Comprehensive identification of PIP3-regulated PH domains from C. elegans to H. sapiens by model prediction and live imaging.

Phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol(3,4,5)-trisphosphate (PIP3) control cell growth, migration, and other processes by recruiting proteins with pleckstrin homology (PH) domains and possibly other domains to the plasma membrane (PM). However, previous experimental and structural work with PH domains left conflicting evidence about which ones are PIP3 regulated. Here we used live-cell confocal imaging of 130 YFP-conjugated mouse PH domains and found that 20% translocated to the PM in response to receptor-generated PIP3 production. We developed a recursive-learning algorithm to predict PIP3 regulation of 1200 PH domains from different eukaryotes and validated that it accurately predicts PIP3 regulation. Strikingly, this algorithm showed that PIP3 regulation is specified by amino acids across the PH domain, not just the PIP3-binding pocket, and must have evolved several times independently from PIP3-insensitive ancestral PH domains. Finally, our algorithm and live-cell experiments provide a functional survey of PH domains in different species, showing that PI3K regulation increased from approximately two C. elegans and four Drosophila to 40 vertebrate proteins.

Population genetic structure of gray wolves (Canis lupus) in a marine archipelago suggests island-mainland differentiation consistent with dietary niche.

BACKGROUND: Emerging evidence suggests that ecological heterogeneity across space can influence the genetic structure of populations, including that of long-distance dispersers such as large carnivores. On the central coast of British Columbia, Canada, wolf (Canis lupus L., 1758) dietary niche and parasite prevalence data indicate strong ecological divergence between marine-oriented wolves inhabiting islands and individuals on the coastal mainland that interact primarily with terrestrial prey. Local holders of traditional ecological knowledge, who distinguish between mainland and island wolf forms, also informed our hypothesis that genetic differentiation might occur between wolves from these adjacent environments. RESULTS: We used microsatellite genetic markers to examine data obtained from wolf faecal samples. Our results from 116 individuals suggest the presence of a genetic cline between mainland and island wolves. This pattern occurs despite field observations that individuals easily traverse the 30 km wide study area and swim up to 13 km among landmasses in the region. CONCLUSIONS: Natal habitat-biased dispersal (i.e., the preference for dispersal into familiar ecological environments) might contribute to genetic differentiation. Accordingly, this working hypothesis presents an exciting avenue for future research where marine resources or other components of ecological heterogeneity are present.

Human disturbance in riparian areas disrupts predator-prey interactions between grizzly bears and salmon.

Wildlife must increasingly balance trade-offs between the need to access important foods and the mortality risks associated with human-dominated landscapes. Human disturbance can profoundly influence wildlife behavior, but managers know little about the relationship between disturbance-behavior dynamics and associated consequences for foraging. We address this gap by empirically investigating the consequences of human activity on a keystone predator-prey interaction in a region with limited but varied industrial disturbance. Using stable isotope data from 226 hair samples of grizzly bears (Ursus arctos horribilis) collected from 1995 to 2014 across 22 salmon-bearing watersheds (88,000 km2) in British Columbia, Canada, we examined how human activity influenced their consumption of spawning salmon (Oncorhynchus spp.), a fitness-related food. Accounting for the abundance of salmon and other foods, salmon consumption strongly decreased (up to 59% for females) with increasing human disturbance (as measured by the human footprint index) in riparian zones of salmon-bearing rivers. Declines in salmon consumption occurred with disturbance even in watersheds with low footprints. In a region currently among the least influenced by industrial activity, intensification of disturbance in river valleys is predicted to increasingly decouple bears from salmon, possibly driving associated reductions in population productivity and provisioning of salmon nutrients to terrestrial ecosystems. Accordingly, we draw on our results to make landscape-scale and access-related management recommendations beyond current streamside protection buffers. This work illustrates the interaction between habitat modification and food security for wildlife, highlighting the potential for unacknowledged interactions and cumulative effects in increasingly modified landscapes.

Humanity's diverse predatory niche and its ecological consequences.

