Multi-Level Effects of Low Dose Rate Ionizing Radiation on Southern Toad,Anaxyrus [Bufo] terrestris

Despite their potential vulnerability to contaminants from exposure at multiple life stages, amphibians are one of the least studied groups of vertebrates in ecotoxicology, and research on radiation effects in amphibians is scarce. We used multiple endpoints to assess the radiosensitivity of the southern toad (Anaxyrus [Bufo] terrestris) during its pre-terrestrial stages of development –embryonic, larval, and metamorphic. Toads were exposed, from several hours after oviposition through metamorphosis (up to 77 days later), to four low dose rates of ¹³⁷Cs at 0.13, 2.4, 21, and 222 mGy d^-1, resulting in total doses up to 15.8 Gy. Radiation treatments did not affect hatching success of embryos, larval survival, or the length of the larval period. The individual family variation in hatching success of embryos was larger than the radiation response. In contrast, newly metamorphosed individuals from the higher dose-rate treatments had higher mass and mass/length body indices, a measure which may relate to higher post-metamorphic survival. The increased mass and index at higher dose rates may indicate that the chronic, low dose rate radiation exposures triggered secondary responses. Additionally, the increases in growth were linked to a decrease in DNA damage (as measured by the Comet Assay) in red blood cells at a dose rate of 21 mGy d^-1 and a total dose of 1.1 Gy. In conclusion, the complex effects of low dose rates of ionizing radiation may trigger growth and cellular repair mechanisms in amphibian larvae.     

Hatching plasticity in a Southeast Asian tree frog mitigates submergence-induced mortality

Environmentally cued hatching has been well-documented in amphibians in response to a wide range of abiotic and biotic factors. The hatching of terrestrial amphibian eggs in response to flooding may be basal within the group, but amphibian lineages in tropical Asia and sub-Saharan Africa have not received as much attention as their Neotropical counterparts. We investigated submergence-induced hatching in Feihyla hansenae, a Rhacophorid tree frog with terrestrial eggs. We quantified natural rates of clutch submergence at our study site in Thailand. Using submergence experiments, we found that embryos are capable of hatching early to escape flooding, and that failure to hatch results in mortality. Among the embryos that were able to hatch early, only the earliest, youngest hatchlings experienced a trade-off in body size that persisted for 6 days, while later, older hatchlings were not significantly smaller than spontaneous hatchlings under control conditions. By incorporating our natural and experimental data into Monte Carlo methods to simulate and compare survival probabilities with and without hatching plasticity, we found an overall 3.1% increase in submergence survival due to hatching plasticity. Our findings support the idea that flooding-induced hatching is widespread across amphibians with terrestrial eggs and highlight the importance of researching understudied tropical regions. As climate change is projected to affect rainfall patterns, the ability of embryos to escape abiotic egg-stage threats may be an indicator of species' ability to flexibly navigate a changing environment.     

An identification of invariants in life history traits of amphibians and reptiles

While many morphological, physiological, and ecological characteristics of organisms scale with body size, some do not change under size transformation. They are called invariant. A recent study recommended five criteria for identifying invariant traits. These are based on that a trait exhibits a unimodal central tendency and varies over a limited range with body mass (type I), or that it does not vary systematically with body mass (type II). We methodologically improved these criteria and then applied them to life history traits of amphibians, Anura, Caudata (eleven traits), and reptiles (eight traits). The numbers of invariant traits identified by criteria differed across amphibian orders and between amphibians and reptiles. Reproductive output (maximum number of reproductive events per year), incubation time, length of larval period, and metamorphosis size were type I and II invariant across amphibians. In both amphibian orders, reproductive output and metamorphosis size were type I and II invariant. In Anura, incubation time and length of larval period and in Caudata, incubation time were further type II invariant. In reptiles, however, only number of clutches per year was invariant (type II). All these differences could reflect that in reptiles body size and in amphibians, Anura, and Caudata metamorphosis (neotenic species go not through it) and the trend toward independence of egg and larval development from water additionally constrained life history evolution. We further demonstrate that all invariance criteria worked for amphibian and reptilian life history traits, although we corroborated some known and identified new limitations to their application.     

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.     

