Functional Redundancy of Batrachochytrium dendrobatidis Inhibition in Bacterial Communities Isolated from Lithobates clamitans Skin.

The cutaneous microbial community can influence the health of amphibians exposed to Batrachochytrium dendrobatidis (Bd), a fungal pathogen that has contributed to recent amphibian declines. Resistance to Bd in amphibian populations is correlated with the presence of anti-Bd cutaneous microbes, which confer disease resistance by inhibiting Bd growth. I aimed to determine if green frogs (Lithobates clamitans), an abundant and widely distributed species in New Jersey, harbored bacteria that inhibit Bd and whether the presence and identity of these microbes varied among sites. I used in vitro challenge assays to determine if bacteria isolated from green frog skin could inhibit or enhance the growth of Bd. I found that green frogs at all sites harbored anti-Bd bacteria. However, there were differences in Bd inhibition capabilities among bacterial isolates identified as the same operational taxonomic unit (OTU), lending support to the idea that phylogenetic relatedness does not always predict Bd inhibition status. Additionally, anti-Bd bacterial richness did not vary by site, but the composition of anti-Bd bacterial taxa was distinct at each site. This suggests that there is functional redundancy of Bd inhibition across unique communities of anti-Bd symbionts found on frogs at different sites. These findings highlight the need to better elucidate the structure-function relationship of microbiomes and their role in disease resistance.

Mechanistic basis for loss of water balance in green tree frogs infected with a fungal pathogen.

Chytridiomycosis, a lethal skin disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), disrupts skin function of amphibians, interfering with ionic and osmotic regulation. To regulate fungal loads, amphibians increase their rate of skin sloughing. However, sloughing also causes a temporary loss of ionic and osmotic homeostasis due to disruption of the skin, a key osmoregulatory organ. The combined effects of increased sloughing frequency and chytridiomycosis contribute to the high rates of mortality from Bd infections. However, the mechanisms responsible for the loss of cutaneous osmotic regulation remain unknown. We measured the changes in whole animal water uptake rates, in vitro transcutaneous water fluxes across the ventral skin, and the mRNA expression of epithelial water transport proteins (aquaporins, AQPs) and junctional proteins in Bd-infected and uninfected Litoria caerulea skin. We hypothesize that infected frogs would show reduction/inhibition in cutaneous water transporters responsible for regulating water balance, and sloughing would exacerbate cutaneous water fluxes. We found that infected, nonsloughing frogs had an impaired rate of water uptake and showed increased rates of in vitro water efflux across the ventral skin. In uninfected frogs, the expression of AQPs and junction genes increased significantly with sloughing, which may assist in regulating cutaneous water movements and barrier function in the newly exposed skin. In contrast, infected frogs did not show this postsloughing increase in AQP gene expression. The combination of increased sloughing frequency, impaired water uptake rates, and increased rates of water loss likely contributes to the loss of osmotic homeostasis in frogs infected with Bd.

Seasonal infection rates of Batrachochytrium dendrobatidis in populations of northern green frog Lithobates clamitans melanota tadpoles.

Few studies have documented seasonal variation of Batrachochytrium dendrobatidis (Bd) infection rates in larval amphibians. We identified 4 natural populations of northern green frogs Lithobates clamitans melanota in Pennsylvania (USA) that contained Bd-infected tadpoles during post-wintering collections in May and June, after hibernating tadpoles had overwintered in wetlands. However, we failed to detect infected tadpoles at those wetlands when pre-wintering collections were made in late July through early September. We observed 2 cohorts of tadpoles that appeared to lack Bd-infected individuals in pre-wintering collections, yet contained Bd-infected individuals the following spring. We also observed 4 cohorts of pre-wintering tadpoles that were Bd-free, even though post-wintering tadpoles collected earlier in the year were infected with Bd. Our results suggest that tadpoles either reduce Bd infections during the summer months, and/or infections proliferate sometime prior to (or shortly after) tadpoles emerge from hibernation. It is unlikely that pre-wintering tadpoles were too small to detect Bd zoospores because (1) there was no correlation between Bd zoospore levels and tadpole size or stage, and (2) size was not a significant predictor of infection status. These results suggest that, while sampling larvae can be an effective means of collecting large sample sizes, investigators in our Mid-Atlantic region should conduct sampling by early summer to maximize the chances of detecting Bd. Further research is warranted to determine whether wetland topography and warm, shallow microhabitats within wetlands contribute to a population's ability to drastically reduce Bd prevalence prior to overwintering at ponds.

Direct and Indirect Horizontal Transmission of the Antifungal Probiotic Bacterium Janthinobacterium lividum on Green Frog (Lithobates clamitans) Tadpoles.

