Faecal bacterial communities differ amongst discrete foraging populations of dugongs along the east Australian coast.

Gut bacterial communities play a vital role in a host's digestion, fermentation of complex carbohydrates, absorption of nutrients and energy harvest/storage. Dugongs are obligate seagrass grazers with an expanded hindgut and associated microbiome. Here, we characterised and compared the faecal bacterial communities of dugongs from genetically distinct populations along the east coast of Australia, between subtropical Moreton Bay and tropical Cleveland Bay. Amplicon sequencing of fresh dugong faecal samples (n=47) revealed Firmicutes (62%) dominating the faecal bacterial communities across all populations. Several bacterial genera (Bacteroides, Clostridium sensu stricto 1, Blautia and Polaribacter) were detected in samples from all locations, suggesting their importance in seagrass digestion. Principal coordinate analysis showed the three southern-most dugong populations having different faecal bacterial community compositions from northern populations. The relative abundances of the genera Clostridium sensu stricto 13 and dgA-11 gut group were higher, but Bacteroides was lower, in the southern dugong populations, compared to the northern populations, suggesting potential adaptive changes associated with location. This study contributes to our knowledge of the faecal bacterial communities of dugongs inhabiting Australian coastal waters. Future studies of diet selection in relation to seagrass availability throughout the dugong's range will help to advance our understanding of the roles that seagrass species may play in affecting the dugong's faecal bacterial community composition.

A chromosome-level genome assembly for the dugong (Dugong dugon).

The dugong (Dugong dugon) is a marine mammal widely distributed throughout the Indo-Pacific and the Red Sea, with a Vulnerable conservation status, and little is known about many of the more peripheral populations, some of which are thought to be close to extinction. We present a de novo high-quality genome assembly for the dugong from an individual belonging to the well-monitored Moreton Bay population in Queensland, Australia. Our assembly uses long-read PacBio HiFi sequencing and Omni-C data following the Vertebrate Genome Project pipeline to reach chromosome-level contiguity (24 chromosome-level scaffolds; 3.16 Gbp) and high completeness (97.9% complete BUSCOs). We observed relatively high genome-wide heterozygosity, which likely reflects historical population abundance before the last interglacial period, approximately 125,000 yr ago. Demographic inference suggests that dugong populations began declining as sea levels fell after the last interglacial period, likely a result of population fragmentation and habitat loss due to the exposure of seagrass meadows. We find no evidence for ongoing recent inbreeding in this individual. However, runs of homozygosity indicate some past inbreeding. Our draft genome assembly will enable range-wide assessments of genetic diversity and adaptation, facilitate effective management of dugong populations, and allow comparative genomics analyses including with other sirenians, the oldest marine mammal lineage.

A new DNA extraction method (HV-CTAB-PCI) for amplification of nuclear markers from open ocean-retrieved faeces of an herbivorous marine mammal, the dugong.

Non-invasively collected faecal samples are an alternative source of DNA to tissue samples, that may be used in genetic studies of wildlife when direct sampling of animals is difficult. Although several faecal DNA extraction methods exist, their efficacy varies between species. Previous attempts to amplify mitochondrial DNA (mtDNA) markers from faeces of wild dugongs (Dugong dugon) have met with limited success and nuclear markers (microsatellites) have been unsuccessful. This study aimed to establish a tool for sampling both mtDNA and nuclear DNA (nDNA) from dugong faeces by modifying approaches used in studies of other large herbivores. First, a streamlined, cost-effective DNA extraction method that enabled the amplification of both mitochondrial and nuclear markers from large quantities of dugong faeces was developed. Faecal DNA extracted using a new 'High Volume- Cetyltrimethyl Ammonium Bromide- Phenol-Chloroform-Isoamyl Alcohol' (HV-CTAB-PCI) method was found to achieve comparable amplification results to extraction of DNA from dugong skin. As most prevailing practices advocate sampling from the outer surface of a stool to maximise capture of sloughed intestinal cells, this study compared amplification success of mtDNA between the outer and inner layers of faeces, but no difference in amplification was found. Assessment of the impacts of faecal age or degradation on extraction, however, demonstrated that fresher faeces with shorter duration of environmental (seawater) exposure amplified both markers better than eroded scats. Using the HV-CTAB-PCI method, nuclear markers were successfully amplified for the first time from dugong faeces. The successful amplification of single nucleotide polymorphism (SNP) markers represents a proof-of-concept showing that DNA from dugong faeces can potentially be utilised in population genetic studies. This novel DNA extraction protocol offers a new tool that will facilitate genetic studies of dugongs and other large and cryptic marine herbivores in remote locations.

