Alytes Muletensis Research Articles

Invasive predator snake induces oxidative stress responses in insular amphibian species

The presence of predators induces physiological stress responses in preys to avoid being captured. A stressful situation enhances reactive oxygen species production with potential damage to macromolecules and alterations in oxidant defences levels. The antioxidant enzyme response of the endemic Majorcan Midwife toad (Alytes muletensis) and the Balearic green toad (Bufotes balearicus) tadpoles against an invasive predator, the viperine snake (Natrix maura) was investigated. Tadpoles were introduced in aquaria containing N. maura exudates during 24h. Antioxidant enzyme activities – catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) – and reduced glutathione (GSH) and malondialdehyde (MDA) concentrations were measured in tadpoles. The presence of snake exudates induced a significant increase in CAT and GR activities and in GSH levels (p<0.05) in A. muletensis tadpoles, whereas no significant differences were reported in any of the parameters analysed in B. balearicus tadpoles. In conclusion, the presence of N. maura exudates is capable to induce an antipredatory response in the endemic A. muletensis tadpoles but not in B. balearicus.

Ageing and growth of the endangered midwife toad Alytes muletensis

A better understanding of the demography of endangered amphibians is important for the development of suitable management and recovery plans, and for building population via- bility models. Our work presents, for the first time, growth curves and measurements of mean longevity, growth rates and age at maturity for the Vulnerable midwife toad Alytes muletensis. Von Bertalanffy growth models were used to estimate longevity and growth rate parameters. Females had a mean (±SD) longevity of 4.70 ± 0.19 yr, significantly higher than that of males (3.24 ± 0.10 yr). The maximum estimated longevity was 18 yr for both males and females. The age distribution indicated that males reached sexual maturity at the age of 1 yr, and most females at 2 yr. There were significant differences in growth rate between sexes, with higher values in females during the first 4 yr of life, and similar values in both sexes thereafter. These life-history traits were compared with equivalent measures in the closely related amphibian genera Bombina and Discoglossus.

Context‐dependent amphibian host population response to an invading pathogen

Amphibian chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), is an emerging infectious disease that widely threatens amphibian biodiversity. However, population-level outcomes following the introduction of the pathogen are highly context dependent and are mediated by a broad suite of biotic and abiotic variables. Here, we examine the effect of the introduction of Bd on native island populations of the IUCN red-listed amphibian species Alytes muletensis, the Mallorcan midwife toad. We show that the outcome of pathogen introduction is not only dependent on biotic factors, but is also dependent on environmental factors that vary across local scales. Our experimental infections confirm that the genotype of Bd occurring on Mallorca is hypovirulent in A. muletensis when compared against the lineage found occurring on mainland Iberia. Long-term population data show that A. muletensis populations on the island are increasing overall, but trends in highly infected populations are conflicting. We use mathematical models and field data to demonstrate that this divergence in population response to infection can be explained by local environmental differences between infected sites, whereas pathogen genetics, host genetics, and intrinsic epidemiological dynamics driven by fungal load are less likely to be the cause of these differing population trajectories. Our results illustrate the need to take into account the appropriate environmental scale and context when assessing the risk that an emerging pathogen presents to a naive population or species.

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.

Developing a safe antifungal treatment protocol to eliminateBatrachochytrium dendrobatidisfrom amphibians

Batrachochytrium dendrobatidis is one of the most pathogenic microorganisms affecting amphibians in both captivity and in nature. The establishment of B. dendrobatidis free, stable, amphibian captive breeding colonies is one of the emergency measures that is being taken to save threatened amphibian species from extinction. For this purpose, in vitro antifungal susceptibility testing and the development of efficient and safe treatment protocols are required. In this study, we evaluated the use of amphotericin B and voriconazole to treat chytridiomycosis in amphibians. The concentration at which the growth of five tested B. dendrobatidis strains was inhibited was 0.8 μg/ml for amphotericin B and 0.0125 μg/ml for voriconazole. To completely eliminate a mixture of sporangia and zoospores of strain IA042 required 48 h of exposure to 8 μg/ml of amphotericin B or 10 days to 1.25 μg/ml of voriconazole. Zoospores were killed within 0.5 h by 0.8 μg/ml of amphotericin B, but even after 24 h exposure to 1.25 μg/ml of voriconazole they remained viable. Amphotericin B was acutely toxic for Alytes muletensis tadpoles at 8 μg/ml, whereas toxic side effects were not noticed during a seven-day exposure to voriconazole at concentrations as high as 12.5 μg/ml. The voriconazole concentrations remained stable in water during this exposure period. On the basis of this data, experimentally inoculated postmetamorphic Alytes cisternasii were sprayed once daily for 7 days with a 1.25 μg/ml solution of voriconazole in water which eliminated the B. dendrobatidis infection from all treated animals. Finally, treatment of a naturally infected colony of poison dart frogs (Dendrobatidae) using this protocol, combined with environmental disinfection, cleared the infection from the colony.

