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The webinar series and workshop titled "Trust Your Gut: Establishing Confidence in Gastrointestinal Models - An Overview of the State of the Science and Contexts of Use" was co-organized by NICEATM, NIEHS, FDA, EPA, CPSC, DoD, and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and hosted at the National Institutes of Health in Bethesda, MD, USA on October 11-12, 2023. New approach methods (NAMs) for assessing issues of gastrointestinal tract (GIT)related toxicity offer promise in addressing some of the limitations associated with animal-based assessments. GIT NAMs vary in complexity, from two-dimensional monolayer cell line-based systems to sophisticated 3-dimensional organoid systems derived from human primary cells. Despite advances in GIT NAMs, challenges remain in fully replicating the complex interactions and processes occurring within the human GIT. Presentations and discussions addressed regulatory needs, challenges, and innovations in incorporating NAMs into risk assessment frameworks; explored the state of the science in using NAMs for evaluating systemic toxicity, understanding absorption and pharmacokinetics, evaluating GIT toxicity, and assessing potential allergenicity; and discussed strengths, limitations, and data gaps of GIT NAMs as well as steps needed to establish confidence in these models for use in the regulatory setting.

Many marine food webs are “wasp-waist” in which energy funnels through one, or few, intermediate trophic level prey species, such as forage fish. However, marine prey are patchy and ephemeral, a challenge for marine predators. Ideal Free Distribution posits that individuals will disperse “ideally” among patches, whereby patches persistently supporting more individuals can be inferred to be high quality. Understanding the habitat types that support high abundances of animals is critical for effective marine conservation efforts. In this thesis, I focused from the bottom up on areas of high forage fish (capelin, Mallotus villosus) density in Newfoundland and from the top down on areas of high marine predator density in the California Current Ecosystem (CCE). To understand habitat associations of forage fish in Atlantic Canada, I interviewed fishers to map the spatial extent of subtidal spawning sites of capelin, a key foraging area for marine predators and used the interview results to conduct at-sea surveys. In the CCE, I used archived marine bird and mammal count surveys and a variety of statistical and spatial methods, such as zero-inflated negative binomial models and canonical correlation analyses, to examine predator habitat associations and niche partitioning. My findings provide an increased understanding of the factors that influence, and limit, habitat selection of key marine predators and their prey during especially vulnerable life-history stages, including breeding and over-wintering. To continue monitoring capelin subtidal spawning dynamics, future studies in Newfoundland will greatly benefit from establishing professional relationships with fishers as citizen scientists. In the CCE, my results emphasize the need for survey coverage with in-situ oceanography monitors in all four seasons in the highly seasonal CCE.

The Earth's polar regions are low rates of inter- and intraspecific diversification. An extreme mammalian example is the Arctic ringed seal (Pusa hispida hispida), which is assumed to be panmictic across its circumpolar Arctic range. Yet, local Inuit communities in Greenland and Canada recognize several regional variants; a finding supported by scientific studies of body size variation. It is however unclear whether this phenotypic variation reflects plasticity, morphs or distinct ecotypes. Here, we combine genomic, biologging and survey data, to document the existence of a unique ringed seal ecotype in the Ilulissat Icefjord (locally ‘Kangia’), Greenland; a UNESCO World Heritage site, which is home to the most productive marine-terminating glacier in the Arctic. Genomic analyses reveal a divergence of Kangia ringed seals from other Arctic ringed seals about 240 kya, followed by secondary contact since the Last Glacial Maximum. Despite ongoing gene flow, multiple genomic regions appear under strong selection in Kangia ringed seals, including candidate genes associated with pelage coloration, growth and osmoregulation, potentially explaining the Kangia seal's phenotypic and behavioural uniqueness. The description of ‘hidden’ diversity and adaptations in yet another Arctic species merits a reassessment of the evolutionary processes that have shaped Arctic diversity and the traditional view of this region as an evolutionary freezer. Our study highlights the value of indigenous knowledge in guiding science and calls for efforts to identify distinct populations or ecotypes to understand how these might respond differently to environmental change.