Taste organ growth and development in the direct developing frog Eleutherodactylus coqui (Lissamphibia: Eleutherodactylidae)

Previous research on amphibian taste organs concerned amphibians with a biphasic life history, that is, with larval period and metamorphosis. Direct developing frog species, such as Eleutherodactylus coqui, undergo a cryptic metamorphosis before hatching, and many larval‐specific features are vestigial or have been lost entirely from their ontogeny. Taste buds are present in larval stages of biphasically developing anurans and are replaced by taste discs during metamorphosis. One goal of this study was to characterize the ontogeny of taste buds and/or discs in E. coqui. The other goal was to examine correlations between body size and taste organ density and size in different regions of oral epithelium. The research reveals the presence of only one type of taste organ, characteristic of metamorphs of biphasic amphibians, namely taste disc. In addition, taste disc density and the area of the taste disc sensory zone change dramatically during growth.     

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.     

The response of amphibian larvae to environmental change is both consistent and variable

Many environments are undergoing rapid environmental change and there is a need to understand the mechanisms by which species can persist in altered environments. Model systems, such as amphibian metamorphosis, which can be generalized across many types of environmental change and across many species, are a powerful tool for understanding mechanisms that facilitate persistence in altered and disturbed environments. Amphibian larvae respond to environmental change by varying age at metamorphosis, or size at metamorphosis. Differential selection pressures on age or size at metamorphosis may result in a differential response among taxa to environmental change. Using a meta‐analysis, we investigated whether age at metamorphosis, size at metamorphosis, and larval growth rate vary within and among taxonomic families of amphibians in experiments that modified the environmental temperature, density of individuals, food, hydroperiod and the presence of predators. For all environmental factors except predators, the direction of the response was consistent across most of the studied taxa. However, there was considerable variation in effect size both within and among families. Results demonstrate that amphibian metamorphosis is a valuable model system for studying the effects of environmental change. Yet, we stress the need for caution in making generalizations about how individuals respond to environmental factors that have an indirect effect on physiology and require the perception of an environmental cue, such as the presence of predators. Synthesis As the current conditions of the environment are rapidly changing there is a need to understand how organisms respond to environmental change, and whether response of one species can be generalized to other species. Using a meta‐analyses, we tested whether the phenotypic response of amphibian larvae to five types of environmental change is consistent among and within taxonomic families. The phenotypic response to changes in environmental factors was consistent when the environmental factor has a direct effect on physiology, but varies among and within family if the environmental factor has an indirect effect on physiology or requires the perception of an environmental cue.     

Combined Effects of Pesticides and Trematode Infections on Hourglass Tree Frog <Emphasis Type="Italic">Polypedates cruciger</Emphasis>

The impact of widespread and common environmental factors, such as chemical contaminants, on infectious disease risk in amphibians is particularly important because both chemical contaminants and infectious disease have been implicated in worldwide amphibian declines. Here we report on the lone and combined effects of exposure to parasitic cercariae (larval stage) of the digenetic trematode, Acanthostomum burminis, and four commonly used pesticides (insecticides: chlorpyrifos, dimethoate; herbicides: glyphosate, propanil) at ecologically relevant concentrations on the survival, growth, and development of the common hourglass tree frog, Polypedates cruciger Blyth 1852. There was no evidence of any pesticide-induced mortality on cercariae because all the cercariae successfully penetrated each tadpole host regardless of pesticide treatment. In isolation, both cercarial and pesticide exposure significantly decreased frog survival, development, and growth, and increased developmental malformations, such as scoliosis, kyphosis, and also edema and skin ulcers. The combination of cercariae and pesticides generally posed greater risk to frogs than either factor alone by decreasing survival or growth or increasing time to metamorphosis or malformations. The exception was that lone exposure to chlorpyrifos had higher mortality without than with cercariae. Consistent with mathematical models that suggest that stress should increase the impact of generalist parasites, the weight of the evidence from the field and laboratory suggests that ecologically relevant concentrations of agrochemicals generally increase the threat that trematodes pose to amphibians, highlighting the importance of elucidating interactions between anthropogenic activities and infectious disease in taxa of conservation concern.     