Using itraconazole to clear Batrachochytrium dendrobatidis infection, and subsequent depigmentation of Alytes muletensis tadpoles

Batrachochytrium dendrobatidis (Bd) is a global threat to amphibian biodiversity. Current calls for conservation through captive breeding require that efficient and reliable antifungal treatments be developed for target species. Here we confirm that the antifungal itraconazole is an effective treatment for infection in larval Alytes muletensis. Exceptionally low doses applied as few as 7 times were effective at clearing infection from tadpoles for up to 28 d after treatment. However, we cannot recommend itraconazole as a treatment for this species as depigmentation of tadpoles was observed. Further research is required to determine the putative hepatotoxicity of this treatment.

Individual identification and sexual dimorphism in the endangered Balearic Midwife Toad, Alytes muletensis (Sanchíz and Adrover, 1981)

AbstractThe Majorcan Midwife Toad (Alytes muletensis) is and endemic and threatened species of the island of Mallorca which has been the subject of a large number of studies. However, there is a lack of information about the adult fraction of wild populations. Here we show the first step to increase the knowledge of adult Majorcan midwife toads, how to recognize them individually and how to distinguish between males and females. We tested with satisfactory results a photoidentification method that can be used to identify adults individually. In order to sex individuals in the wild we carry out a morphological study with fourteen variables building a discriminant function which allow us to sex all captured adults.

Saving the Majorcan midwife toad

Saving the Majorcan midwife toad

Captive breeding and the fitness of reintroduced species: a test of the responses to predators in a threatened amphibian

Summary Although captive breeding and reintroduction is a high‐profile management tool for many threatened species, it is unclear how long‐term captive breeding can influence fitness attributes such as natural defences to predators. Induced defences that have evolved in the Mallorcan midwife toad Alytes muletensis in response to introduced predators were compared in natural and reintroduced populations that had a common ancestry, and in short‐term and long‐term captive populations that differed in ancestry. Defences against predators were maintained in a reintroduced population derived from stock that had passed through three to eight generations of captive breeding prior to release into a predator‐free area. Heterozygosity did not differ between natural and reintroduced populations, but the reintroduced population displayed lower allelic richness. A comparison between populations maintained for different lengths of time in captivity revealed a significant reduction in one defensive trait in stock maintained for more than eight generations. Neutral genetic variation (i.e. heterozygosity and allelic richness) did not differ between the short‐term captive population and a natural population, but there was a significant loss of genetic variation in the long‐term captive population. Synthesis and applications. The results suggest that relatively high levels of heterozygosity and important fitness attributes can be maintained for a few generations in breeding programmes for threatened species despite small numbers of founders and the absence of natural selection. Nevertheless, both fitness and heterozygosity may eventually start to deteriorate in the long term, and this may have implications for reintroduction strategies.

Introduced Alien or Persecuted Native? Resolving the Origin of the Viperine Snake (Natrix Maura) on Mallorca

The viperine snake (Natrix maura) is an important agent of decline of the threatened midwife toad (Alytes muletensis) of Mallorca. However, there is a paucity of biological data to support the notion that the viperine snake is an introduced species to the island. Here we compare mitochondrial cytochrome b gene sequences and genomic ISSR-PCR fingerprints from Mallorcan and mainland European viperine snakes. Identical or nearly identical haplotypes and very similar ISSR-PCR profiles provide strong evidence that N. maura arrived only recently to Mallorca. There is no indication of a recent natural colonization of the island by transmarine dispersal. The data therefore support historical information that N. maura was introduced to Mallorca by human agency comparatively recently, and that management measures to reduce the impact of the snake on toad populations are justified.