Amphibian populations worldwide are being threatened by the disease chytridiomycosis, which is caused by Batrachochytrium dendrobatidis To mitigate the effects of B. dendrobatidis, bioaugmentation of antifungal bacteria has been shown to be a promising strategy. One way to implement bioaugmentation is through indirect horizontal transmission, defined as the transfer of bacteria from a host to the environment and to another host. In addition, direct horizontal transmission among individuals can facilitate the spread of a probiotic in a population. In this study, we tested whether the antifungal bacterium Janthinobacterium lividum could be horizontally transferred, directly or indirectly, in a laboratory experiment using Lithobates clamitans tadpoles. We evaluated the ability of J. lividumto colonize the tadpoles' skin and to persist through time using culture-dependent and culture-independent techniques. We also tested whether the addition of J. lividum affected the skin community in L. clamitans tadpoles. We found that transmission occurred rapidly by direct and indirect horizontal transmission, but indirect transmission that included a potential substrate was more effective. Even though J. lividum colonized the skin, its relative abundance on the tadpole skin decreased over time. The inoculation of J. lividum did not significantly alter the skin bacterial diversity of L. clamitans tadpoles, which was dominated by Pseudomonas Our results show that indirect horizontal transmission can be an effective bioaugmentation method. Future research is needed to determine the best conditions, including the presence of substrates, under which a probiotic can persist on the skin so that bioaugmentation becomes a successful strategy to mitigate chytridiomycosis.

Disease dynamics of red-spotted newts and their anuran prey in a montane pond community.

Long-term monitoring of amphibians is needed to clarify population-level effects of ranaviruses (Rv) and the fungal pathogen Batrachochytrium dendrobatidis (Bd). We investigated disease dynamics of co-occurring amphibian species and potential demographic consequences of Rv and Bd infections at a montane site in the Southern Appalachians, Georgia, USA. Our 3-yr study was unique in combining disease surveillance with intensive population monitoring at a site where both pathogens are present. We detected sub-clinical Bd infections in larval and adult red-spotted newts Notophthalmus viridescens viridescens, but found no effect of Bd on body condition of adult newts. Bd infections also occurred in larvae of 5 anuran species that bred in our fishless study pond, and we detected co-infections with Bd and Rv in adult newts and larval green frogs Lithobates clamitans. However, all mortality and clinical signs in adult newts and larval anurans were most consistent with ranaviral disease, including a die-off of larval wood frogs Lithobates sylvaticus in small fish ponds located near our main study pond. During 2 yr of drift fence monitoring, we documented high juvenile production in newts, green frogs and American bullfrogs L. catesbeianus, but saw no evidence of juvenile recruitment in wood frogs. Larvae of this susceptible species may have suffered high mortality in the presence of both Rv and predators. Our findings were generally consistent with results of Rv-exposure experiments and support the purported role of red-spotted newts, green frogs, and American bullfrogs as common reservoirs for Bd and/or Rv in permanent and semi-permanent wetlands.

Temperature alone does not explain patterns of Batrachochytrium dendrobatidis infections in the green frog Lithobates clamitans.

Batrachochytrium dendrobatidis (Bd) is an invasive, waterborne fungal pathogen that has caused significant declines and extinctions of amphibian species worldwide. Temperature is a major factor impacting the growth and spread of Bd, but little is known regarding the associated patterns in natural habitats. This study analyzed the temperature-associated trends, as correlated with season and microhabitat, of Bd prevalence and infection intensity in green frogs Lithobates clamitans in a temperate environment (central Ohio, USA). Bd was widely distributed at the study sites and found in more than half of the frogs sampled. Bd prevalence was significantly higher in the spring and in forested stream habitats compared to emergent wetland habitats. In contrast, Bd infection intensities tended to be higher in summer. Given the known temperature sensitivity of Bd as demonstrated in laboratory studies, these findings suggest that temperature may be an important factor determining Bd prevalence in green frogs at our study sites, but that factors other than temperature are more important in determining infection intensity. Our findings suggest that future monitoring of Bd among vulnerable species in regions experiencing seasonal temperature variation should study a range of environmental variables to better understand the dynamic relationship between Bd and its amphibian hosts.

Laboratory and field exposure of two species of juvenile amphibians to a glyphosate-based herbicide and Batrachochytrium dendrobatidis.

Herbicides are commonly used in agriculture and silviculture to reduce interspecific competition among plants and thereby enhance crop growth, quality, and volume. Internationally, formulations of glyphosate-based herbicides are the most widely used herbicides in both these sectors. A large amount of work has focused on the effects of these herbicides on amphibians. Several laboratory and mesocosm studies have demonstrated that various formulations of glyphosate herbicides can be acutely toxic to larval and juvenile amphibians at concentrations at the upper end of environmental realism. However, to date there has been little work done investigating such effects in natural systems, limited work on juvenile amphibians, and only a few studies have investigated interactions with other stressors. We conducted a 16 day field experiment in which juveniles of two amphibian species (Lithobates clamitans and Lithobates pipiens) were exposed to the herbicide Roundup WeatherMax™ at four application rates (0, 2.16, 4.32 and 8.64 kg a.e./ha) to investigate effects on survival, liver somatic index (LSI), body condition, and incidence of disease caused by Batrachochytrium dendrobatidis (Bd). In a separate 16 day laboratory experiment, we exposed juvenile L. clamitans to both the herbicide and Bd. Results of our studies showed that this particular herbicide formulation had no effect on juvenile survival, LSI, body condition, or disease incidence, nor was there an interaction between exposure to herbicide and exposure to the disease in tests which closely mimic real world exposure scenarios. These experiments suggest that Roundup WeatherMax as typically used in agriculture is unlikely to cause significant deleterious effects on juvenile amphibians under real world exposure conditions.