Haematological and immunological characteristics of eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) infected and co-infected with endo- and ectoparasites.

Disease is among the leading causes of the global decline in amphibian populations. In North America, parasites and pathogens are among the factors implicated in precipitous population declines of the giant hellbender salamander (Cryptobranchus alleganiensis), but the incidence of infections and the responses of hellbenders to infections remain poorly studied. Here, we document the prevalence of leech and trypanosome infections in a wild population of eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) and describe haematological and immunological characteristics of hellbenders harbouring these infections. We hypothesized that hellbenders parasitized by trypanosomes would be anaemic, that individuals infected with either or both parasites would exhibit shifts in white blood cell counts and that hellbenders infected with leeches would exhibit altered plasma bactericidal capacity. We found that 24 and 68% of hellbenders in our sample population were infected with leeches and trypanosomes, respectively, and 20% were co-infected with both parasites. We found no evidence suggestive of anaemia among infected individuals. However, hellbenders infected with either or both parasites exhibited marked shifts in circulating white blood cells that were consistent with predictable responses to parasitic infection. Additionally, we found that hellbenders harbouring leeches had much higher plasma bactericidal capacity than individuals without leeches, and we offer multiple potential mechanistic explanations for this observation. We also found evidence that cellular and serological immune responses to parasites were less robust in juvenile than adult hellbenders. This finding warrants further investigation in light of the demographic characteristics, specifically the scarcity of juvenile age classes, of hellbender populations where disease is a possible contributor to declines. Finally, we describe two methodological advances that will improve future studies seeking to diagnose trypanosome infections and to test the bactericidal capacity of hellbenders and perhaps other amphibians. Our study provides fundamental insights into how hellbenders respond physiologically to endo- and ectoparasites, which could ultimately prove useful for their conservation.

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.