Beluga whales (Delphinapterus leucas) are an Arctic cetacean with circumpolar distribution. They are known to have distinct, lengthy and consistent migration paths to and from summer and wintering grounds. The Western Hudson Bay beluga population, the largest summering aggregation in the world, inhabits three main estuaries in the summer season. Little protection is afforded to them here, and little is known of the specific details of their summer distribution and factors affecting that distribution. Using a combination of satellite telemetry, aerial photos, satellite data, and visual observations and historical reports, investigations into factors affecting beluga distribution in Western Hudson Bay were conducted. An examination into beluga age class distribution near the Churchill and Seal River estuaries, using aerial survey imagery, suggested that belugas may not be segregating by age in summer, however the Seal River estuary may be more important from a calf-rearing perspective. Belugas use a greater home range than we would expect, based on historical range data, in the face of predation by their main predator, the killer whale (Orcinus orca). Killer whales are sighted more frequently in Hudson Bay than historically, and results presented here show that ice entrapments, occurring with higher frequency in the future, may impact distribution of beluga as killer whales expand their range and occurrence in Hudson Bay. Finally, an assessment of beluga behaviour in the presence of whale-watching vessels showed that beluga response to vessels varied but the probability of travelling behaviour was significantly greater with distance from vessels; belugas also appear to be spending more time interacting with vessels now, as compared to 15 years ago when there were fewer whale-watching vessels. The results found through this research are useful in informing climate change implications, marine-protected area boundaries, policy and marine spatial planning, and subsistence

Ringed seals (Pusa hispida) are opportunistic predators but primarily feed on Arctic cod (Boreogadus saida) and lipid-rich pelagic crustaceans. Recently, shifts in diet have been observed. We used semi-structured interviews and stomach contents analysis to describe ringed seal diet and foraging ecology. Interviews (n=23) were conducted with hunters from Arctic Bay, Pangnirtung, and Pond Inlet, Nunavut. Ringed seal stomach samples (n=201) were harvested from Admiralty Inlet, Cumberland Sound, and Eclipse Sound and samples containing prey contents (n=166) were analysed. Hunters observed ugak (cod) and kinguk (amphipods) in the stomachs of ringed seals, year-round, across spatial scales, and within all age classes. The flavour of ringed seal meat was variable across space and time, with some associating the flavour with ringed seal prey. Some participants identified specific areas where ringed seals foraged, for example, the floe edge, polynyas and in areas of current, while most responses pointed to widespread feeding. Ringed seals were described as feeding year-round, except for the moulting and basking period when their stomachs were typically empty or had fewer contents. Some age-related differences in diet were observed by interviewees, specifically that juveniles consumed more kinguk than other prey types when they were first weaned. Stomach contents revealed Arctic cod and Hyperiidae were most abundant and frequently occurring of fishes and invertebrates, respectively. Multivariate tests found a significant effect for region, year of harvest, month of harvest, age class, and the interaction between age class and region. Univariate tests indicated significant differences between regions in the prey counts of Arctic cod and Euphausiacea. Arctic cod had higher abundance and biomass in both northern regions than in Cumberland Sound, whereas Euphausiacea was not present in either of the northern regions. Temporal differences were driven by Euphausiacea among years an

Climate change is predicted to affect ringed seals negatively. The first objective of my thesis was to assess trends in abundance of ringed seals using genetics. I used genetic profiles to estimate the effective number of breeders (Nb) and assessed relationships with counts from aerial surveys (Ns). I did not detect a relationship between Nb and Ns or a temporal trend in either variable when the entire time series was assessed. However, Nb fluctuated over time, with a negative trend from 1983 to 1994 and becoming stable from 1995 to 2012. My second objective was to determine which variables predicted Nb. My analysis suggested that spring rainfall, snow depth, and the date of fall sea-ice freezeup had a greater influence on Nb than the timing of spring breakup. This research helps address the lack of long-term monitoring data for ringed seals, while improving our understanding of population dynamics.

With climate change, temperate species are becoming increasingly able to occupy Arctic regions that were previously unsuitable. In Cumberland Sound, Nunavut, the forage fish capelin has become abundant in recent decades, as has the seasonally-migrant harp seal during the summer months. Coincident have been reported decreases to resident ringed seal numbers. While capelin foraging is well studied in other regions, it is less so in Cumberland Sound. In addition, no research has yet examined whether harp seals may act as competitors to Cumberland Sound ringed seals. The objectives of this thesis were to (1) quantify capelin diet and isotopic niche characteristics, and (2) examine ringed and harp seal diet and isotopic niches and determine the degree of overlap. Results from this thesis have provided insight into trophic interactions within the Cumberland Sound food web and provide a baseline for future studies examining the impacts of climate change in Arctic ecosystems.

