Lipid extraction has tissue-dependent effects on isotopic values (δ34 S, δ13 C, and δ15 N) from different marine predators.

RATIONALE: The use of sulfur isotopes to study trophic ecology in marine ecosystems has increased in the past decade. Unlike other commonly used isotopes (e.g., carbon), sulfur can better discriminate benthic and pelagic productivity. However, how lipid extraction affects sulfur isotopic values has not been assessed, despite its frequent use to remove lipid effects on δ13 C values. METHODS: We used white muscle and liver samples from two species of sharks and skin samples from two species of pinnipeds (sea lion and fur seal) to assess the effects of lipid extraction on stable isotope values for δ34 S, δ13 C, and δ15 N. Isotopic values were determined using a continuous flow-isotope ratio mass spectrometer coupled to an elemental analyzer. RESULTS: Lipid extraction significantly decreased δ34 S values in shark tissues, more so for liver than muscle (-4.6 ± 0.9‰ vs -0.8 ± 0.3‰, average change), with nearly no change in their standard deviations. Lipid extraction did not affect δ34 S values from pinniped skin samples (0.2 ± 0.8‰, average change). After lipid extraction, consistent increases in δ13 C values (0.2‰-7.3‰) were detected as expected, especially in tissue with high lipid content (C:N >4). After lipid extraction, significant increases in δ15 N values (0.5‰-1.4‰) were found in shark muscle and liver tissues. For pinniped skin samples, δ15 N values were not significantly lower after lipid extraction (-0.4‰ to -0.1‰). CONCLUSIONS: Lipid extraction did not have a strong impact on δ34 S values of shark muscle and pinniped skin (≤1‰). However, our results suggest it is essential to consider the effects of lipid extraction when interpreting results from δ34 S values of shark liver tissue, as they significantly depleted values relative to bulk tissue (~5‰). This may reflect selective removal of sulfolipids and glutathione present in higher concentrations in the liver than in muscle and skin and requires further investigation.

A shark's eye view: testing the 'mistaken identity theory' behind shark bites on humans.

Shark bites on humans are rare but are sufficiently frequent to generate substantial public concern, which typically leads to measures to reduce their frequency. Unfortunately, we understand little about why sharks bite humans. One theory for bites occurring at the surface, e.g. on surfers, is that of mistaken identity, whereby sharks mistake humans for their typical prey (pinnipeds in the case of white sharks). This study tests the mistaken identity theory by comparing video footage of pinnipeds, humans swimming and humans paddling surfboards, from the perspective of a white shark viewing these objects from below. Videos were processed to reflect how a shark's retina would detect the visual motion and shape cues. Motion cues of humans swimming, humans paddling surfboards and pinnipeds swimming did not differ significantly. The shape of paddled surfboards and human swimmers was also similar to that of pinnipeds with their flippers abducted. The difference in shape between pinnipeds with abducted versus adducted flippers was bigger than between pinnipeds with flippers abducted and surfboards or human swimmers. From the perspective of a white shark, therefore, neither visual motion nor shape cues allow an unequivocal visual distinction between pinnipeds and humans, supporting the mistaken identity theory behind some bites.

Pelagic and benthic ecosystems drive differences in population and individual specializations in marine predators.

Individual specialization, which describes whether populations are comprised of dietary generalists or specialists, has profound ecological and evolutionary implications. However, few studies have quantified individual specialization within and between sympatric species that are functionally similar but have different foraging modes. We assessed the relationship between individual specialization, isotopic niche metrics and foraging behaviour of two marine predators with contrasting foraging modes: pelagic foraging female South American fur seals (Arctocephalus australis) and benthic foraging female southern sea lions (Otaria byronia). Stable isotope analysis of carbon and nitrogen was conducted along the length of adult female vibrissae to determine isotopic niche metrics and the degree of individual specialization. Vibrissae integrated time ranged between 1.1 and 5.5 years, depending on vibrissae length. We found limited overlap in dietary niche-space. Broader population niche sizes were associated with higher degrees of individual specialization, while narrower population niches with lower degrees of individual specialization. The degree of individual specialization was influenced by pelagic and benthic foraging modes. Specifically, South American fur seals, foraging in dynamic pelagic environments with abundant but similar prey, comprised specialist populations composed of generalist individuals. In contrast, benthic southern sea lions foraging in habitats with diverse but less abundant prey had more generalist populations composed of highly specialized individuals. We hypothesize that differences in specialization within and between populations were related to prey availability and habitat differences. Our study supports growing body of literature highlighting that individual specialization is a critical factor in shaping the ecological niche of higher marine predators.