Plasticity of the duration of metamorphosis in the African clawed toad

In organisms with complex life cycles, such as amphibians, selection is thought to have minimized the duration of metamorphosis, because this is the stage at which predation risk is presumed to be highest. Consequently, metamorphic duration is often assumed to show little if any environmentally induced plasticity, because the elevation in the extrinsic mortality risk associated with prolonging metamorphosis is presumed to have selected for a duration as short as is compatible with normal development. We examined the extent to which metamorphic duration in the anuran amphibian Xenopus laevis was sensitive to environmental temperature. Metamorphic duration was influenced by body size, but independent of this effect, it was strongly influenced by environmental temperature: the duration at 18 °C was more than double that at 24 and 30 °C. We also compared the vulnerability of larval, metamorphosing and post metamorphic Xenopus to predators by measuring their burst swimming speeds. Burst swim speed increased through development and while we found no evidence that it was reduced during metamorphosis, it did increase sharply on completion of metamorphosis. We therefore found no evidence of a substantial increase in vulnerability to predators during metamorphosis compared with larval stages, and hence the slowing of metamorphosis in response to temperature may not be as costly as has been assumed.     

Physiological,Behavioral and Maternal Factors That Contribute to Size Variation in Larval Amphibian Populations

Size variance among similarly aged individuals within populations is a pattern common to many organisms that is a result of interactions between intrinsic and extrinsic traits of individuals. While genetic and maternal effects, as well as physiological and behavioral traits have been shown to contribute to size variation in animal populations, teasing apart the influence of such factors on individual growth rates remain a challenge. Furthermore, tracing the effects of these interactions across life stages and in shaping adult phenotypes also requires further exploration. In this study we investigated the relationship between genetics, hatching patterns, behaviors, neuroendocrine stress axis activity and variance in growth and metamorphosis among same-aged larval amphibians. Through parallel experiments we found that in the absence of conspecific interactions, hatch time and to a lesser extent egg clutch identity (i.e. genetics and maternal effects) influenced the propensity for growth and development in individual tadpoles and determined metamorphic traits. Within experimental groups we found that variance in growth rates was associated with size-dependent foraging behaviors and responses to food restriction. We also found an inverse relationship between glucocorticoid (GC) hormone levels and body mass and developmental stage among group-reared tadpoles, which suggests that GC expression plays a role in regulating differing within-population growth trajectories in response to density-dependent conditions. Taken together these findings suggest that factors that influence hatching conditions can have long-term effects on growth and development. These results also raise compelling questions regarding the extent to which maternal and genetic factors influence physiological and behavioral profiles in amphibians.     

Effects of chemical pollutants on reproductive and developmental processes in Italian amphibians

It is a widely held belief that environmental contaminants contribute to the decline of amphibian populations. By spending most of their early life in water and later stages on the land, amphibians face a constant risk of exposure to pesticides and other chemical pollutants in both aquatic and terrestrial environments. This review presents an overview of the studies carried out in Italian amphibians to highlight hazardous effects of bioaccumulation of chemical pollutants in juveniles and adults in various contaminated environments. Further, the studies in the laboratory setting assessing the effects of chemical pollutants on reproductive and developmental processes are reported. These studies and their relative references have been summarized in a tabular form. Three prominent contaminant groups were identified: herbicides, insecticides, and fungicides; and only a few works reported the effects of other chemical pollutants. Each pollutant group has been delegated to a section. All through the literature survey, it is seen that interest in this topic in Italy is very recent and sparse, where only a few anuran and caudata species and only some chemical pollutants have been studied.     

Ultraviolet radiation, toxic chemicals and amphibian population declines

Abstract. As part of an overall ‘biodiversity crisis’, many amphibian populations are in decline throughout the world. Numerous factors have contributed to these declines, including habitat destruction, pathogens, increasing ultraviolet (UV) radiation, introduced non‐native species and contaminants. In this paper we review the contribution of increasing UV radiation and environmental contamination to the global decline of amphibian populations. Both UV radiation and environmental contaminants can affect amphibians at all life stages. Exposure to UV radiation and to certain contaminants can kill amphibians and induce sublethal affects in embryos, larvae and adults. Moreover, UV radiation and contaminants may interact with one another synergistically. Synergistic interactions of UV radiation with contaminants can enhance the detrimental effects of the contaminant and UV radiation.     

