Effects of exogenous elevation of corticosterone on immunity and the skin microbiome of eastern newts (Notophthalmus viridescens).

The amphibian chytrid fungus, Batrachochytrium salamandrivorans (Bsal) threatens salamander biodiversity. The factors underlying Bsal susceptibility may include glucocorticoid hormones (GCs). The effects of GCs on immunity and disease susceptibility are well studied in mammals, but less is known in other groups, including salamanders. We used Notophthalmus viridescens (eastern newts) to test the hypothesis that GCs modulate salamander immunity. We first determined the dose required to elevate corticosterone (CORT; primary GC in amphibians) to physiologically relevant levels. We then measured immunity (neutrophil lymphocyte ratios, plasma bacterial killing ability (BKA), skin microbiome, splenocytes, melanomacrophage centres (MMCs)) and overall health in newts following treatment with CORT or an oil vehicle control. Treatments were repeated for a short (two treatments over 5 days) or long (18 treatments over 26 days) time period. Contrary to our predictions, most immune and health parameters were similar for CORT and oil-treated newts. Surprisingly, differences in BKA, skin microbiome and MMCs were observed between newts subjected to short- and long-term treatments, regardless of treatment type (CORT, oil vehicle). Taken together, CORT does not appear to be a major factor contributing to immunity in eastern newts, although more studies examining additional immune factors are necessary. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

In Vitro Investigation of the Antibacterial Activity of Salamander Skin Peptides.

Given the current and future costs of antibiotic-resistant bacteria to human health and economic productivity, there is an urgent need to develop new antimicrobial compounds. Antimicrobial peptides are a promising alternative to conventional antibiotics and other antimicrobials. Amphibian skin is a rich source of bioactive compounds, but the antibacterial properties of salamander skin peptides have been neglected. Here, we examined the in vitro ability of skin peptides from 9 species of salamander representing 6 salamander families to inhibit the growth of ESKAPE pathogens, which are bacteria that have developed resistance to conventional antibiotics. We also examined whether the skin peptides caused lysis of human red blood cells. Skin peptides from Amphiuma tridactylum had the greatest antimicrobial properties, completely inhibiting the growth of all bacterial strains except for Enterococcus faecium. Likewise, skin peptides from Cryptobranchus alleganiensis completely inhibited the growth of several of the bacterial strains. In contrast, skin peptide mixtures from Ambystoma maculatum, Desmognathus fuscus, Eurycea bislineata, E. longicauda, Necturus beyeri, N. maculosus, and Siren intermedia did not completely inhibit bacterial growth even at the highest concentrations. Finally, none of the skin peptide mixtures caused lysis of human red blood cells. Together, we demonstrate that salamander skin produces peptides with potent antibacterial properties. It remains to elucidate the peptide sequences and their antibacterial mechanisms.

External nasal gland morphology of Eurycea bislineata (Amphibia, Urodela, Plethodontidae).

Plethodontid salamanders possess numerous courtship glands. Previous studies have shown that the glands are more prominent in male individuals than females, and often experience periods of atrophy and hypertrophy throughout the year that correlate to the nonmating and mating seasons, respectively. We sampled male and female Eurycea bislineata throughout the year to test the hypothesis that external nasal glands are courtship glands. External nasal glands are paired, branched tubular glands that extend from excretory ducts dorsal to the nares to terminal secretory units posterior to the eyes. We found that the glands hypertrophy and stain/react more intensely with histochemical procedures during the mating season. Hypertrophy of the glands is more pronounced in males, and seasonal variation in epithelial height of external nasal glands of males is significantly correlated to that of seasonal variation in mental gland epithelial height, a known courtship gland found in males, when compared throughout the year. This correlation was not as strong in females, confirming sexual dimorphism of external nasal glands in terms of seasonal variation. We found no ultrastructural differences between male and female external nasal glands. In all specimens, the glandular tubules were lined by a simple, columnar epithelium that was packed with secretory granules that often obscured other cytoplasmic contents.

Isolation and maintenance of Batrachochytrium salamandrivorans cultures.

Discovered in 2013, the chytrid fungus Batrachochytrium salamandrivorans (Bsal) is an emerging amphibian pathogen that causes ulcerative skin lesions and multifocal erosion. A closely related pathogen, B. dendrobatidis (Bd), has devastated amphibian populations worldwide, suggesting that Bsal poses a significant threat to global salamander biodiversity. To expedite research into this emerging threat, we seek to standardize protocols across the field so that results of laboratory studies are reproducible and comparable. We have collated data and experience from multiple labs to standardize culturing practices of Bsal. Here we outline common culture practices including a medium for standardized Bsal growth, standard culturing protocols, and a method for isolating Bsal from infected tissue.

Skin glands of an aquatic salamander vary in size and distribution and release antimicrobial secretions effective against chytrid fungal pathogens.

Amphibian skin is unique among vertebrate classes, containing a large number of multicellular exocrine glands that vary among species and have diverse functions. The secretions of skin glands contain a rich array of bioactive compounds including antimicrobial peptides (AMPs). Such compounds are important for amphibian innate immune responses and may protect some species from chytridiomycosis, a lethal skin disease caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). While the bioactivity of skin secretions against Bd has been assessed for many amphibian taxa, similar studies are lacking for Bsal, a chytrid fungus that is especially pathogenic for salamanders. We studied the skin glands and their potential functions in an aquatic salamander, the three-toed amphiuma (Amphiuma tridactylum). Skin secretions of captive adult salamanders were analyzed by RP-HPLC and tested against the growth of Bd and Bsal using in vitro assays. We found that compounds within collected skin secretions were similar between male and female salamanders and inhibited the growth of Bd and Bsal. Thus, skin secretions that protect against Bd may also provide protection against Bsal. Histological examination of the skin glands of preserved salamanders revealed the presence of enlarged granular glands concentrated within caudal body regions. A site of potential gland specialization was identified at the tail base and may indicate specialized granular glands related to courtship and communication.