Osmoregulation and electrolyte balance in a fully marine mammal, the dugong (Dugong dugon).

Dugongs (Dugong dugon) are fully marine mammals that live independently of fresh water so must balance water and electrolytes in a hyperosmotic environment. To investigate osmoregulation, matched plasma and urine from 51 live wild dugongs were analysed for osmolality, major electrolytes (Na+, Cl-, K+), urea, creatinine, and glucose. Maximum urine osmolality (1468 mOsm kg -1) and Na+, K+, and Cl- concentrations (757, 131.3, 677 mmol L-1, respectively) indicate that dugongs are capable of concentrating urine above seawater and could potentially realise a net gain of free water from drinking seawater. However, mean urine osmolality of 925.4 (± 46.6) mOsm kg-1 suggests that mariposia is unlikely to be an important osmoregulatory mechanism. Dugongs may obtain enough preformed water from their seagrass diet and metabolic oxidation to maintain homeostasis. Mean plasma osmolality of 339.6 (± 1.8) mOsm kg-1 is higher than in the related manatees but within the range for fully marine cetaceans. Relatively high mean plasma Na+ (175.5 ± 1.7 mmol L-1) and K+ (6.9 ± 0.1 mmol L-1), as well as mean urinary Na+ (469.6 ± 22.5 mmol L-1) and K+ levels (32.5 ± 4.5 mmol L-1) may reflect a salt-rich seagrass diet. Pregnant females had higher mean plasma osmolality (355.3 ± 4.9 mmol L-1) than non-pregnant females and males (337.9 ± 1.7 mOsm kg-1), suggesting that fluid retention was not a feature of pregnancy. Further research on water intake and endocrinology will enhance our understanding of osmoregulation in dugongs.

Reproductive Science Methods for Wild, Fully-Marine Mammals: Current Approaches and Future Applications.

Determining reproductive rates of marine mammal populations can give insight into their persistence and resilience in changing environments. As our marine environments continue to degrade along developed coastal fringes and as mankind's influences extend across even our widest oceans, there is a concern that the reproductive functioning of marine mammals may be affected adversely. Since many marine mammal species and populations are still in the recovery phase post-commercial harvest, and yet others are endangered or threatened by ongoing pressures, further environmental changes may represent direct or indirect threats to their reproductive potential. In this chapter, we review the current methods employed to investigate various aspects of reproductive science in fully-marine mammals, including direct observation of reproductive behavior and output, endocrinology to determine reproductive state, and assisted reproductive technologies to enhance reproductive outcomes. In particular, we focus on the most recent developments and innovations to reproductively sample marine mammals. Two case studies are presented to illustrate the challenges thrown up to researchers studying free-ranging marine mammals, and to highlight diversity in research approach. The North Atlantic right whale is on the brink of extinction through historic overharvest and present-day entanglement and ship strike. Environmental disruption to their migration routes and declining population health has resulted in reduced reproductive rates. In contrast, the main current threats to the reproductive success and survival of the vulnerable dugong are extreme weather events that affect availability of its seagrass diet. Climate disruption with increasingly severe coastal storms and flooding threaten the health of coastal seagrass beds, and consequently reproductive success and survival of this species. It is anticipated that climate change may have diverse and often serious effects on marine mammal reproduction in populations around the globe.

Ancient convergent losses of Paraoxonase 1 yield potential risks for modern marine mammals.

Mammals diversified by colonizing drastically different environments, with each transition yielding numerous molecular changes, including losses of protein function. Though not initially deleterious, these losses could subsequently carry deleterious pleiotropic consequences. We have used phylogenetic methods to identify convergent functional losses across independent marine mammal lineages. In one extreme case, Paraoxonase 1 (PON1) accrued lesions in all marine lineages, while remaining intact in all terrestrial mammals. These lesions coincide with PON1 enzymatic activity loss in marine species' blood plasma. This convergent loss is likely explained by parallel shifts in marine ancestors' lipid metabolism and/or bloodstream oxidative environment affecting PON1's role in fatty acid oxidation. PON1 loss also eliminates marine mammals' main defense against neurotoxicity from specific man-made organophosphorus compounds, implying potential risks in modern environments.

