Michelle P. Stockwell

Sodium Chloride Inhibits the Growth and Infective Capacity of the Amphibian Chytrid Fungus and Increases Host Survival Rates

The amphibian chytrid fungus Batrachochytrium dendrobatidis is a recently emerged pathogen that causes the infectious disease chytridiomycosis and has been implicated as a contributing factor in the global amphibian decline. Since its discovery, research has been focused on developing various methods of mitigating the impact of chytridiomycosis on amphibian hosts but little attention has been given to the role of antifungal agents that could be added to the hosts environment. Sodium chloride is a known antifungal agent used routinely in the aquaculture industry and this study investigates its potential for use as a disease management tool in amphibian conservation. The effect of 0–5 ppt NaCl on the growth, motility and survival of the chytrid fungus when grown in culture media and its effect on the growth, infection load and survivorship of infected Perons tree frogs (Litoria peronii) in captivity, was investigated. The results reveal that these concentrations do not negatively affect the survival of the host or the pathogen. However, concentrations greater than 3 ppt significantly reduced the growth and motility of the chytrid fungus compared to 0 ppt. Concentrations of 1–4 ppt NaCl were also associated with significantly lower host infection loads while infected hosts exposed to 3 and 4 ppt NaCl were found to have significantly higher survival rates. These results support the potential for NaCl to be used as an environmentally distributed antifungal agent for the prevention of chytridiomycosis in susceptible amphibian hosts. However, further research is required to identify any negative effects of salt exposure on both target and non-target organisms prior to implementation.

Evidence of a salt refuge: chytrid infection loads are suppressed in hosts exposed to salt

With the incidence of emerging infectious diseases on the rise, it is becoming increasingly important to identify refuge areas that protect hosts from pathogens and therefore prevent population declines. For the chytrid fungus Batrachochytrium dendrobatidis, temperature and humidity refuge areas for amphibian hosts exist but are difficult to manipulate. Other environmental features that may affect the outcome of infection include water quality, drying regimes, abundance of alternate hosts and isolation from other hosts. We identified relationships between water bodies with these features and infection levels in the free-living hosts inhabiting them. Where significant relationships were identified, we used a series of controlled experiments to test for causation. Infection loads were negatively correlated with the salt concentration of the aquatic habitat and the degree of water level fluctuation and positively correlated with fish abundance. However, only the relationship with salt was confirmed experimentally. Free-living hosts inhabiting water bodies with mean salinities of up to 3.5 ppt had lower infection loads than those exposed to less salt. The experiment confirmed that exposure to sodium chloride concentrations >2 ppt significantly reduced host infection loads compared to no exposure (0 ppt). These results suggest that the exposure of amphibians to salt concentrations found naturally in lentic habitats may be responsible for the persistence of some susceptible species in the presence of B. dendrobatidis. By manipulating the salinity of water bodies, it may be possible to create refuges for declining amphibians, thus allowing them to be reintroduced to their former ranges.

Do invasive cane toads affect the parasite burdens of native Australian frogs

Graphical abstract

Effects of pond salinization on survival rate of amphibian hosts infected with the chytrid fungus

The chytrid fungus Batrachochytrium dendrobatidis has been implicated in the decline and extinction of amphibian populations worldwide, but management options are limited. Recent studies show that sodium chloride (NaCl) has fungicidal properties that reduce the mortality rates of infected hosts in captivity. We investigated whether similar results can be obtained by adding salt to water bodies in the field. We increased the salinity of 8 water bodies to 2 or 4 ppt and left an additional 4 water bodies with close to 0 ppt and monitored salinity for 18 months. Captively bred tadpoles of green and golden bell frog (Litoria aurea) were released into each water body and their development, levels of B. dendrobatidis infection, and survival were monitored at 1, 4, and 12 months. The effect of salt on the abundance of nontarget organisms was also investigated in before and after style analyses. Salinities remained constant over time with little intervention. Hosts in water bodies with 4 ppt salt had a significantly lower prevalence of chytrid infection and higher survival, following metamorphosis, than hosts in 0 ppt salt. Tadpoles in the 4 ppt group were smaller in length after 1 month in the release site than those in the 0 and 2 ppt groups, but after metamorphosis body size in all water bodies was similar . In water bodies with 4 ppt salt, the abundance of dwarf tree frogs (Litoria fallax), dragonfly larvae, and damselfly larvae was lower than in water bodies with 0 and 2 ppt salt, which could have knock-on effects for community structure. Based on our results, salt may be an effective field-based B. dendrobatidis mitigation tool for lentic amphibians that could contribute to the conservation of numerous susceptible species. However, as in all conservation efforts, these benefits need to be weighed against negative effects on both target and nontarget organisms.

