DNA methylation networks underlying mammalian traits.

Using DNA methylation profiles (n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species.

Epidemiology of skin changes in endangered Southern Resident killer whales (Orcinus orca).

Photographic identification catalogs of individual killer whales (Orcinus orca) over time provide a tool for remote health assessment. We retrospectively examined digital photographs of Southern Resident killer whales in the Salish Sea to characterize skin changes and to determine if they could be an indicator of individual, pod, or population health. Using photographs collected from 2004 through 2016 from 18,697 individual whale sightings, we identified six lesions (cephalopod, erosions, gray patches, gray targets, orange on gray, and pinpoint black discoloration). Of 141 whales that were alive at some point during the study, 99% had photographic evidence of skin lesions. Using a multivariate model including age, sex, pod, and matriline across time, the point prevalence of the two most prevalent lesions, gray patches and gray targets, varied between pods and between years and showed small differences between stage classes. Despite minor differences, we document a strong increase in point prevalence of both lesion types in all three pods from 2004 through 2016. The health significance of this is not clear, but the possible relationship between these lesions and decreasing body condition and immunocompetence in an endangered, non-recovering population is a concern. Understanding the etiology and pathogenesis of these lesions is important to better understand the health significance of these skin changes that are increasing in prevalence.

Divergent foraging strategies between populations of sympatric matrilineal killer whales.

In cooperative species, human-induced rapid environmental change may threaten cost-benefit tradeoffs of group behavioral strategies that evolved in past environments. Capacity for behavioral flexibility can increase population viability in novel environments. Whether the partitioning of individual responsibilities within social groups is fixed or flexible across populations is poorly understood, despite its relevance for predicting responses to global change at the population and species levels and designing successful conservation programs. We leveraged bio-logging data from two populations of fish-eating killer whales (Orcinus orca) to quantify patterns of fine-scale foraging movements and their relationships with demography. We reveal striking interpopulation differences in patterns of individual foraging behavior. Females from the endangered Southern Resident (SRKW) population captured less prey and spent less time pursuing prey than SRKW males or Northern Resident (NRKW) females, whereas NRKW females captured more prey than NRKW males. The presence of a calf (≤3 years) reduced the number of prey captured by adult females from both populations, but disproportionately so for SRKW. SRKW adult males with a living mother captured more prey than those whose mother had died, whereas the opposite was true for NRKW adult males. Across populations, males foraged in deeper areas than females, and SRKW captured prey deeper than NRKW. These population-level differences in patterns of individual foraging behavior challenge the existing paradigm that females are the disproportionate foragers in gregarious resident killer whales, and demonstrate considerable variation in the foraging strategies across populations of an apex marine predator experiencing different environmental stressors.

DNA methylation-based biomarkers for ageing long-lived cetaceans.

Epigenetic approaches for estimating the age of living organisms are revolutionizing studies of long-lived species. Molecular biomarkers that allow age estimates from small tissue biopsies promise to enhance studies of long-lived whales, addressing a fundamental and challenging parameter in wildlife management. DNA methylation (DNAm) can affect gene expression, and strong correlations between DNAm patterns and age have been documented in humans and nonhuman vertebrates and used to construct "epigenetic clocks". We present several epigenetic clocks for skin samples from two of the longest-lived cetaceans, killer whales and bowhead whales. Applying the mammalian methylation array to genomic DNA from skin samples we validate four different clocks with median errors of 2.3-3.7 years. These epigenetic clocks demonstrate the validity of using cytosine methylation data to estimate the age of long-lived cetaceans and have broad applications supporting the conservation and management of long-lived cetaceans using genomic DNA from remote tissue biopsies.

Bacterial microbiomes from mucus and breath of southern resident killer whales (Orcinus orca).