Development of a polyclonal anti-dugong immunoglobulin G (IgG) antibody with evaluation of total plasma IgG in a living dugong (Dugong dugon) population.

Species-specific antibodies (Ab) for the measurement of immunoglobulins (Ig) are valuable tools for determining the humoral immune status of threatened and endangered wildlife species such as dugongs. However, no studies have reported antibody reagents against dugong immunoglobulin. The object of this study was to develop an Ab with specificity for dugong IgG and apply this tool to survey total IgG levels in plasma samples from a live wild population of dugongs in southern Queensland, Australia. Dugong IgG was isolated from plasma by protein A/G column chromatography and a polyclonal antiserum was successfully raised against the dugong IgG through immunization of mice. The anti-dugong antiserum was reactive with dugong serum but not immunoglobulin from other species such as rats and humans. When tested against a panel of dugong plasma samples, relative IgG levels from dugongs (n = 116) showed biologically relevant relationships with pregnancy status and a principal component of Body Mass Index (BMI)/globulin/fecal glucocorticosteroid (chronic stress) levels combined, which together accounted for 9.2% of the variation in total Ig levels. Together these data suggest that dugongs show variation in total IgG and that this correlates with some physiological parameters of dugong health.

Hematology of dugongs (Dugong dugon) in southern Queensland.

BACKGROUND: Little is known of the hematology of the dugong (Dugong dugon), a secretive and endangered coastal marine mammal. OBJECTIVES: This paper reports hematologic reference intervals (RI) for dugongs and characterizes morphologic, cytochemical, and ultrastructural features of dugong leukocytes. METHODS: Blood was collected from live, apparently healthy dugongs and analyzed using Cell-Dyn 3700 or Sysmex XT-2000iV hematology analyzers. Blood films were subjected to a series of cytochemical stains, and leukocyte structure was examined using transmission electron microscopy. RESULTS: Reference intervals were established for 14 hematologic variables, total solids, and fibrinogen for 92 dugongs. Significant differences in some variables were found for animal size class, sex, and pregnancy status, and between analyzers. Subadults had higher leukocyte and lymphocyte counts than adults. Males had higher total solids and fibrinogen than females. Pregnant females had higher HCT, MCV, and circulating nucleated RBC, and lower platelet counts than nonpregnant females. Lymphocytes were usually the predominant circulating leukocyte. Heterophil cytoplasmic granules were abundant, fine, round to ovoid, and intensely eosinophilic, and round to ovoid or rod-shaped, and variably electron dense in electron microscopy. Eosinophils contained larger round eosinophilic to orange cytoplasmic granules, which ultrastructurally were bicompartmental with a round eccentric electron-dense core. Cytochemical staining of dugong heterophils suggests biochemical similarity to those of manatees and elephants, and for eosinophils, similarity to those of elephants, ruminants, and equids. CONCLUSIONS: Generation of hematologic RI and characterization of leukocyte morphology improves evaluation of dugong health across this population and serves as a reference for other populations outside southern Queensland.

Serum biochemistry reference intervals of live wild dugongs (Dugong dugon) from urban coastal Australia.

BACKGROUND: Little is known about the baseline clinical pathology of the dugong (Dugong dugon), a vulnerable marine mammal found in tropical coastal marine systems. OBJECTIVES: The purpose of the study was to collect and determine reference intervals (RI) for select serum biochemical variables for dugongs, and to analyze differences between males and females and different age groups. METHODS: Reference intervals were established from 103 apparently healthy, wild-caught dugongs for 31 analytes using a Beckman Coulter AU400 Automated Chemistry Analyzer and an Olympus AU680 Chemistry-Immuno Analyzer. RESULTS: Significant differences (P < .05) in some of the variables were found related to size class, sex, and pregnancy status. Adult dugongs had higher serum sodium, potassium, bicarbonate, glucose, and l-lactate concentrations and higher anion gap, compared to sub-adults. Male dugongs had higher triglyceride and l-lactate concentrations than females. Pregnant females displayed higher l-lactate levels compared to nonpregnant animals. Statistical differences in variables within the population contributed to better understanding of the physiologic differences between cohorts. Some serum biochemistry changes observed in this study here also potentially include some effects of pursuit on dugongs (eg, higher l-lactate); however, as all dugongs were subject to similar capture and handling, serum biochemistry RI should be considered as normal for captured dugongs. CONCLUSIONS: The serum biochemical RI documented here are considered representative of a population of healthy captured dugongs. They provide a baseline for health surveillance of this and other dugong populations.

