Constant-temperature predictions underestimate growth of a fungal amphibian pathogen under individual host thermal profiles.

Ectotherm body temperatures fluctuate with environmental variability and host behavior, which may influence host-pathogen interactions. Fungal pathogens are a major threat to ectotherms and may be highly responsive to the fluctuating thermal profiles of individual hosts, especially cool-loving fungi exposed to high host temperatures. However, most studies estimate pathogen thermal performance based on averages of host or surrogate environmental temperatures, potentially missing effects of short-term host temperature shifts such as daily or hourly heat spikes. We recorded individual thermal profiles of Australian rainforest frogs using temperature-sensitive radio-transmitters. We then reproduced a subset of individual thermal profiles in growth chambers containing cultures of the near-global amphibian pathogen Batrachochytrium dendrobatidis (Bd) to investigate how realistic host temperature profiles affect Bd growth. We focused on thermal profiles that exceed the thermal optimum of Bd because the effects of realistic heat spikes on Bd growth are unresolved. Our laboratory incubation experiment revealed that Bd growth varied in response to relatively small differences in heat spike characteristics of individual frog thermal profiles, such as a single degree or a few hours, highlighting the importance of individual host behaviors in predicting population-level disease dynamics. The fungus also grew better than predicted under the most extreme and unpredictable frog temperature profile, recovering from two days of extreme (nearly 32 °C) heat spikes without negative effects on overall growth, suggesting we are underestimating the growth potential of the pathogen in nature. Combined with the previous finding that Bd reduces host heat tolerance, our study suggests that this pathogen may carry a competitive edge over hosts in the face of anthropogenic climate change.

Increased rates of dispersal of free-ranging cane toads (Rhinella marina) during their global invasion.

Invasions often accelerate through time, as dispersal-enhancing traits accumulate at the expanding range edge. How does the dispersal behaviour of individual organisms shift to increase rates of population spread? We collate data from 44 radio-tracking studies (in total, of 650 animals) of cane toads (Rhinella marina) to quantify distances moved per day, and the frequency of displacement in their native range (French Guiana) and two invaded areas (Hawai'i and Australia). We show that toads in their native-range, Hawai'i and eastern Australia are relatively sedentary, while toads dispersing across tropical Australia increased their daily distances travelled from 20 to 200 m per day. That increase reflects an increasing propensity to change diurnal retreat sites every day, as well as to move further during each nocturnal displacement. Daily changes in retreat site evolved earlier than did changes in distances moved per night, indicating a breakdown in philopatry before other movement behaviours were optimised to maximise dispersal.

Mistaken identity may explain why male sea snakes (Aipysurus laevis, Elapidae, Hydrophiinae) "attack" scuba divers.

Scuba-divers on tropical coral-reefs often report unprovoked "attacks" by highly venomous Olive sea snakes (Aipysurus laevis). Snakes swim directly towards divers, sometimes wrapping coils around the diver's limbs and biting. Based on a focal animal observation study of free-ranging Olive sea snakes in the southern Great Barrier Reef, we suggest that these "attacks" are misdirected courtship responses. Approaches to divers were most common during the breeding season (winter) and were by males rather than by female snakes. Males also made repeated approaches, spent more time with the diver, and exhibited behaviours (such as coiling around a limb) also seen during courtship. Agitated rapid approaches by males, easily interpreted as "attacks", often occurred after a courting male lost contact with a female he was pursuing, after interactions between rival males, or when a diver tried to flee from a male. These patterns suggest that "attacks" by sea snakes on humans result from mistaken identity during sexual interactions. Rapid approaches by females occurred when they were being chased by males. Divers that flee from snakes may inadvertently mimic the responses of female snakes to courtship, encouraging males to give chase. To prevent escalation of encounters, divers should keep still and avoid retaliation.

Predicting the growth of the amphibian chytrid fungus in varying temperature environments.