This thesis examines the genetic diversity, population structure and phylogeography of belugas (Delphinapterus leucas) in Canadian waters that encounter multiple stressors throughout their seasonal distributions. Data were collected from multiple scales, including: samples covering a temporal scale of 25 years; broad geographic comparisons to finer-scale, within-group comparisons; and varying amounts of genetic information from partial mtDNA sequences to whole mitogenome sequences, and multiple nuclear microsatellite loci. At least nine genetically distinct summer aggregations, with an additional distinct winter sample collection of unknown summer distribution, were identified. This information contributed to the identification of Designatable Units (DUs) of belugas for future assessments by the Committee on the Status of Endangered Wildlife in Canada (COSEWIC). Phylogenetic analyses of mtDNA sequences revealed that the most divergent lineages are found at the east, west and southern edges of the Canadian distribution, with the central area characteristic of a contact zone displaying an admixture of lineages marked by incomplete lineage sorting. The geographic distribution of these lineages suggests multiple glacial refugia as sources of ancestral beluga populations that recolonized Canadian Arctic and sub-Arctic waters. Preliminary tests of selection detected the presence of purifying selection on all mtDNA protein-coding genes of belugas. However, no signals of adaptive selection were detected among genetic lineages or geographic groups. Within nearshore summer aggregations of Beaufort Sea belugas, three distinct maternal lineages were identified and patterns of genetic relatedness suggest clusters of related females form in the overall area. However, these clusters of related belugas did not form fine-scale kin structure corresponding to aggregation/harvesting locations. Thus, disturbances and subsistence harvesting in particular areas where belugas are aggregati

Climate change is reducing the Arctic sea ice concentration and extent and it has been thought that narwhal will be poorly able to adjust. The goal of this thesis was to (a) analyzing narwhal year-round movement, and to (b) examine winter habitat selection in relation to sea ice and bathymetry. Narwhal from Admiralty Inlet and Eclipse Sound were equipped with satellite transmitters between 2009 and 2012. Narwhal conducted multiple late-summer movement patterns with three stocks overlapping, had a delayed fall migration compared to a tagging studies a decade earlier, and had decreased summer site fidelity. During the winter narwhal selected 1500 to 2000 m depths, which likely have higher prey densities, regardless of the mobile pack ice structure. They also conducted extensive movements coinciding with a delayed growth in sea ice extent. These results indicate that narwhal may be more able to adjust to habitat changes than previously believed.

Ringed seals (Phoca hispida) in the Canadian Arctic are subject to a variety of environmental and anthropogenic stressors that stand to potentially compromise population health and survival. Typically, animals exposed to chronic stressors initiate a stress response resulting in cortisol production, which results in physiological and behavioural changes designed to maintain homeostasis under the influence of the stressor. Cortisol extraction techniques were developed for Ringed seal blubber and fur samples. Blubber cortisol was found to be a reliable indicator of the condition factor ratio of blubber depth to core diameter. Ringed seal blubber cortisol concentration and condition was shown to alter depending on season and age class. The findings of this study are an important first step in developing an understanding of how this ice obligate species has and may respond to environmental stressors and will assist with developing conservation strategies.

The study characterized the morphological differences among Cisco between geographic arms and depth strata, and determined the trophic positioning of various morphs, based on twelve years of capture data and specimens using morphological measurements, meristic counts, stomach contents, stable isotopes and fatty acids. Two main Cisco morpho-types were identified, a shallow water form and a deep water form that mainly differed in traits associated with swimming and feeding. These results were reinforced by the results on trophic ecology of morphs that demonstrated that each morph occupied a distinct niche. Results showed that shallow water groups had a more varied diet with a high percentage of copepods being ingested whereas deep-water groups showed preferences towards mysids. Although a consistent pattern in differences among shallow and deep morphs was observed, there was some degree of variation among arms of the lake suggesting a degree of isolation and adaptation to local conditions in each area of the lake. For example, in Smith Arm, morphological and dietary differences were found such as narrower body depth and a dietary preference towards daphnia.