Methods to examine reproductive biology in free-ranging, fully-marine mammals.

Historical overexploitation of marine mammals, combined with present-day pressures, has resulted in severely depleted populations, with many species listed as threatened or endangered. Understanding breeding patterns of threatened marine mammals is crucial to assessing population viability, potential recovery and conservation actions. However, determining reproductive parameters of wild fully-marine mammals (cetaceans and sirenians) is challenging due to their wide distributions, high mobility, inaccessible habitats, cryptic lifestyles and in many cases, large body size and intractability. Consequently, reproductive biologists employ an innovative suite of methods to collect useful information from these species. This chapter reviews historic, recent and state-of-the-art methods to examine diverse aspects of reproduction in fully-aquatic mammals.

Development and testing of a genetic marker-based pedigree reconstruction system 'PR-genie' incorporating size-class data.

For wildlife populations, it is often difficult to determine biological parameters that indicate breeding patterns and population mixing, but knowledge of these parameters is essential for effective management. A pedigree encodes the relationship between individuals and can provide insight into the dynamics of a population over its recent history. Here, we present a method for the reconstruction of pedigrees for wild populations of animals that live long enough to breed multiple times over their lifetime and that have complex or unknown generational structures. Reconstruction was based on microsatellite genotype data along with ancillary biological information: sex and observed body size class as an indicator of relative age of individuals within the population. Using body size-class data to infer relative age has not been considered previously in wildlife genealogy and provides a marked improvement in accuracy of pedigree reconstruction. Body size-class data are particularly useful for wild populations because it is much easier to collect noninvasively than absolute age data. This new pedigree reconstruction system, PR-genie, performs reconstruction using maximum likelihood with optimization driven by the cross-entropy method. We demonstrated pedigree reconstruction performance on simulated populations (comparing reconstructed pedigrees to known true pedigrees) over a wide range of population parameters and under assortative and intergenerational mating schema. Reconstruction accuracy increased with the presence of size-class data and as the amount and quality of genetic data increased. We provide recommendations as to the amount and quality of data necessary to provide insight into detailed familial relationships in a wildlife population using this pedigree reconstruction technique.

Variation in the hindgut microbial communities of the Florida manatee, Trichechus manatus latirostris over winter in Crystal River, Florida.

The Florida manatee, Trichechus manatus latirostris, is a hindgut-fermenting herbivore. In winter, manatees migrate to warm water overwintering sites where they undergo dietary shifts and may suffer from cold-induced stress. Given these seasonally induced changes in diet, the present study aimed to examine variation in the hindgut bacterial communities of wild manatees overwintering at Crystal River, west Florida. Faeces were sampled from 36 manatees of known sex and body size in early winter when manatees were newly arrived and then in mid-winter and late winter when diet had probably changed and environmental stress may have increased. Concentrations of faecal cortisol metabolite, an indicator of a stress response, were measured by enzyme immunoassay. Using 454-pyrosequencing, 2027 bacterial operational taxonomic units were identified in manatee faeces following amplicon pyrosequencing of the 16S rRNA gene V3/V4 region. Classified sequences were assigned to eight previously described bacterial phyla; only 0.36% of sequences could not be classified to phylum level. Five core phyla were identified in all samples. The majority (96.8%) of sequences were classified as Firmicutes (77.3 ± 11.1% of total sequences) or Bacteroidetes (19.5 ± 10.6%). Alpha-diversity measures trended towards higher diversity of hindgut microbiota in manatees in mid-winter compared to early and late winter. Beta-diversity measures, analysed through PERMANOVA, also indicated significant differences in bacterial communities based on the season.

Reproductive hormone monitoring of dugongs in captivity: detecting the onset of sexual maturity in a cryptic marine mammal.