Although humans have long been predators with enduring nutritive and cultural relationships with their prey, seldom have conservation ecologists considered the divergent predatory behavior of contemporary, industrialized humans. Recognizing that the number, strength and diversity of predator-prey relationships can profoundly influence biodiversity, here we analyze humanity's modern day predatory interactions with vertebrates and estimate their ecological consequences. Analysing IUCN 'use and trade' data for ~47,000 species, we show that fishers, hunters and other animal collectors prey on more than a third (~15,000 species) of Earth's vertebrates. Assessed over equivalent ranges, humans exploit up to 300 times more species than comparable non-human predators. Exploitation for the pet trade, medicine, and other uses now affects almost as many species as those targeted for food consumption, and almost 40% of exploited species are threatened by human use. Trait space analyses show that birds and mammals threatened by exploitation occupy a disproportionally large and unique region of ecological trait space, now at risk of loss. These patterns suggest far more species are subject to human-imposed ecological (e.g., landscapes of fear) and evolutionary (e.g., harvest selection) processes than previously considered. Moreover, continued overexploitation will likely bear profound consequences for biodiversity and ecosystem function.

The Drosophila FTZ-F1 nuclear receptor mediates juvenile hormone activation of E75A gene expression through an intracellular pathway.

Juvenile hormone (JH) regulates a wide variety of biological activities in holometabolous insects, ranging from vitellogenesis and caste determination in adults to the timing of metamorphosis in larvae. The mechanism of JH signaling in such a diverse array of processes remains either unknown or contentious. We previously found that the nuclear receptor gene E75A is activated in S2 cells as a primary response to JH. Here, by expressing an intracellular form of JH esterase, we demonstrate that JH must enter the cell in order to activate E75A. To find intracellular receptors involved in the JH response, we performed an RNAi screen against nuclear receptor genes expressed in this cell line and identified the orphan receptor FTZ-F1. Removal of FTZ-F1 prevents JH activation of E75A, whereas overexpression enhances activation, implicating FTZ-F1 as a critical component of the JH response. FTZ-F1 is bound in vivo to multiple enhancers upstream of E75A, suggesting that it participates in direct JH-mediated gene activation. To better define the role of FTZ-F1 in JH signaling, we investigated interactions with candidate JH receptors and found that the bHLH-PAS proteins MET and GCE both interact with FTZ-F1 and can activate transcription through the FTZ-F1 response element. Removal of endogenous GCE, but not MET, prevents JH activation of E75A. We propose that FTZ-F1 functions as a competence factor by loading JH signaling components to the promoter, thus facilitating the direct regulation of E75A gene expression by JH.

Seasonal and biogeographical patterns of gastrointestinal parasites in large carnivores: wolves in a coastal archipelago.

Parasites are increasingly recognized for their profound influences on individual, population and ecosystem health. We provide the first report of gastrointestinal parasites in gray wolves from the central and north coasts of British Columbia, Canada. Across 60 000 km(2), wolf feces were collected from 34 packs in 2005-2008. At a smaller spatial scale (3300 km(2)), 8 packs were sampled in spring and autumn. Parasite eggs, larvae, and cysts were identified using standard flotation techniques and morphology. A subset of samples was analysed by PCR and sequencing to identify tapeworm eggs (n=9) and Giardia cysts (n=14). We detected ≥14 parasite taxa in 1558 fecal samples. Sarcocystis sporocysts occurred most frequently in feces (43·7%), followed by taeniid eggs (23·9%), Diphyllobothrium eggs (9·1%), Giardia cysts (6·8%), Toxocara canis eggs (2·1%), and Cryptosporidium oocysts (1·7%). Other parasites occurred in ≤1% of feces. Genetic analyses revealed Echinococcus canadensis strains G8 and G10, Taenia ovis krabbei, Diphyllobothrium nehonkaiense, and Giardia duodenalis assemblages A and B. Parasite prevalence differed between seasons and island/mainland sites. Patterns in parasite prevalence reflect seasonal and spatial resource use by wolves and wolf-salmon associations. These data provide a unique, extensive and solid baseline for monitoring parasite community structure in relation to environmental change.

Seroepidemiology of respiratory (group 2) canine coronavirus, canine parainfluenza virus, and Bordetella bronchiseptica infections in urban dogs in a humane shelter and in rural dogs in small communities.

