon their three-dimensional foraging paths. We then isolate all benthic phases from their foraging trips and calculate a range of dive metrics to characterise their bottom usage. Finally, k-means cluster analysis is used to identify core benthic areas utilised by sea lions. Backwards stepwise regressions are then iteratively performed to identify the most parsimonious model for describing bottom usage and its included predictor variables. RESULTS: Our results show distinct spatial partitioning in benthic habitat-use by Australian sea lions. This method has also identified individual differences in benthic habitat-use. Here, the application of high-resolution magnetometer/accelerometer data has helped reveal the tortuous foraging movements Australian sea lions use to exploit key benthic marine habitats and features. CONCLUSIONS: This study has illustrated how magnetometer and accelerometer data can provide a fine-scale description of the underwater movement of diving species, beyond GPS and depth data alone, For endangered species like Australian sea lions, management of populations must be spatially targeted. Here, this method demonstrates a fine-scale analysis of benthic habitat-use which can help identify key areas for both marine and terrestrial species. Future integration of this method with concurrent habitat and prey data would further augment its power as a tool for understanding the foraging behaviours of species.

Mitigating disease risk in an endangered pinniped: early hookworm elimination optimizes the growth and health of Australian sea lion pups.

The Australian sea lion (Neophoca cinerea) experiences high pup mortality of seasonally alternating severity, partly attributed to endemic hookworm (Uncinaria sanguinis) infection. To further explore health outcomes of early hookworm elimination, a treatment trial was conducted at Seal Bay Conservation Park, South Australia, over consecutive lower and higher mortality breeding seasons (2019, 19.2%; 2020-1; 28.9%). Pups (n = 322) were stratified into two age cohorts (median 14 d and 24 d recruitment ages) and randomly assigned to treated (topical ivermectin 500 µg/kg) or control (untreated) groups. A younger prepatent cohort <14 d old (median 10 d) was identified a posteriori. A seasonally independent growth benefit resulted from hookworm elimination across all age cohorts. The greatest relative improvements (bodyweight + 34.2%, standard length + 42.1%; p ≤ 0.001) occurred in the month post-treatment, in the youngest prepatent cohort. A significant benefit of lesser magnitude (bodyweight + 8.6-11.6%, standard length + 9.5-18.4%; p ≤ 0.033) persisted up to 3 months across all age cohorts - greatest in the youngest pups. Treatment resulted in immediate improvement in hematological measures of health - decreased anemia and inflammation severity (p ≤ 0.012). These results enhance our understanding of host-parasite-environment interactions within the context of hematological ontogenesis, confirm the seasonally independent benefits of hookworm disease intervention, and further inform conservation recommendations for this endangered species.

Non-essential heavy metals and protective effects of selenium against mercury toxicity in endangered Australian sea lion (Neophoca cinerea) pups with hookworm disease.

The endangered Australian sea lion, Neophoca cinerea, faces ongoing population decline. Identification of key threats to N. cinerea population recovery, including disease and pollutants, is an objective of the species' recovery plan. Previous studies have identified Uncinaria sanguinis, an intestinal nematode, as a significant cause of disease and mortality in N. cinerea pups. Given the impact of heavy metals on the immune response, investigation of these pollutants is critical. To this end, the concentrations of arsenic (As), total mercury (Hg), cadmium (Cd), chromium (Cr), lead (Pb) and selenium (Se) were determined in blood collected from N. cinerea pups sampled during the 2017/18, 2019 and 2020/21 breeding seasons at Seal Bay Conservation Park, South Australia. Significant differences (p < 0.05) in Hg, As, Cr, and Se concentrations and molar ratio of Se:Hg were seen between breeding seasons. Pup age, maternal parity and inter-individual foraging behaviour were considered factors driving these differences. The concentrations of Hg (357, 198 and 241 µg/L) and As (225, 834 and 608 µg/L) were high in 2017/18, 2019 and 2020/21 respectively with Hg concentrations in the blood of N. cinerea pups above toxicological thresholds reported for marine mammals. The concentration of Se (1332, 647, 763 µg/L) and molar ratio of Se:Hg (9.47, 7.98 and 6.82) were low compared to other pinniped pups, indicating potential vulnerability of pups to the toxic effects of Hg. Significant (p < 0.05) negative associations for Pb and Cd with several red blood cell parameters suggest they could be exacerbating the anaemia caused by hookworm disease. Temporal (age-related) changes in element concentrations were also seen, such that pup age needs to be considered when interpreting bioaccumulation patterns. Further investigation of the role of elevated heavy metal concentrations on N. cinerea pup health, disease and development is recommended, particularly with respect to immunological impacts.