Efficacy of SYBR 14/propidium iodide viability stain for the amphibian chytrid fungus Batrachochytrium dendrobatidis

The amphibian chytrid fungus Batrachochytrium dendrobatidis is a recently described pathogen that has been implicated as a causal agent in the global decline in amphibians. Research into its biology and epidemiology has frequently involved in vitro experimentation. However, this research is currently limited by the inability to differentiate between viable and inviable zoospores. Stains are frequently used to determine cell viability, and this study tested a 2-colour fluorescence assay for the detection and quantification of viable B. dendrobatidis zoospores. The results show that the nucleic acid stains SYBR 14 and propidium iodide are effective in distinguishing live from dead zoospores, and a protocol has been optimized for their use. This viability assay provides an efficient and reliable tool that will have applications in B. dendrobatidis challenge and amphibian exposure experiments.

Finding a place to live: conspecific attraction affects habitat selection in juvenile green and golden bell frogs

Conspecific attraction plays an important role in habitat selection of several taxa and can affect and determine distribution patterns of populations. The behaviour is largely studied and widespread among birds, but in amphibians, its occurrence seems limited to breeding habitats of adults and gregarious tadpoles. The Australian green and golden bell frogs (Litoria aurea) have suffered considerable shrinking of their original distribution in south-eastern Australia since the 1970s. Currently, with only about 40 populations remaining, the species is considered nationally threatened. In natural conditions, these frogs are aggregated in the landscape and do not seem to occupy all suitable ponds within the occurrence area. To date, studies focusing on the frogs’ habitat have failed in finding a general habitat feature that explains current or past occupancy. This led us to the hypothesis that social cues may play a key role in habitat selection in this species. Using two choice experiments, we tested the preference of juvenile green and golden bell frogs for habitats containing cues of conspecifics of similar size versus habitats without conspecific cues. Tested frogs did not show a preference for habitats containing only scent from conspecifics but did prefer habitats where conspecifics were present. Our results show that conspecific attraction is a determining factor in juvenile green and golden bell frog habitat selection. To our knowledge, this is the first time the behaviour is shown to occur in juvenile frogs in the habitat selection context. From a conservation management point of view, the behaviour may help to explain the failure of reintroductions to areas where the frogs have been extinct, and the non-occupation of suitable created habitats in areas where they still inhabit and develop appropriated management strategies.

Estimates of sex ratio require the incorporation of unequal catchability between sexes

Abstract Context. Estimates of the sex ratio of a population are a common summary statistic used for ecological studies and conservation planning. However, methods to determine the sex ratio often ignore capture probability, which can lead to a perceived bias in the sex ratio when the sexes are detected at different rates. Aims. To illustrate the bias from conventional count-based analysis methods for determining sex ratio by comparison with analytical methods that include capture probability. Methods. Closed-population mark–recapture analysis was used to determine the population size of each sex within a population of green and golden bell frogs (Litoria aurea). This was then compared with the traditional count-based methods of estimating sex ratio to determine the effect of incorporating capture probability on the sex ratio estimate. Key results. More males than females were detected during surveys, producing a male-biased sex ratio when there was no incorporation of capture probability. Mark–recapture results indicated a similar population size between the two sexes, suggesting that the sex ratio is closer to even. Conclusions. Methods to estimate sex ratio that incorporate capture probability can significantly reduce the bias obtained from count data. Implications. We suggest that population studies must incorporate capture probability to determine the sex ratio of a population.

