A species-specific digital PCR assay for the endangered blue racer (Coluber constrictor foxii) in Canada.

The only population of the endangered blue racer (Coluber constrictor foxii) in Canada occurs on Pelee Island, Ontario. The species is threatened by multiple factors, including habitat degradation and loss, road mortality, persecution, and potentially predation. We designed and evaluated the performance of an environmental DNA droplet digital PCR assay that can be used for multiple facets of conservation of this species. We tested the assay in silico and in vitro using DNA of blue racers and co-occurring snake species and estimated the LOD and LOQ using synthetic DNA. As wild turkey predation has been suggested to negatively affect racers, we tested the assay on eight wild turkey faecal samples. Our assay is specific, can detect the target species at very low levels of concentration (0.002 copies/µL), and can accurately quantify copy numbers ≥ 0.26 copies/µL. We detected no racer DNA in any wild turkey faecal sample. More faecal samples collected at strategic locations during snake peak activity on Pelee Island would enable a more thorough assessment of the possibility of turkey predation. Our assay should be effective for other environmental samples and can be used for investigating other factors negatively affecting blue racers, for example, helping to quantify blue racer habitat suitability and site occupancy.

WEST NILE VIRUS SEROCONVERSION IN EASTERN LOGGERHEAD SHRIKE (LANIUS LUDOVICIANUS MIGRANS) AFTER VACCINATION WITH A KILLED VACCINE.

The loggerhead shrike (Lanius ludovicianus migrans) is a migratory songbird that has undergone massive population declines in Ontario since the 1950s. As part of a broad strategy of recovery, a captive breeding population was established in the late 1990s. This species appears to be extremely sensitive to West Nile virus (WNV) infection, with prior outbreaks at Ontario breeding facilities reaching a 100% mortality rate. This study aimed to investigate the humoral response to vaccination in juvenile birds given single versus serial booster vaccinations, as well as to assess the duration of protective virus-neutralizing titers in annually vaccinated adult birds, by measuring WNV-neutralizing antibodies via the Plaque Reduction Neutralization Test. Twenty-two adult birds and forty 18-22-day-old chicks were included in the study. Annual vaccination resulted in serum neutralizing antibody against WNV for only 59% of adult individuals 1 yr following vaccination. These results, coupled with the death of one vaccinated adult individual due to WNV infection, suggest that a second booster vaccination may be required to adequately protect adult individuals throughout the WNV transmission season. The results of the trial involving juvenile birds indicate that vaccination does not effectively stimulate the immune system of naïve juveniles to produce serum-neutralizing antibodies against WNV in the majority of tested birds, although serial booster vaccination appears to provide a level of improved seroconversion. However, the loss of 19% of naïve juveniles to natural WNV infection versus a less than 3% loss of juveniles that received at least one vaccination suggests some level of cell-mediated immunity and protection against infection takes place in juvenile birds postvaccination. The deaths of several nonvaccinated juveniles and one vaccinated adult at this study facility suggest that WNV continues to be a pathogen of high risk in this species in captivity, and likely in the wild as well.

Visually Sexing Loggerhead Shrike (Lanius Ludovicianus) Using Plumage Coloration and Pattern.

The loggerhead shrike is a small sexually monomorphic passerine bird using grassland habitats across North America. Based on Breeding Bird Survey data, the species has undergone a drastic decline since the mid-1960s. The cause of decline is unknown, and research is actively underway to address this knowledge gap. These efforts are hindered by an inability to sex the species in hand, which to date was only possible using molecular markers. Here, we present a protocol to sex loggerhead shrikes by visually analyzing the coloration and pattern in the sixth primary feather. The application of the method will facilitate our ability to identify threats on a finer scale than has been possible to date and to address various ecological and evolutionary hypotheses. The methodology is simple and results reliable-we encourage including this method for research of both in situ and ex situ populations.

Migratory connectivity in the Loggerhead Shrike (Lanius ludovicianus).

AIM: We combine genetic and stable isotope data to quantify migration patterns in Loggerhead Shrike (Lanius ludovicianus), a species of conservation concern in North America, to assess how connectivity differs and impacts population evolution, ecology, and conservation. LOCATION: We sampled shrikes across the majority of their nonbreeding range, from the Atlantic Coast to the western United States east of the Rocky Mountains and throughout Mexico. METHODS: Our study used a Bayesian framework using δ2Hf from a breeding season origin feather and nuclear genetic microsatellite markers to distinguish between co-occurring migratory and nonmigratory individuals on the wintering grounds and, for migrants, to assign individuals to a breeding ground origin and genetic group. RESULTS: Migratory shrikes were present throughout the nonbreeding range but the proportion differed among sample areas. Four main wintering areas were identified. Connectivity ranged from weakly negative in birds wintering on the Atlantic Coast to strongly positive between wintering grounds in the southwestern United States and Mexico and northwestern breeding populations. Connectivity was weakest in L. l. migrans, and strongest in L. l. mexicanus and L. l. excubitorides. Although believed to be nonmigratory, long-distance movements of individuals were observed in L. ludovicianus and L. l. mexicanus. Our data support a pattern of chain migration, again most notable in the western half of the species nonbreeding range, and differential migration based on age. MAIN CONCLUSIONS: Our study provides of one such of the first quantitative measures of migratory connectivity and is among the first studies of a short-distance migratory passerine in North America. The higher migratory connectivity among western, versus eastern populations, and less severe population declines attributable to habitat loss or reproductive success, may result in more localized and/or less severe limiting factors for western populations and more severe on the Atlantic coast and Mississippi Alluvial Valley wintering grounds.

Advances in linking wintering migrant birds to their breeding-ground origins using combined analyses of genetic and stable isotope markers.

An enduring problem in avian ecology and conservation is linking breeding and wintering grounds of migratory species. As migratory species and populations vary in the degree to which individuals from distinct breeding locales mix on stop-over sites and wintering grounds, establishing migratory connectivity informs our understanding of population demography and species management. We present a new Bayesian approach for inferring breeding grounds of wintering birds of unknown origins in North America. We incorporate prior information from analysis of genetic markers into geographic origin assignment based upon stable-hydrogen isotope analysis of feathers (δ(2)H(f)), using the Loggerhead Shrike (Lanius ludovicianus). Likely geographic origins derived from analyses of DNA microsatellites were used as priors for Bayesian analyses in which birds were assigned to a breeding-ground origin using their δ(2)H(f) values. As with most applications of Bayesian methods, our approach greatly improved the results (i.e. decreased the size of the potential area of origin). Area of origin decreased by 3 to 5-fold on average, but ranged up to a 10-fold improvement. We recommend this approach in future studies of migratory connectivity and suggest that our methodology could be applied more broadly to the study of dispersal, sources of productivity of migratory populations, and a range of evolutionary phenomena.