Opportunities to assess odontocete health are restricted due to their limited time at the surface, relatively quick movements and large geographic ranges. For endangered populations such as the southern resident killer whales (SKRWs) of the northeast Pacific Ocean, taking advantage of non-invasive samples such as expelled mucus and exhaled breath is appealing. Over the past 12 years, such samples were collected, providing a chance to analyse and assess their bacterial microbiomes using amplicon sequencing. Based on operational taxonomic units, microbiome communities from SRKW and transient killer whales showed little overlap between mucus, breath and seawater from SRKW habitats and six bacterial phyla were prominent in expelled mucus but not in seawater. Mollicutes and Fusobacteria were common and abundant in mucus, but not in breath or seawater, suggesting these bacterial classes may be normal constituents of the SRKW microbiome. Out of 134 bacterial families detected, 24 were unique to breath and mucus, including higher abundances of Burkholderiaceae, Moraxellaceae and Chitinophagaceae. Although there were multiple bacterial genera in breath or mucus that include pathogenic species (e.g. Campylobacter, Hemophilus, Treponema), the presence of these bacteria is not necessarily evidence of disease or infection. Future emphasis on genotyping mucus samples to the individual animal will allow further assessment in the context of that animal's history, including body condition index and prior contaminants burden. This study is the first to examine expelled mucus from cetaceans for microbiomes and demonstrates the value of analysing these types of non-invasive samples.

Vessels and their sounds reduce prey capture effort by endangered killer whales (Orcinus orca).

Vessel traffic is prevalent throughout marine environments. However, we often have a limited understanding of vessel impacts on marine wildlife, particularly cetaceans, due to challenges of studying fully-aquatic species. To investigate vessel and acoustic effects on cetacean foraging behavior, we attached suction-cup sound and movement tags to endangered Southern Resident killer whales in their summer habitat while collecting geo-referenced proximate vessel data. We identified prey capture dives using whale kinematic signatures and found that the probability of capturing prey increased as salmon abundance increased, but decreased as vessel speed increased. When vessels emitted navigational sonar, whales made longer dives to capture prey and descended more slowly when they initiated these dives. Finally, whales descended more quickly when noise levels were higher and vessel approaches were closer. These findings advance a growing understanding of vessel and sound impacts on marine wildlife and inform efforts to manage vessel impacts on endangered populations.

Endangered predators and endangered prey: Seasonal diet of Southern Resident killer whales.

Understanding diet is critical for conservation of endangered predators. Southern Resident killer whales (SRKW) (Orcinus orca) are an endangered population occurring primarily along the outer coast and inland waters of Washington and British Columbia. Insufficient prey has been identified as a factor limiting their recovery, so a clear understanding of their seasonal diet is a high conservation priority. Previous studies have shown that their summer diet in inland waters consists primarily of Chinook salmon (Oncorhynchus tshawytscha), despite that species' rarity compared to some other salmonids. During other times of the year, when occurrence patterns include other portions of their range, their diet remains largely unknown. To address this data gap, we collected feces and prey remains from October to May 2004-2017 in both the Salish Sea and outer coast waters. Using visual and genetic species identification for prey remains and genetic approaches for fecal samples, we characterized the diet of the SRKWs in fall, winter, and spring. Chinook salmon were identified as an important prey item year-round, averaging ~50% of their diet in the fall, increasing to 70-80% in the mid-winter/early spring, and increasing to nearly 100% in the spring. Other salmon species and non-salmonid fishes, also made substantial dietary contributions. The relatively high species diversity in winter suggested a possible lack of Chinook salmon, probably due to seasonally lower densities, based on SRKW's proclivity to selectively consume this species in other seasons. A wide diversity of Chinook salmon stocks were consumed, many of which are also at risk. Although outer coast Chinook samples included 14 stocks, four rivers systems accounted for over 90% of samples, predominantly the Columbia River. Increasing the abundance of Chinook salmon stocks that inhabit the whales' winter range may be an effective conservation strategy for this population.

Sounds associated with foraging and prey capture in individual fish-eating killer whales, Orcinus orca.