Determining the reproductive status of long-term captive animals is essential because the onset of sexual maturity and reproductive activity may necessitate changes in husbandry requirements. This study reports on the first multi-year reproductive hormone monitoring program for captive dugongs of both sexes using feces. Fecal samples were collected from one male (Pig) over 9 years (4-13.2y of age; n=288 samples, 0.8±0.1 samples per week from July 2007 to February 2012) and one female (Wuru) over 7 years (from neonate to 6.9 y; n=171 samples, 0.5±0.1 samples per week from July 2007 to February 2012), and from one solitary female dugong (Gracie) over 10 months (10.5-11.3y of age; n=54 samples, 1.1±0.2 sample per week from September 2008 to June 2009). Using enzyme-immunoassay, fecal progesterone (fP) and estradiol-17ß (fE) concentrations were assayed in the two captive females, and testosterone (fT) concentration in the captive male, and compared these to concentrations in wild dugongs. Female Wuru exhibited increasing fP concentrations at 5+ y, indicating early onset of ovarian cycling typical of non-pregnant adult females. Female Gracie maintained basal fP concentrations consistent with wild immature dugongs, indicating that she had not reached puberty by 11y. Nutritional plane may account for differences in age at sexual maturity in these female dugongs. At age 3-4y, Wuru had fE concentrations 1.4 times greater than maximum concentrations recorded in all wild females, and these concentrations were coincident with a period of rapid weight gain. For the male Pig, increasing fT concentrations at 9y provided early indications of puberty. Pig's tusks erupted by 11y, and sexual maturity (indicated by spermatic semen) was confirmed by 12.8y. Identification of sexual maturation prompted two trials of a male contraceptive treatment using the GnRH agonist, deslorelin (9.4mg administered in 2010 and 15.6mg in 2011). Testosterone production was not significantly suppressed by these dosages, and treatment did not terminate sperm production at week 10-11 post-implantation, even at the larger dose tested. Routine analysis of fecal hormones was helpful for making reproductive management decisions regarding individual captives and in guiding the long-term captive management of this cryptic species.

Monitoring dolphins in an urban marine system: total and effective population size estimates of Indo-Pacific bottlenose dolphins in Moreton Bay, Australia.

Moreton Bay, Queensland, Australia is an area of high biodiversity and conservation value and home to two sympatric sub-populations of Indo-Pacific bottlenose dolphins (Tursiops aduncus). These dolphins live in close proximity to major urban developments. Successful management requires information regarding their abundance. Here, we estimate total and effective population sizes of bottlenose dolphins in Moreton Bay using photo-identification and genetic data collected during boat-based surveys in 2008-2010. Abundance (N) was estimated using open population mark-recapture models based on sighting histories of distinctive individuals. Effective population size (Ne ) was estimated using the linkage disequilibrium method based on nuclear genetic data at 20 microsatellite markers in skin samples, and corrected for bias caused by overlapping generations (Ne c). A total of 174 sightings of dolphin groups were recorded and 365 different individuals identified. Over the whole of Moreton Bay, a population size N of 554 ± 22.2 (SE) (95% CI: 510-598) was estimated. The southern bay sub-population was small at an estimated N = 193 ± 6.4 (SE) (95% CI: 181-207), while the North sub-population was more numerous, with 446 ± 56 (SE) (95% CI: 336-556) individuals. The small estimated effective population size of the southern sub-population (Ne c = 56, 95% CI: 33-128) raises conservation concerns. A power analysis suggested that to reliably detect small (5%) declines in size of this population would require substantial survey effort (>4 years of annual mark-recapture surveys) at the precision levels achieved here. To ensure that ecological as well as genetic diversity within this population of bottlenose dolphins is preserved, we consider that North and South sub-populations should be treated as separate management units. Systematic surveys over smaller areas holding locally-adapted sub-populations are suggested as an alternative method for increasing ability to detect abundance trends.

Sex, scarring, and stress: understanding seasonal costs in a cryptic marine mammal.