This thesis examines the biogeography of world pinnipeds, a unique group of marine mammals that have adapted to marine foraging while maintaining terrestrial (land or ice) habitat links. Comparative analyses of species range sizes controlled for phylogenetic relationships using a multi-gene supertree with divergence dates estimated using fossil calibrations. Adaptations to aquatic mating and especially sea ice parturition have influenced range size distribution, and ranges are larger than those of terrestrially mating and/or pupping species. Small range size is endangering for many taxa, and most at risk pinnipeds are terrestrial species with small ranges. Ancestral state reconstructions suggest that pinnipeds had a long association with sea ice, an adaptation that would have allowed early seals to expand into novel habitats and increase their distribution. Range sizes exhibit a strong Rapoport effect (positive relationship between range size and latitude) at the global scale, even after controlling for phylogeny and body size allometry. A latitudinal gradient in species diversity cannot explain the Rapoport effect for global pinniped ranges, as diversity is highest at mid-latitudes in both hemispheres. These regions are characterized by marginal ice zones and variable climates, supporting a mix of pagophilic and temperate species. The climatic variability hypothesis also did not explain the Rapoport effect. Variability is bimodal, and annual sea surface temperature (SST) variability does explain diversity patterns. Range size has a significant negative relationship with annual mean SST, and the largest ranges are found in areas with low mean SST. Temperature responses are possibly related to thermoregulation, sea ice availability, and ecological relationships with other large marine predators. These results agree with other studies and suggest that ocean temperature, and not productivity, drives marine species richness patterns. Future research needs include studie

This dissertation attempts to improve the understanding of spatial distribution and evolution of snow-covered sea ice as related to polar bear habitat. This will be accomplished at both the local (i.e. 1m spatial resolution) and regional scales (i.e. 100 km spatial resolution) for various types of first-year sea ice (FYI) through four primary objectives. The first primary objective (i.e. Chapter 3) examines the observed and modeled temporal evolution of snow over smooth FYI, as well as the estimation of on-ice meteorological conditions. Results suggest that increases in observed snowdrifts and changes to the orientation of the drifts are related to snowfall and drifting events. Modeling these changes over time using a spatially distributed snow model is not able to accurately estimate the snow distribution. As well, only the on-ice temperature and humidity can be estimated from land-based station data, limiting the modeling of snow over sea ice. The second primary objective (i.e. Chapter 4) extends this research to rough FYI types, more relevant to polar bear habitat. More specifically this objective studies the spatial pattern of snow distribution over rough ice and ridges and the relationship between ice roughness and meteorological conditions. Results suggest that ice roughness influences the magnitude of snow depth, while the wind direction during periods of snow deposition and/or blowing snow events will impact the spatial pattern. The third primary objective (i.e. Chapter 5) focuses on developing a more feasible method of deriving surface roughness. This objective attempts to use satellite imagery and texture analysis to derive surface roughness for snow-covered sea ice. Results suggest that a Gamma speckle reduction filter, coupled with a grey-level co-occurrence matrix texture measure (Entropy or Angular Second Moment) is able to account for more than 88% of the variability in the surface roughness. The final primary objective (i.e. Chapter 6) examines the t

An understanding of the adaptability of belugas (Delphinapterus leucas) to changing ice-conditions is required to interpret and predict possible changes in habitat selection in response to projected loss of sea ice throughout the circumpolar Arctic. Beluga spring distribution in the eastern Beaufort Sea was described by analyzing observations from aerial surveys conducted from 1975 to 1979. Repeated surveys along the Franklin Bay fast-ice edge in June 2008 were used to study the distribution and behaviour of belugas and bowheads. Despite inter-annual variability in ice extent, belugas consistently selected areas with water depths of 200-500 m, heavy ice concentrations (8/10 to 10/10) and seafloor slope ≥0.5 degrees in spring 1975 to 1979. While predator avoidance may partially explain the observed distribution, foraging success likely has more influence on beluga habitat selection in the spring. In ice-covered offshore regions, belugas may be engaged in under-ice and deep water foraging on Arctic cod (Boreogadus saida). In lighter ice years, belugas may expand their distribution and shift shoreward to take advantage of high prey densities along fast-ice edges. Both belugas and bowheads appeared to be feeding along the Franklin Bay ice edge in June 2008. More research is required to examine and compare possible changes in distribution since the late 1970s and to investigate the factors driving the patterns described.