Foraging behavior in odontocetes is fundamentally tied to the use of sound. Resident-type killer whales use echolocation to locate and capture elusive salmonid prey. In this investigation, acoustic recording tags were suction cup-attached to endangered Southern Resident killer whales to describe their acoustic behavior during different phases of foraging that, along with detections of prey handling sounds (e.g., crunches) and observed predation events, allow confirmation of prey capture. Echolocation click trains were categorized based on the inter-click interval (ICI) according to hypothesized foraging function. Whales produced slow click trains (ICI >100 ms) at shallowest depths but over the largest change of depth, fast click trains (10 ms < ICI ≤100 ms) at intermediate depths, and buzz trains (ICI ≤10 ms) at deepest depths over the smallest depth change. These results align with hypotheses regarding biosonar use to search, pursue and capture prey. Males exhibited a higher probability of producing slow click trains, buzzes and prey handling sounds, indicating higher levels of prey searching and capture to support the energy requirement of their larger body size. These findings identify relevant acoustic indicators of subsurface foraging behaviors of killer whales, enabling investigations of human impacts on sound use and foraging.

Hidden Markov models reveal temporal patterns and sex differences in killer whale behavior.

Behavioral data can be important for effective management of endangered marine predators, but can be challenging to obtain. We utilized suction cup-attached biologging tags equipped with stereo hydrophones, triaxial accelerometers, triaxial magnetometers, pressure and temperature sensors, to characterize the subsurface behavior of an endangered population of killer whales (Orcinus orca). Tags recorded depth, acoustic and movement behavior on fish-eating killer whales in the Salish Sea between 2010-2014. We tested the hypotheses that (a) distinct behavioral states can be characterized by integrating movement and acoustic variables, (b) subsurface foraging occurs in bouts, with distinct periods of searching and capture temporally separated from travel, and (c) the probabilities of transitioning between behavioral states differ by sex. Using Hidden Markov modeling of two acoustic and four movement variables, we identified five temporally distinct behavioral states. Persistence in the same state on a subsequent dive had the greatest likelihood, with the exception of deep prey pursuit, indicating that behavior was clustered in time. Additionally, females spent more time at the surface than males, and engaged in less foraging behavior. These results reveal significant complexity and sex differences in subsurface foraging behavior, and underscore the importance of incorporating behavior into the design of conservation strategies.

Kinematic signatures of prey capture from archival tags reveal sex differences in killer whale foraging activity.

Studies of odontocete foraging ecology have been limited by the challenges of observing prey capture events and outcomes underwater. We sought to determine whether subsurface movement behavior recorded from archival tags could accurately identify foraging events by fish-eating killer whales. We used multisensor bio-logging tags attached by suction cups to Southern Resident killer whales (Orcinus orca) to: (1) identify a stereotyped movement signature that co-occurred with visually confirmed prey capture dives; (2) construct a prey capture dive detector and validate it against acoustically confirmed prey capture dives; and (3) demonstrate the utility of the detector by testing hypotheses about foraging ecology. Predation events were significantly predicted by peaks in the rate of change of acceleration ('jerk peak'), roll angle and heading variance. Detection of prey capture dives by movement signatures enabled substantially more dives to be included in subsequent analyses compared with previous surface or acoustic detection methods. Males made significantly more prey capture dives than females and more dives to the depth of their preferred prey, Chinook salmon. Additionally, only half of the tag deployments on females (5 out of 10) included a prey capture dive, whereas all tag deployments on males exhibited at least one prey capture dive (12 out of 12). This dual approach of kinematic detection of prey capture coupled with hypothesis testing can be applied across odontocetes and other marine predators to investigate the impacts of social, environmental and anthropogenic factors on foraging ecology.

Modulation in Persistent Organic Pollutant Concentration and Profile by Prey Availability and Reproductive Status in Southern Resident Killer Whale Scat Samples.