Convergent evolution of forelimb-propelled swimming in seals.

Modern pinnipeds (true and eared seals) employ two radically different swimming styles, with true seals (phocids) propelling themselves primarily with their hindlimbs, whereas eared seals (otariids) rely on their wing-like foreflippers.1,2 Current explanations of this functional dichotomy invoke either pinniped diphyly3-5 or independent colonizations of the ocean by related but still largely terrestrial ancestors.6-8 Here, we show that pinniped swimming styles form an anatomical, functional, and behavioral continuum, within which adaptations for forelimb swimming can arise directly from a hindlimb-propelled bauplan. Within phocids, southern seals (monachines) show a convergent trend toward wing-like, hydrodynamically efficient forelimbs used for propulsion during slow swimming, turning, bursts of speed, or when initiating movement. This condition is most evident in leopard seals, which have well-integrated foreflippers with little digit mobility, reduced claws, and hydrodynamic characteristics comparable to those of forelimb-propelled otariids. Using monachines as a model, we suggest that the last common ancestor of modern seals may have been hindlimb-propelled and aquatically adapted, thus resolving the apparent contradiction at the root of pinniped evolution.

Into the sea: Antimicrobial resistance determinants in the microbiota of little penguins (Eudyptula minor).

Terrestrial and aquatic birds have been proposed as sentinels for the spread of antimicrobial resistant bacteria, but few species have been investigated specifically in the context of AMR in the marine ecosystem. This study contrasts the occurrence of class 1 integrons and associated antimicrobial resistance genes in wild and captive little penguins (Eudyptula minor), an Australian seabird with local population declines. PCR screening of faecal samples (n = 448) revealed a significant difference in the prevalence of class 1 integrons in wild and captive groups, 3.2% and 44.7% respectively, with genes that confer resistance to streptomycin, spectinomycin, trimethoprim and multidrug efflux pumps detected. Class 1 integrons were not detected in two clinically relevant bacterial species, Klebsiella pneumoniae or Escherichia coli, isolated from penguin faeces. The presence of class 1 integrons in the little penguin supports the use of marine birds as sentinels of AMR in marine environments.

Using accelerometers to develop time-energy budgets of wild fur seals from captive surrogates.

BACKGROUND: Accurate time-energy budgets summarise an animal's energy expenditure in a given environment, and are potentially a sensitive indicator of how an animal responds to changing resources. Deriving accurate time-energy budgets requires an estimate of time spent in different activities and of the energetic cost of that activity. Bio-loggers (e.g., accelerometers) may provide a solution for monitoring animals such as fur seals that make long-duration foraging trips. Using low resolution to record behaviour may aid in the transmission of data, negating the need to recover the device. METHODS: This study used controlled captive experiments and previous energetic research to derive time-energy budgets of juvenile Australian fur seals (Arctocephalus pusillus) equipped with tri-axial accelerometers. First, captive fur seals and sea lions were equipped with accelerometers recording at high (20 Hz) and low (1 Hz) resolutions, and their behaviour recorded. Using this data, machine learning models were trained to recognise four states-foraging, grooming, travelling and resting. Next, the energetic cost of each behaviour, as a function of location (land or water), season and digestive state (pre- or post-prandial) was estimated. Then, diving and movement data were collected from nine wild juvenile fur seals wearing accelerometers recording at high- and low- resolutions. Models developed from captive seals were applied to accelerometry data from wild juvenile Australian fur seals and, finally, their time-energy budgets were reconstructed. RESULTS: Behaviour classification models built with low resolution (1 Hz) data correctly classified captive seal behaviours with very high accuracy (up to 90%) and recorded without interruption. Therefore, time-energy budgets of wild fur seals were constructed with these data. The reconstructed time-energy budgets revealed that juvenile fur seals expended the same amount of energy as adults of similar species. No significant differences in daily energy expenditure (DEE) were found across sex or season (winter or summer), but fur seals rested more when their energy expenditure was expected to be higher. Juvenile fur seals used behavioural compensatory techniques to conserve energy during activities that were expected to have high energetic outputs (such as diving). DISCUSSION: As low resolution accelerometry (1 Hz) was able to classify behaviour with very high accuracy, future studies may be able to transmit more data at a lower rate, reducing the need for tag recovery. Reconstructed time-energy budgets demonstrated that juvenile fur seals appear to expend the same amount of energy as their adult counterparts. Through pairing estimates of energy expenditure with behaviour this study demonstrates the potential to understand how fur seals expend energy, and where and how behavioural compensations are made to retain constant energy expenditure over a short (dive) and long (season) period.

