Using a keratinase to degrade chicken feathers for improved extraction of glucocorticoids.

Stress in animals is a concern in conservation breeding programs and livestock production facilities. The biological stress response is mediated by the release of glucocorticoids, which can suppress reproduction, growth, and immunity if recurrently activated. Feathers can be used to extract and monitor concentrations of corticosterone, a primary glucocorticoid in birds. However, current techniques for extracting feather corticosterone present challenges, including difficulty assessing extraction efficiency or hormone recovery, inconsistent extraction across feather lengths or pieces, and several uncertainties regarding the mechanisms of hormone deposition into feathers. To overcome such challenges and to provide tools useful for facilitating conservation breeding and livestock production, we developed and validated an alternative procedure for extracting feather glucocorticoids. We first developed a protocol to enzymatically digest the protein matrix of feathers using a keratinase, such that non-protein analytes could be isolated by organic extraction. We then developed an extraction protocol and evaluated techniques by measuring extraction efficiency and by testing parallelism and hormone recovery (accuracy) using radioimmunoassay. Our results demonstrated high and consistent extraction efficiency, as well as high accuracy and reliable parallelism to a standard curve upon measurement of corticosterone concentrations from extracts. By dissolving feather material into solution prior to extraction, we were able to replicate hormone deposition into the feather matrix and ensure consistent extraction across feathers. This work provides additional support for the validity and practicality of extracting glucocorticoids from feathers. Our extraction protocol is likely to extend to other applications as well, including the isolation of numerous non-protein analytes from various keratinized tissues.

A re-evaluation of a case-control model with contaminated controls for resource selection studies.

1. A common sampling design in resource selection studies involves measuring resource attributes at sample units used by an animal and at sample units considered available for use. Few models can estimate the absolute probability of using a sample unit from such data, but such approaches are generally preferred over statistical methods that estimate a relative probability of use. 2. The case-control model that allows for contaminated controls, proposed by Lancaster & Imbens (1996) and Lele (2009), can estimate the absolute probability of using a sample unit from use-availability data. However, numerous misconceptions have likely prevented the widespread application of this model to resource selection studies. We address common misconceptions regarding the case-control model with contaminated controls and demonstrate its ability to estimate the absolute probability of use, prevalence and parameters associated with categorical covariates from use-availability data. 3. We fit the case-control model with contaminated controls to simulated data with varying prevalence (defined as the average probability of use across all sample units) and sample sizes (n1 = 500 used and na = 500 available samples; n1 = 1000 used and na = 1000 available samples). We then applied this model to estimate the probability Ozark hellbenders (Cryptobranchus alleganiensis bishopi) would use a location within a stream as a function of covariates. 4. The case-control model with contaminated controls provided unbiased estimates of all parameters at N = 2000 sample size simulation scenarios, particularly at low prevalence. However, this model produced increasingly variable maximum likelihood estimates of parameters as prevalence increased, particularly at N = 1000 sample size scenarios. We thus recommend at least 500-1000 used samples when fitting the case-control model with contaminated controls to use-availability data. Our application to hellbender data revealed selection for locations with coarse substrate that are close to potential sources of cover. 5. This study unites a disparate literature, addresses and clarifies many commonly held misconceptions and demonstrates that the case-control model with contaminated controls is a viable alternative for estimating the absolute probability of use from use-availability data.

Environmental drivers of autumn migration departure decisions in midcontinental mallards.

