The microbiome at the interface between environmental stress and animal health: an example from the most threatened vertebrate group.

Nitrate pollution and global warming are ubiquitous stressors likely to interact and affect the health and survival of wildlife, particularly aquatic ectotherms. Animal health is largely influenced by its microbiome (commensal/symbiotic microorganisms), which responds to such stressors. We used a crossed experimental design including three nitrate levels and five temperature regimes to investigate their interactive and individual effects on an aquatic ectotherm, the European common frog. We associated health biomarkers in larvae with changes in gut bacteria diversity and composition. Larvae experienced higher stress levels and lower body condition under high temperatures and nitrate exposure. Developmental rate increased with temperature but decreased with nitrate pollution. Alterations in bacteria composition but not diversity are likely to correlate with the observed outcomes in larvae health. Leucine degradation decreased at higher temperatures corroborating accelerated development, nitrate degradation increased with nitrate level corroborating reduced body condition and an increase in lysine biosynthesis may have helped larvae deal with the combined effects of both stressors. These results reinforce the importance of associating traditional health biomarkers with underlying microbiome changes. Therefore, we urge studies to investigate the effects of environmental stressors on microbiome composition and consequences for host health in a world threatened by biodiversity loss.

Exploring water-borne corticosterone collection as a non-invasive tool in amphibian conservation physiology: benefits, limitations and future perspectives.

Global change exposes wildlife to a variety of environmental stressors and is affecting biodiversity worldwide, with amphibian population declines being at the forefront of the global biodiversity crisis. The use of non-invasive methods to determine the physiological state in response to environmental stressors is therefore an important advance in the field of conservation physiology. The glucocorticoid hormone corticosterone (CORT) is one useful biomarker to assess physiological stress in amphibians, and sampling water-borne (WB) CORT is a novel, non-invasive collection technique. Here, we tested whether WB CORT can serve as a valid proxy of organismal levels of CORT in larvae of the common frog (Rana temporaria). We evaluated the association between tissue and WB CORT levels sampled from the same individuals across ontogenetic stages, ranging from newly hatched larvae to froglets at 10 days after metamorphosis. We also investigated how both tissue and WB CORT change throughout ontogeny. We found that WB CORT is a valid method in pro-metamorphic larvae as values for both methods were highly correlated. In contrast, there was no correlation between tissue and WB CORT in newly hatched, pre-metamorphic larvae, metamorphs or post-metamorphic froglets probably due to ontogenetic changes in respiratory and skin morphology and physiology affecting the transdermal CORT release. Both collection methods consistently revealed a non-linear pattern of ontogenetic change in CORT with a peak at metamorphic climax. Thus, our results indicate that WB CORT sampling is a promising, non-invasive conservation tool for studies on late-stage amphibian larvae. However, we suggest considering that different contexts might affect the reliability of WB CORT and consequently urge future studies to validate this method whenever it is used in new approaches. We conclude proposing some recommendations and perspectives on the use of WB CORT that will aid in broadening its application as a non-invasive tool in amphibian conservation physiology.

Complete mitochondrial genome sequence of the high altitude Brazilian treefrog Pithecopus megacephalus (Anura, Phyllomedusidae).

The first complete mitochondrial genome (mtDNA) for the family Phyllomedusidae (genus Pithecopus) is presented. It is a circular molecule with 17713 pb including 13 protein coding genes, 22 tRNA genes, two rRNA genes, and a control region (D-loop). Pithecopus megacephalus was close to the only other phyllomedusid whose complete mtDNA sequence is available, but had the cytb gene 147 pb smaller.

Background choice and immobility as context dependent tadpole responses to perceived predation risk.