Environmental temperature is a crucial abiotic factor that influences the success of ectothermic organisms, including hosts and pathogens in disease systems. One example is the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), which has led to widespread amphibian population declines. Understanding its thermal ecology is essential to effectively predict outbreaks. Studies that examine the impact of temperature on hosts and pathogens often do so in controlled constant temperatures. Although varying temperature experiments are becoming increasingly common, it is unrealistic to test every temperature scenario. Thus, reliable methods that use constant temperature data to predict performance in varying temperatures are needed. In this study, we tested whether we could accurately predict Bd growth in three varying temperature regimes, using a Bayesian hierarchical model fit with constant temperature Bd growth data. We fit the Bayesian hierarchical model five times, each time changing the thermal performance curve (TPC) used to constrain the logistic growth rate to determine how TPCs influence the predictions. We then validated the model predictions using Bd growth data collected from the three tested varying temperature regimes. Although all TPCs overpredicted Bd growth in the varying temperature regimes, some functional forms performed better than others. Varying temperature impacts on disease systems are still not well understood and improving our understanding and methodologies to predict these effects could provide insights into disease systems and help conservation efforts.

Infection dynamics, dispersal, and adaptation: understanding the lack of recovery in a remnant frog population following a disease outbreak.

Emerging infectious diseases can cause dramatic declines in wildlife populations. Sometimes, these declines are followed by recovery, but many populations do not recover. Studying differential recovery patterns may yield important information for managing disease-afflicted populations and facilitating population recoveries. In the late 1980s, a chytridiomycosis outbreak caused multiple frog species in Australia's Wet Tropics to decline. Populations of some species (e.g., Litoria nannotis) subsequently recovered, while others (e.g., Litoria dayi) did not. We examined the population genetics and current infection status of L. dayi, to test several hypotheses regarding the failure of its populations to recover: (1) a lack of individual dispersal abilities has prevented recolonization of previously occupied locations, (2) a loss of genetic variation has resulted in limited adaptive potential, and (3) L. dayi is currently adapting to chytridiomycosis. We found moderate-to-high levels of gene flow and diversity (Fst range: <0.01-0.15; minor allele frequency (MAF): 0.192-0.245), which were similar to previously published levels for recovered L. nannotis populations. This suggests that dispersal ability and genetic diversity do not limit the ability of L. dayi to recolonize upland sites. Further, infection intensity and prevalence increased with elevation, suggesting that chytridiomycosis is still limiting the elevational range of L. dayi. Outlier tests comparing infected and uninfected individuals consistently identified 18 markers as putatively under selection, and several of those markers matched genes that were previously implicated in infection. This suggests that L. dayi has genetic variation for genes that affect infection dynamics and may be undergoing adaptation.

Microbiome diversity and composition varies across body areas in a freshwater turtle.

There is increasing recognition that microbiomes are important for host health and ecology, and understanding host microbiomes is important for planning appropriate conservation strategies. However, microbiome data are lacking for many taxa, including turtles. To further our understanding of the interactions between aquatic microbiomes and their hosts, we used next generation sequencing technology to examine the microbiomes of the Krefft's river turtle (Emydura macquarii krefftii). We examined the microbiomes of the buccal (oral) cavity, skin on the head, parts of the shell with macroalgae and parts of the shell without macroalgae. Bacteria in the phyla Proteobacteria and Bacteroidetes were the most common in most samples (particularly buccal samples), but Cyanobacteria, Deinococcus-thermus and Chloroflexi were also common (particularly in external microbiomes). We found significant differences in community composition among each body area, as well as significant differences among individuals. The buccal cavity had lower bacterial richness and evenness than any of the external microbiomes, and it had many amplicon sequence variants (ASVs) with a low relative abundance compared to other body areas. Nevertheless, the buccal cavity also had the most unique ASVs. Parts of the shell with and without algae also had different microbiomes, with particularly obvious differences in the relative abundances of the families Methylomonaceae, Saprospiraceae and Nostocaceae. This study provides novel, baseline information about the external microbiomes of turtles and is a first step in understanding their ecological roles.

Host thermoregulatory constraints predict growth of an amphibian chytrid pathogen (Batrachochytrium dendrobatidis).