BACKGROUND: The timing of autumn migration in ducks is influenced by a range of environmental conditions that may elicit individual experiences and responses from individual birds, yet most studies have investigated relationships at the population level. We used data from individual satellite-tracked mallards (Anas platyrhynchos) to model the timing and environmental drivers of autumn migration movements at a continental scale. METHODS: We combined two sets of location records (2004-2007 and 2010-2011) from satellite-tracked mallards during autumn migration in the Mississippi Flyway, and identified records that indicated the start of long-range (≥ 30 km) southward movements during the migration period. We modeled selection of departure date by individual mallards using a discrete choice model accounting for heterogeneity in individual preferences. We developed candidate models to predict the departure date, conditional on daily mean environmental covariates (i.e. temperature, snow and ice cover, wind conditions, precipitation, cloud cover, and pressure) at a 32 × 32 km resolution. We ranked model performance with the Bayesian Information Criterion. RESULTS: Departure was best predicted (60% accuracy) by a "winter conditions" model containing temperature, and depth and duration of snow cover. Models conditional on wind speed, precipitation, pressure variation, and cloud cover received lower support. Number of days of snow cover, recently experienced snow cover (snow days) and current snow cover had the strongest positive effect on departure likelihood, followed by number of experienced days of freezing temperature (frost days) and current low temperature. Distributions of dominant drivers and of correct vs incorrect prediction along the movement tracks indicate that these responses applied throughout the latitudinal range of migration. Among recorded departures, most were driven by snow days (65%) followed by current temperature (30%). CONCLUSIONS: Our results indicate that among the tested environmental parameters, the dominant environmental driver of departure decision in autumn-migrating mallards was the onset of snow conditions, and secondarily the onset of temperatures close to, or below, the freezing point. Mallards are likely to relocate southwards quickly when faced with snowy conditions, and could use declining temperatures as a more graduated early cue for departure. Our findings provide further insights into the functional response of mallards to weather factors during the migration period that ultimately determine seasonal distributions.

Transgenic insertion of the cyanobacterial membrane protein ictB increases grain yield in Zea mays through increased photosynthesis and carbohydrate production.

The C4 crop maize (Zea mays) is the most widely grown cereal crop worldwide and is an essential feedstock for food and bioenergy. Improving maize yield is important to achieve food security and agricultural sustainability in the 21st century. One potential means to improve crop productivity is to enhance photosynthesis. ictB, a membrane protein that is highly conserved across cyanobacteria, has been shown to improve photosynthesis, and often biomass, when introduced into diverse C3 plant species. Here, ictB from Synechococcus sp. strain PCC 7942 was inserted into maize using Agrobacterium-mediated transformation. In three controlled-environment experiments, ictB insertion increased leaf starch and sucrose content by up to 25% relative to controls. Experimental field trials in four growing seasons, spanning the Midwestern United States (Summers 2018 & 2019) and Argentina (Winter 2018 & 2019), showed an average of 3.49% grain yield improvement, by as much as 5.4% in a given season and up to 9.4% at certain trial locations. A subset of field trial locations was used to test for modification of ear traits and ФPSII, a proxy for photosynthesis. Results suggested that yield gain in transgenics could be associated with increased ФPSII, and the production of longer, thinner ears with more kernels. ictB localized primarily to the microsome fraction of leaf bundle-sheath cells, but not to chloroplasts. Extramembrane domains of ictB interacted in vitro with proteins involved in photosynthesis and carbohydrate metabolism. To our knowledge, this is the first published evidence of ictB insertion into a species using C4 photosynthesis and the largest-scale demonstration of grain yield enhancement from ictB insertion in planta. Results show that ictB is a valuable yield gene in the economically important crop maize, and are an important proof of concept that transgenic manipulation of photosynthesis can be used to create economically viable crop improvement traits.

Bats in a changing landscape: Linking occupancy and traits of a diverse montane bat community to fire regime.

