Characterization of the Skin Cultivable Microbiota Composition of the Frog Pelophylax perezi Inhabiting Different Environments

Microorganisms that live in association with amphibian skin can play important roles in protecting their host. Within the scenarios of global change, it is important to understand how environmental disturbances, namely, metal pollution, can affect this microbiota. The aim of this study is to recognize core bacteria in the skin cultivable microbiota of the Perez frog (Pelophylax perezi) that are preserved regardless of the environmental conditions in which the frogs live. The characterization of these isolates revealed characteristics that can support their contributions to the ability of frogs to use metal impacted environments. Frog’s skin swabs were collected from P. perezi populations that inhabit a metal-polluted site and three reference (non-metal polluted) sites. Bacterial strains were isolated, identified, and subjected to an acid mine drainage tolerance (AMD) test, collected upstream from a site heavily contaminated with metals, and tested to produce extracellular polymeric substances (exopolysaccharide, EPS). All frog populations had Acinetobacter in their cutaneous cultivable microbiota. Significant growth inhibition was observed in all bacterial isolates exposed to 75% of AMD. EPS production was considered a characteristic of several isolates. The data obtained is a preliminary step but crucial to sustain that the cultivable microbiota is a mechanism for protecting frogs against environmental contamination.

Consequences of Long-term Exposure to Increased Salinity on the Amphibian Skin Bacterium Erwinia Toletana

Amphibian’s skin bacterial community may help them to cope with several types of environmental perturbations, including osmotic stress caused by increased salinity. This work aimed at assessing if an amphibian skin bacterium could increase its tolerance to NaCl after a long-term exposure to this salt. A strain of Erwinia toletana, isolated from the skin of Pelophylax perezi, was exposed to two salinity scenarios (of 18g/L of NaCl): (i) long-term exposure (for 46 days; Et-NaCl) and (ii) long-term exposure followed by a recovery period, (exposure for 30 days to NaCl and then to LB medium for 16 days; Et-R). After exposure, the sensitivity of E. toletana clonal populations to NaCl was assessed by testing 6 NaCl concentrations (LB medium spiked with NaCl) plus a control (LB medium). Genotypic alterations were assessed by PCR-based molecular typing method (BOX-PCR). Results shown that tolerance of E. toletana to NaCl slightly increased after the long-term exposure, EC50 for growth were: 22.5g/L (8.64-36.4) for Et-LB; 30.3g/L (23.2-37.4) for Et-NaCl, and 26.1g/L (19.3-32.9) for Et-R. Differences in metabolic activity were observed between Et-LB and Et-R and Et-NaCl and Et-R suggesting the use of different substrates by this bacterium when exposed to salinized environments. NaCl-induced genotypic alterations were not detected. This work suggests that E. toletana exposed to low levels of salinity, activate different metabolic pathways to cope with osmotic stress. Which may be further explored to be used in bioaugmentation procedures in natural populations of amphibians exposed to salinization.

Morphological determinants of jumping performance in the Iberian green frog

Predation is one of the main selective forces in nature, frequently selecting potential prey for developing escape strategies. Escape ability is typically influenced by several morphological parameters, such as morphology of the locomotor appendices, muscular capacity, body mass, or fluctuating asymmetry, and may differ between sexes and age classes. In this study, we tested the relationship among these variables and jumping performance in 712 Iberian green frogs Pelophylax perezi from an urban population. The results suggest that the main determinant of jumping capacity was body size (explaining 48% of variance). Larger frogs jumped farther, but jumping performance reached an asymptote for the largest frogs. Once controlled by structural body size, the heaviest frogs jumped shorter distances, suggesting a trade-off between fat storage and jumping performance. Relative hind limb length also determined a small but significant percentage of variance (2.4%) in jumping performance—that is, the longer the hind limbs, the greater the jumping capacity. Juveniles had relatively shorter and less muscular hind limbs than adults (for a given body size), and their jumping performance was poorer. In our study population, the hind limbs of the frogs were very symmetrical, and we found no effect of fluctuating asymmetry on jumping performance. Therefore, our study provides evidence that jumping performance in frogs is not only affected by body size, but also by body mass and hind limb length, and differ between age classes.

Life cycle of Alytes dickhilleni Arntzen and García-París, 1995 and sympatric amphibian community in ponds with high conservation interest in Region of Murcia (SE Spain)

Se ha estudiado el ciclo de vida durante un periodo anual (diciembre 2016–noviembre 2017) de los anfibios en dos cuerpos de agua artificiales en el límite oriental de la distribución de la especie amenazada Alytes dickhilleni Arntzen y García-París, 1995. En ambos sistemas, se reproduce la especie objetivo y dos más, Bufo spinosus Daudin, 1803, y Pelophylax perezi (López-Seoane, 1885). Los ciclos reproductivos de A. dickhilleni y P. perezi muestran semejanzas con lo descrito en medios naturales y artificiales en la Región de Murcia; por el contrario, B. spinosus presentó un adelanto significativo. Este estudio pone de manifiesto el importante papel de los cuerpos de agua artificiales ligados a prácticas ganaderas tradicionales para la conservación de A. dickhilleni en el sureste ibérico semiárido. Life cycle of amphibian community inhabiting two artificial ponds on the eastern edge of the native distribution of the endangered species Alytes dickhilleni Arntzen and García- Paris, 1995, was studied over a complete annual cycle (from December 2016 to November 2017). We confirm the breeding status of the target species and two more, Bufo spinosus Daudin, 1803 and Pelophylax perezi (López-Seoane, 1885). Breeding cycles of A. dickhilleni and P. perezi showed similar phenology to available data from other natural and manmade ponds on Region of Murcia; conversely, B. spinosus showed a slightly advanced cycle. This study highlights the important role of artificial and traditional livestock ponds to conservation of A. dickhilleni population in Iberian southeast.

Effects of the invasive crayfish Procambarus clarkii on growth and development of Pelophylax perezi tadpoles in field conditions

Introduced predatory aquatic invertebrates may contribute to the global decline of amphibians as their larval are extremely vulnerable to predators. The objective of this study was to examine the effects of the predatory invasive crayfish Procambarus clarkii on the growth and development of native Iberian green frog tadpoles, Pelophylax perezi, in field conditions. We hypothesized that P. clarkii might affect P. perezi development by (a) inducing a delay in its metamorphosis and (b) reducing survival and mass of metamorphs. The experiment was developed in two ponds (with and without P. clarkii’s presence) in the Natural Park of Aiguamolls de l’Empordà (NE of the Iberian Peninsula). For each pond, groups of 10 tadpoles were randomly assigned to 15 cylindrical field enclosures. These enclosures avoided direct contact (i.e. predation) between both species. Our results suggest that, in field conditions, the presence of P. clarkii accelerates metamorphosis of P. perezi tadpoles. The higher growth rate of P. perezi through shorter larval periods could be the result of behavioural plasticity in response to the strong pressure imposed by P. clarkii. This conclusion would be in accordance with the hypothesis that phenotypic plasticity plays an important role in the conservation of P. perezi in front of biological invasions.