In many organisms, exposure to stressors during embryonic or prenatal stages can result in both direct and latent effects on subsequent developmental stages. For example, in birds, conditions experienced at early life are determinants of fitness in adults (1); in snapping turtles (Chelydra serpentina), hatchling from eggs incubated on a wet substrate have an improved locomotor performance in comparison to hatchlings from drier substrates (2) and in fishes, embryos of pink salmon (Oncorhynchus gorbuscha) exposed to oil have a reduction in juvenile growth and survival (3). In amphibians, these effects can be on individual growth rates, behavior, locomotion or immunology (4–7). Thus, exposure to predator cues in the pinewoods tree frog Hyla femoralis slowed larval growth and development, resulting in metamorphs with relatively smaller body sizes (8). In amphibians, repeated exposure at early life stages to other environmental stressors, such as contaminants, predator cues, and pathogens, can produce latent effects in juvenile and adult amphibians (4, 9, 10). Particularly in amphibian embryos, environmental cues can cause significant changes in hatching traits (11–14). Time of hatching can change in response to risks and opportunities. For example, embryos can hatch early to escape predators and pathogens, and this life history shift can have effects that persist through later life stages (15–17). Additionally, embryos infected with water molds can suffer differential mortality rates relative to the timing of exposure to the pathogen (18). As such, the timing of pathogen exposure might play a critical role on host susceptibility to infection (19).

Host ontogeny is a key factor for examining or predicting disease dynamics. There is precedence in different systems than minimum changes in the history of exposure to pathogens during a particular developmental stage can drastically change host life history. Changes in individual susceptibility to pathogens occur throughout ontogeny in many organisms, including plants (20), insects (21), birds (22), reptiles (23), mammals (24) and amphibians (25, 26). The key, however, to understanding temporal association between pathogens and susceptibility is to empirically discern latent and direct effects within and across life history stages. We posit that amphibians can be model systems for testing these questions as they are a taxon of conservation concern, have complex life histories, and are susceptible to multiple emerging infectious diseases.

One of the most researched amphibian pathogens is the fungus Batrachochytrium dendrobatidis (Bd), which has been implicated in the decline of numerous amphibian species worldwide (27–29). Differential susceptibility to Bd has been documented across species (30–35), populations (36, 37), life stages (38–42), and Bd strains (29, 33, 43–46). However, how exposure to Bd in one developmental stage can produce latent affects in a later life stage is unclear. Information regarding direct Bd impacts on embryos is also lacking as Bd mainly affects keratinized structures, which are absent in embryos. Further, the importance of evolutionary relationships between Bd strain and the embryonic host may also have significant implications.

We explored the direct and latent effects of Bd exposure on both the embryonic and larval stages using three amphibian species with differential susceptibility to native and invasive Bd strains (33, 34, 46, 47). We posit that amphibian embryos will be susceptible to the chytrid fungus as Bd can produce enzymes that can destroy tissue (48–50). Further, the release of fungal toxin (30, 51) could impact embryos by delaying growth, triggering key transitions resulting in ontogenetic shifts or latent effects on life history trajectories. Finally, we hypothesized that Bd can impact embryos by depletion of oxygen. The lack of dissolved oxygen slows down the development in Bufo bufo (52) and hypoxia can kill early life stages (13). Direct or latent effects may also vary with Bd strain and with host species, therefore evaluating different strains is critical to disentangle intrinsic aspects of the pathogen as virulence and how it changes among hosts. We also examined the influence of Bd exposure on larval survival predicting that repeated exposure to Bd across the embryonic/larval transition would result in decreased survival.

We studied three anuran species found in the US Pacific Northwest (PNW). These are the Pacific chorus frog (Pseudacris regilla) a common species throughout its PNW range found in a variety of habitats from sea level to montane regions, the Western toad (Anaxyrus boreas) whose populations have experienced declines across much of its historic range and American bullfrogs (Lithobates catesbeianus) an introduced species in the PNW (53–55). Twenty clutches of P. regilla were collected from Little Three creeks on 19 June 2014 (44°06′03.5″ N, 121°38′34.7″ WGS84 Deschutes County, OR, elevation = 2,000 m) and 600 eggs of A. boreas were collected from 20 different egg masses at Todd Lake (44°01′44.5″ N, 121°41′07.6″W WGS84 Deschutes County, OR, elevation = 1,870 m) on 29 May 2015. We collected 600 newly laid eggs from six distinct L. catesbeianus egg masses from William L. Finley National Wildlife Refuge on 20 May 2014 (44°25′23.6″ N, 123°18′41.8″W WGS84 Benton County, OR, elevation = 276 m). After collection, eggs were immediately transported to a climate-controlled environment at Oregon State University and held under constant temperature (14°–15.5°C) and photoperiod (12L: 12D) conditions. Less than 6 h after arrival, every clutch of P. regilla or group of eggs of A. boreas and L. catesbeianus were divided into three groups and each group for P. regilla and A. boreas contained ~10 eggs (±1.95 eggs), and 20 eggs for L. catesbeianus.

