Diet- and salinity-induced modifications of the gut microbiota are associated with differential physiological responses to ranavirus infection in Rana sylvatica.

Greater knowledge of how host-microbiome interactions vary with anthropogenic environmental change and influence pathogenic infections is needed to better understand stress-mediated disease outcomes. We investigated how increasing salinization in freshwaters (e.g. due to road de-icing salt runoff) and associated increases in growth of nutritional algae influenced gut bacterial assembly, host physiology and responses to ranavirus exposure in larval wood frogs (Rana sylvatica). Elevating salinity and supplementing a basic larval diet with algae increased larval growth and also increased ranavirus loads. However, larvae given algae did not exhibit elevated kidney corticosterone levels, accelerated development or weight loss post-infection, whereas larvae fed a basic diet did. Thus, algal supplementation reversed a potentially maladaptive stress response to infection observed in prior studies in this system. Algae supplementation also reduced gut bacterial diversity. Notably, we observed higher relative abundances of Firmicutes in treatments with algae-a pattern consistent with increased growth and fat deposition in mammals-that may contribute to the diminished stress responses to infection via regulation of host metabolism and endocrine function. Our study informs mechanistic hypotheses about the role of microbiome mediation of host responses to infection that can be tested in future experiments in this host-pathogen system. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Site-specific spatiotemporal occurrence and molecular congener distributions of naphthenic acids in Athabasca oil sands wetlands of Alberta, Canada.

The Athabasca oil sands region (AOSR) of Alberta, Canada is notable for its considerable unconventional petroleum extraction projects, where bitumen is extracted from naturally-occurring oil sands ore. The large scale of these heavy crude oil developments raises concerns because of their potential to distribute and/or otherwise influence the occurrence, behaviour, and fate of environmental contaminants. Naphthenic acids (NAs) are one such contaminant class of concern in the AOSR, so studies have examined the occurrence and molecular profiles of NAs in the region. We catalogued the spatiotemporal occurrence and characteristics of NAs in boreal wetlands in the AOSR over a 7-year period, using derivatized liquid chromatography-tandem mass spectrometry (LC-MS/MS). Comparing median concentrations of NAs across these wetlands revealed a pattern of NAs suggesting NAs in surface waters derived from oil sands deposits. Opportunistic wetlands that formed adjacent to reclaimed overburden and other reclamation activities had the highest concentrations of NAs and consistent patterns suggestive of bitumen-derived inputs. However, similar patterns in the occurrence of NAs were also observed in undeveloped natural wetlands located above the known surface-mineable oil sands deposit that underlies the region. Intra-annual sampling results along with inter-annual comparisons across wetlands demonstrated that differences in the spatial and temporal NA concentrations were dependent on local factors, particularly when naturally occurring oil sands ores were observed in the wetland or drainage catchment.

Ranavirus Amplification in Low-Diversity Amphibian Communities.

In an era where emerging infectious diseases are a serious threat to biodiversity, epidemiological patterns need to be identified, particularly the complex mechanisms driving the dynamics of multi-host pathogens in natural communities. Many amphibian species have faced unprecedented population declines associated with diseases. Yet, specific processes shaping host-pathogen relationships within and among communities for amphibian pathogens such as ranaviruses (RV) remain poorly understood. To address this gap, we conducted a comprehensive study of RV in low-diversity amphibian communities in north-western Canada to assess the effects of biotic factors (species identity, species richness, abundance) and abiotic factors (conductivity, pH) on the pathogen prevalence and viral loads. Across 2 years and 18 sites, with communities of up to three hosts (wood frog, Rana sylvatica; boreal chorus frog, Pseudacris maculata; Canadian toad, Anaxyrus hemiophrys), we observed that RV prevalence nearly doubled with each additional species in a community, suggesting an amplification effect in aquatic, as well as terrestrial life-history stages. Infection intensity among infected wood frogs and boreal chorus frogs also significantly increased with an increase in species richness. Interestingly, we did not observe any effects of host abundance or abiotic factors, highlighting the importance of including host identity and species richness when investigating multi-host pathogens. Ultimately, only such a comprehensive approach can improve our understanding of complex and often highly context-dependent host-pathogen interactions.

Transformation of bitumen-derived naphthenic acid fraction compounds across surface waters of wetlands in the Athabasca Oil Sands region.