1. The course and outcome of many wildlife diseases are context-dependent, and therefore change depending on the behaviour of hosts and environmental response of the pathogen. 2. Contemporary declines in amphibian populations are widely attributed to chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis. Despite the thermal sensitivity of the pathogen and its amphibian hosts, we do not understand how host thermal regimes experienced by frogs in the wild directly influence pathogen growth. 3. We tested how thermal regimes experienced by the rainforest frog Litoria rheocola in the wild influence pathogen growth in the laboratory, and whether these responses differ from pathogen growth under available environmental thermal regimes. 4. Frog thermal regimes mimicked in the laboratory accelerated pathogen growth during conditions representative of winter at high elevations more so than if temperatures matched air or stream water temperatures. By contrast, winter frog thermal regimes at low elevations slowed pathogen growth relative to air temperatures, but not water temperatures. 5. The growth pattern of the fungus under frog thermal regimes matches field prevalence and intensity of infections for this species (high elevation winter > high elevation summer > low elevation winter > low elevation summer), whereas pathogen growth trajectories under environmental temperatures did not match these patterns. 6. If these laboratory results translate into field responses, tropical frogs may be able to reduce disease impacts by regulating their body temperatures to limit pathogen growth (e.g., by using microhabitats that facilitate basking to reach high temperatures); in other cases, the environment may limit the ability of frogs to thermoregulate such that individuals are more vulnerable to this pathogen (e.g., in dense forests at high elevations). 7. Species-specific thermoregulatory behaviour, and interactions with and constraints imposed by the environment, are therefore essential to understanding and predicting the spatial and temporal impacts of this global disease.

Spinal arthritis in invasive cane toads is linked to rate of dispersal as well as to latitude.

Initial research on the spread of cane toads (Rhinella marina) through tropical Australia reported a high incidence of spinal arthritis (spondylosis) in toads at the invasion front (where toads disperse rapidly), but not in areas colonized earlier (where toads are more sedentary). The idea that spondylosis was a cost of rapid dispersal was challenged by wider spatial sampling which linked rates of spondylosis to hot (tropical) climates rather than to dispersal rates. Here, the authors of these competing interpretations collaborate to reinterpret the data. Our reanalysis supports both previous hypotheses; rates of spondylosis are higher in populations established by fast-dispersing toads, and are higher in tropical than in temperate environments; they are also higher in larger toads. The functional reason for climatic effects is unclear, but might involve effects on the soil-living bacteria involved in the induction of spondylosis; and/or may reflect higher movement (as opposed to dispersal) or more pronounced dry-season aggregation rates of toads in tropical conditions.

The return of the frogs: The importance of habitat refugia in maintaining diversity during a disease outbreak.

Recent decades have seen the emergence and spread of numerous infectious diseases, often with severe negative consequences for wildlife populations. Nevertheless, many populations survive the initial outbreaks, and even undergo recoveries. Unfortunately, the long-term effects of these outbreaks on host population genetics are poorly understood; to increase this understanding, we examined the population genetics of two species of rainforest frogs (Litoria nannotis and Litoria serrata) that have largely recovered from a chytridiomycosis outbreak at two national parks in the Wet Tropics of northern Australia. At the wetter, northern park there was little evidence of decreased genetic diversity in either species, and all of the sampled sites had high minor allele frequencies (mean MAF = 0.230-0.235), high heterozygosity (0.318-0.325), and few monomorphic markers (1.4%-4.0%); however, some recovered L. nannotis populations had low Ne values (59.3-683.8) compared to populations that did not decline during the outbreak (1,537.4-1,756.5). At the drier, southern park, both species exhibited lower diversity (mean MAF = 0.084-0.180; heterozygosity = 0.126-0.257; monomorphic markers = 3.7%-43.5%; Ne  = 18.4-676.1). The diversity patterns in this park matched habitat patterns, with both species having higher diversity levels and fewer closely related individuals at sites with higher quality habitat. These patterns were more pronounced for L. nannotis, which has lower dispersal rates than L. serrata. These results suggest that refugia with high quality habitat are important for retaining genetic diversity during disease outbreaks, and that gene flow following disease outbreaks is important for re-establishing diversity in populations where it was reduced.

Disentangling causes of seasonal infection prevalence patterns: tropical tadpoles and chytridiomycosis as a model system.