Wildfires are increasing in incidence and severity across coniferous forests of the western United States, leading to changes in forest structure and wildlife habitats. Knowledge of how species respond to fire-driven habitat changes in these landscapes is limited and generally disconnected from our understanding of adaptations that underpin responses to fire.We aimed to investigate drivers of occupancy of a diverse bat community in a fire-altered landscape, while identifying functional traits that underpinned these relationships.We recorded bats acoustically at 83 sites (n = 249 recording nights) across the Plumas National Forest in the northern Sierra Nevada over 3 summers (2015-2017). We investigated relationships between fire regime, physiographic variables, forest structure and probability of bat occupancy for nine frequently detected species. We used fourth-corner regression and RLQ analysis to identify ecomorphological traits driving species-environment relationships across 17 bat species. Traits included body mass; call frequency, bandwidth, and duration; and foraging strategy based on vegetation structure (open, edge, or clutter).Relationships between bat traits and fire regime were underpinned by adaptations to diverse forest structure. Bats with traits adapting them to foraging in open habitats, including emitting longer duration and narrow bandwidth calls, were associated with higher severity and more frequent fires, whereas bats with traits consistent with clutter tolerance were negatively associated with fire frequency and burn severity. Relationships between edge-adapted bat species and fire were variable and may be influenced by prey preference or habitat configuration at a landscape scale.Predicted increases in fire frequency and severity in western US coniferous forests are likely to shift dominance in the bat community to open-adapted species and those able to exploit postfire resource pulses (aquatic insects, beetles, and snags). Managing for pyrodiversity within the western United States is likely important for maintaining bat community diversity, as well as diversity of other biotic communities.

Phenological mismatch in Arctic-breeding shorebirds: Impact of snowmelt and unpredictable weather conditions on food availability and chick growth.

The ecological consequences of climate change have been recognized in numerous species, with perhaps phenology being the most well-documented change. Phenological changes may have negative consequences when organisms within different trophic levels respond to environmental changes at different rates, potentially leading to phenological mismatches between predators and their prey. This may be especially apparent in the Arctic, which has been affected more by climate change than other regions, resulting in earlier, warmer, and longer summers. During a 7-year study near Utqiagvik (formerly Barrow), Alaska, we estimated phenological mismatch in relation to food availability and chick growth in a community of Arctic-breeding shorebirds experiencing advancement of environmental conditions (i.e., snowmelt). Our results indicate that Arctic-breeding shorebirds have experienced increased phenological mismatch with earlier snowmelt conditions. However, the degree of phenological mismatch was not a good predictor of food availability, as weather conditions after snowmelt made invertebrate availability highly unpredictable. As a result, the food available to shorebird chicks that were 2-10 days old was highly variable among years (ranging from 6.2 to 28.8 mg trap-1 day-1 among years in eight species), and was often inadequate for average growth (only 20%-54% of Dunlin and Pectoral Sandpiper broods on average had adequate food across a 4-year period). Although weather conditions vary among years, shorebirds that nested earlier in relation to snowmelt generally had more food available during brood rearing, and thus, greater chick growth rates. Despite the strong selective pressure to nest early, advancement of nesting is likely limited by the amount of plasticity in the start and progression of migration. Therefore, long-term climatic changes resulting in earlier snowmelt have the potential to greatly affect shorebird populations, especially if shorebirds are unable to advance nest initiation sufficiently to keep pace with seasonal advancement of their invertebrate prey.

Functional visual sensitivity to ultraviolet wavelengths in the Pileated Woodpecker (Dryocopus pileatus), and its influence on foraging substrate selection.

Most diurnal birds are presumed visually sensitive to near ultraviolet (UV) wavelengths, however, controlled behavioral studies investigating UV sensitivity remain few. Although woodpeckers are important as primary cavity excavators and nuisance animals, published work on their visual systems is limited. We developed a novel foraging-based behavioral assay designed to test UV sensitivity in the Pileated Woodpecker (Dryocopus pileatus). We acclimated 21 wild-caught woodpeckers to foraging for frozen mealworms within 1.2m sections of peeled cedar (Thuja spp.) poles. We then tested the functional significance of UV cues by placing frozen mealworms behind UV-reflective covers, UV-absorptive covers, or decayed red pine substrates within the same 1.2m poles in independent experiments. Behavioral responses were greater toward both UV-reflective and UV-absorptive substrates in three experiments. Study subjects therefore reliably differentiated and attended to two distinct UV conditions of a foraging substrate. Cue-naïve subjects showed a preference for UV-absorptive substrates, suggesting that woodpeckers may be pre-disposed to foraging from such substrates. Behavioral responses were greater toward decayed pine substrates (UV-reflective) than sound pine substrates suggesting that decayed pine can be a useful foraging cue. The finding that cue-naïve subjects selected UV-absorbing foraging substrates has implications for ecological interactions of woodpeckers with fungi. Woodpeckers transport fungal spores, and communication methods analogous to those of plant-pollinator mutualisms (i.e. UV-absorbing patterns) may have evolved to support woodpecker-fungus mutualisms.