Bitumen is extracted from oil sands in the Athabasca Oil Sands region (AOSR) of Alberta, Canada. Much of the bitumen-derived toxicity in mine waste is attributable to naphthenic acid fraction compounds (NAFCs). Mines in the AOSR are required to be returned to a natural state after closure; thus, cost-effective strategies are needed to reduce toxicity from NAFCs. Previous studies have demonstrated the capability of constructed wetlands to attenuate NAFCs. However, the capacity of wetlands in the natural environment to degrade and transform NAFCs to less toxic components is poorly understood. To better understand the spatial distribution and fate of NAFCs in natural wetlands, samples were collected across the surfaces of two mature opportunistic wetlands near active oil sands mines. The first wetland has a well-defined surface flow pathway and inflows affected by overburden containing lean bitumen ore. The second wetland, in contrast, is a stagnant water body with raw bitumen visible along its edges. For the wetland with a well defined flow path, NAFCs decreased in concentration down gradient, while oxidized NAFCs constituted a greater proportion of NAFCs with increase in flow path. Likewise there was a decrease in the molecular weights of NAFCs, similar to trends observed in constructed wetland treatment systems. In comparison, NAFCs were more uniformly distributed across the relatively stagnant wetland. Overall, these data provide new evidence that mature opportunistic wetlands in the AOSR can promote the degradation and oxidation of bitumen-derived naphthenic acids into less toxic compounds.

Characterization of naphthenic acid fraction compounds in water from Athabasca oil sands wetlands by Orbitrap high-resolution mass spectrometry.

Classical naphthenic acids (NAs) are known to be primary aquatic toxicants of concern in the Athabasca oil sands region (AOSR), and are a component of naphthenic acid fraction compounds (NAFCs). Recent studies conducted in the AOSR have examined metals and polycyclic aromatic hydrocarbons in regional wetlands. However, few studies have described NAs and/or NAFCs in AOSR wetlands. To address this gap, we examined NAFC profiles in the water of different wetlands in the AOSR, including naturalized borrow pits (i.e., abandoned pits created by excavation of road-building materials), and opportunistically-formed wetlands associated with reclamation activities. For comparison, NAFC profiles from these wetlands were compared to an opportunistic wetland formed from tailings pond dyke seepage. Samples were prepared using solid-phase extraction and analyzed using negative-ion high-resolution Orbitrap mass spectrometry. Principal component analyses (PCA) revealed patterns to the NAFC profiles in the wetlands. The first distinct grouping of wetlands included water bodies associated with reclamation activities that are located on and/or adjacent to mining overburden. One other wetland, HATS5w, separated from all other wetlands during PCA, and had a unique NAFC profile; detailed examination of NAFCs revealed HATS5w contained the heaviest (i.e., high m/z components) and most unsaturated NAFCs among study locations, demonstrating the usefulness of high-resolution mass spectrometry for characterizing individual wetlands. The NAFCs of HATS5w are also substantially different from bitumen-derived inputs in overburden-adjacent opportunistic wetlands. Collectively, the NAFC profiles presented provide new information on background levels of polar bitumen-derived organics in AOSR wetlands.

Using wood frog (Lithobates sylvaticus) tadpoles and semipermeable membrane devices to monitor polycyclic aromatic compounds in boreal wetlands in the oil sands region of northern Alberta, Canada.

Several recent studies have reported evidence that surface mining operations of bitumen in northern Alberta's oil sands (OS) region contribute significantly to the atmospheric deposition of metals and polycyclic aromatic compounds (PACs) within the vicinity of OS development. The present study examines the accumulation of PACs in boreal wetlands at varying distance from OS industrial activities with the use of semipermeable membrane devices (SPMDs) and wood frog (Lithobates sylvaticus) tadpoles. SPMDs were deployed in shallow lentic waterbodies adjacent to wood frog egg masses and were retrieved, along with tadpoles, approximately 35-45 days later. The highest concentrations of PACs were detected in SPMDs deployed within a 25 km radius of surface mining activity, consistent with snow deposition studies of PACs in the region. In wetlands located within the vicinity of surface mining activity, PAC profiles of SPMDs and wood frog tadpoles were dominated by C1-C4 alkylated PACs, including alkylated dibenzothiophenes, which are strongly indicative of petrogenic sources. Contrary to differences seen in the SPMD PAC concentrations, there were no obvious differences in the ∑PACs in wood frog tissue between wetland study sites, although alkylated fluorenes were found to be higher in tadpoles collected from a wetland located within 10 km of two bitumen upgrading facilities. The use of SPMDs in tandem with wood frog tadpoles can help assess the potential exposure of aquatic organisms to PACs in boreal wetlands.

