Douglas C. Woodhams

LIFE-HISTORY TRADE-OFFS INFLUENCE DISEASE IN CHANGING CLIMATES: STRATEGIES OF AN AMPHIBIAN PATHOGEN

Life-history trade-offs allow many animals to maintain reproductive fitness across a range of climatic conditions. When used by parasites and pathogens, these strategies may influence patterns of disease in changing climates. The chytrid fungus, Batrachochytrium dendrobatidis, is linked to global declines of amphibian populations. Short-term growth in culture is maximal at 17 degrees-25 degrees C. This has been used in an argument that global warming, which increases the time that amphibians spend at these temperatures in cloud-covered montane environments, has led to extinctions. Here we show that the amphibian chytrid responds to decreasing temperatures with trade-offs that increase fecundity as maturation rate slows and increase infectivity as growth decreases. At 17 degrees-25 degrees C, infectious zoospores encyst (settle and develop a cell wall) and develop into the zoospore-producing stage (zoosporangium) faster, while at 7 degrees-10 degrees C, greater numbers of zoospores are produced per zoosporangium; these remain infectious for a longer period of time. We modeled the population growth of B. dendrobatidis through time at various temperatures using delayed differential equations and observational data for four parameters: developmental rate of thalli, fecundity, rate of zoospore encystment, and rate of zoospore survival. From the models, it is clear that life-history trade-offs allow B. dendrobatidis to maintain a relatively high long-term growth rate at low temperatures, so that it maintains high fitness across a range of temperatures. When a seven-day cold shock is simulated, the outcome is intermediate between the two constant temperature regimes, and in culture, a sudden drop in temperature induces zoospore release. These trade-offs can be ecologically important for a variety of organisms with complex life histories, including pathogenic microorganisms. The effect of temperature on amphibian mortality will depend on the interaction between fungal growth and host immune function and will be modified by host ecology, behavior, and life history. These results demonstrate that B. dendrobatidis populations can grow at high rates across a broad range of environmental temperatures and help to explain why it is so successful in cold montane environments.

Population trends associated with skin peptide defenses against chytridiomycosis in Australian frogs

Many species of amphibians in the wet tropics of Australia have experienced population declines linked with the emergence of a skin-invasive chytrid fungus, Batrachochytrium dendrobatidis. An innate defense, antimicrobial peptides produced by granular glands in the skin, may protect some species from disease. Here we present evidence that supports this hypothesis. We tested ten synthesized peptides produced by Australian species, and natural peptide mixtures from five Queensland rainforest species. Natural mixtures and most peptides tested in isolation inhibited growth of B. dendrobatidis in vitro. The three most active peptides (caerin 1.9, maculatin 1.1, and caerin 1.1) were found in the secretions of non-declining species (Litoria chloris, L. caerulea, and L. genimaculata). Although the possession of a potent isolated antimicrobial peptide does not guarantee protection from infection, non-declining species (L. lesueuri and L. genimaculata) inhabiting the rainforest of Queensland possess mixtures of peptides that may be more protective than those of the species occurring in the same habitat that have recently experienced population declines associated with chytridiomycosis (L. nannotis, L. rheocola, and Nyctimystes dayi). This study demonstrates that in vitro effectiveness of skin peptides correlates with the degree of decline in the face of an emerging pathogen. Further research is needed to assess whether this non-specific immune defense may be useful in predicting disease susceptibility in other species.

Predicted disease susceptibility in a Panamanian amphibian assemblage based on skin peptide defenses.

Chytridiomycosis is an emerging infectious disease of amphibians caused by a chytrid fungus, Batrachochytrium dendrobatidis. This panzootic does not equally affect all amphibian species within an assemblage; some populations decline, others persist. Little is known about the factors that affect disease resistance. Differences in behavior, life history, biogeography, or immune function may impact survival. We found that an innate immune defense, antimicrobial skin peptides, varied significantly among species within a rainforest stream amphibian assemblage that has not been exposed to B. dendrobatidis. If exposed, all amphibian species at this central Panamanian site are at risk of population declines. In vitro pathogen growth inhibition by peptides from Panamanian species compared with species with known resistance (Rana pipiens and Xenopus laevis) or susceptibility (Bufo boreas) suggests that of the nine species examined, two species (Centrolene prosoblepon and Phyllomedusa lemur) may demonstrate strong resistance, and the other species will have a higher risk of disease-associated population declines. We found little variation among geographically distinct B. dendrobatidis isolates in sensitivity to an amphibian skin peptide mixture. This supports the hypothesis that B. dendrobatidis is a generalist pathogen and that species possessing an innate immunologic defense at the time of disease emergence are more likely to survive.