Mother-pup recognition mechanisms in Australia sea lion (Neophoca cinerea) using uni- and multi-modal approaches.

Communication is the process by which one emitter conveys information to one or several receivers to induce a response (behavioral or physiological) by the receiver. Communication plays a major role in various biological functions and may involve signals and cues from different sensory modalities. Traditionally, investigations of animal communication focused on a single sensory modality, yet communication is often multimodal. As these different processes may be quite complex and therefore difficult to disentangle, one approach is to first study each sensorial modality separately. With this refined understanding of individual senses, revealing how they interact becomes possible as the characteristics and properties of each modality can be accounted for, making a multimodal approach feasible. Using this framework, researchers undertook systematic, experimental investigations on mother-pup recognition processes in a colonial pinniped species, the Australian sea lion Neophoca cinerea. The research first assessed the abilities of mothers and pups to identify each other by their voice using playback experiments. Second, they assessed whether visual cues are used by both mothers and pups to distinguish them from conspecifics, and/or whether females discriminate the odor of their filial pup from those from non-filial pups. Finally, to understand if the information transmitted by different sensory modalities is analyzed synergistically or if there is a hierarchy among the sensory modalities, experiments were performed involving different sensory cues simultaneously. These findings are discussed with regards to the active space of each sensory cue, and of the potential enhancements that may arise by assessing information from different modalities.

Developing Immune Profiles of Endangered Australian Sea Lion (Neophoca cinerea) Pups Within the Context of Endemic Hookworm (Uncinaria sanguinis) Infection.

As a top predator, the endangered Australian sea lion (Neophoca cinerea) is a sentinel of ecosystem change, where population trends can reflect broader shifts in the marine environment. The population of this endemic pinniped was historically diminished by commercial sealing, and recovery has been slowed by fishery interactions, disease and, potentially, pollutants. Hookworm infects 100% of neonatal pups and has been identified as a contributor to population decline. Here, a multivariable approach using traditional serological and novel molecular tools such as qPCR and ddPCR was used to examine immune phenotypes of developing Australian sea lion pups infected with the endemic hookworm (Uncinaria sanguinis) from two South Australian colonies. Results show changing immunophenotypes throughout the patent period of infection represented by pro-inflammatory cytokines (IL-6), IgG and acute-phase proteins. Although cytokines may prove useful as markers of resistance, in this study, IL-6 is determined to be an early biomarker of inflammation in Australian sea lion pups, excluding the alternative hypothesis. Additionally, immunological differences between animals from high- and low-intensity hookworm seasons, as well as ivermectin-treated animals, indicate hookworm infection modulation of the host immune response, as evidenced by a lower IL-6 mRNA expression in the non-treated groups. This study of the Australian sea lion is an example of an ecoimmunological approach to disease investigation, which can be applied to evaluate the impact of environmental and anthropogenic factors on susceptibility to infectious diseases in free-ranging species.

Topical ivermectin is a highly effective seal 'spot-on': A randomised trial of hookworm and lice treatment in the endangered Australian sea lion (Neophoca cinerea).

The Australian sea lion (Neophoca cinerea) is an endangered and declining otariid species, with a high rate of pup mortality associated with endemic hookworm (Uncinaria sanguinis) infection a suspected contributor to this decline. Injected ivermectin is an effective treatment for Uncinaria sp. in otariids, with optimal outcomes achieved by the early treatment of pups prior to disease development. This randomised controlled trial evaluated the effectiveness of the novel use of a topical ivermectin formulation against hookworm infection and lice (Antarctophthirus microchir) infestation, in comparison with injected ivermectin. During the 2017 breeding season at Dangerous Reef, South Australia, pups ≤ 70 cm in standard length (≤ 2 weeks of age; n = 85) were randomised to single dose topical (500 µg/kg spot-on; n = 27) or injected (200 µg/kg subcutaneous; n = 29) ivermectin treatment groups, or to an untreated control group (n = 29). Topical ivermectin was highly effective for U. sanguinis elimination, and not significantly different to the injected formulation (estimated effectiveness 96.4% and 96.8%, respectively; P > 0.05). Its application resulted in an 81.6% reduction and 62.7% additional clearance for A. microchir infestation by 15-24 days post-treatment, compared with untreated control pups (also not significantly different to injected ivermectin; 83.1% and 59.4%, respectively; P > 0.05). Treatment with either ivermectin formulation significantly ameliorated increases in inflammatory markers detected in the blood of untreated control pups - peripheral blood eosinophil counts (persisting to 36-41 days post-recruitment P < 0.05) and increased plasma protein concentrations (15-24 days post-recruitment; P < 0.05). Further, an initial short-term decrease in body condition in the control group was not observed in either of the treatment groups. This study demonstrates that topical ivermectin is an effective antiparasitic treatment in N. cinerea. It offers an alternative administration method for ivermectin delivery to a young pup cohort in this species, and an alternative, minimally invasive management tool for species conservation.