Oslerus osleri (metastrongyloidea; filaroididae) in gray wolves (Canis lupus) from Banff National Park, Alberta, Canada.

Oslerus osleri is a filaroid parasite of the respiratory tract of canids. In North America, it is most commonly reported from coyotes (Canis latrans) and domestic dogs (Canis lupus familiaris), but reports in gray wolves (Canis lupus) are infrequent. We report a new geographic record for O. osleri in four gray wolves from Banff National Park, Alberta, Canada. Adult nematodes found in nodules in the submucosa of the trachea and bronchi were identified as O. osleri based on morphometry of spicules of males. We sequenced two segments of the genome of adult nematodes: a 1,111-base pair (bp) segment of the 18S region that was 100% identical to the 18S region of O. osleri from a coyote in California and a 537-bp segment that included the ITS-2 region and partial 5.8S and 28S genes. Histopathologically, there were submucosal nodules of adult nematodes surrounded by fibrosis and lymphoplasmacytic inflammation. These findings are consistent with O. osleri infections in other canids. The importance of this parasite in wild canid populations is unknown, but prevalence may be underestimated because many studies have focused on gastrointestinal parasites or used routine fecal flotation that has low sensitivity for this parasite. The ecology and population genetics of this parasite where multiple species of canids are sympatric warrant closer investigation to determine potential for interspecies transmission.

Amphibian chytrid fungus and ranaviruses in the Northwest Territories, Canada.

Pathogens can cause serious declines in host species, and knowing where pathogens associated with host declines occur facilitates understanding host-pathogen ecology. Suspected drivers of global amphibian declines include infectious diseases, with 2 pathogens in particular, Batrachochytrium dendrobatidis (Bd) and ranaviruses, causing concern. We explored the host range and geographic distribution of Bd and ranaviruses in the Taiga Plains ecoregion of the Northwest Territories, Canada, in 2007 and 2008. Both pathogens were detected, greatly extending their known geographic distributions. Ranaviruses were widespread geographically, but found only in wood frogs. In contrast, Bd was found at a single site, but was detected in all 3 species of amphibians in the survey area (wood frogs, boreal chorus frogs, western toads). The presence of Bd in the Northwest Territories is not congruent with predicted distributions based on niche models, even though findings from other studies at northern latitudes are consistent with those same models. Unexpectedly, we also found evidence that swabs routinely used to collect samples for Bd screening detected fewer infections than toe clips. Our use and handling of the swabs was consistent with other studies, and the cause of the apparent lack of integrity of swabs is unknown. The ranaviruses detected in our study were confirmed to be Frog Virus 3 by sequence analysis of a diagnostic 500 bp region of the major capsid protein gene. It is unknown whether Bd or ranaviruses are recent arrivals to the Canadian north. However, the genetic analyses required to answer that question can inform larger debates about the origin of Bd in North America as well as the potential effects of climate change and industrial development on the distributions of these important amphibian pathogens.

Mortality rates differ among amphibian populations exposed to three strains of a lethal ranavirus.

Infectious diseases are a growing threat to biodiversity, in many cases because of synergistic effects with habitat loss, environmental contamination, and climate change. Emergence of pathogens as new threats to host populations can also arise when novel combinations of hosts and pathogens are unintentionally brought together, for example, via commercial trade or wildlife relocations and reintroductions. Chytrid fungus (Batrachochytrium dendrobatidis) and amphibian ranaviruses (family Iridoviridae) are pathogens implicated in global amphibian declines. The emergence of disease associated with these pathogens appears to be at least partly related to recent translocations over large geographic distances. We experimentally examined the outcomes of novel combinations of host populations and pathogen strains using the amphibian ranavirus Ambystoma tigrinum virus (ATV) and barred tiger salamanders (Ambystoma mavortium, formerly considered part of the Ambystoma tigrinum complex). One salamander population was highly resistant to lethal infections by all ATV strains, including its own strain, and mortality rates differed among ATV strains according to salamander population. Mortality rates in novel pairings of salamander population and ATV strain were not predictable based on knowledge of mortality rates when salamander populations were exposed to their own ATV strain. The underlying cause(s) for the differences in mortality rates are unknown, but local selection pressures on salamanders, viruses, or both, across the range of this widespread host-pathogen system are a plausible hypothesis. Our study highlights the need to minimize translocations of amphibian ranaviruses, even among conspecifc host populations, and the importance of considering intraspecific variation in endeavors to manage wildlife diseases.