Winter is coming-Temperature affects immune defenses and susceptibility to Batrachochytrium salamandrivorans.

Environmental temperature is a key factor driving various biological processes, including immune defenses and host-pathogen interactions. Here, we evaluated the effects of environmental temperature on the pathogenicity of the emerging fungal pathogen, Batrachochytrium salamandrivorans (Bsal), using controlled laboratory experiments, and measured components of host immune defense to identify regulating mechanisms. We found that adult and juvenile Notophthalmus viridescens died faster due to Bsal chytridiomycosis at 14°C than at 6 and 22°C. Pathogen replication rates, total available proteins on the skin, and microbiome composition likely drove these relationships. Temperature-dependent skin microbiome composition in our laboratory experiments matched seasonal trends in wild N. viridescens, adding validity to these results. We also found that hydrophobic peptide production after two months post-exposure to Bsal was reduced in infected animals compared to controls, perhaps due to peptide release earlier in infection or impaired granular gland function in diseased animals. Using our temperature-dependent susceptibility results, we performed a geographic analysis that revealed N. viridescens populations in the northeastern United States and southeastern Canada are at greatest risk for Bsal invasion, which shifted risk north compared to previous assessments. Our results indicate that environmental temperature will play a key role in the epidemiology of Bsal and provide evidence that temperature manipulations may be a viable disease management strategy.

Multi-tissue microarray analysis identifies a molecular signature of regeneration.

The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine.

Parasite age-intensity relationships in red-spotted newts: does immune memory influence salamander disease dynamics?

Acquired immune memory in vertebrates influences transmission and persistence of infections, with consequences for parasite dynamics at both the individual and population levels. The potential impact of acquired immunity is of particular interest for salamanders, whose acquired immune systems are thought to be less effective than those of frogs and other tetrapods. One way to examine the importance of acquired immunity to parasite dynamics at the population level is by examining the relationship between host age and parasite infection intensity. Acquired immunity reduces infection rates in older animals, causing decreased parasite intensity in older age classes and leading to curvilinear age-intensity relationships for persistent parasites and convex age-intensity relationships for transient parasites. We used age-intensity relationships to look for the signature of acquired immunity for 12 parasite taxa of red-spotted newts (Notophthalmus viridescens), using data from a 2-year parasitological survey of six newt populations. We estimated ages from snout-vent length (SVL) based on the relationship between SVL and skeletochronologically-derived ages in a subset of newts. We found evidence of acquired immunity to two parasite taxa, bacterial pathogens and the protist Amphibiocystidium viridescens, whose convex age-intensity relationships could not be easily explained by alternative mechanisms. Our results suggest that the acquired immune response of newts is sufficient to influence the dynamics of at least some parasites.

Appearance and regulation of an antigen associated with limb regeneration in Notophthalmus viridescens.

Previous reports from this laboratory have documented a cellular antigen, 22/18, associated with regeneration of forelimbs in the newt Notophthalmus viridescens (Kintner and Brockes: Nature 308:67-69, 1984; Journal of Embryology and Experimental Morphology 89:37-55, 1985; Brockes: Science 225:1280-1287, 1984; Kintner et al.: Molecular Basis of Neural Development, 1985). The antigen is expressed at various stages of regeneration in several types of cells including Schwann cells and muscle cells. We report here that, in addition to the previous results, the expression of 22/18 occurs very early in the regeneration process and is initially confined to an apparently nonmuscle, non-neural population of cells. The well-defined context of the antigen's first appearance has allowed us to investigate the regulation of its expression. In particular, injury is sufficient to elicit the appearance of 22/18. It can appear prior to mitosis and despite denervation. In circumstances where regeneration is inhibited, expression of 22/18 can persist.

The substitution of carrier priming of helper function in the common American newt, Notophthalmus viridescens by lectins and human lymphokines.

There is no clear evidence that helper function is thymus dependent in the Common American newt, Notophthalmus viridescens. Here we test the capacity of concanavalin A, wheat germ agglutinin, human rIL-1 and rIL-2, reagents which stimulate T cell activities in other species, to substitute for carrier priming in the newt. Cytofluorimetric analyses have been used to demonstrate specific IL-2 receptor binding sites on newt splenocytes. Competitive pre-binding with rIL-2 tested whether anti-IL-2 receptor antibody binding sites would bind rIL-2. While Con A can substitute for carrier priming in the newt only when it is presented on Sepharose or agarose particles, wheat germ agglutinin cannot, even when it is injected in particulate form. Additionally, human rIL-1 can serve as an effective substitute for carrier priming, but rIL-2 cannot. The cytofluorimetric data are in agreement with the functional data in that they suggest that human rIL-2 may not bind newt splenocytes. Our data which show shared lectin specificities with T cell regulated helper function in another amphibian species are consistant with the possibility that T-like cells are responsible for helper function in this species.

Hapten, carrier recognition and response by immunocytes of the primitive vertebrate, Notophthalmus viridescens.

These experiments were designed to test whether newt (Notophthalmus viridescens) immunocytes are able to recognize the hapten, 2,4,6-trinitrophenyl (TNP), separately from a heterologous erythrocyte (RBC) carrier. While a single priming injection with an erythrocyte species which is unrelated to the carrier of the TNP fails to stimulate amplification of the anti-TNP response, sequential (2X) priming provides about 50% as much amplification as a single priming injection of RBC homologous with the carrier. The anti-carrier response is not increased by sequential priming with the unrelated RBC. Moreover, sequential priming with RBC homologous with the carrier species initiates a suppression of the anti-hapten response without affecting anti-carrier activity levels. Thus the recognition and regulation of responses to a hapten and its carrier may be unlinked in this species.

Limitations in response capacity of the newt, Notophthalmus viridescens, to soluble and particulate antigens.

We have investigated the cellular basis of the failure of primitive extant vertebrates, e.g. Notophthalmus viridescens, the American common newt, to respond to soluble thymus-dependent (TD) antigens, e.g. keyhole limpet hemocyanin (KLH). We have found that KLH can be used to prime for amplification of a response to hapten, 2,4,6-trinitrophenyl (TNP) when it is conjugated to KLH, only if both the unconjugated and conjugated KLH are adsorbed onto bentonite particles. The response is monitored in terms of antigen binding cells in the spleen. Moreover, prior injection of colloidal carbon, which is actively engulfed by phagocytes throughout the body, will prevent the response from taking place. Injection of colloidal carbon diminished responses to heterologous erythrocytes and to soluble or bentonite-coated 2,4-dinitrophenylated (DNP) dextran. Comparable colloidal carbon treatment did not reduce response levels to either soluble or bentonite adsorbed TNP-lipopolysaccharide (LPS) of E. coli. We suggest that the limitation in response capacity to soluble TD antigens may be associated with regulatory phagocytes. Moreover, the phagocytic cells of this primitive vertebrate appear to mediate responses to naturally particulate TD antigens and certain thymus-independent (TI) carriers, e.g. dextran, but not to LPS.