We investigated variation in adrenal activity in the dugong, a vulnerable cryptic marine mammal species, in a population inhabiting the lower thermal limit of their range. We sampled 319 live wild dugongs and examined faecal glucocorticoid (fGC) concentrations in relationship to sex, reproductive state (immature, sexually mature, or pregnant), and season, as well as associations with body condition, male-induced aggressive conspecific interactions, and environmental temperature. Physiological validations with apparently healthy and unhealthy animals indicated that fGC levels reliably reflect adrenal activation in dugongs. Glucocorticoid levels and body condition varied seasonally, with individuals expressing lowest fGC levels and an improving body condition over summer and autumn. Best body condition was observed in winter, which may have enhanced the ability of the animals to cope with thermal and nutritional challenges and helped to sustain the subsequent costs of reproduction (including mating activity in spring). Immature dugongs, especially those recently estranged from their mothers, may be particularly vulnerable to stress during winter, and also at risk of injury from adult conspecifics. During the spring mating period, mature and sexually active males with large erupted tusks had high fGC and poorer body condition compared with all dugongs, except pregnant females, in all other seasons. This finding was consistent with high levels of body scarring on adults of both sexes, resulting from agonistic behaviour by mature males. Competitive reproductive behaviour, inherent in a promiscuous mating system, may have stressful consequences for male dugongs, especially given that strenuous mating activity follows unfavourable thermal and nutritional conditions. Overall, fGC levels appear to be a good proxy for stressors in dugongs associated with season and/or temperature, intra-specific aggression, and pregnancy, but not lactation and/or maternal care. Understanding baseline fGC levels will allow wildlife managers to identify additional and/or unusual stressors that may threaten wild dugong populations.

Fine-scale genetic population structure in a mobile marine mammal: inshore bottlenose dolphins in Moreton Bay, Australia.

Highly mobile marine species in areas with no obvious geographic barriers are expected to show low levels of genetic differentiation. However, small-scale variation in habitat may lead to resource polymorphisms and drive local differentiation by adaptive divergence. Using nuclear microsatellite genotyping at 20 loci, and mitochondrial control region sequencing, we investigated fine-scale population structuring of inshore bottlenose dolphins (Tursiops aduncus) inhabiting a range of habitats in and around Moreton Bay, Australia. Bayesian structure analysis identified two genetic clusters within Moreton Bay, with evidence of admixture between them (F(ST) = 0.05, P = 0.001). There was only weak isolation by distance but one cluster of dolphins was more likely to be found in shallow southern areas and the other in the deeper waters of the central northern bay. In further analysis removing admixed individuals, southern dolphins appeared genetically restricted with lower levels of variation (AR = 3.252, π = 0.003) and high mean relatedness (r = 0.239) between individuals. In contrast, northern dolphins were more diverse (AR = 4.850, π = 0.009) and were mixing with a group of dolphins outside the bay (microsatellite-based STRUCTURE analysis), which appears to have historically been distinct from the bay dolphins (mtDNA Φ(ST) = 0.272, P < 0.001). This study demonstrates the ability of genetic techniques to expose fine-scale patterns of population structure and explore their origins and mechanisms. A complex variety of inter-related factors including local habitat variation, differential resource use, social behaviour and learning, and anthropogenic disturbances are likely to have played a role in driving fine-scale population structure among bottlenose dolphins in Moreton Bay.

Testosterone and tusks: maturation and seasonal reproductive patterns of live, free-ranging male dugongs (Dugong dugon) in a subtropical population.

Knowledge of male reproductive status and activity in free-ranging animals is vital to understanding reproductive patterns and population dynamics. Until now, almost all information regarding reproductive behavior of the dugong, a cryptic marine mammal, has relied on post-mortem examination. We examined the relationships between body length, tusk eruption (secondary sexual characteristic), seasonality, and group association on fecal testosterone metabolite concentrations in 322 free-ranging dugongs (159 males, 163 females) in subtropical Moreton Bay, Australia. Fecal testosterone concentrations demonstrated biologically meaningful differences in testicular activity between sexes and across reproductive/age classes, and were correlated with circulating concentrations in serum. Male dugongs have a pre-reproductive period that persists until a body length of 240 cm is achieved. Puberty apparently occurs between 240 and 260 cm body length when fecal testosterone levels increase fourfold (>500 ng/g) over juvenile levels, and is associated with tusk eruption. However, social maturity may be delayed until male dugongs are larger than 260 cm with well-developed tusks. In mature males, the lowest (<500 ng/g) fecal testosterone concentrations occur in the austral autumn months with maximal concentrations in September-October, coincident with the onset of a spring mating season. During spring, solitary mature males had fecal testosterone concentrations double those of mature males sampled within groups, potentially suggesting a mating strategy involving roving of reproductively active males. This study demonstrates that single-point physiological data from individuals across a population have value as indicators of reproductive processes. Our approach provides an efficacious non-lethal method for the census of reproductive status and seasonality in live male dugongs.