Identifying the factors that affect pathogen prevalence is critical to understanding the effects of wildlife diseases. We aimed to examine drivers of seasonal changes in the prevalence of infection by the amphibian chytrid fungus Batrachochytrium dendrobatidis in tadpoles. Because tadpoles may be important reservoirs for this disease, examining them will aid in understanding how chytridiomycosis affects entire amphibian populations. We hypothesized that temperature is a strong driver of prevalence of Bd in tadpoles, and the accumulation of infection as tadpoles become larger and older also drives prevalence in this system. We studied Litoria rheocola, a tropical rainforest stream frog with seasonal recruitment of annual tadpoles, and surveyed 6 streams in northeastern Queensland, Australia. Comparisons among models relating infection status to stream type, season, their interaction, tadpole age, and water temperature showed that age explained a large portion of the variance in infection status. Across sites and seasons, larger, older tadpoles had increased mean probabilities of infection, indicating that a large component of the variation among individuals was related to age, and thus to cumulative infection risk. Our results indicate that in systems with annual tadpoles, seasonal changes in infection prevalence may be strongly affected by seasonal patterns of tadpole growth and development in addition to stream type, season, and water temperature. These effects may then influence prevalence of infection in terrestrial individuals in species that have relatively frequent contact with water. This reinforces the need to integrate studies of the drivers of pathogen prevalence across all host life history stages.

Spinal arthritis in cane toads across the Australian landscape.

Loss of fitness can be a consequence of selection for rapid dispersal ability in invasive species. Increased prevalence of spinal arthritis may occur in cane toad populations at the invasion front as a cost of increased invasiveness, but our knowledge of the ecological drivers of this condition is lacking. We aimed to determine the factors explaining the prevalence of spinal arthritis in populations across the Australian landscape. We studied populations across a gradient of invasion histories. We collected 2415 toads over five years and determined the presence and size of spondylosis for each individual. We examined the effect of host size, leg length and invasion history on the prevalence of spondylosis. Host size was a significant predictor of spondylosis across populations. Contrary to our expectation, the overall prevalence of spondylosis was not positively related to invasion history and did not correlate with toad relative leg length. Rather than invasion age, the latitude at which populations were sampled provided an alternate explanation for the prevalence of spondylosis in cane toad populations and suggested that the incidence of this condition did not increase as a physiological cost of invasion, but is instead related to physical variables, such as climate.

Increased Numbers of Culturable Inhibitory Bacterial Taxa May Mitigate the Effects of Batrachochytrium dendrobatidis in Australian Wet Tropics Frogs.

Symbiotic bacterial communities resident on amphibian skin can benefit their hosts. For example, antibiotic production by community members can control the pathogen Batrachochytrium dendrobatidis (Bd) and it is possible for these community members to be used as probiotics to reduce infection levels. In the early 1990s, the emergence of Bd caused declines and disappearances of frogs in the Australian Wet Tropics; the severity of its effects varied among species and sites. Some species have since recolonized despite enzootic Bd within their populations. This variation in history among species and sites provided an opportunity to investigate the role of anti-fungal cutaneous bacteria in protecting frogs against Bd infection. We collected cutaneous swab samples from three species of frogs at two upland and two lowland sites in the Wet Tropics, and used in vitro challenge assays to identify culturable Bd-inhibitory bacterial isolates for further analysis. We sequenced DNA from cultured inhibitory isolates to identify taxa, resulting in the classification of 16 Bd-inhibitory OTUs, and determined whether inhibitory taxa were associated with frog species, site, or intensity of infection. We present preliminary results showing that the upper limit of Bd infection intensity was negatively correlated with number of inhibitory OTUs present per frog indicating that increased numbers of Bd-inhibiting taxa may play a role in reducing the intensity of Bd infections, facilitating frog coexistence with enzootic Bd. One upland site had a significantly lower prevalence of Bd infection, a significantly higher proportion of frogs with one or more culturable Bd-inhibitory OTUs, a greater number of inhibitory bacterial genera present per frog, and statistically significant clustering of individual frogs with similar Bd-inhibitory signatures when compared to all other sites. This suggests that Bd-inhibitory taxa are likely to be particularly important to frogs at this site and may have played a role in their ability to recolonize following population declines. Our findings suggest that the use of multi-taxon Bd-inhibitory probiotics to support at-risk amphibian populations may be more effective than single-taxon alternatives.

White blood cell profiles in amphibians help to explain disease susceptibility following temperature shifts.

