Interacting symbionts and immunity in the amphibian skin mucosome predict disease risk and probiotic effectiveness.

Pathogenesis is strongly dependent on microbial context, but development of probiotic therapies has neglected the impact of ecological interactions. Dynamics among microbial communities, host immune responses, and environmental conditions may alter the effect of probiotics in human and veterinary medicine, agriculture and aquaculture, and the proposed treatment of emerging wildlife and zoonotic diseases such as those occurring on amphibians or vectored by mosquitoes. Here we use a holistic measure of amphibian mucosal defenses to test the effects of probiotic treatments and to assess disease risk under different ecological contexts. We developed a non-invasive assay for antifungal function of the skin mucosal ecosystem (mucosome function) integrating host immune factors and the microbial community as an alternative to pathogen exposure experiments. From approximately 8500 amphibians sampled across Europe, we compared field infection prevalence with mucosome function against the emerging fungal pathogen Batrachochytrium dendrobatidis. Four species were tested with laboratory exposure experiments, and a highly susceptible species, Alytes obstetricans, was treated with a variety of temperature and microbial conditions to test the effects of probiotic therapies and environmental conditions on mucosome function. We found that antifungal function of the amphibian skin mucosome predicts the prevalence of infection with the fungal pathogen in natural populations, and is linked to survival in laboratory exposure experiments. When altered by probiotic therapy, the mucosome increased antifungal capacity, while previous exposure to the pathogen was suppressive. In culture, antifungal properties of probiotics depended strongly on immunological and environmental context including temperature, competition, and pathogen presence. Functional changes in microbiota with shifts in temperature provide an alternative mechanistic explanation for patterns of disease susceptibility related to climate beyond direct impact on host or pathogen. This nonlethal management tool can be used to optimize and quickly assess the relative benefits of probiotic therapies under different climatic, microbial, or host conditions.

Genetic attributes of midwife toad (Alytes obstetricans) populations do not correlate with degree of species decline.

Genetic diversity is crucial for long-term population persistence. Population loss and subsequent reduction in migration rate among the most important processes that are expected to lead to a reduction in genetic diversity and an increase in genetic differentiation. While the theory behind this is well-developed, empirical evidence from wild populations is inconsistent. Using microsatellite markers, we compared the genetic structure of populations of an amphibian species, the midwife toad (Alytes obstetricans), in four Swiss regions where the species has suffered variable levels of subpopulation extirpation. We also quantified the effects of several geographic factors on genetic structure and used a model selection approach to ascertain which of the variables were important for explaining genetic variation. Although subpopulation pairwise F ST-values were highly significant even over small geographic scales, neither any of the geographic variables nor loss of subpopulations were important factors for predicting spatial genetic structure. The absence of a signature of subpopulation loss on genetic differentiation may suggest that midwife toad subpopulations function as relatively independent units.

Surface microstructures on planar substrates and textile fibers guide neurite outgrowth: a scaffold solution to push limits of critical nerve defect regeneration?

The treatment of critical size peripheral nerve defects represents one of the most serious problems in neurosurgery. If the gap size exceeds a certain limit, healing can't be achieved. Connection mismatching may further reduce the clinical success. The present study investigates how far specific surface structures support neurite outgrowth and by that may represent one possibility to push distance limits that can be bridged. For this purpose, growth cone displacement of fluorescent embryonic chicken spinal cord neurons was monitored using time-lapse video. In a first series of experiments, parallel patterns of polyimide ridges of different geometry were created on planar silicon oxide surfaces. These channel-like structures were evaluated with and without amorphous hydrogenated carbon (a-C:H) coating. In a next step, structured and unstructured textile fibers were investigated. All planar surface materials (polyimide, silicon oxide and a-C:H) proved to be biocompatible, i.e. had no adverse effect on nerve cultures and supported neurite outgrowth. Mean growth cone migration velocity measured on 5 minute base was marginally affected by surface structuring. However, surface structure variability, i.e. ridge height, width and inter-ridge spacing, significantly enhanced the resulting net velocity by guiding the growth cone movement. Ridge height and inter-ridge distance affected the frequency of neurites crossing over ridges. Of the evaluated dimensions ridge height, width, and inter-ridge distance of respectively 3, 10, and 10 µm maximally supported net axon growth. Comparable artificial grooves, fabricated onto the surface of PET fibers by using an excimer laser, showed similar positive effects. Our data may help to further optimize surface characteristics of artificial nerve conduits and bioelectronic interfaces.