Evaluating monitoring methods to guide adaptive management of a threatened amphibian (Litoria aurea)

Prompt detection of declines in abundance or distribution of populations is critical when managing threatened species that have high population turnover. Population monitoring programs provide the tools necessary to identify and detect decreases in abundance that will threaten the persistence of key populations and should occur in an adaptive management framework which designs monitoring to maximize detection and minimize effort. We monitored a population of Litoria aurea at Sydney Olympic Park over 5 years using mark–recapture, capture encounter, noncapture encounter, auditory, tadpole trapping, and dip-net surveys. The methods differed in the cost, time, and ability to detect changes in the population. Only capture encounter surveys were able to simultaneously detect a decline in the occupancy, relative abundance, and recruitment of frogs during the surveys. The relative abundance of L. aurea during encounter surveys correlated with the population size obtained from mark–recapture surveys, and the methods were therefore useful for detecting a change in the population. Tadpole trapping and auditory surveys did not predict overall abundance and were therefore not useful in detecting declines. Monitoring regimes should determine optimal survey times to identify periods where populations have the highest detectability. Once this has been achieved, capture encounter surveys provide a cost-effective method of effectively monitoring trends in occupancy, changes in relative abundance, and detecting recruitment in populations.

We Made Your Bed, Why Won’t You Lie in It? Food Availability and Disease May Affect Reproductive Output of Reintroduced Frogs

Mitigation to offset the impacts of land development is becoming increasingly common, with reintroductions and created habitat programs used as key actions. However, numerous reviews cite high rates of poor success from these programs, and a need for improved monitoring and scientific testing to evaluate outcomes and improve management actions. We conducted extensive monitoring of a released population of endangered green and golden bell frogs, Litoria aurea, within a created habitat, as well as complementary surveys of a surrounding wild population. We then compared differences between the created habitat and natural ponds where extant frogs either bred or didn’t breed in order to determine factors that contributed to the breeding failure within the created habitat. We evaluated differences of L. aurea abundance, abundance of other fauna, vegetation, water quality, habitat structure, invasive fish, and disease between the three pond types (created habitat, breeding ponds, non-breeding ponds). We discovered that vegetation and invertebrate diversity were low within the created habitat, potentially reducing energy and nutritional resources required for breeding. Also, a greater proportion of frogs in the created habitat were carrying the chytrid fungal pathogen, Batrachochytrium dendrobatidis, compared to the wild populations. In addition to causing the potentially fatal disease, chytridiomycosis, this pathogen has been shown to reduce reproductive functioning in male L. aurea, and subsequently may have reduced reproductive activities in the created habitat. Conspecific attraction, pond hydrology, and aquatic vegetation may also have had some influence on breeding behaviours, whilst the presence of the invasive mosquitofish, Gambusia holbrooki, and heterospecific tadpoles were unlikely to have deterred L. aurea from breeding within the created habitat. Through the use of scientific testing and monitoring, this study is able to make recommendations for future amphibian breed and release programs, and suggests planting a diversity of plant species to attract invertebrates, creating some permanent ponds, connecting habitat with existing populations, trialling artificial mating calls, and following recommendations to reduce the prevalence of disease within the population.

Low disease-causing threshold in a frog species susceptible to chytridiomycosis

A simple diagnosis of the presence or absence of an infection is an uninformative metric when individuals differ considerably in their tolerance to different infection loads or resistance to rates of disease progression. Models that incorporate the relationship between the progression of the infection with the potential alternate outcomes provide a far more powerful predictive tool than diagnosis alone. The global decline of amphibians has been amplified by Batrachochytrium dendrobatidis, a pathogen that can cause the fatal disease chytridiomycosis. We measured the infection load and observed signs of disease in Litoria aurea Receiver operating characteristic curves were used to quantify the dissimilarity between the infection loads of L. aurea that showed signs associated with chytridiomycosis and those that did not. Litoria aurea had a 78% probability of developing chytridiomycosis past a threshold of 68 zoospore equivalents (ZE) per swab and chytridiomycosis occurred within a variable range of 0.5-490 ZE. Studies should incorporate a species-specific threshold as a predictor of chytridiomycosis, rather than a binary diagnosis. Measures of susceptibility to chytridiomycosis must account not only for the ability of B. dendrobatidis to increase its abundance on the skin of amphibians but also to determine how each species tolerates these infection loads.