Temperature variability, and in particular temperature decreases, can increase susceptibility of amphibians to infections by the fungus Batrachochytrium dendrobatidis (Bd). However, the effects of temperature shifts on the immune systems of Bd-infected amphibians are unresolved. We acclimated frogs to 16 °C and 26 °C (baseline), simultaneously transferred them to an intermediate temperature (21 °C) and inoculated them with Bd (treatment), and tracked their infection levels and white blood cell profiles over six weeks. Average weekly infection loads were consistently higher in 26°C-history frogs, a group that experienced a 5 °C temperature decrease, than in 16°C-history frogs, a group that experienced a 5 °C temperature increase, but this pattern only approached statistical significance. The 16°C-acclimated frogs had high neutrophil:lymphocyte (N:L) ratios (suggestive of a hematopoietic stress response) at baseline, which were conserved post-treatment. In contrast, the 26°C-acclimated frogs had low N:L ratios at baseline which reversed to high N:L ratios post-treatment (suggestive of immune system activation). Our results suggest that infections were less physiologically taxing for the 16°C-history frogs than the 26°C-history frogs because they had already adjusted immune parameters in response to challenging conditions (cold). Our findings provide a possible mechanistic explanation for observations that amphibians are more susceptible to Bd infection following temperature decreases compared to increases and underscore the consensus that increased temperature variability associated with climate change may increase the impact of infectious diseases.

Infection increases vulnerability to climate change via effects on host thermal tolerance.

Unprecedented global climate change and increasing rates of infectious disease emergence are occurring simultaneously. Infection with emerging pathogens may alter the thermal thresholds of hosts. However, the effects of fungal infection on host thermal limits have not been examined. Moreover, the influence of infections on the heat tolerance of hosts has rarely been investigated within the context of realistic thermal acclimation regimes and potential anthropogenic climate change. We tested for effects of fungal infection on host thermal tolerance in a model system: frogs infected with the chytrid Batrachochytrium dendrobatidis. Infection reduced the critical thermal maxima (CTmax) of hosts by up to ~4 °C. Acclimation to realistic daily heat pulses enhanced thermal tolerance among infected individuals, but the magnitude of the parasitism effect usually exceeded the magnitude of the acclimation effect. In ectotherms, behaviors that elevate body temperature may decrease parasite performance or increase immune function, thereby reducing infection risk or the intensity of existing infections. However, increased heat sensitivity from infections may discourage these protective behaviors, even at temperatures below critical maxima, tipping the balance in favor of the parasite. We conclude that infectious disease could lead to increased uncertainty in estimates of species' vulnerability to climate change.

Robust calling performance in frogs infected by a deadly fungal pathogen.

Reproduction is an energetically costly behavior for many organisms, including species with mating systems in which males call to attract females. In these species, calling males can often attract more females by displaying more often, with higher intensity, or at certain frequencies. Male frogs attract females almost exclusively by calling, and we know little about how pathogens, including the globally devastating fungus, Batrachochytrium dendrobatidis, influence calling effort and call traits. A previous study demonstrated that the nightly probability of calling by male treefrogs, Litoria rheocola, is elevated when they are in good body condition and are infected by B. dendrobatidis. This suggests that infections may cause males to increase their present investment in mate attraction to compensate for potential decreases in future reproduction. However, if infection by B. dendrobatidis decreases the attractiveness of their calls, infected males might experience decreased reproductive success despite increases in calling effort. We examined whether calls emitted by L. rheocola infected by B. dendrobatidis differed from those of uninfected individuals in duration, pulse rate, dominant frequency, call rate, or intercall interval, the attributes commonly linked to mate choice. We found no effects of fungal infection status or infection intensity on any call attribute. Our results indicate that infected males produce calls similar in all the qualities we measured to those of uninfected males. It is therefore likely that the calls of infected and uninfected males should be equally attractive to females. The increased nightly probability of calling previously demonstrated for infected males in good condition may therefore lead to greater reproductive success than that of uninfected males. This could reduce the effectiveness of natural selection for resistance to infection, but could increase the effectiveness of selection for infection tolerance, the ability to limit the harm caused by infection, such as reductions in body condition.

Mixed population genomics support for the central marginal hypothesis across the invasive range of the cane toad (Rhinella marina) in Australia.