Understanding meta-population trends of the Australian fur seal, with insights for adaptive monitoring.

Effective ecosystem-based management requires estimates of abundance and population trends of species of interest. Trend analyses are often limited due to sparse or short-term abundance estimates for populations that can be logistically difficult to monitor over time. Therefore it is critical to assess regularly the quality of the metrics in long-term monitoring programs. For a monitoring program to provide meaningful data and remain relevant, it needs to incorporate technological improvements and the changing requirements of stakeholders, while maintaining the integrity of the data. In this paper we critically examine the monitoring program for the Australian fur seal (AFS) Arctocephalus pusillus doriferus as an example of an ad-hoc monitoring program that was co-ordinated across multiple stakeholders as a range-wide census of live pups in the Austral summers of 2002, 2007 and 2013. This 5-yearly census, combined with historic counts at individual sites, successfully tracked increasing population trends as signs of population recovery up to 2007. The 2013 census identified the first reduction in AFS pup numbers (14,248 live pups, -4.2% change per annum since 2007), however we have limited information to understand this change. We analyse the trends at breeding colonies and perform a power analysis to critically examine the reliability of those trends. We then assess the gaps in the monitoring program and discuss how we may transition this surveillance style program to an adaptive monitoring program than can evolve over time and achieve its goals. The census results are used for ecosystem-based modelling for fisheries management and emergency response planning. The ultimate goal for this program is to obtain the data we need with minimal cost, effort and impact on the fur seals. In conclusion we identify the importance of power analyses for interpreting trends, the value of regularly assessing long-term monitoring programs and proper design so that adaptive monitoring principles can be applied.

Proxies of energy expenditure for marine mammals: an experimental test of "the time trap".

Direct measures of energy expenditure are difficult to obtain in marine mammals, and accelerometry may be a useful proxy. Recently its utility has been questioned as some analyses derived their measure of activity level by calculating the sum of accelerometry-based values and then comparing this summation to summed (total) energy expenditure (the so-called "time trap"). To test this hypothesis, we measured oxygen consumption of captive fur seals and sea lions wearing accelerometers during submerged swimming and calculated total and rate of energy expenditure. We compared these values with two potential proxies of energy expenditure derived from accelerometry data: flipper strokes and dynamic body acceleration (DBA). Total number of strokes, total DBA, and submergence time all predicted total oxygen consumption [Formula: see text] ml kg-1). However, both total DBA and total number of strokes were correlated with submergence time. Neither stroke rate nor mean DBA could predict the rate of oxygen consumption ([Formula: see text] ml min-1 kg-1). The relationship of total DBA and total strokes with total oxygen consumption is apparently a result of introducing a constant (time) into both sides of the relationship. This experimental evidence supports the conclusion that proxies derived from accelerometers cannot estimate the energy expenditure of marine mammals.

Intrinsic and extrinsic influences on standard metabolic rates of three species of Australian otariid.

The study of marine mammal energetics can shed light on how these animals might adapt to changing environments. Their physiological potential to adapt will be influenced by extrinsic factors, such as temperature, and by intrinsic factors, such as sex and reproduction. We measured the standard metabolic rate (SMR) of males and females of three Australian otariid species (two Australian fur seals, three New Zealand fur seals and seven Australian sea lions). Mean SMR ranged from 0.47 to 1.05 l O2 min-1, which when adjusted for mass was from 5.33 to 7.44 ml O2 min-1 kg-1. We found that Australian sea lion mass-specific SMR (sSMR; in millilitres of oxygen per minute per kilogram) varied little in response to time of year or moult, but was significantly influenced by sex and water temperature. Likewise, sSMR of Australian and New Zealand fur seals was also influenced by sex and water temperature, but also by time of year (pre-moult, moult or post-moult). During the moult, fur seals had significantly higher sSMR than at other times of the year, whereas there was no discernible effect of moult for sea lions. For both groups, females had higher sSMR than males, but sea lions and fur seals showed different responses to changes in water temperature. The sSMR of fur seals increased with increasing water temperature, whereas sSMR of sea lions decreased with increasing water temperature. There were no species differences when comparing animals of the same sex. Our study suggests that fur seals have more flexibility in their physiology than sea lions, perhaps implying that they will be more resilient in a changing environment.