This prospective study evaluated seroepidemiologic features of canine respiratory coronavirus (CRCoV), canine parainfluenza virus (CPIV), and Bordetella bronchiseptica infections in dogs in an urban humane shelter and in rural/small community dog populations in western Canada. Seroprevalence of CRCoV and CPIV was low compared with other countries; seroprevalence of B. bronchiseptica was moderate to high in most populations examined. Rural dogs were 0.421 times (P ≤ 0.0001) less likely to be positive for CRCoV than dogs admitted to the shelter. There were no statistical differences in prevalence of antibodies to B. bronchiseptica and CPIV between urban and rural populations. Dogs from Fort Resolution, NWT were significantly (P < 0.05) less likely to have moderate or high antibody titers to the 3 agents than dogs in the shelter. Seroconversion to CRCoV was common in dogs in the shelter, but was not associated (P = 0.18) with respiratory disease. Antibodies to CRCoV, CPIV, or B. bronchiseptica on arrival were not significantly (P > 0.05) associated with disease-sparing after entry into the shelter.

Cross-continental comparison of parasite communities in a wide-ranging carnivore suggests associations with prey diversity and host density.

Parasites are integral to ecosystem functioning yet often overlooked. Improved understanding of host-parasite associations is important, particularly for wide-ranging species for which host range shifts and climate change could alter host-parasite interactions and their effects on ecosystem function.Among the most widely distributed mammals with diverse diets, gray wolves (Canis lupus) host parasites that are transmitted among canids and via prey species. Wolf-parasite associations may therefore influence the population dynamics and ecological functions of both wolves and their prey. Our goal was to identify large-scale processes that shape host-parasite interactions across populations, with the wolf as a model organism.By compiling data from various studies, we examined the fecal prevalence of gastrointestinal parasites in six wolf populations from two continents in relation to wolf density, diet diversity, and other ecological conditions.As expected, we found that the fecal prevalence of parasites transmitted directly to wolves via contact with other canids or their excreta was positively associated with wolf density. Contrary to our expectations, the fecal prevalence of parasites transmitted via prey was negatively associated with prey diversity. We also found that parasite communities reflected landscape characteristics and specific prey items available to wolves.Several parasite taxa identified in this study, including hookworms and coccidian protozoans, can cause morbidity and mortality in canids, especially in pups, or in combination with other stressors. The density-prevalence relationship for parasites with simple life cycles may reflect a regulatory role of gastrointestinal parasites on wolf populations. Our result that fecal prevalence of parasites was lower in wolves with more diverse diets could provide insight into the mechanisms by which biodiversity may regulate disease. A diverse suite of predator-prey interactions could regulate the effects of parasitism on prey populations and mitigate the transmission of infectious agents, including zoonoses, spread via trophic interactions.

Physiological responses and accumulation ability of Microcystis aeruginosa to zinc and cadmium: Implications for bioremediation of heavy metal pollution.

Algae has potential to remediate heavy metals. However, the physiological responses of live algae to heavy metals are not well studied. In this study, the physiological responses of Microcystis aeruginosa to zinc (Zn) and cadmium (Cd) ions and its ability to accumulate ions were investigated. Low concentrations (<0.1 mg/l) of Zn and Cd had little influence on algal growth and physiological processes, whereas concentrations above 0.1 mg/l increased the esterase activity (from 42.5% to 621.9%), superoxide dismutase activity (from 12.8% to 45.4%), and malondialdehyde content (from 18.2% to 103.9%), and dramatically inhibited the cell division (from 12.6% to 70.0%) and photosynthetic performance (from 7.1% to 53.1%) of M. aeruginosa. The accumulation of Zn or Cd ions by M. aeruginosa increased exponentially with the initial concentration of metallic ions. Collectively, these findings reveal that M. aeruginosa has considerable potential in the remediation of freshwater lakes with heavy metal contamination during cyanobacterial blooms, where metallic ions are lower than 0.1 mg/l.

Raising the bar: Recovery ambition for species at risk in Canada and the US.