Life stage, time of exposure, and Bd strain influenced susceptibility to Bd in the embryo-larvae life history transition for three anuran species: P. regilla, A. boreas, and L. catesbeianus. We detected direct effects of Bd on embryonic and larval mortality, latent effects across the embryo/larval transition, and additive effects when double-exposed to Bd across both life stages. Exposure of embryos to Bd resulted in direct impacts on hatchling survivorship. We found direct, negative impacts of Bd strain and time of exposure on embryonic survival and proportion of hatching success for P. regilla and L. catesbeianus. Embryos of P. regilla were drastically affected by the non-native East Bd strain, resulting in more than 90% mortality when exposed later in embryonic development. Interestingly, embryos of invasive L. catesbeianus died when exposed to either Bd strain (East or West). When exposed early in embryonic development to the East strain, the number of viable hatchlings was zero and we detected a mortality of 90% in hatchlings after early exposure to the West Bd strain. When exposed later in development (East or West strains), only 50% of embryos hatched. The influence of time of exposure may be explained by changes in the thickness of the jelly layer surrounding the embryo through development. This jelly layer becomes thinner over development, thus late-stage embryos may be more affected by exposure to a pathogen (15, 18). Amphibian species-specific egg deposition forms (films, strings, clusters), as well as morphology of egg structure, provide differential protection from pathogens (67).

Post-hatching exposure resulted in both direct and latent impacts on larval survivorship. Direct effects on larvae are reported mainly as an increase in the odds of mortality for all three anuran species. P. regilla was negatively affected by exposure to the non-native East Bd strain, while A. boreas and L. catesbeianus were affected by both strains (East and West). Odds of mortality in A. boreas were higher when exposed to the West strain (19.56) than when exposed to the East strain (14.38). On the contrary, the odds of mortality in L. catesbeianus were higher when exposed to the East strain (9.9) than when exposed to the West strain (5.39). This result was not wholly unexpected as larvae mortality has been reported in experimental studies exposing these same species to Bd. A. boreas has been identified as particularly susceptible to Bd (30, 46) while P. regilla and L. catesbeianus larvae have relatively high survivorship (30, 33, 46, 68). In this study, we found a direct effect of Bd on larval survivorship for all three species. The increase in the odds of mortality in P. regilla and L. catesbeianus larvae can be explained by the origin and characteristics of the East strain. Isolated from L. catesbeianus in Maine (USA), this strain has been identified as hypervirulent (57, 69, 70) and categorized as part of the North American clade in the Global Pandemic Lineage (GPL) (57). As such, we anticipated an increase in larval mortality due to a lack of evolutionary relationship with this strain. However, L. catesbeianus larvae were also susceptible to the East strain even though it was isolated from their conspecifics within their native range.

We detected an increase in the odds of mortality in P. regilla when exposed early in embryonic development to Bd while the odds ratio for larval mortality were lower for later exposure. We could not evaluate potential latent effects after late exposure of P. regilla embryos to the East strain. Effects of Bd differed in embryos according to the timing of exposure. However, these effects of timing of exposure and duration of stressors such as pathogens and its relation to critical window and disease is not broadly tested (71, 72). We found an increase in the odds of larval mortality of both A. boreas and L. catesbeianus as a function of Bd strain and timing of embryonic exposure. In A. boreas, embryos exposed early to the West strain showed a decrease in the odds of mortality. Conversely, when A. boreas were exposed to the East strain late in embryonic development, larvae were almost 10 times more likely to die than control individuals. There was a similar increase in the odds of larval mortality in L. catesbeianus when exposed as embryos to any of the Bd strains. The high mortality rates in L. catesbeianus when exposed early to Bd prevented us from understanding potential latent effects for this invasive species. Our results support the hypothesis that timing of pathogen exposure is a major factor that influences host survivorship. Exposures of amphibian embryos to stressors such as pathogens at early stages of development can trigger effects over ontogeny (73). This exposure can later modify other characteristics, such as growth rates (74), mortality rates (75), mass (5), and development of immune response (76).

We also found effects of double Bd exposure (exposed in both the embryonic and larval stages) in all three anuran species. All species showed an increase in the odds of larval mortality when the first Bd exposure occurred at a later embryonic developmental stage (Gosner stage 18). In P. regilla, odds of mortality increased after double exposure to the West strain. Exposure to either strain (East or West) increased the odds of mortality in A. boreas and L. catesbeianus. Double exposure effects have been reported in experiments examining the larval/metamorph transition (77), thus our experiment provides additional information concerning other life history transitions providing a comprehensive view of the jeopardy through life.

The differential response of A. boreas to Early/Late and East/West Bd treatments may be explained by the presence of a potential critical window of vulnerability for this species and by the virulence of the Bd strain. Late exposure of A. boreas embryos to the East strain increased the odds of larval mortality of this species. Fernandez-Beneitez et al. (18) found that embryos of toads (Bufo calamita and Pelobates cultripes) exposed to Saprolegnia spp. at an early developmental stage (Gosner stage 12) suffered no increase in mortality, while embryos challenged at later stages of embryonic development (Gosner stages 15 and 19) were sensitive to the pathogen dying 72 h after exposure. Understanding which species experience latent effects will help target management efforts by identifying how exposure in particular life history stages can change host response. If a species is identified as being particularly susceptible to exposure to Bd as embryos, actions such as ex situ protection could be useful for its conservation.

Our findings complement the information on susceptibility of P. regilla to Bd as larvae of this species had previously been reported to be tolerant to certain Bd strains (30, 46). Interestingly, we found that this tolerance can change with an individual's previous exposure regime to non-native strains. Our experimental evaluation revealed that Bd strains from an invasive species can have harmful consequences on native and even invasive conspecific hosts. Our findings for A. boreas support previous work showing species as being susceptible to both the East and West strains of Bd (30, 46, 47). In L. catesbeianus, larvae and adults have been reported as able to withstand infection loads of the chytrid in different regions (78) and this species is suggested as an asymptomatic carrier or reservoir of Bd (79, 80). Our results indicate that larvae can also be susceptible to Bd but this response will be mediated by previous exposure in an early life stage. Individuals that were re-exposed to Bd were about 50 times more likely to die than individuals kept as controls. This contrasts with previous experimental studies reporting this species as a carrier of Bd (30, 32, 60). Generally, those studies directly exposed individuals in the larval stage [Gosner stage (26–30)] without considering previous exposure regimes. In our study, L. catesbeianus were vulnerable to Bd exposure in response to direct exposure and across life history transitions.

We found species-specific embryonic mortality after exposure to Bd. Many pathogens impact anuran embryos, including ranavirus (76), oomycetes (81–84), filamentous ascomycetes (85) and microsporidia (86). The mechanisms of protection offered by the vitelline membrane against ranavirus are unknown. However, bacteriostatic activity of the egg membrane or the capsule (87) or just its role as structural barrier when exposed to contaminants have been proposed (88, 89). In the case of Oomycetes, zoospores are chemotactic and move toward suitable substrates where they can germinate and grow. Similarly, ascomycetes are able to grow through the jelly. Few studies have suggested how Bd affects anuran embryos. Bd enzymatic action is one mechanism that could explain this result, as it can cause damage in skin tissue of hosts after exposure (30, 49, 90, 91). A complex mix of proteolytic and hydrolyzing enzymes (esterases) that degrade amphibian tissue have been described from different Bd isolates (90–92). In addition, many hatching anurans release enzymes to assist with degradation of the egg capsule at the moment of hatching (93, 94); this could potentially facilitate the enzymatic action of Bd to degrade tissues. Recently, dose-dependent mortality and proliferation in zebrafish (Danio rerio) tissue was reported with toxins secreted after the establishment of Bd sporangia (95).

The present study offers useful information about the complexity of host response to a pathogen, particularly with multiple exposures across life stages. Our study provides information about direct effects of Bd on anuran embryos, with significant impacts on mortality and the proportion of hatching success. Our results also quantified latent effects of Bd exposure over ontogeny (96). Despite being a relatively brief period, exposure to Bd in the egg led to increased mortality after hatching and species-specific differences were due to the timing of embryonic exposure and re-exposure in the larval stage. As we increase our understanding of how Bd impacts amphibians through direct and even latent effects, we are recognizing that the effects Bd may have on population dynamics and conservation of amphibians have been underestimated in wild populations.

Additional research exploring the mechanisms protecting the embryos is needed to better understand the susceptibility of this developmental stage to disease. Characteristics such as jelly thickness and composition, or size of the capsule, can be involved in resistance to chytrid. As eggs received material from their parents during oviposition, evaluating the role of parents in the immune response of their offspring can help us to understand more about embryonic immunity. As continued survey efforts have located Bd in populations of amphibians around the world, there is growing evidence that the risk of Bd cannot be simplified to species susceptibility. Instead, Bd risk includes strain, host life-stage, and specific exposure scenarios. Further studies are also required to better understand how variation in other environmental and biological parameters can affect the outcome of repeated Bd exposure in anuran species. Our results add information to the growing body of evidence concerning differential susceptibility to pathogens among amphibian species and across life stages.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Institutional Animal Care and Use committee Oregon State University.

JU, AB, and TG conceptualized the manuscript. JU and EB performed the field work, experiments and data analysis. JU, EB, AB, and TG have made a significant, direct and intellectual contribution to the work. All authors contributed to the article and approved the submitted version.

Publication of this paper was supported, in part, by the Henry Mastin Graduate Student Fund (Department of Fisheries, Wildlife and Conservation Sciences, Oregon State University) and the US Department of Agriculture, Forest Service, Pacific Northwest Research Station through Deanna H. Olson.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.