Low gene flow but high genetic diversity in the threatened Mallorcan midwife toad Alytes muletensis

We investigated fine-scale genetic structuring in the rare and vulnerable Mallorcan midwife toad Alytes muletensis using eight polymorphic microsatellite markers. The current range of this amphibian is restricted to some 19 sites of which six are derived from reintroductions, all located in the mountain ranges of Mallorca. We sampled tadpoles from 14 pools covering 10 natural sites and two reintroduction sites for microsatellite DNA analyses. Relatively high levels of genetic variation were found in most pools (H(E) = 0.38-0.71, allelic richness = 2.6-6.2). Only at one pool has the population recently gone through a bottleneck. Dispersal between pools in different torrents does not occur whereas downstream dispersal between pools within the same torrent does happen at low frequencies. This occasional exchange of individuals does not lead to neighbouring pools in the same torrent being panmictic. This can be concluded because all F(ST) values (0.12-0.53) differ significantly from zero and STRUCTURE analyses identified neighbouring pools as separate populations. Furthermore, assignment and migration tests showed little exchange between neighbouring pools. If upstream locations or complete torrents go extinct, they are unlikely to be recolonized naturally. For conservation purposes, reintroductions of tadpoles to sites where local extinctions have occurred may therefore be advisable.

Distribution of the Mallorcan midwife toad ( Alytes muletensis) in relation to landscape topography and introduced predators

The endemic midwife toad of Mallorca ( Alytes muletensis) is restricted to a small number of breeding populations in the mountainous northwest of the island. The decline of the species has been attributed to the impacts of introduced species such as the viperine snake ( Natrix maura) and green frog ( Rana perezi), and toads may be surviving only in areas that are suboptimal for these predators. The influence of landscape features (elevation, aspect and maximum slope) on the distribution of toads and associated predators was therefore investigated using GIS. The presence of toads was positively associated with steep slopes. At sites where they occurred with toads, the distribution of predators was negatively associated with elevation. Reproductive success within toad populations was strongly associated with the number of pools at each site, while reproductive success within individual pools was positively associated with elevation. These findings may be used to optimise the design and location of future reintroduction sites.

Microsatellite markers for the Mallorcan midwife toad Alytes muletensis

AbstractThe highly endangered midwife toad Alytes muletensis occurs only on the Mediterranean island of Mallorca. Little is known about genetic variability and dispersal among the 19 breeding sites (six of which stem from reintroductions) of this amphibian. The value of such information should be high for successful management of this species. We report here the characterization of eight polymorphic microsatellite loci in A. muletensis using individuals from a single population.

The mating strategy of Alytes muletensis: Some males are less ready to mate than females

A laboratory experiment was set up to examine mating success in male Mallorcan midwife toads, Alytes muletensis. Mating appeared to be unrelated to male size, condition OF asymmetry. This was despite potential benefits to females of mating with males of a large size (and avoiding small males), good condition or high symmetry, and/or despite advantages that these males hypothetically have in maintaining courtship or amplexus. There was a skew in male mating success but there was no clear evidence to suggest that this was a result of direct male-male competition or overt or cryptic female choice. Instead, males appeared to become receptive at different times over an extended breeding season and we suggest that, generally, male inclination to mate was low. This is evidenced by the fact that only one quarter of the males mated and, despite a sex ratio of five males to one female, females still dropped (oviposited) one third of their egg clutches without mating. Male inclination to mate may vary across time in this species because of the heavy burden of brooding the eggs, or some artefact of the captive environment may have affected it.

The functionality of female reciprocal calls in the Iberian midwife toad (Alytes cisternasii): female-female acoustic competition?

Female midwife toads (genus Alytes) emit highly variable reciprocal calls of unclear function prior to and during courtship. In some species, female-female competition, expressed as physical fighting, has been reported. Males of Majorcan midwife toads (Alytes muletensis) show phonotactic response to female calls, and females of Iberian midwife toads (Alytes cisternasii) respond differently according to the male call characteristics. In this study, I test the hypothesis of female-female acoustic competition as an additional function of female reciprocal calls. Playback tests indicate that female calls are not clearly involved in female acoustic competition in the Iberian midwife toad, therefore female calls could be directed at males rather than towards competitive females.