Local adaptation of an anuran amphibian to osmotically stressful environments

Abstract. Water salinity is an intense physiological stress for amphibians. However, some species, such as Bufo calamita, breed in both brackish and freshwater environments. Because selection under environmentally stressful conditions can promote local adaptation of populations, we examined the existence of geographic variation in water salinity tolerance among B. calamita populations from either fresh or brackish water ponds in Southern Spain. Comparisons were made throughout various ontogenetic stages. A combination of field transplant and common garden experiments showed that water salinity decreased survival probability of individuals in all populations, prolonged their larval period, and reduced their mass at metamorphosis. However, significant population X salinity interactions indicated that the population native to brackish water (Saline) had a higher salinity tolerance than the freshwater populations, suggesting local adaptation. Saline individuals transplanted to freshwater environments showed similar survival probabilities, length of larval period, and mass at metamorphosis than those native to freshwater. This indicates that increased tolerance to osmotic stress does not imply a loss of performance in freshwater, at least during the larval and juvenile phases. Despite the adaptive process apparently undergone by Saline, all populations still shared the same upper limit of embryonic stress tolerance (around 10 g/l), defining a window of salinity range within which selection can act. Significant differences in embryonic and larval survival in brackish water among sibships for all populations suggest the existence of a genetic basis for the osmotic tolerance.     

Differences in the Phenotypic Mean and Variance Between Two Geographically Separated Populations of Wood Frog (Lithobates sylvaticus)

Intraspecific phenotypic variation between populations separated by large geographic distances is common. Differences in the mean and variance of traits among populations can be used to infer the relative strength, direction, and type of selection on traits. Patterns in the mean provide information on the type of selection, and patterns in variance provide information on the strength of selection. However, interpretation of mean/variance patterns is difficult when two traits are linked and strongly correlated to fitness because it is unlikely that each trait will reach phenotypic optima. In amphibians time to metamorphosis and size at metamorphosis are positively related both phenotypically and genetically. Using a common-garden experiment we investigated whether selection favours shorter time to metamorphosis or increased mass at metamorphosis between two populations which differ in the length of the post-metamorphic growing season by 2–4 weeks. Animals from the population a shorter growing season took longer to reach and metamorphosed at a greater mass, while animals from the population with a longer period for post metamorphic growth reached metamorphosis faster, but at a smaller mass. Greater phenotypic variance was observed in both traits in the population with the shorter growing season. These data suggest that animals from the population with a restricted growth period maximise mass at metamorphosis at the expense of longer larval periods while animals from population with the longer post-metamorphic growth period sacrifice mass at metamorphosis to shorten the larval period and maximise larval survival. Differences in phenotypic variance among populations suggest either directional or diversifying selection has acted on both traits.     

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.     

Qualitative risk analysis of introducing Batrachochytrium dendrobatidis to the UK through the importation of live amphibians

The international amphibian trade is implicated in the emergence and spread of the amphibian fungal disease chytridiomycosis, which has resulted in amphibian declines and extinctions globally. The establishment of the causal pathogen, Batrachochytrium dendrobatidis (Bd), in the UK could negatively affect the survival of native amphibian populations. In recognition of the ongoing threat that it poses to amphibians, Bd was recently included in the World Organisation for Animal Health Aquatic Animal Health Code, and therefore is in the list of international notifiable diseases. Using standardised risk analysis guidelines, we investigated the likelihood that Bd would be introduced to and become established in wild amphibians in the UK through the importation of live amphibians. We obtained data on the volume and origin of the amphibian trade entering the UK and detected Bd infection in amphibians being imported for the pet and private collection trade and also in amphibians already held in captive pet, laboratory and zoological collections. We found that current systems for recording amphibian trade into the UK underestimate the volume of non-European Union trade by almost 10-fold. We identified high likelihoods of entry, establishment and spread of Bd in the UK and the resulting major overall impact. Despite uncertainties, we determined that the overall risk estimation for the introduction of Bd to the UK through the importation of live amphibians is high and that risk management measures are required, whilst ensuring that negative effects on legal trade are minimised.     

An examination of amphibian sensitivity to environmental contaminants: are amphibians poor canaries?