The utility of accelerometers to predict stroke rate in captive fur seals and sea lions.

The energy expenditure of free-living fur seals and sea lions is difficult to measure directly, but may be indirectly derived from flipper stroke rate. We filmed 10 captive otariids swimming with accelerometers either attached to a harness (Daily Diary: sampling frequency 32 Hz, N=4) or taped to the fur (G6a+: 25 Hz, N=6). We used down sampling to derive four recording rates from each accelerometer (Daily Diary: 32, 16, 8, 4 Hz; G6a+: 25, 20, 10, 5 Hz). For each of these sampling frequencies, we derived 20 combinations of two parameters (RMW, the window size used to calculate the running mean; and m, the minimum number of points smaller than a local maxima used to detect a peak) from the dynamic acceleration of x, z and x+z, to estimate stroke rate from the accelerometers. These estimates differed by up to ∼20% in comparison to the actual number of foreflipper strokes counted from videos. RMW and the choice of axis used to make the calculations (x, z or x+z) had little effect on the overall differences, though the variability was reduced when using x+z. The best m varied depending on the axis used and the sampling frequency; a larger m was needed for higher sampling frequencies. This study demonstrates that when parameters are appropriately tuned, accelerometers are a simple yet valid tool for estimating the stroke rates of swimming otariids.

Swimming metabolic rates vary by sex and development stage, but not by species, in three species of Australian otariid seals.

Physiology may limit the ability for marine mammals to adapt to changing environments. Depth and duration of foraging dives are a function of total available oxygen stores, which theoretically increase as animals grow, and metabolic costs. To evaluate how physiology may influence the travelling costs for seals to foraging patches in the wild, we measured metabolic rates of a cross-section of New Zealand fur seals, Australian fur seals and Australian sea lions representing different foraging strategies, development stages, sexes and sizes. We report values for standard metabolic rate, active metabolic rate (obtained from submerged swimming), along with estimates of cost of transport (COT), measured via respirometry. We found a decline in mass-specific metabolic rate with increased duration of submerged swimming. For most seals mass-specific metabolic rate increased with speed and for all seals mass-specific COT decreased with speed. Mass-specific metabolic rate was higher for subadult than adult fur seals and sea lions, corresponding to an overall higher minimum COT. Some sex differences were also apparent, such that female Australian fur seals and Australian sea lions had higher mass-specific metabolic rates than males. There were no species differences in standard or active metabolic rates for adult males or females. The seals in our study appear to operate at their physiological optimum during submerged swimming. However, the higher metabolic rates of young and female fur seals and sea lions may limit their scope for increasing foraging effort during times of resource limitation.

Seeing It All: Evaluating Supervised Machine Learning Methods for the Classification of Diverse Otariid Behaviours.

Constructing activity budgets for marine animals when they are at sea and cannot be directly observed is challenging, but recent advances in bio-logging technology offer solutions to this problem. Accelerometers can potentially identify a wide range of behaviours for animals based on unique patterns of acceleration. However, when analysing data derived from accelerometers, there are many statistical techniques available which when applied to different data sets produce different classification accuracies. We investigated a selection of supervised machine learning methods for interpreting behavioural data from captive otariids (fur seals and sea lions). We conducted controlled experiments with 12 seals, where their behaviours were filmed while they were wearing 3-axis accelerometers. From video we identified 26 behaviours that could be grouped into one of four categories (foraging, resting, travelling and grooming) representing key behaviour states for wild seals. We used data from 10 seals to train four predictive classification models: stochastic gradient boosting (GBM), random forests, support vector machine using four different kernels and a baseline model: penalised logistic regression. We then took the best parameters from each model and cross-validated the results on the two seals unseen so far. We also investigated the influence of feature statistics (describing some characteristic of the seal), testing the models both with and without these. Cross-validation accuracies were lower than training accuracy, but the SVM with a polynomial kernel was still able to classify seal behaviour with high accuracy (>70%). Adding feature statistics improved accuracies across all models tested. Most categories of behaviour -resting, grooming and feeding-were all predicted with reasonable accuracy (52-81%) by the SVM while travelling was poorly categorised (31-41%). These results show that model selection is important when classifying behaviour and that by using animal characteristics we can strengthen the overall accuracy.