Persistent organic pollutants (POPs), specifically PCBs, PBDEs, and DDTs, in the marine environment are well documented, however accumulation and mobilization patterns at the top of the food-web are poorly understood. This study broadens the understanding of POPs in the endangered Southern Resident killer whale population by addressing modulation by prey availability and reproductive status, along with endocrine disrupting effects. A total of 140 killer whale scat samples collected from 54 unique whales across a 4 year sampling period (2010-2013) were analyzed for concentrations of POPs. Toxicant measures were linked to pod, age, and birth order in genotyped individuals, prey abundance using open-source test fishery data, and pregnancy status based on hormone indices from the same sample. Toxicant concentrations were highest and had the greatest potential for toxicity when prey abundance was the lowest. In addition, these toxicants were likely from endogenous lipid stores. Bioaccumulation of POPs increased with age, with the exception of presumed nulliparous females. The exceptional pattern may be explained by females experiencing unobserved neonatal loss. Transfer of POPs through mobilization of endogenous lipid stores during lactation was highest for first-borns with diminished transfer to subsequent calves. Contrary to expectation, POP concentrations did not demonstrate an associated disruption of thyroid hormone, although this association may have been masked by impacts of prey abundance on thyroid hormone concentrations. The noninvasive method for measuring POP concentrations in killer whales through scat employed in this study may improve toxicant monitoring in the marine environment and promote conservation efforts.

Estimation of a Killer Whale (Orcinus orca) Population's Diet Using Sequencing Analysis of DNA from Feces.

Estimating diet composition is important for understanding interactions between predators and prey and thus illuminating ecosystem function. The diet of many species, however, is difficult to observe directly. Genetic analysis of fecal material collected in the field is therefore a useful tool for gaining insight into wild animal diets. In this study, we used high-throughput DNA sequencing to quantitatively estimate the diet composition of an endangered population of wild killer whales (Orcinus orca) in their summer range in the Salish Sea. We combined 175 fecal samples collected between May and September from five years between 2006 and 2011 into 13 sample groups. Two known DNA composition control groups were also created. Each group was sequenced at a ~330bp segment of the 16s gene in the mitochondrial genome using an Illumina MiSeq sequencing system. After several quality controls steps, 4,987,107 individual sequences were aligned to a custom sequence database containing 19 potential fish prey species and the most likely species of each fecal-derived sequence was determined. Based on these alignments, salmonids made up >98.6% of the total sequences and thus of the inferred diet. Of the six salmonid species, Chinook salmon made up 79.5% of the sequences, followed by coho salmon (15%). Over all years, a clear pattern emerged with Chinook salmon dominating the estimated diet early in the summer, and coho salmon contributing an average of >40% of the diet in late summer. Sockeye salmon appeared to be occasionally important, at >18% in some sample groups. Non-salmonids were rarely observed. Our results are consistent with earlier results based on surface prey remains, and confirm the importance of Chinook salmon in this population's summer diet.

Persistent Organic Pollutant Determination in Killer Whale Scat Samples: Optimization of a Gas Chromatography/Mass Spectrometry Method and Application to Field Samples.

Biologic sample collection in wild cetacean populations is challenging. Most information on toxicant levels is obtained from blubber biopsy samples; however, sample collection is invasive and strictly regulated under permit, thus limiting sample numbers. Methods are needed to monitor toxicant levels that increase temporal and repeat sampling of individuals for population health and recovery models. The objective of this study was to optimize measuring trace levels (parts per billion) of persistent organic pollutants (POPs), namely polychlorinated-biphenyls (PCBs), polybrominated-diphenyl-ethers (PBDEs), dichlorodiphenyltrichloroethanes (DDTs), and hexachlorocyclobenzene, in killer whale scat (fecal) samples. Archival scat samples, initially collected, lyophilized, and extracted with 70 % ethanol for hormone analyses, were used to analyze POP concentrations. The residual pellet was extracted and analyzed using gas chromatography coupled with mass spectrometry. Method detection limits ranged from 11 to 125 ng/g dry weight. The described method is suitable for p,p'-DDE, PCBs-138, 153, 180, and 187, and PBDEs-47 and 100; other POPs were below the limit of detection. We applied this method to 126 scat samples collected from Southern Resident killer whales. Scat samples from 22 adult whales also had known POP concentrations in blubber and demonstrated significant correlations (p < 0.01) between matrices across target analytes. Overall, the scat toxicant measures matched previously reported patterns from blubber samples of decreased levels in reproductive-age females and a decreased p,p'-DDE/∑PCB ratio in J-pod. Measuring toxicants in scat samples provides an unprecedented opportunity to noninvasively evaluate contaminant levels in wild cetacean populations; these data have the prospect to provide meaningful information for vital management decisions.