Are Isolated Indigenous Populations Headed toward Extinction?

At least 50 indigenous groups spread across lowland South America remain isolated and have only intermittent and mostly hostile interactions with the outside world. Except in emergency situations, the current policy of governments in Brazil, Colombia, and Peru towards isolated tribes is a "leave them alone" strategy, in which isolated groups are left uncontacted. However, these no-contact policies are based on the assumption that isolated populations are healthy and capable of persisting in the face of mounting external threats, and that they can maintain population viability in the long-term. Here, we test this assumption by tracking the sizes and movements of cleared horticultural areas made by 8 isolated groups over the last 10-14 years. We used deforestation data derived from remote sensing Landsat satellite sensors to identify clearings, and those were then validated and assessed with high-resolution imagery. We found only a single example of a relatively large and growing population (c. 50 cleared ha and 400 people), whereas all of the other 7 groups exhibited much smaller villages and gardens with no sizable growth through time. These results indicated that the smaller groups are critically endangered, and it prompts an urgent re-thinking of policies toward isolated populations, including plans for well-organized contacts that may help save lives and rescue isolated indigenous populations from imminent extinction.

Geographic distribution of isolated indigenous societies in Amazonia and the efficacy of indigenous territories.

The headwaters of the Amazon Basin harbor most of the world's last indigenous peoples who have limited contact with encroaching colonists. Knowledge of the geographic distribution of these isolated groups is essential to assist with the development of immediate protections for vulnerable indigenous settlements. We used remote sensing to document the locations of 28 isolated villages within the four Brazilian states of Acre, Amazonas, Roraima, and Rondônia. The sites were confirmed during previous over-flights and by image evidence of thatched-roof houses; they are estimated to host over 1,700 individuals. Locational data were used to train maximum entropy models that identified landscape and anthropogenic features associated with the occurrence of isolated indigenous villages, including elevation, proximity to streams of five different orders, proximity to roads and settlements, proximity to recent deforestation, and vegetation cover type. Isolated villages were identified at mid elevations, within 20 km of the tops of watersheds and at greater distances from existing roads and trails. We further used model results, combined with boundaries of the existing indigenous territory system that is designed to protect indigenous lands, to assess the efficacy of the existing protected area network for isolated peoples. Results indicate that existing indigenous territories encompass all of the villages we identified, and 50% of the areas with high predicted probabilities of isolated village occurrence. Our results are intended to help inform policies that can mitigate against future external threats to isolated peoples.

Crash and rebound of indigenous populations in lowland South America.

Lowland South America has long been a battle-ground between European colonization and indigenous survival. Initial waves of European colonization brought disease epidemics, slavery, and violence that had catastrophic impacts on indigenous cultures. In this paper we focus on the demography of 238 surviving populations in Brazil. We use longitudinal censuses from all known indigenous Brazilian societies to quantify three demographic metrics: 1) effects of European contact on indigenous populations; 2) empirical estimates of minimum viable population sizes; and 3) estimates of post-contact population growth rates. We use this information to conduct population viability analysis (PVA). Our results show that all surviving populations suffered extensive mortality during, and shortly after, contact. However, most surviving populations exhibit positive growth rates within the first decade post-contact. Our findings paint a positive demographic outlook for these indigenous populations, though long-term survival remains subject to powerful externalities, including politics, economics, and the pervasive illegal exploitation of indigenous lands.

The role of wildfire, prescribed fire, and mountain pine beetle infestations on the population dynamics of black-backed woodpeckers in the black hills, South Dakota.