Phonotaxis to advertisement calls by midwife toads (Alytes muletensis) is not necessarily related to mating

AbstractPrevious studies with the Mallorcan midwife toad (Alytes muletensis) found that gravid females show inconsistent phonotactic preferences for male calls unless they are highly motivated to mate; and, non-gravid females will occasionally show phonotaxis to male advertisement calls. These results suggest that sometimes phonotaxis may not be related to seeking a mating partner. In this article we report on experiments that give further support to a non-mating function of phonotaxis in this species. Males and metamorphs were played conspecific male and female advertisement calls. Males that were expected to be unmotivated to mate were not less likely to respond to, or less consistent in their choice of, female calls than males who were expected to be motivated to mate. Metamorphs who were not yet in breeding condition nevertheless showed positive phonotaxis. We suggest that Mallorcan midwife toads may sometimes show phonotaxis simply to seek out refuges where toads aggregate.

Calling by male midwife toads stimulates females to maintain reproductive condition

We investigated the effect of the calls of male Mallorcan midwife toads, Alytes muletensis, on the reproductive condition of females. We used three groups of gravid laboratory-reared females. One group was kept in silence, one group was played heterospecific male calls and one group was played conspecific male calls. Only the females that heard conspecific male calls continued to ripen and mature their eggs while females in the other two groups reabsorbed resources from their eggs. We conclude that conspecific male calls have a stimulatory effect on the reproductive physiology of females, such that gravid females hearing the calls maintain their reproductive condition. The calls of male frogs have previously been shown to act only in male–male competition and to attract females. This is the first experiment to show that the call of a male frog affects the reproductive status of females.

Reproductive stage and history affect the phonotactic preferences of female midwife toads, Alytes muletensis

We investigated whether the reproductive stage or reproductive history of females affects their preferences for male calls that differ in frequency. We repeatedly tested gravid female midwife toads prior to and immediately after mating, and during ovulation. When females were ovulating they were much more consistent in their choice of call than when they were gravid but not ovulating. When females had recently mated they still showed positive phonotaxis, but did not reliably discriminate between alternative calls. This study is the first to test gravid female anurans repeatedly at different stages in their reproductive cycle (ovulating or not; pre- and postmating). It highlights the need to treat with caution the results from population-based preference studies in which individuals are tested only once, and in which variation in reproductive state is not taken into account.

Activity patterns and microhabitat selection of Mallorcan midwife toad (Alytes muletensis) tadpoles in natural torrent pools

Abstract Tadpoles of the Mallorcan midwife toad (Alytes muletensis) displayed a clear diel pattern of activity and microhabitat selection in torrent pools in Mallorca. Tadpoles spent much of the day foraging in a non-aggregated fashion in warm shallow areas, and activity peaked at 1700-1900 hr. In mid-afternoon, however, when much of the water surface was in direct sunlight, tadpoles moved away from the shallows and formed aggregations under rock overhangs at the sides of the pool. Both activity and tadpole density in the shallows dropped dramatically after dark, when there was a movement of the population back into deeper water or hiding places.

Phonotaxis by Female Majorcan Midwife Toads, Alytes Muletensis

Female choice based on male call characteristics has been experimentally demonstrated in a number of species of anurans. In the Majorcan midwife toad, the male performs parental care by carrying the eggs in a string around his hind legs until they are ready to hatch. Both sexes produce calls to advertise sexual receptivity and both show phonotaxis towards the calls of the opposite sex. We used two-choice phonotaxis experiments to investigate whether females assess male caretaking ability using variation in call characteristics. Both call frequency and call duration are good indicators of male size, a characteristic that potentially influences the ability of males to successfully brood a clutch. Variation in call repetition rate and call intensity may also reflect differences in male caretaking ability, given the high energetic demands of calling behaviour in anurans. We predicted that females should prefer to mate with larger males and that this would be manifest in selective phonotaxis to low frequency and/or long duration calls. We also predicted that they would prefer calls produced at a fast rate and a high intensity. Contrary to our predictions females did not prefer lower frequency calls, longer calls or louder calls. They did however, selectively approach calls produced at a faster rate. These results are discussed in the context of sexual selection theory.