The Relationship between Vessel Traffic and Noise Levels Received by Killer Whales (Orcinus orca).

Whale watching has become increasingly popular as an ecotourism activity around the globe and is beneficial for environmental education and local economies. Southern Resident killer whales (Orcinus orca) comprise an endangered population that is frequently observed by a large whale watching fleet in the inland waters of Washington state and British Columbia. One of the factors identified as a risk to recovery for the population is the effect of vessels and associated noise. An examination of the effects of vessels and associated noise on whale behavior utilized novel equipment to address limitations of previous studies. Digital acoustic recording tags (DTAGs) measured the noise levels the tagged whales received while laser positioning systems allowed collection of geo-referenced data for tagged whales and all vessels within 1000 m of the tagged whale. The objective of the current study was to compare vessel data and DTAG recordings to relate vessel traffic to the ambient noise received by tagged whales. Two analyses were conducted, one including all recording intervals, and one that excluded intervals when only the research vessel was present. For all data, significant predictors of noise levels were length (inverse relationship), number of propellers, and vessel speed, but only 15% of the variation in noise was explained by this model. When research-vessel-only intervals were excluded, vessel speed was the only significant predictor of noise levels, and explained 42% of the variation. Simple linear regressions (ignoring covariates) found that average vessel speed and number of propellers were the only significant correlates with noise levels. We conclude that vessel speed is the most important predictor of noise levels received by whales in this study. Thus, measures that reduce vessel speed in the vicinity of killer whales would reduce noise exposure in this population.

Assessing the coastal occurrence of endangered killer whales using autonomous passive acoustic recorders.

Using moored autonomous acoustic recorders to detect and record the vocalizations of social odonotocetes to determine their occurrence patterns is a non-invasive tool in the study of these species in remote locations. Acoustic recorders were deployed in seven locations on the continental shelf of the U.S. west coast from Cape Flattery, WA to Pt. Reyes, CA to detect and record endangered southern resident killer whales between January and June of 2006-2011. Detection rates of these whales were greater in 2009 and 2011 than in 2006-2008, were most common in the month of March, and occurred with the greatest frequency off the Columbia River and Westport, which was likely related to the presence of their most commonly consumed prey, Chinook salmon. The observed patterns of annual and monthly killer whale occurrence may be related to run strength and run timing, respectively, for spring Chinook returning to the Columbia River, the largest run in this region at this time of year. Acoustic recorders provided a unique, long-term, dataset that will be important to inform future consideration of Critical Habitat designation for this U.S. Endangered Species Act listed species.

Distinguishing the impacts of inadequate prey and vessel traffic on an endangered killer whale (Orcinus orca) population.