Routinely crossing international borders and/or persisting in populations across multiple countries, species are commonly subject to a patchwork of endangered species legislation. Canada and the United States share numerous endangered species; their respective acts, the Species at Risk Act (SARA) and the Endangered Species Act (ESA), require documents that outline requirements for species recovery. Although there are many priorities for improving endangered species legislation effectiveness, species recovery goals are a crucial component. We compared recovery goal quality, as measured by goal quantitativeness and ambition, for species listed under SARA and ESA. By comparing across ESA and SARA, the intent of the study was to identify differences and similarities that could support the development of stronger species' recovery goals under both legislations. Our results indicated that: (1) overall, only 38% of recovery goals were quantitative, 41% had high ambition, and 26% were both quantitative and with high ambition; (2) recovery goals had higher quantitativeness and ambition under ESA than SARA; (3) recovery goals for endangered species had higher ambition than threatened species under ESA and SARA, and; (4) no recovery goal aimed to restore populations to historic levels. Combined, these findings provide guidance to strengthen recovery goals and improve subsequent conservation outcomes. In particular, species at risk planners should seek to attain higher recovery goal ambition, particularly for SARA-listed species, and include quantitative recovery goals wherever possible.

HUMAN IMPACTS. The unique ecology of human predators.

Paradigms of sustainable exploitation focus on population dynamics of prey and yields to humanity but ignore the behavior of humans as predators. We compared patterns of predation by contemporary hunters and fishers with those of other predators that compete over shared prey (terrestrial mammals and marine fishes). Our global survey (2125 estimates of annual finite exploitation rate) revealed that humans kill adult prey, the reproductive capital of populations, at much higher median rates than other predators (up to 14 times higher), with particularly intense exploitation of terrestrial carnivores and fishes. Given this competitive dominance, impacts on predators, and other unique predatory behavior, we suggest that humans function as an unsustainable "super predator," which­unless additionally constrained by managers­will continue to alter ecological and evolutionary processes globally.

Stress and reproductive hormones reflect inter-specific social and nutritional conditions mediated by resource availability in a bear-salmon system.

Food availability can influence the nutritional and social dynamics within and among species. Our investigation focused on grizzly and black bears in coastal British Columbia, Canada, where recent and dramatic declines in their primary prey (salmon) raise concerns about potentially negative effects on bear physiology. We examined how salmon availability relates to stress and reproductive hormones in coastal grizzly (n = 69) and black bears (n = 68) using cortisol and testosterone. In hair samples from genotyped individuals, we quantified salmon consumption using stable isotope analysis and hormone levels by enzyme immunoassay. To estimate the salmon biomass available to each bear, we developed a spatially explicit approach based on typical bear home-range sizes. Next, we compared the relative importance of salmon consumption and salmon availability on hormone levels in male bears using an information theoretical approach. Cortisol in grizzly bears was higher in individuals that consumed less salmon, possibly reflecting nutritional stress. In black bears, cortisol was better predicted by salmon availability than salmon consumption; specifically, individuals in areas and years with low salmon availability showed higher cortisol levels. This indicates that cortisol in black bears is more strongly influenced by the socially competitive environment mediated by salmon availability than by nutritional requirements. In both species, testosterone generally decreased with increasing salmon availability, possibly reflecting a less competitive environment when salmon were abundant. Differences between species could relate to different nutritional requirements, social densities and competitive behaviour and/or habitat use. We present a conceptual model to inform further investigations in this and other systems. Our approach, which combines data on multiple hormones with dietary and spatial information corresponding to the year of hair growth, provides a promising tool for evaluating the responses of a broad spectrum of wildlife to changes in food availability or other environmental conditions.

Stress and reproductive hormones in grizzly bears reflect nutritional benefits and social consequences of a salmon foraging niche.

Physiological indicators of social and nutritional stress can provide insight into the responses of species to changes in food availability. In coastal British Columbia, Canada, grizzly bears evolved with spawning salmon as an abundant but spatially and temporally constrained food source. Recent and dramatic declines in salmon might have negative consequences on bear health and ultimately fitness. To examine broadly the chronic endocrine effects of a salmon niche, we compared cortisol, progesterone, and testosterone levels in hair from salmon-eating bears from coastal BC (n = 75) with the levels in a reference population from interior BC lacking access to salmon (n = 42). As predicted, testosterone was higher in coastal bears of both sexes relative to interior bears, possibly reflecting higher social density on the coast mediated by salmon availability. We also investigated associations between the amount of salmon individual bears consumed (as measured by stable isotope analysis) and cortisol and testosterone in hair. Also as predicted, cortisol decreased with increasing dietary salmon and was higher after a year of low dietary salmon than after a year of high dietary salmon. These findings at two spatial scales suggest that coastal bears might experience nutritional or social stress in response to on-going salmon declines, providing novel insights into the effects of resource availability on fitness-related physiology.