Understanding factors that cause species' geographic range limits is a major focus in ecology and evolution. The central marginal hypothesis (CMH) predicts that species cannot adapt to conditions beyond current geographic range edges because genetic diversity decreases from core to edge due to smaller, more isolated edge populations. We employed a population genomics framework using 24 235-33 112 SNP loci to test major predictions of the CMH in the ongoing invasion of the cane toad (Rhinella marina) in Australia. Cane toad tissue samples were collected along broad-scale, core-to-edge transects across their invasive range. Geographic and ecological core areas were identified using GIS and habitat suitability indices from ecological niche modelling. Bayesian clustering analyses revealed three genetic clusters, in the northwest invasion-front region, northeast precipitation-limited region and southeast cold temperature-limited region. Core-to-edge patterns of genetic diversity and differentiation were consistent with the CMH in the southeast, but were not supported in the northeast and showed mixed support in the northwest. Results suggest cold temperatures are a likely contributor to southeastern range limits, consistent with CMH predictions. In the northeast and northwest, ecological processes consisting of a steep physiological barrier and ongoing invasion dynamics, respectively, are more likely explanations for population genomic patterns than the CMH.

Rapid differentiation of sexual signals in invasive toads: call variation among populations.

Advertisement calls tend to differ among populations, based on morphological and environmental factors, or simply geographic distance, in many taxa. Invasive cane toads (Rhinella marina) were introduced to Australia in 1935 and their distribution has expanded at increasing rates over time. Rapid evolution occurred in morphological and behavioural characters that accelerate dispersal, but the effects of rapid expansion on sexual signals have not been examined. We collected advertisement calls from four populations of different ages since invasion, and analysed the geographic differentiation of seven call parameters. Our comparisons indicate that the calls of R. marina differ among Australian populations. The signal variation was not simply clinal with respect to population age, climate, or morphological differentiation. We suggest that selection on signalling among populations has been idiosyncratic and may reflect local female preferences or adaptation to environmental factors that are not clinal such as energy availability.

Patterns of Batrachochytrium dendrobatidis transmission between tadpoles in a high-elevation rainforest stream in tropical Australia.

The highly virulent fungal pathogen Batrachochytrium dendrobatidis (Bd) poses a global threat to amphibian biodiversity. Streams and other water bodies are central habitats in the ecology of the disease, particularly in rainforests where they may transport and transmit the pathogen and harbor infected tadpoles that serve as reservoir hosts. We conducted an experiment using larval green-eyed tree frogs Litoria serrata in semi-natural streamside channels to test the hypotheses that (1) the fungus can be transmitted downstream in stream habitats and (2) infection affects tadpole growth and mouthpart loss. Our results showed that transmission can occur downstream in flowing water with no contact between individuals, that newly infected tadpoles suffered increased mouthpart loss in comparison with controls that were never infected and that infected tadpoles grew at reduced rates. Although recently infected tadpoles showed substantial loss of mouthparts, individuals with longstanding infections did not, suggesting that mouthparts may re-grow following initial loss. Our study suggests that any management efforts that can reduce the prevalence of infections in tadpoles may be particularly effective if applied in headwater areas, as their effects are likely to be felt downstream.

Infection dynamics in frog populations with different histories of decline caused by a deadly disease.

Pathogens can drive host population dynamics. Chytridiomycosis is a fungal disease of amphibians that is caused by the fungus Batrachochytrium dendrobatidis (Bd). This pathogen has caused declines and extinctions in some host species whereas other host species coexist with Bd without suffering declines. In the early 1990s, Bd extirpated populations of the endangered common mistfrog, Litoria rheocola, at high-elevation sites, while populations of the species persisted at low-elevation sites. Today, populations have reappeared at many high-elevation sites where they presently co-exist with the fungus. We conducted a capture-mark-recapture (CMR) study of six populations of L. rheocola over 1 year, at high and low elevations. We used multistate CMR models to determine which factors (Bd infection status, site type, and season) influenced rates of frog survival, recapture, infection, and recovery from infection. We observed Bd-induced mortality of individual frogs, but did not find any significant effect of Bd infection on the survival rate of L. rheocola at the population level. Survival and recapture rates depended on site type and season. Infection rate was highest in winter when temperatures were favourable for pathogen growth, and differed among site types. The recovery rate was high (75.7-85.8%) across seasons, and did not differ among site types. The coexistence of L. rheocola with Bd suggests that (1) frog populations are becoming resistant to the fungus, (2) Bd may have evolved lower virulence, or (3) current environmental conditions may be inhibiting outbreaks of the fatal disease.