Nearly two decades ago, the global biodiversity crisis was catapulted to the front pages of newspapers with the recognition of worldwide amphibian declines. Amphibians earned their appellation, 'canaries in a coal mine', because of apparent high sensitivity to human-mediated environmental change. The most frequently cited causes for high susceptibility include permeable skin, a dual aquatic-terrestrial life cycle and a relatively rudimentary immune system. While some researchers have questioned the basis for the canary assertion, there has been no systematic evaluation of amphibian sensitivity to environmental challenges relative to other taxa. Here, we apply a database representing thousands of toxicity tests to compare the responses of amphibians relative to that of other taxonomic groups. The use of standardized methods combined with large numbers of identical challenges enables a particularly powerful test of relative effect size. Overall, we found that amphibians only exhibit moderate relative responses to water-borne toxins. Our findings imply that, as far as chemical contaminants are concerned, amphibians are not particularly sensitive and might more aptly be described as 'miners in a coal mine'. To the extent that amphibian declines have been mediated by chemical contaminants, our findings suggest that population losses and extinctions may have already occurred in a variety of taxa much more sensitive than amphibians.
Ecology Letters (2010) 13: 60–67     

Larval Environment Alters Amphibian Immune Defenses Differentially across Life Stages and Populations

Recent global declines, extirpations and extinctions of wildlife caused by newly emergent diseases highlight the need to improve our knowledge of common environmental factors that affect the strength of immune defense traits. To achieve this goal, we examined the influence of acidification and shading of the larval environment on amphibian skin-associated innate immune defense traits, pre and post-metamorphosis, across two populations of American Bullfrogs (Rana catesbeiana), a species known for its wide-ranging environmental tolerance and introduced global distribution. We assessed treatment effects on 1) skin-associated microbial communities and 2) post-metamorphic antimicrobial peptide (AMP) production and 3) AMP bioactivity against the fungal pathogen Batrachochytrium dendrobatidis (Bd). While habitat acidification did not affect survival, time to metamorphosis or juvenile mass, we found that a change in average pH from 7 to 6 caused a significant shift in the larval skin microbial community, an effect which disappeared after metamorphosis. Additionally, we found shifts in skin-associated microbial communities across life stages suggesting they are affected by the physiological or ecological changes associated with amphibian metamorphosis. Moreover, we found that post-metamorphic AMP production and bioactivity were significantly affected by the interactions between pH and shade treatments and interactive effects differed across populations. In contrast, there were no significant interactions between treatments on post-metamorphic microbial community structure suggesting that variation in AMPs did not affect microbial community structure within our study. Our findings indicate that commonly encountered variation in the larval environment (i.e. pond pH and degree of shading) can have both immediate and long-term effects on the amphibian innate immune defense traits. Our work suggests that the susceptibility of amphibians to emerging diseases could be related to variability in the larval environment and calls for research into the relative influence of potentially less benign anthropogenic environmental changes on innate immune defense traits.     

Sublethal Effects of Nitrite on Eastern Tiger Salamander (<Emphasis Type="Italic">Ambystoma tigrinum tigrinum</Emphasis>) and Wood Frog (<Emphasis Type="Italic">Rana sylvatica</Emphasis>) Embryos and Larvae: Implications for Field Populations

Ephemeral pools, which can have high animal biomass and low dissolved oxygen, may be prone to nitrite accumulation. As such, it is important to understand how exposure to nitrite might affect development and growth of amphibians that breed in these ephemeral pools. Wood frog (Rana sylvatica) and eastern tiger salamander (Ambystoma tigrinum tigrinum) embryos and tadpoles and young larvae were exposed to elevated concentrations of nitrite derived from sodium nitrite: 0, 0.3, 0.6, 1.2, 2.1, 4.6, and 6.1 mg l⁻¹ NO₂–N. Increasing nitrite exposure slowed embryonic and larval development in both the eastern tiger salamander and the wood frog, reduced growth in tiger salamander embryos and larvae, and delayed metamorphosis in the wood frog. At concentrations less than 2 mg l⁻¹ NO₂–N nitrite delayed hatching, and at concentrations above 2 mg l⁻¹ time to hatching decreased causing more individuals to hatch at less developed stages. Nitrite also increased asynchrony in tiger salamander hatching. The sublethal effects of nitrite on amphibian development, growth and hatching could have serious repercussions on amphibian fitness in ephemeral environments. Potential increases in mortality on field populations caused by sublethal effects of nitrite are discussed.