Hair as a meaningful measure of baseline cortisol levels over time in dogs.

Cortisol measurements of hair are becoming a valuable tool in monitoring chronic stress. To further validate this approach in domestic dogs, we compared the variability of cortisol immunoreactivity in hair with that in saliva and feces of dogs housed under constant social and physical conditions. Fecal (n = 268), and hair (n = 21) samples were collected over 3 mo from 7 dogs housed in a kennel and kept for training veterinary students in minimally invasive procedures. Salivary samples (n = 181) were collected 3 times daily twice weekly during the last month of the study. Hair and salivary samples were analyzed by enzyme immunoassay and feces by radioimmunoassay. HPLC coupled with tandem mass spectrometry was used to confirm the presence of cortisol in 3 hair samples. Variability of cortisol was compared across sample types by using repeated-measures ANOVA followed by paired t tests. Within dogs, cortisol immunoreactivity was less variable in hair than in saliva or feces. Averaged over time, the variability of fecal samples approached that of hair when feces were collected at least 4 times monthly. As predicted, the stable social and environmental condition of the dogs maintained repeatability over time and supported the hypothesis that data from hair samples reflect baseline cortisol levels. These findings indicate that determining cortisol immunoreactivity in hair is a more practical approach than is using samples of saliva or feces in monitoring the effects of long-term stressors such as social or physical environments and disease progression.

Exposure to infectious agents in dogs in remote coastal British Columbia: Possible sentinels of diseases in wildlife and humans.

Ranked among the top threats to conservation worldwide, infectious disease is of particular concern for wild canids because domestic dogs (Canis familiaris) may serve as sources and reservoirs of infection. On British Columbia's largely undeveloped but rapidly changing central and north coasts, little is known about diseases in wolves (Canis lupus) or other wildlife. However, several threats exist for transfer of diseases among unvaccinated dogs and wolves. To gain baseline data on infectious agents in this area, including those with zoonotic potential, we collected blood and stool samples from 107 dogs in 5 remote communities in May and September 2007. Serology revealed that the dogs had been exposed to canine parvovirus, canine distemper virus, Bordetella bronchiseptica, canine respiratory coronavirus, and Leptospira interrogans. No dogs showed evidence of exposure to Ehrlichia canis, Anaplasma phagocytophilum, Borrelia burgdorferi, Dirofilaria immitis, or Cryptococcus gattii. Of 75 stool samples, 31 contained at least 1 parasitic infection, including Taeniid tapeworms, the nematodes Toxocara canis and Toxascaris leonina, and the protozoans Isospora sp., Giardia sp., Cryptosporidium sp., and Sarcocystis sp. This work provides a sound baseline for future monitoring of infectious agents that could affect dogs, sympatric wild canids, other wildlife, and humans.

Identification of Parelaphostrongylus odocoilei (Nematoda: Protostrongylidae) first-stage larvae in the feces of gray wolves (Canis lupus) by molecular methods.

First-stage nematode larvae with a dorsal-spine (DSL) were detected in five of 1,565 fecal samples from gray wolves (Canis lupus) collected in British Columbia, Canada, between 2005 and 2008. Molecular techniques were used to identify the DSL because it was not possible to determine their species identity using morphologic characters. The DSL were identified as Parelaphostrongylus odocoilei based on the results of single-strand conformation polymorphism (SSCP) analyses and DNA sequencing of the ribosomal DNA first and second internal transcribed spacers. Finding DSL of P. odocoilei in the feces of gray wolves was unexpected because P. odocoilei adults are parasites of cervids and bovids. The most likely explanation for the presence of DSL in wolf feces is that they were ingested along with the viscera of recently consumed prey. This was probably black-tailed deer (Odocoileus hemionus columbianus), which are known in the sampling area to be hosts of P. odocoilei. The present study demonstrates the use of SSCP and DNA sequencing for the identification, to the species level, of parasitic nematode larvae in feces.

The alliance for cellular signaling plasmid collection: a flexible resource for protein localization studies and signaling pathway analysis.

Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines.