Development and Infectious Disease in Hosts with Complex Life Cycles

Metamorphosis is often characterized by profound changes in morphology and physiology that can affect the dynamics of species interactions. For example, the interaction between a pathogen and its host may differ depending on the life stage of the host or pathogen. One pathogen that infects hosts with complex life cycles is the emerging fungal pathogen of amphibians, Batrachochytrium dendrobatidis (Bd). We sought to determine how conditions at the larval stage can affect variation in development and patterns of Bd infection across amphibian life stages. We used outdoor experimental mesocosms to simulate natural pond habitats and manipulated the presence of Bd, the larval density, and the number of host species in larvae of two co-occurring amphibian species (Rana cascadae and Pseudacris regilla). We found that infection differed between species throughout development; P. regilla consistently had higher infection severity compared to R. cascadae. Additionally, while up to 100% of larvae were infected, only 18.2% of R. cascadae and 81.5% of P. regilla were infected after metamorphosis. This indicates that amphibians have the ability to recover from Bd infection as they undergo metamorphosis. Higher larval densities in P. regilla led to a shorter larval period, and individuals with a shorter larval period had lower infection severity. This led to a trend where P. regilla larvae reared at high densities tended to have lower infection prevalence after metamorphosis. We also found that exposure to Bd increased larval mortality and prolonged the larval period in P. regilla, indicating that P. regilla are susceptible to the negative effects of Bd as larvae. This study demonstrates that host density, species composition, and pathogen exposure may all interact to influence development and infection in hosts with complex life cycles.     

Temperature, hydric environment, and prior pathogen exposure alter the experimental severity of chytridiomycosis in boreal toads

Prevalence of the pathogen Batrachochytrium dendrobatidis (Bd), implicated in amphibian population declines worldwide, is associated with habitat moisture and temperature, but few studies have varied these factors and measured the response to infection in amphibian hosts. We evaluated how varying humidity, contact with water, and temperature affected the manifestation of chytridiomycosis in boreal toads Anaxyrus (Bufo) boreas boreas and how prior exposure to Bd affects the likelihood of survival after re-exposure, such as may occur seasonally in long-lived species. Humidity did not affect survival or the degree of Bd infection, but a longer time in contact with water increased the likelihood of mortality. After exposure to approximately 10(6) Bd zoospores, all toads in continuous contact with water died within 30 d. Moreover, Bd-exposed toads that were disease-free after 64 d under dry conditions, developed lethal chytridiomycosis within 70 d of transfer to wet conditions. Toads in unheated aquaria (mean = 15 degrees C) survived less than 48 d, while those in moderately heated aquaria (mean = 18 degrees C) survived 115 d post-exposure and exhibited behavioral fever, selecting warmer sites across a temperature gradient. We also found benefits of prior Bd infection: previously exposed toads survived 3 times longer than Bd-na?ve toads after re-exposure to 106 zoospores (89 vs. 30 d), but only when dry microenvironments were available. This study illustrates how the outcome of Bd infection in boreal toads is environmentally dependent: when continuously wet, high reinfection rates may overwhelm defenses, but periodic drying, moderate warming, and previous infection may allow infected toads to extend their survival.     

Development and disease: how susceptibility to an emerging pathogen changes through anuran development

Ranaviruses have caused die-offs of amphibians across the globe. In North America, these pathogens cause more amphibian mortality events than any other pathogen. Field observations suggest that ranavirus epizootics in amphibian communities are common during metamorphosis, presumably due to changes in immune function. However, few controlled studies have compared the relative susceptibility of amphibians to ranaviruses across life stages. Our objectives were to measure differences in mortality and infection prevalence following exposure to ranavirus at four developmental stages and determine whether the differences were consistent among seven anuran species. Based on previous studies, we hypothesized that susceptibility to ranavirus would be greatest at metamorphosis. Our results did not support this hypothesis, as four of the species were most susceptible to ranavirus during the larval or hatchling stages. The embryo stage had the lowest susceptibility among species probably due to the protective membranous layers of the egg. Our results indicate that generalizations should be made cautiously about patterns of susceptibility to ranaviruses among amphibian developmental stages and species. Further, if early developmental stages of amphibians are susceptible to ranaviruses, the impact of ranavirus epizootic events may be greater than realized due to the greater difficulty of detecting morbid hatchlings and larvae compared to metamorphs.     

Effect of temperature on host response to Batrachochytrium dendrobatidis infection in the mountain yellow-legged frog (Rana muscosa)

The pathogenic chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis, has been implicated in declines of amphibian populations throughout the world, including declines and extinctions of local populations of mountain yellow-legged frogs, Rana muscosa, in the California Sierra Nevada. Previous studies have shown B. dendrobatidis achieves its maximum growth rate in culture in the temperature range of 17-25 C, and exposure to very high temperatures can clear frogs of B. dendrobatidis infection. Here we present the results of a laboratory experiment in which experimentally infected R. muscosa tadpoles were followed through metamorphosis at temperatures of 17 and 22 C. All infected animals developed clinical disease within a similar time frame. However, frogs housed at 22 C exhibited a significantly lower mortality than those housed at 17 C. Within 35 days after metamorphosis, 50% of the frogs housed at 22 C died, while 95% of the frogs housed at 17 C died. Clinical signs subsided in the surviving frogs at 22 C, despite persistent infection. Because both temperatures are within the optimal thermal range for growth of B. dendrobatidis, we propose that the difference in outcome indicates the effect of temperature on the host's resistance to chytridiomycosis, rather than an effect on the fungus alone.     

Relationships among size, development, and Batrachochytrium dendrobatidis infection in African tadpoles

The fungal pathogen Batrachochytrium dendrobatidis contributes to the global decline of amphibians. Although mortality from B. dendrobatidis infections occurs primarily in postmetamorphic individuals, infected tadpoles may suffer reduced growth and developmental rates as a result of oral chytridiomycosis, possibly affecting adult fitness. We conducted a field study in which we examined South African tadpoles for oral chytridiomycosis and compared the body sizes of infected and uninfected individuals of 2 species, Heleophryne natalensis and Strongylopus hymenopus. Presence of B. dendrobatidis was determined by microscopic inspection of mouthparts. Infection prevalence was high in both species, 62.5 and 38.6%, respectively, and infected individuals were significantly larger in both species. The inclusion of developmental stage in the analysis of S. hymenopus body size eliminated the relationship between body size and infection status, suggesting that differences in body size were not due to differences in growth, but to differences in developmental stage of infected larvae. These results suggest that larvae at more advanced developmental stages are more likely to be infected with B. dendrobatidis and that infection in larval amphibians may be dependent on time or developmental status of larvae. Contrary to the results of past studies, there was no evidence that oral chytridiomycosis resulted in decreased growth of tadpoles, despite the occurrence of oral abnormalities in infected individuals of 1 species. Because tadpole performance can subsequently affect the health of anuran populations and because tadpoles can act as reservoirs of infection, the study of B. dendrobatidis in larval amphibians is important to understanding the effects of this emerging disease.     

Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses

Innate immune mechanisms of defense are especially important to ectothermic vertebrates in which adaptive immune responses may be slow to develop. One innate defense in amphibian skin is the release of abundant quantities of antimicrobial peptides. Chytridiomycosis is an emerging infectious disease of amphibians caused by the skin fungus, Batrachochytrium dendrobatidis . Susceptibility to chytridiomycosis varies among species, and mechanisms of disease resistance are not well understood. Previously, we have shown that Australian and Panamanian amphibian species that possess skin peptides that effectively inhibit the growth of B. dendrobatidis in vitro tend to survive better in the wild or are predicted to survive the first encounter with this lethal pathogen. For most species, it has been difficult to experimentally infect individuals with B. dendrobatidis and directly evaluate both survival and antimicrobial peptide defenses. Here, we demonstrate differences in susceptibility to chytridiomycosis among four Australian species ( Litoria caerulea, Litoria chloris, Mixophyes fasciolatus and Limnodynastes tasmaniensis ) after experimental infection with B. dendrobatidis , and show that the survival rate increases with the in vitro effectiveness of the skin peptides. We also observed that circulating granulocyte, but not lymphocyte, counts differed between infected and uninfected Lit. chloris . This suggests that innate granulocyte defenses may be activated by pathogen exposure. Taken together, our data suggest that multiple innate defense mechanisms are involved in resistance to chytridiomycosis, and the efficacy of these defenses varies by amphibian species.     

All hosts are not equal: explaining differential patterns of malformations in an amphibian community

1. Within a community, different host species often exhibit broad variation in sensitivity to infection and disease. Because such differences can influence the strength and outcome of community interactions, it is essential to understand differential disease patterns and identify the mechanisms responsible. 2. In North American wetlands, amphibian species often exhibit extraordinary differences in the frequency of limb malformations induced by the digenetic trematode, Ribeiroia ondatrae. By coupling field studies with parasite exposure experiments, we evaluated whether such patterns were due to differences in (i) parasite encounter rate, (ii) infection establishment, or (iii) parasite persistence within hosts. 3. Field results underscored the broad variation in malformations and infection between host species; while nearly 60% (n = 618) of emerging American toads exhibited severe limb deformities such as bony triangles, skin webbings and missing limbs, fewer than 4% (n = 251) of Eastern gray treefrogs from the same pond were abnormal. Despite similarities in the phenology and larval development period of these species, they differed sharply in Ribeiroia infection. On average, toads supported 75x more metacercariae than did metamorphic treefrogs. 4. Experimental exposures of larval toads and treefrogs to a realistic range of Ribeiroia cercariae revealed strong differences in the sensitivity of these species to infection; exposed toads suffered elevated mortality (up to 95%), delayed metamorphosis, and severe limb malformations consistent with field observations. Treefrogs, in contrast, exhibited limited mortality and no malformations, regardless of exposure level. Ribeiroia cercariae were substantially less successful in locating and infecting Hyla versicolor larvae. 5. Our results indicate that the observed differences in infection and malformations owe to a lower ability of Ribeiroia cercariae to both find and establish within larval treefrogs, possibly stemming from a heightened immune response to infection. Because Ribeiroia is a highly pathogenic parasite with negative effects on larval and metamorphic amphibian survival, variation in infection resistance among species could have important implications for understanding patterns of species co-occurrence, competition, and community diversity.     

Effects of an infectious fungus, Batrachochytrium dendrobatidis, on amphibian predator-prey interactions

The effects of parasites and pathogens on host behaviors may be particularly important in predator-prey contexts, since few animal behaviors are more crucial for ensuring immediate survival than the avoidance of lethal predators in nature. We examined the effects of an emerging fungal pathogen of amphibians, Batrachochytrium dendrobatidis, on anti-predator behaviors of tadpoles of four frog species. We also investigated whether amphibian predators consumed infected prey, and whether B. dendrobatidis caused differences in predation rates among prey in laboratory feeding trials. We found differences in anti-predator behaviors among larvae of four amphibian species, and show that infected tadpoles of one species (Anaxyrus boreas) were more active and sought refuge more frequently when exposed to predator chemical cues. Salamander predators consumed infected and uninfected tadpoles of three other prey species at similar rates in feeding trials, and predation risk among prey was unaffected by B. dendrobatidis. Collectively, our results show that even sub-lethal exposure to B. dendrobatidis can alter fundamental anti-predator behaviors in some amphibian prey species, and suggest the unexplored possibility that indiscriminate predation between infected and uninfected prey (i.e., non-selective predation) could increase the prevalence of this widely distributed pathogen in amphibian populations. Because one of the most prominent types of predators in many amphibian systems is salamanders, and because salamanders are susceptible to B. dendrobatidis, our work suggests the importance of considering host susceptibility and behavioral changes that could arise from infection in both predators and prey.     

Unlikely Remedy: Fungicide Clears Infection from Pathogenic Fungus in Larval Southern Leopard Frogs (Lithobates sphenocephalus)

Amphibians are often exposed to a wide variety of perturbations. Two of these, pesticides and pathogens, are linked to declines in both amphibian health and population viability. Many studies have examined the separate effects of such perturbations; however, few have examined the effects of simultaneous exposure of both to amphibians. In this study, we exposed larval southern leopard frog tadpoles (Lithobates sphenocephalus) to the chytrid fungus Batrachochytrium dendrobatidis and the fungicide thiophanate-methyl (TM) at 0.6 mg/L under laboratory conditions. The experiment was continued until all larvae completed metamorphosis or died. Overall, TM facilitated increases in tadpole mass and length. Additionally, individuals exposed to both TM and Bd were heavier and larger, compared to all other treatments. TM also cleared Bd in infected larvae. We conclude that TM affects larval anurans to facilitate growth and development while clearing Bd infection. Our findings highlight the need for more research into multiple perturbations, specifically pesticides and disease, to further promote amphibian heath.     

Survival of three species of anuran metamorphs exposed to UV-B radiation and the pathogenic fungus Batrachochytrium dendrobatidis

When exploring the possible factors contributing to population declines, it is necessary to consider multiple, interacting environmental stressors. Here, we investigate the impact of 2 factors, ultraviolet radiation and disease, on the survival of anuran amphibians. Exposure to ultraviolet-B (UV-B) radiation increases mortality and results in various sub-lethal effects for many amphibian species. Infectious diseases can also negatively impact amphibian populations. In this study, we exposed metamorphic individuals (metamorphs) to both UV-B and Batrachochytrium dendrobatidis (BD), a fungal pathogen and cause of the disease chytridiomycosis, and monitored survival for 3 wk. We tested for possible interactions between UV-B and BD in 3 species: the Cascades frog Rana cascadae; the Western toad Bufo boreas; and the Pacific treefrog Hyla regilla. We found strong interspecific differences in susceptibility to BD. For example, R. cascadae suffered a large increase in mortality when exposed to BD; B. boreas also experienced mortality, but this effect was small relative to the R. cascadae response. H. regilla did not show any decrease in survival when exposed to either factor. No synergistic interactions between UV-B and BD were found for any of the test species. A previous study investigating the impact of BD on larval amphibians showed different species responses (Blaustein et al. 2005a). Our results highlight the importance of studying multiple life history stages when determining the impact of environmental stressors. The contrast between these 2 studies emphasizes how vulnerability to a pathogen can vary between life history stages within a single species.     

Transmission of Batrachochytrium dendrobatidis within and between amphibian life stages

Chytridiomycosis is an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis, which has been implicated in amphibian declines worldwide. The mountain yellow-legged frog Rana muscosa is a declining amphibian species that can be infected by B. dendrobatidis; however, transmission between conspecifics has not been documented. Here, we present experimental evidence that R. muscosa tadpoles can be infected by fungal zoospores and that they can transmit infection to each other and to postmetamorphic animals. We compared several techniques for detecting B. dendrobatidis transmission and found that histology with serial sectioning was able to detect infection before cytology or visual inspections. We also show that R. muscosa tadpoles appear healthy with B. dendrobatidis infection, while postmetamorphic animals experience mortality. In addition, we provide guidelines for visually detecting B. dendrobatidis in R. muscosa tadpoles, which may be useful in other affected species. Field surveys of infected and uninfected populations verify this identification technique.     

Predators and trematode parasites jointly affect larval anuran functional traits and corticosterone levels

Non‐consumptive predator effects may have dramatic consequences for host–parasite interactions by influencing the ability of prey items to avoid, resist, or tolerate infection. Both predators and parasites can affect host traits, such as growth rates and behavior, and these effects may in part be mediated through shared physiological pathways (e.g. the glucocorticoid stress hormone, corticosterone [CORT]). Here, we examined the effects of trematode parasites (Digena: Echinostomatidae) and predator (larval odonate) exposure on larvae of two amphibian species (Rana sylvatica and R. clamitans) in laboratory experiments. First, we measured behavior and CORT responses of tadpoles exposed to predator chemical cue in combination with parasite cue or under direct exposure to parasites. We then measured the combined effects of predator cue and parasite infection on survival and traits. Evidence for effects of parasite cue in our study was equivocal, but we found novel interactive effects of parasites and predators on larval frogs. Parasites and predators had antagonistic effects on CORT, behavior, and morphology, and negative synergistic effects on development. In addition, parasite infection and predator cues additively reduced activity levels of both species and growth in wood frogs. Negative effects of parasite infection on survival and traits were dose‐dependent for both species, although wood frogs generally experienced stronger effects of infection than green frogs. Our results emphasize the importance of considering effects of parasites as well as predators, since both can have strong effects on survival and the combination can have both additive and non‐additive effects on key traits. These effects likely have important implications for amphibian population dynamics, community structure, and conservation.     

Carry‐over Effects of Size at Metamorphosis in Red‐eyed Treefrogs: Higher Survival but Slower Growth of Larger Metamorphs

Most animals have complex life histories, composed of a series of ecologically distinct stages, and the transitions between stages are often plastic. Anurans are models for research on complex life cycles. Many species exhibit plastic timing of and size at metamorphosis, due to both environmental constraints on larval growth and development and adaptive plastic responses to environmental variation. Models predicting optimal timing of metamorphosis balance cost/benefit ratios across stages, assuming that size affects growth and mortality rates in each stage. Much research has documented such effects in the larval period, but we lack an equal understanding of juvenile growth and mortality. Here, we examine how variation in size at metamorphosis in the Neotropical red‐eyed treefrog, Agalychnis callidryas, affects post‐metamorphic growth, foraging, and behavior in the lab as well as growth and survival in the field. Surprisingly, many individuals lost mass for weeks after metamorphosis. In the lab, larger metamorphs lost more mass following metamorphosis, ate similar amounts, had lower food conversion efficiencies, and grew more slowly after mass loss ceased than did smaller ones. In field cages larger metamorphs were more likely to survive than smaller ones; just one froglet died in the lab. Our data suggest that size‐specific differences in physiology and behavior influence these trends. Comparing across species and studies, large size at metamorphosis generally confers higher survival; size effects on growth rates vary substantially among species, in both magnitude and direction, but may be stronger in the tropics.     

Will climate change reduce the effects of a pesticide on amphibians?: partitioning effects on exposure and susceptibility to contaminants

Several studies suggest that global climate change could increase the toxicity of contaminants, but none of these studies explicitly integrate the effects of climate change on both susceptibility and duration of exposure to pollution. For many amphibian and aquatic insect species, exposure to contaminants is probably greatest during their fully aquatic embryonic and larval stages because these stages cannot readily escape water bodies where many contaminants accumulate and concentrate. Hence, by accelerating embryonic and larval development, global warming might reduce the duration of contaminant exposure for these taxa. To test this hypothesis, we isolated the effects of a temperature gradient (13–25 °C) on susceptibility (toxicity at a controlled exposure duration) and exposure of the streamside salamander, Ambystoma barbouri, to the herbicide atrazine (0, 4, 40, and 400 μg L^?1) by quantifying growth, survival, hatching, and metamorphosis under an atrazine exposure duration that was either constant or that depended on time to metamorphosis (and thus temperature). Increasing atrazine concentrations reduced growth, delayed hatching and metamorphosis, and decreased embryonic and larval survival. Increasing temperatures enhanced growth, accelerated development, and reduced survival for embryos but not larvae. With the exception of growth, increasing temperatures generally did not enhance the toxicity of atrazine, but they did generally ameliorate the adverse effects of atrazine by accelerating development and reducing the duration of atrazine exposure. The actual effects of climate change on contaminants remains difficult to predict because temperature changes can affect chemical use, uptake, excretion, biotransformation, fate, transport, and bioavailability. However, this work highlights the importance of explicitly considering how climate change will affect both exposure and toxicity to contaminants to accurately assess risk.     

Malformations and mortality in the Asian Common Toad induced by exposure to pleurolophocercous cercariae (Trematoda: Cryptogonimidae)

Malformations and increased mortality due to infection by the digenetic trematode, Riberioa ondatrae have been reported for many species of amphibians. Severe malformations have also been reported in the Common Hourglass Tree Frog, Polypedates cruciger induced by pleurolophocercous cercariae in Sri Lanka in addition to the changes in the behaviour, development and survival of the host. We exposed pre-limb bud stage tadpoles (Gosner stages 25–26) of the Asian Common Toad, Duttaphrynus melanostictus to the same pleurolophocercous type cercariae under laboratory conditions. Molecular and morphological identification showed that these cercariae belonged Acanthostomum burminis infecting freshwater snakes as definitive hosts. These cercariae induced malformations (27.8%) and reduced survival to metamorphosis (53.8%). The magnitude of the effects increased with the dose of cercariae. Types of malformations were mainly axial, such as scoliosis and kyphosis. Severe limb malformations such as extra or missing limbs as reported for amphibians exposed to R. ondatrae were not observed in the D. melanostictus. Same authors reported a higher percentage of malformations previously when P. cruciger was exposed to the cercariae A. burminis compared to D. melanostictus. However, tadpoles of D. melanostictus, which are smaller compared to those of P. cruciger, experienced higher mortality than P. cruciger tadpoles. Trematode induced malformations and mortality in amphibians are highly variable and depend on multiple factors such as host species differences such as resistance to infection and tolerance, life-history characteristics such as size at metamorphosis and length of the metamorphosis period, and other factors such as size of the amphibian at the time of trematode exposure.     

Germ Tube Mediated Invasion of Batrachochytrium dendrobatidis in Amphibian Skin Is Host Dependent

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, a fungal skin disease in amphibians and driver of worldwide amphibian declines.We focussed on the early stages of infection by Bd in 3 amphibian species with a differential susceptibility to chytridiomycosis. Skin explants of Alytes muletensis, Litoria caerulea and Xenopus leavis were exposed to Bd in an Ussing chamber for 3 to 5 days. Early interactions of Bd with amphibian skin were observed using light microscopy and transmission electron microscopy. To validate the observations in vitro, comparison was made with skin from experimentally infected frogs. Additional in vitro experiments were performed to elucidate the process of intracellular colonization in L. caerulea.Early interactions of Bd with amphibian skin are: attachment of zoospores to host skin, zoospore germination, germ tube development, penetration into skin cells, invasive growth in the host skin, resulting in the loss of host cell cytoplasm. Inoculation of A. muletensis and L. caerulea skin was followed within 24 h by endobiotic development, with sporangia located intracellularly in the skin. Evidence is provided of how intracellular colonization is established and how colonization by Bd proceeds to deeper skin layers. Older thalli develop rhizoid-like structures that spread to deeper skin layers, form a swelling inside the host cell to finally give rise to a new thallus.In X. laevis, interaction of Bd with skin was limited to an epibiotic state, with sporangia developing upon the skin. Only the superficial epidermis was affected. Epidermal cells seemed to be used as a nutrient source without development of intracellular thalli. The in vitro data agreed with the results obtained after experimental infection of the studied frog species. These data suggest that the colonization strategy of B. dendrobatidis is host dependent, with the extent of colonization most likely determined by inherent characteristics of the host epidermis.     

Sequential predator effects across three life stages of the African tree frog, <Emphasis Type="Italic">Hyperolius spinigularis</Emphasis>

While theoretical studies of the timing of key switch points in complex life cycles such as hatching and metamorphosis have stressed the importance of considering multiple stages, most empirical work has focused on a single life stage. However, the relationship between the fitness components of different life stages may be complex. Ontogenetic switch points such as hatching and metamorphosis do not represent new beginnings—carryover effects across stages can arise when environmental effects on the density and/or traits of early ontogenetic stages subsequently alter mortality or growth in later stages. In this study, I examine the effects of egg- and larval-stage predators on larval performance, size at metamorphosis, and post-metamorphic predation in the African tree frog Hyperolius spinigularis. I monitored the density and survival of arboreal H. spinigularis clutches in the field to estimate how much egg-stage predation reduced the input of tadpoles into the pond. I then conducted experiments to determine: (1) how reductions in initial larval density due to egg predators affect larval survival and mass and age at metamorphosis in the presence and absence of aquatic larval predators, dragonfly larvae, and (2) how differences in mass or age at metamorphosis arising from predation in the embryonic and larval environments affect encounters with post-metamorphic predators, fishing spiders. Reduction in larval densities due to egg predation tended to increase per capita larval survival, decrease larval duration and increase mass at metamorphosis. Larval predators decreased larval survival and had density-dependent effects on larval duration and mass at metamorphosis. The combined effects of embryonic and larval-stage predators increased mass at metamorphosis of survivors by 91%. Larger mass at metamorphosis may have immediate fitness benefits, as larger metamorphs had higher survival in encounters with fishing spiders. Thus, the effects of predators early in ontogeny can alter predation risk even two life stages later.     

Effects of Tail Clipping on Larval Performance and Tail Regeneration Rates in the Near Eastern Fire Salamander,Salamandra infraimmaculata

Tail-tip clipping is a common technique for collecting tissue samples from amphibian larvae and adults. Surprisingly, studies of this invasive sampling procedure or of natural tail clipping – i.e., bites inflicted by predators including conspecifics - on the performance and fitness of aquatic larval stages of urodeles are scarce. We conducted two studies in which we assessed the effects of posterior tail clipping (~30 percent of tail) on Near Eastern fire salamander (Salamandra infraimmaculata) larvae. In a laboratory study, we checked regeneration rates of posterior tail-tip clipping at different ages. Regeneration rates were hump-shaped, peaking at the age of ~30 days and then decreasing. This variation in tail regeneration rates suggests tradeoffs in resource allocation between regeneration and somatic growth during early and advanced development. In an outdoor artificial pond experiment, under constant larval densities, we assessed how tail clipping of newborn larvae affects survival to, time to, and size at metamorphosis. Repeated measures ANOVA on mean larval survival per pond revealed no effect of tail clipping. Tail clipping had correspondingly no effect on larval growth and development expressed in size (mass and snout-vent length) at, and time to, metamorphosis. We conclude that despite the given variation in tail regeneration rates throughout larval ontogeny, clipping of 30% percent of the posterior tail area seems to have no adverse effects on larval fitness and survival. We suggest that future use of this imperative tool for the study of amphibian should take into account larval developmental stage during the time of application and not just the relative size of the clipped tail sample.     

Whether larval amphibians school does not affect the parasite aggregation rule: testing the effects of host spatial heterogeneity in field and experimental studies

Almost all macroparasites show over‐dispersed infections within natural host populations such that most parasites are distributed among a few heavily‐infected individuals. Despite the importance of parasite aggregation for understanding system stability, the potential for population regulation, and super‐spreading events, many questions persist about its underlying drivers. Theoretically, aggregation results from heterogeneity in host exposure, resistance, and tolerance. However, few studies have examined how host spatial arrangement – which likely affects both parasite encounter and density‐dependent interactions – influences infection and dispersion, representing a critical gap in our current knowledge regarding the possible drivers of parasite aggregation. Using field data from over 165 ponds and 8000 hosts, we evaluated how the spatial clustering of amphibian larvae within ponds 1) varied among different amphibian species, and 2), affected the distribution of parasites within the host population using Taylor's power law. A complementary mesocosm experiment used field‐guided manipulations of the spatial arrangement of larval amphibians to create a gradient in host clustering while controlling host density, thereby testing for spatial effects on both infection success and aggregation by three different trematode species. Our field data indicated that larval amphibians exhibited significant spatial clustering that was well captured by Taylor's power law (R² 0.92 to 0.97 for different host species), but the residual variation only weakly correlated with observed patterns of trematode parasite over‐dispersion. Correspondingly, experimental manipulation of host clustering had no effects on parasite infection success or the degree of parasite aggregation among cages or mesocosms. Given the importance of parasite over‐dispersion for host populations and disease dynamics, we advocate for further investigations of host and parasite spatial aggregation, particularly studies that incorporate and/or control for heterogeneity in exposure and susceptibility.