Skin peptides protect juvenile leopard frogs (Rana pipiens) against chytridiomycosis

Abstract

SUMMARYOne issue of great concern for the scientific community is the continuing loss of diverse amphibian species on a global scale. Amphibian populations around the world are experiencing serious losses due to the chytrid fungus, Batrachochytrium dendrobatidis. This pathogen colonizes the skin, leading to the disruption of ionic balance and eventual cardiac arrest. In many species, antimicrobial peptides secreted into the mucus are thought to contribute to protection against colonization by skin pathogens. Although it is generally thought that antimicrobial peptides are an important component of innate immune defenses against B. dendrobatidis, much of the current evidence relies on correlations between effective antimicrobial peptide defenses and species survival. There have been few studies to directly demonstrate that antimicrobial peptides play a role. Using the northern leopard frog, Rana pipiens, we show here that injection of noradrenaline (norepinephrine) brings about a long-term depletion of skin peptides (initial concentrations do not recover until after day 56). When peptide stores recovered, the renewed peptides were similar in composition to the initial peptides as determined by MALDI-TOF mass spectrometry and in activity against B. dendrobatidis as determined by growth inhibition assays. Newly metamorphosed froglets depleted of their peptide stores and exposed to B. dendrobatidis died more rapidly than B. dendrobatidis-exposed froglets with their peptides intact. Thus, antimicrobial peptides in the skin mucus appear to provide some resistance to B. dendrobatidis infections, and it is important for biologists to recognize that this defense is especially important for newly metamorphosed frogs in which the adaptive immune system is still immature.

Evaluation of Amphotericin B and Chloramphenicol as Alternative Drugs for Treatment of Chytridiomycosis and Their Impacts on Innate Skin Defenses

“…Pharmacological doses of norepinephrine can be used to completely eliminate amphibian peptide stores from dermal granular glands (56). Following injection of norepinephrine, peptides should recover to their preinjection levels in leopard frogs by 50 days postinjection under normal physiological conditions (50). To assess whether continual exposure to chloramphenicol and amphotericin B impacts this innate skin defense, we induced peptides from R. sphenocephala at day 0 and again at day 56.…”

“…The second group was maintained in dechlorinated tap water for the same period to serve as controls. While this amount of time is longer than a typical treatment, it was necessary in this instance, as 50 days is the reported length of time over which peptide levels recover to their preinjection concentrations following depletion by norepinephrine (50). On day 56, frogs were injected again with 20 nmol/ gbw norepinephrine-HCl to induce peptides.…”

“…Further, many species produce AMPs in dermal granular glands that are an important part of their innate skin defenses (reviewed in references 47 and 48). These peptides are constitutively secreted onto the skin (49) and act as a first line of defense against a variety of infectious microorganisms, including B. dendrobatidis (50)(51)(52)(53). Thus, it is important to investigate whether proposed antifungal treatments would impair these natural innate skin defenses.…”

Interactive effects of competition and predator cues on immune responses of leopard frogs at metamorphosis

“…For example, frogs chased by a researcher's hand released the same amount of peptides as frogs injected with 2 nmol noradrenaline g −1 frog and significantly more peptides than resting frogs (Ramsey et al, 2010;Pask et al, 2012). Maximum peptide release is achieved with an injection of 40 nmol noradrenaline g −1 frog for this species (Pask et al, 2013). As metamorphs do not release as many peptides as adult frogs (L.A.R.-S., unpublished data), we used 20 nmol g −1 frog of noradrenaline to allow the release of a detectable amount of peptides without approaching a maximum level of release (Ramsey et al, 2010;Pask et al, 2013).…”

“…Maximum peptide release is achieved with an injection of 40 nmol noradrenaline g −1 frog for this species (Pask et al, 2013). As metamorphs do not release as many peptides as adult frogs (L.A.R.-S., unpublished data), we used 20 nmol g −1 frog of noradrenaline to allow the release of a detectable amount of peptides without approaching a maximum level of release (Ramsey et al, 2010;Pask et al, 2013).…”