Diagnosing pregnancy in free-ranging dugongs using fecal progesterone metabolite concentrations and body morphometrics: a population application.

Assessing reproductive status and monitoring reproductive rates is important in the effective management of vulnerable marine mammal species such as the dugong (Dugong dugon). Knowledge of the reproductive physiology of this species is limited, and determining reproductive parameters (e.g., sexual maturation, pregnancy, and reproductive senescence) has been restricted by a lack of non-lethal methods for assessing reproductive status in free-ranging individuals. The aim of this study was to develop a method to identify pregnant individuals in a wild dugong population. Using an enzymeimmunoassay, we quantified concentrations of fecal progesterone metabolites (fP) in 322 dugongs, including confirmed pregnant females (n=10), presumed non-pregnant adult females (n=25), juvenile females (n=24), subadult females (n=41), adult females of unknown pregnancy state (n=63), and males of all sizes (n=159). External body morphometrics of each dugong were measured, and confirmation of pregnancy in adult female dugongs was determined by ultrasonography or observation of subsequent neonates. Concentrations of fP were different between sexes and reproductive size classes (P<0.001), and ∼30-fold higher in confirmed pregnant dugongs (2017-7760 ng/g) compared to presumed non-pregnant females (30-221 ng/g), juvenile females (29-195 ng/g), and males (24-261 ng/g) (P<0.001). Body measures of maximum and anal girths, and teat length were all greater in confirmed pregnant females than presumed non-pregnant females (all P<0.05). We evaluated a Discriminant Function Analysis (DFA) to provide a model for predicting pregnant and non-pregnant dugongs. Cross-validated results showed that the DFA correctly classified 100% of pregnant and non-pregnant females using fP concentrations, body length, fineness ratio (an index of body shape), and teat length (a female reproductive trait). Using the DFA model, we classified the pregnancy status of all female dugongs and identified a total of 30 females as pregnant and 133 females as non-pregnant from the sampled population over the sample period. Pregnant dugongs in the Moreton Bay population are characterized by fecal progesterone metabolite concentrations > 1000 ng/g, body length ≥ 260 cm, maximum girth ≥ 215 cm, anal girth ≥ 126 cm, and teat length ≥ 5 cm long. In summary, analysis of fP concentrations in combination with body morphometrics may be used to diagnose pregnancy in free-ranging dugongs, and provides a new tool to monitor breeding rates of wild sirenian populations.

Evaluation of exertion and capture stress in serum of wild dugongs (Dugong dugon).

Seven hundred fifty-one dugongs (Dugong dugon) were pursued, captured, and handled for up to 20 min for population sampling. Fifty of these dugongs were then removed from the water for up to 55 min for comprehensive medical examination. Fifty whole blood and separated serum samples were analyzed for potassium, sodium, chloride, creatinine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), urea, creatinine, glucose, anion gap, and total blood CO2. Serum biochemical variables of the dugong were compared with those obtained in previous studies of the related West Indian manatee, a mammal that does not appear to experience capture myopathy based on available data. Differences between these species included higher blood sodium and chloride in dugongs, which may reflect differences in salt balance and renal function, and higher blood lactate and CO2. Some biochemical analytes such as CK and AST, which may be indicative of rhabdomyolysis associated with capture stress myopathy (a potentially fatal condition for which dugongs have been thought to be highly susceptible) were high compared with levels previously measured in wild West Indian manatees (Trichechus latirostris). One of the 50 dugongs had marked elevations of CK and AST but showed no other clinical indications of rhabdomyolysis associated with capture myopathy such as hyperthermia. Rather, generally high levels of lactate, CK, and AST most probably reflect metabolic acidosis resulting from the exertion involved in the pursuit prior to capture. Earlier observations suggesting that dugongs were probably susceptible to capture stress myopathy (based on high serum potassium levels) were not supported by this study. Capture and handling methods currently used on dugongs in this research program do not appear to result in acute capture stress.

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers.

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.