Managing endangered species often involves evaluating the relative impacts of multiple anthropogenic and ecological pressures. This challenge is particularly formidable for cetaceans, which spend the majority of their time underwater. Noninvasive physiological approaches can be especially informative in this regard. We used a combination of fecal thyroid (T3) and glucocorticoid (GC) hormone measures to assess two threats influencing the endangered southern resident killer whales (SRKW; Orcinus orca) that frequent the inland waters of British Columbia, Canada and Washington, U.S.A. Glucocorticoids increase in response to nutritional and psychological stress, whereas thyroid hormone declines in response to nutritional stress but is unaffected by psychological stress. The inadequate prey hypothesis argues that the killer whales have become prey limited due to reductions of their dominant prey, Chinook salmon (Oncorhynchus tshawytscha). The vessel impact hypothesis argues that high numbers of vessels in close proximity to the whales cause disturbance via psychological stress and/or impaired foraging ability. The GC and T3 measures supported the inadequate prey hypothesis. In particular, GC concentrations were negatively correlated with short-term changes in prey availability. Whereas, T3 concentrations varied by date and year in a manner that corresponded with more long-term prey availability. Physiological correlations with prey overshadowed any impacts of vessels since GCs were lowest during the peak in vessel abundance, which also coincided with the peak in salmon availability. Our results suggest that identification and recovery of strategic salmon populations in the SRKW diet are important to effectively promote SRKW recovery.

Effects of noise levels and call types on the source levels of killer whale calls.

Accurate parameter estimates relevant to the vocal behavior of marine mammals are needed to assess potential effects of anthropogenic sound exposure including how masking noise reduces the active space of sounds used for communication. Information about how these animals modify their vocal behavior in response to noise exposure is also needed for such assessment. Prior studies have reported variations in the source levels of killer whale sounds, and a more recent study reported that killer whales compensate for vessel masking noise by increasing their call amplitude. The objectives of the current study were to investigate the source levels of a variety of call types in southern resident killer whales while also considering background noise level as a likely factor related to call source level variability. The source levels of 763 discrete calls along with corresponding background noise were measured over three summer field seasons in the waters surrounding the San Juan Islands, WA. Both noise level and call type were significant factors on call source levels (1-40 kHz band, range of 135.0-175.7 dB(rms) re 1 [micro sign]Pa at 1 m). These factors should be considered in models that predict how anthropogenic masking noise reduces vocal communication space in marine mammals.

Inferred Paternity and Male Reproductive Success in a Killer Whale (Orcinus orca) Population.

We used data from 78 individuals at 26 microsatellite loci to infer parental and sibling relationships within a community of fish-eating ("resident") eastern North Pacific killer whales (Orcinus orca). Paternity analysis involving 15 mother/calf pairs and 8 potential fathers and whole-pedigree analysis of the entire sample produced consistent results. The variance in male reproductive success was greater than expected by chance and similar to that of other aquatic mammals. Although the number of confirmed paternities was small, reproductive success appeared to increase with male age and size. We found no evidence that males from outside this small population sired any of the sampled individuals. In contrast to previous results in a different population, many offspring were the result of matings within the same "pod" (long-term social group). Despite this pattern of breeding within social groups, we found no evidence of offspring produced by matings between close relatives, and the average internal relatedness of individuals was significantly less than expected if mating were random. The population's estimated effective size was <30 or about 1/3 of the current census size. Patterns of allele frequency variation were consistent with a population bottleneck.

Effects of age, sex and reproductive status on persistent organic pollutant concentrations in "Southern Resident" killer whales.

"Southern Resident" killer whales (Orcinus orca) that comprise three fish-eating "pods" (J, K and L) were listed as "endangered" in the US and Canada following a 20% population decline between 1996 and 2001. Blubber biopsy samples from Southern Resident juveniles had statistically higher concentrations of certain persistent organic pollutants than were found for adults. Most Southern Resident killer whales, including the four juveniles, exceeded the health-effects threshold for total PCBs in marine mammal blubber. Maternal transfer of contaminants to the juveniles during rapid development of their biological systems may put these young whales at greater risk than adults for adverse health effects (e.g., immune and endocrine system dysfunction). Pollutant ratios and field observations established that two of the pods (K- and L-pod) travel to California to forage. Nitrogen stable isotope values, supported by field observations, indicated possible changes in the diet of L-pod over the last decade.