Wildfire and mountain pine beetle infestations are naturally occurring disturbances in western North American forests. Black-backed woodpeckers (Picoides arcticus) are emblematic of the role these disturbances play in creating wildlife habitat, since they are strongly associated with recently-killed forests. However, management practices aimed at reducing the economic impact of natural disturbances can result in habitat loss for this species. Although black-backed woodpeckers occupy habitats created by wildfire, prescribed fire, and mountain pine beetle infestations, the relative value of these habitats remains unknown. We studied habitat-specific adult and juvenile survival probabilities and reproductive rates between April 2008 and August 2012 in the Black Hills, South Dakota. We estimated habitat-specific adult and juvenile survival probability with Bayesian multi-state models and habitat-specific reproductive success with Bayesian nest survival models. We calculated asymptotic population growth rates from estimated demographic rates with matrix projection models. Adult and juvenile survival and nest success were highest in habitat created by summer wildfire, intermediate in MPB infestations, and lowest in habitat created by fall prescribed fire. Mean posterior distributions of population growth rates indicated growing populations in habitat created by summer wildfire and declining populations in fall prescribed fire and mountain pine beetle infestations. Our finding that population growth rates were positive only in habitat created by summer wildfire underscores the need to maintain early post-wildfire habitat across the landscape. The lower growth rates in fall prescribed fire and MPB infestations may be attributed to differences in predator communities and food resources relative to summer wildfire.

Quantitative and qualitative approaches to identifying migration chronology in a continental migrant.

The degree to which extrinsic factors influence migration chronology in North American waterfowl has not been quantified, particularly for dabbling ducks. Previous studies have examined waterfowl migration using various methods, however, quantitative approaches to define avian migration chronology over broad spatio-temporal scales are limited, and the implications for using different approaches have not been assessed. We used movement data from 19 female adult mallards (Anas platyrhynchos) equipped with solar-powered global positioning system satellite transmitters to evaluate two individual level approaches for quantifying migration chronology. The first approach defined migration based on individual movements among geopolitical boundaries (state, provincial, international), whereas the second method modeled net displacement as a function of time using nonlinear models. Differences in migration chronologies identified by each of the approaches were examined with analysis of variance. The geopolitical method identified mean autumn migration midpoints at 15 November 2010 and 13 November 2011, whereas the net displacement method identified midpoints at 15 November 2010 and 14 November 2011. The mean midpoints for spring migration were 3 April 2011 and 20 March 2012 using the geopolitical method and 31 March 2011 and 22 March 2012 using the net displacement method. The duration, initiation date, midpoint, and termination date for both autumn and spring migration did not differ between the two individual level approaches. Although we did not detect differences in migration parameters between the different approaches, the net displacement metric offers broad potential to address questions in movement ecology for migrating species. Ultimately, an objective definition of migration chronology will allow researchers to obtain a comprehensive understanding of the extrinsic factors that drive migration at the individual and population levels. As a result, targeted conservation plans can be developed to support planning for habitat management and evaluation of long-term climate effects.

Taxonomic considerations in listing subspecies under the U.S. Endangered Species Act.

The U.S. Endangered Species Act (ESA) allows listing of subspecies and other groupings below the rank of species. This provides the U.S. Fish and Wildlife Service and the National Marine Fisheries Service with a means to target the most critical unit in need of conservation. Although roughly one-quarter of listed taxa are subspecies, these management agencies are hindered by uncertainties about taxonomic standards during listing or delisting activities. In a review of taxonomic publications and societies, we found few subspecies lists and none that stated standardized criteria for determining subspecific taxa. Lack of criteria is attributed to a centuries-old debate over species and subspecies concepts. Nevertheless, the critical need to resolve this debate for ESA listings led us to propose that minimal biological criteria to define disjunct subspecies (legally or taxonomically) should include the discreteness and significance criteria of distinct population segments (as defined under the ESA). Our subspecies criteria are in stark contrast to that proposed by supporters of the phylogenetic species concept and provide a clear distinction between species and subspecies. Efforts to eliminate or reduce ambiguity associated with subspecies-level classifications will assist with ESA listing decisions. Thus, we urge professional taxonomic societies to publish and periodically update peer-reviewed species and subspecies lists. This effort must be paralleled throughout the world for efficient taxonomic conservation to take place.