A Novel Presentation of Tuberculosis with Intestinal Perforation in a Free-Ranging Australian Sea Lion (Neophoca cinerea).

We detail a novel presentation of tuberculosis associated with intestinal perforation in an endangered Australian sea lion (Neophoca cinerea) from South Australian waters and confirm the presence of this disease in the region of highest pup production. In February 2017, a 3-yr-old juvenile male died shortly after hauling out at the Kingscote beach on Kangaroo Island. On postmortem examination, we found a mid-jejunal intestinal perforation and partial obstruction (from a strangulating fibrous and granulomatous mesenteric mass), a marked multicentric abdominal fibrosing granulomatous lymphadenitis, and a large volume serosanguinous peritoneal effusion. Acid-fast bacteria were detected postmortem in cytologic preparations of the mesenteric lymph node and in histologic sections of jejunum and the encircling mass. Mycobacterial infection was confirmed by positive culture after 3 wk. Molecular typing using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing with 12-locus analysis identified Mycobacterium pinnipedii. This case highlights the need for vigilance of zoonotic disease risk when handling pinnipeds, including in the absence of specific respiratory signs or grossly apparent pulmonary pathology. Increased serologic population surveillance is recommended to assess the species' risk from this and other endemic diseases, especially given its endangered status.

Characteristic "neck collar" injuries in Australian sea lions (Neophoca cinerea) caused by marine debris.

An adult male Australian sea lion (Neophoca cinerea) presented with a deep, almost circumferential, incised wound around the neck caused by a noose formed by a single strand of green nylon rope. The wound extended 4-5 cm deeply through the skin and blubber into skeletal muscle, predominantly on the dorsal surface and left side, but had not involved the airway or major vessels. The edges of the wound were sharply incised and oozing blood with a granulating infected base. The rope was removed and the wound debrided of necrotic tissues and irrigated with disinfectant. Antibiotic and rehydration therapy were successful and the seal was released after 2 weeks of treatment. Entanglement injuries such as "neck collars" have characteristic features, are increasing in incidence and often have lethal outcomes. This type of injury may significantly impact upon populations of pinnipeds in the wild.

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.

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.

Whisker spot patterns: a noninvasive method of individual identification of Australian sea lions (Neophoca cinerea).

Reliable methods for identification of individual animals are advantageous for ecological studies of population demographics and movement patterns. Photographic identification, based on distinguishable patterns, unique shapes, or scars, is an effective technique already used for many species. We tested whether photographs of whisker spot patterns could be used to discriminate among individual Australian sea lion (Neophoca cinerea). Based on images of 53 sea lions, we simulated 5,000 patterns before calculating the probability of duplication in a study population. A total of 99% (± 1.5 SD) of patterns were considered reliable for a population of 50, 98% (± 1.7 SD) for 100, 92% (± 4.7 SD) for 500, and 88% (± 5.7 SD) for 1,000. We tested a semiautomatic approach by matching 16 known individuals at 3 different angles (70°, 90°, and 110°), 2 distances (1 and 2 m), and 6 separate times over a 1-year period. A point-pattern matching algorithm for pairwise comparisons produced 90% correct matches of photographs taken on the same day at 90°. Images of individuals at 1 and 2 m resulted in 89% correct matches, those photographed at different angles and different times (at 90°) resulted in 48% and 73% correct matches, respectively. Our results show that the Chamfer distance transform can effectively be used for individual identification, but only if there is very little variation in photograph angle. This point-pattern recognition application may also work for other otariid species.

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.