The association of immobility and camouflage is widespread as a defensive mechanism in prey from varied taxa. However, many experiments assessing the reaction of prey to predator cues are conducted under artificial laboratory conditions. In a previous experiment we observed the tadpoles of Ololygon machadoi (Hylidae) to respond to predator visual and/or chemical cues by choosing backgrounds that improve their disruptive properties, but detected no associated reduction of movement. Here we experimentally demonstrate this response in the species' natural habitat, on backgrounds where the tadpoles are likely to achieve their best camouflage. We also tested whether previous experiences could influence both background choice and immobility in O. machadoi tadpoles. These novel experimental results suggest that a defensive behavior-i.e., reduction of movement-in these tadpoles is more strongly expressed under the natural conditions where they evolved, compared to laboratory conditions where prey and predator were brought into closer contact. Besides, previous experiences are likely to play an important role in expressed defensive responses.

Past vicariance promoting deep genetic divergence in an endemic frog species of the Espinhaço Range in Brazil: The historical biogeography of Bokermannohyla saxicola (Hylidae).

The highland endemic frog Bokermannohyla saxicola occurs within the Espinhaço Range, the most extensive and continuous orogenic belt of the Brazilian territory, located in southeastern Brazil. We used mitochondrial DNA markers to test for spatial structure, to investigate the likely influence of past vicariant events, to evaluate demographic dynamics along the species range, and to understand the role of habitat discontinuities in promoting connectivity and diversity along the range. We found four major monophyletic lineages, each one associated with distinct mountain tops. The divergence time found between the four main clades clearly pre-dated the Pleistocene, except for the most recent separation. We observed no signs of population expansion for most of the sampling sites along the range, and a higher genetic diversity in the most continuous and central highland plateau, compared to smaller marginal regions. The Espinhaço Range harbors four deeply divergent lineages of B. saxicola within areas restricted by barriers for millions of years. These relatively isolated populations were kept apart by discontinuities represented by lowland habitats between mountain tops. Most of the lineage divergences occurred earlier than the Pleistocene, thus they cannot be solely explained by climatic oscillations of this epoch. However, within-lineage divergence times were all dated from the Pleistocene, suggesting an important effect in population dynamics. We also suggest that some marginal populations like those from Serra Negra and Serra de Itacambira can be the result of recent colonization events. Finally, in the southern Espinhaço region, the most continuous central highland area shows greater genetic diversity than the marginal discontinuous areas, where we have also observed a significant correlation between genetic and geographic distances. Bokermannohyla saxicola is a good model to study the biogeography of the Espinhaço Range because its high genetic structure reflects ancient as well as recent geological/climatic events, with important implications for conservation.

Tadpoles respond to background colour under threat.

The ability to respond to background colour is an important feature of species that might benefit from background matching camouflage. Tadpole colour patterns vary and could be associated with several functions, including defense. Because tadpoles are exposed to a wide array of visually oriented predators, they represent good models to study defensive colouration and associated behaviours. We tested whether a potentially disruptively camouflaged tadpole with a dark body crossed by yellow bars (Ololygon machadoi) is able to respond differently to matching light and dark natural background colours and an artificial blue contrasting background. We used a syntopic contrasting black tadpole (Bokermannohyla martinsi) as a control, expecting it not to respond to background colour in search for camouflage. Ololygon machadoi tadpoles chose light over blue backgrounds under threat, as expected, however they did not show preferential use of dark vs. blue backgrounds. Bokermannohyla martinsi did not respond to any combination of background colours. Our results suggest that O. machadoi tadpoles are able to respond to background colour, and may favor matching backgrounds under some circumstances. The potentially disruptive colouration of O. machadoi tadpoles may increase their repertoire of escape strategies, background matching being one of the options to escape predation.

Complete mitochondrial genome sequence of the high-altitude Brazilian tree frog Bokermannohyla alvarengai (Anura, Hylidae).

The first complete mitochondrial genome (mtDNA) for the genus Bokermannohyla (Hylidae) is presented. The mtDNA contains 17,325 bp and is similar in size, gene content, and gene location to other hylid mitochondrial genomes described, with 2 rRNA, 22 tRNA, and 13 PCGs. The control region (D-loop) is shorter than in mtDNAs of hylids from Asia. A phylogenetic tree based on homologous genes did not corroborate the monophyly of Hylidae neither the recently proposed monophyly of Hyla and Dryophytes.