Antimicrobial Peptide defenses in amphibian skin.

Abstract One of the most urgent problems in conservation biology today is the continuing loss of amphibian populations on a global scale. Recent amphibian population declines in Australia, Central America, the western United States, Europe, and Africa have been linked to a pathogenic chytrid fungus, Batrachochytrium dendrobatidis, which infects the skin. The skin of amphibians is critical for fluid balance, respiration, and transport of essential ions; and the immune defense of the skin must be integrated with these physiological responses. One of the natural defenses of the skin is production of antimicrobial peptides in granular glands. Discharge of the granular glands is initiated by stimulation of sympathetic nerves. To determine whether antimicrobial skin peptides play a role in protection from invasive pathogens, purified antimicrobial peptides and natural peptide mixtures recovered from the skin secretions of a number of species have been assayed for growth inhibition of the chytrid fungus. The general findings are that most species tested have one or more antimicrobial peptides with potent activity against the chytrid fungus, and natural mixtures of peptides are also effective inhibitors of chytrid growth. This supports the hypothesis that antimicrobial peptides produced in the skin are an important defense against skin pathogens and may affect survival of populations. We also report on initial studies of peptide depletion using norepinephrine and the kinetics of peptide recovery following induction. Approximately 80 nmoles/g of norepinephrine is required to deplete peptides, and peptide stores are not fully recovered at three weeks following this treatment. Because many species have defensive peptides and yet suffer chytrid-associated population declines, it is likely that other factors (temperature, conditions of hydration, “stress,” or pesticides) may alter normal defenses and allow for uncontrolled infection.

Variations in the expressed antimicrobial peptide repertoire of northern leopard frog (Rana pipiens) populations suggest intraspecies differences in resistance to pathogens.

The northern leopard frog (Rana pipiens or Lithobates pipiens) is historically found in most of the provinces of Canada and the northern and southwest states of the United States. In the last 50 years, populations have suffered significant losses, especially in the western regions of the species range. Using a peptidomics approach, we show that the pattern of expressed antimicrobial skin peptides of frogs from three geographically separated populations are distinct, and we report the presence of four peptides (brevinin-1Pg, brevinin-1Pl, ranatuerin-2Pb, and ranatuerin-2Pc) that have not previously been found in skin secretions. The differences in expressed peptides reflect differences in the distribution of alleles for the newly described Brevinin1.1 locus in the three populations. When enriched peptide mixtures were tested for their ability to inhibit growth of the pathogenic amphibian chytrid (Batrachochytrium dendrobatidis), peptides from Minnesota or Vermont frogs were more effective that peptides from Michigan frogs. Four of the purified peptides were tested for their ability to inhibit growth of two bacterial pathogens (Aeromonas hydrophila and Staphylococcus epidermidis) and B. dendrobatidis. Three of the four were effective inhibitors of B. dendrobatidis and S. epidermidis, but none inhibited A. hydrophila. We interpret these differences in expression and activity of antimicrobial peptides as evidence to suggest that each population may have been selected to express a suite of peptides that reflects current and past encounters with skin microbes.

A peptide of the phylloseptin family from the skin of the frog Hylomantis lemur (Phyllomedusinae) with potent in vitro and in vivo insulin-releasing activity

A peptide with the ability to release insulin from the rat BRIN-BD11 clonal beta cell line was isolated from norepinephrine-stimulated skin secretions of the Lemur leaf frog Hylomantis lemur Boulenger,1882. Determination of the primary structure (FLSLIPHVISALSSL.NH(2)) demonstrated that the peptide belongs to the phylloseptin family whose members have previously been identified in other Phyllomedusinae species. A synthetic replicate of the peptide, termed phylloseptin-L2, produced a significant stimulation of insulin release (134% of basal rate, P<0.01) from BRIN-BD11 cells at a concentration of 30 nM, with a maximum response (301% of basal rate, P<0.001) at a concentration of 3 microM. Phylloseptin-L2 did not stimulate release of the cytosolic enzyme, lactate dehydrogenase at concentrations up to 3 microM, indicating that the integrity of the plasma membrane had been preserved. The stimulatory action was maintained in the absence of extracellular Ca(2+) and in the presence of verapamil (50 microM) and diazoxide (300 microM) suggesting that mechanism of action of the peptide did not primarily involve influx of Ca(2+) or closure of ATP-sensitive K(+) channels. Administration of phylloseptin-L2 (50 nmol/kgbody weight) into mice significantly (P<0.05) increased total release of insulin and improved glucose tolerance during the 60 min period following an intraperitoneal injection of glucose (18 mmol/kgbody weight). It is concluded that the peptide shows potential for development into a therapeutically valuable agent for the treatment of Type 2 diabetes.