Populations of a susceptible amphibian species can grow despite the presence of a pathogenic chytrid fungus.

Disease can be an important driver of host population dynamics and epizootics can cause severe host population declines. Batrachochytrium dendrobatidis (Bd), the pathogen causing amphibian chytridiomycosis, may occur epizootically or enzootically and can harm amphibian populations in many ways. While effects of Bd epizootics are well documented, the effects of enzootic Bd have rarely been described. We used a state-space model that accounts for observation error to test whether population trends of a species highly susceptible to Bd, the midwife toad Alytes obstetricans, are negatively affected by the enzootic presence of the pathogen. Unexpectedly, Bd had no negative effect on population growth rates from 2002-2008. This suggests that negative effects of disease on individuals do not necessarily translate into negative effects at the population level. Populations of amphibian species that are susceptible to the emerging disease chytridiomycosis can persist despite the enzootic presence of the pathogen under current environmental conditions.

Within- and among-population variation in chytridiomycosis-induced mortality in the toad Alytes obstetricans.

BACKGROUND: Chytridiomycosis is a fungal disease linked to local and global extinctions of amphibians. Susceptibility to chytridiomycosis varies greatly between amphibian species, but little is known about between- and within-population variability. However, this kind of variability is the basis for the evolution of tolerance and resistance evolution to disease. METHODOLOGY/PRINCIPAL FINDINGS: In a common garden experiment, we measured mortality after metamorphosis of Alytes obstetricans naturally infected with Batrachochytrium dendrobatidis. Mortality rates differed significantly among populations and ranged from 27 to 90%. Within populations, mortality strongly depended on mass at and time through metamorphosis. CONCLUSIONS/SIGNIFICANCE: Although we cannot rule out that the differences observed resulted from differences in skin microbiota, different pathogen strains or environmental effects experienced by the host or the pathogen prior to the start of the experiment, we argue that genetic differences between populations are a likely source of at least part of this variation. To our knowledge, this is the first study showing differences in survival between and within populations under constant laboratory conditions. Assuming that some of this intraspecific variation has a genetic basis, this may suggest that there is the potential for the evolution of resistance or tolerance, which might allow population persistence.

Effects of carbon nanotubes on primary neurons and glial cells.

Carbon nanotubes (CNTs) are among the most promising novel nanomaterials and their unique chemical and physical properties suggest an enormous potential for many areas of research and applications. As a consequence, the production of CNT-based material and thus the occupational and public exposure to CNTs will increase steadily. Although there is evidence that nanoparticles (NPs) can enter the nervous system via the blood stream, olfactory nerves or sensory nerves in the skin, there is still only little knowledge about possible toxic effects of CNTs on cells of the nervous system. The goal of the present study was to analyse the influences of single-walled CNTs (SWCNTs) with different degrees of agglomeration on primary cultures derived from chicken embryonic spinal cord (SPC) or dorsal root ganglia (DRG). As measured by the Hoechst assay treatment of mixed neuro-glial cultures with up to 30mug/mL SWCNTs significantly decreased the overall DNA content. This effect was more pronounced if cells were exposed to highly agglomerated SWCNTs as compared to better dispersed SWCNT-bundles. Using a cell-based ELISA we found that SWCNTs reduce the amount of glial cells in both peripheral nervous system (PNS) and central nervous system (CNS) derived cultures. Neurons were only affected in DRG derived cultures, where SWCNT treatment resulted in a decreased number of sensory neurons, as measured by ELISA. Additionally, whole-cell patch recordings revealed a diminished inward conductivity and a more positive resting membrane potential of SWCNT treated DRG derived neurons compared to control samples. The SWCNT suspensions used in this study induced acute toxic effects in primary cultures from both, the central and peripheral nervous system of chicken embryos. The level of toxicity is at least partially dependent on the agglomeration state of the tubes. Thus if SWCNTs can enter the nervous system at sufficiently high concentrations, it is likely that adverse effects on glial cells and neurons might occur.