Evaluating eDNA-based monitoring of fire salamander larvae under field conditions

Amphibians globally belong to the most threatened animal groups and monitoring their populations is of critical importance for their conservation. The fire salamander (Salamandra salamandra) is one of the European amphibian species which has been experiencing drastic regional population declines due to the spread of the invasive chytrid fungus Batrachochytrium salamandrivorans, making it a key species for monitoring efforts. Here, we evaluated how the sampling and analysis of eDNA can aid the monitoring of larval salamander populations in small streams under field conditions. Nine small streams with known adult and larval fire salamander populations were investigated in Tirol (Austria). Per steam a stretch of 30 m downstream from its source was divided into 10 m sections where salamander larvae were counted. Water samples were taken at the end of each section and filtered on site. The DNA extracted from these filters was tested by a new PCR assay developed for the detection of mitochondrial DNA of S. salamandra. This assay combines endpoint PCR with capillary electrophoresis, allowing to relatively quantify the amount of fire salamander eDNA present in the water samples. In two of the nine streams no eDNA of S. Salamandra could be detected. The outcomes of an analysis of how larval densities, discharge and volume of filtered water affected the detection of salamander eDNA will be presented. Finally, we will conclude on the practical implications of the current findings for eDNA-based monitoring of fire salamander populations.

Significant reductions of host abundance weakly impact infection intensity of Batrachochytrium dendrobatidis

Infectious diseases are considered major threats to biodiversity, however strategies to mitigate their impacts in the natural world are scarce and largely unsuccessful. Chytridiomycosis is responsible for the decline of hundreds of amphibian species worldwide, but an effective disease management strategy that could be applied across natural habitats is still lacking. In general amphibian larvae can be easily captured, offering opportunities to ascertain the impact of altering the abundance of hosts, considered to be a key parameter affecting the severity of the disease. Here, we report the results of two experiments to investigate how altering host abundance affects infection intensity in amphibian populations of a montane area of Central Spain suffering from lethal amphibian chytridiomycosis. Our laboratory-based experiment supported the conclusion that varying density had a significant effect on infection intensity when salamander larvae were housed at low densities. Our field experiment showed that reducing the abundance of salamander larvae in the field also had a significant, but weak, impact on infection the following year, but only when removals were extreme. While this suggests adjusting host abundance as a mitigation strategy to reduce infection intensity could be useful, our evidence suggests only heavy culling efforts will succeed, which may run contrary to objectives for conservation.

Behavioural responses to chemical cues of predators differ between fire salamander larvae from two different habitats

Predation is one of the strongest selection pressures, forcing prey organisms to detect predators and to display various antipredator behaviours, such as refuge-use or decreased activity. To recognise predators, chemosensory cues play a pivotal role, particularly in aquatic ecosystems. However, it is less known whether the ability to use these cues to respond with adequate antipredator behaviour varies between individuals occupying different habitats that are dissimilar in predation risk. Using field experiments, we examined antipredator behaviour of larval fire salamanders (Salamandra salamandra) from two different habitats, ponds and streams. Among other differences, ponds and streams are inhabited by habitat-specific predators, such as alpine newts (Ichthyosaura alpestris) occurring in ponds. We exposed larvae from both habitats to either chemical cues from alpine newts or a blank control (tap water) and investigated potential differences in their behavioural responses in two experiments. Pond larvae, but not stream larvae, became significantly less active when faced with chemical cues from newts compared to those faced with a control stimulus. Moreover, larvae from both habitats tested in water containing chemical cues spent significantly less time outside a shelter than those in control water. Our results demonstrate that larval fire salamanders recognise predatory newts through kairomones and alter their behaviour accordingly. However, experience with predatory newts may not be necessary to differentiate kairomones from control water, but may be beneficial for larvae to further develop their antipredator behaviour, thus representing conformance to a niche.

Developmental costs of yellow colouration in fire salamanders and experiments to test the efficiency of yellow as a warning colouration

Warning colouration reduces predation risk by signalling or mimicking the unpleasantness of prey and therefore increases survival. We tested in two experiments the evolutionary costs and benefits of the yellow colour pattern in fire salamanders (Salamandra salamandra), which display a yellow/black colour pattern usually associated with toxic alkaloids. Our first experiment aimed to test whether the development of colouration is condition dependent and thus related to developmental costs, i.e. influenced by resource availability during the developmental process. Therefore, we reared fire salamander larvae under different nutritional conditions and compared the relative amount of yellow they developed after metamorphosis. Fire salamander larvae reared under limited food conditions had a lower proportion of yellow following metamorphosis than control larvae reared under superior food conditions. In a second experiment we tested whether the proportion of yellow has an impact on the risk of being attacked using artificial models. We tested, in salamander-free and salamander-occupied natural habitats, whether artificial clay models with different proportions of yellow and black receive different attack rates from potential predators (birds, mammals, insects). In clay models the proportion of yellow and the site had a significant effect on predation risk. Models with larger amounts of yellow had fewer bite marks from predators such as carabid beetles and birds, but only in sympatry with salamanders. In conclusion, the early expression of conspicuous colouration seems to be condition dependent and therefore potentially costly. Furthermore, the yellow colouration of fire salamanders act as a signal that potentially reduces their risk of being attacked by predators. Thus, the yellow colouration of fire salamanders seems to represent an adaptive trait that reduces the risk of predation, which can be expressed in higher quantity by individuals of a certain condition.

Do Salamanders Limit the Abundance of Groundwater Invertebrates in Subterranean Habitats?

Several species of surface salamanders exploit underground environments; in Europe, one of the most common is the fire salamander (Salamandra salamandra). In this study, we investigated if fire salamander larvae occurring in groundwater habitats can affect the abundance of some cave-adapted species. We analyzed the data of abundance of three target taxa (genera Niphargus (Amphipoda; Niphargidae), Monolistra (Isopoda; Sphaeromatidae) and Dendrocoelum (Tricladida; Dedrocoelidae)) collected in 386 surveys performed on 117 sites (pools and distinct subterranean stream sectors), within 17 natural and 24 artificial subterranean habitats, between 2012 and 2019. Generalized linear mixed models were used to assess the relationship between target taxa abundance, fire salamander larvae occurrence, and environmental features. The presence of salamander larvae negatively affected the abundance of all the target taxa. Monolistra abundance was positively related with the distance from the cave entrance of the sites and by their surface. Our study revealed that surface salamanders may have a negative effect on the abundance of cave-adapted animals, and highlited the importance of further investigations on the diet and on the top-down effects of salamanders on the subterranean communities.

Circumventing surface tension: tadpoles suck bubbles to breathe air

The surface tension of water provides a thin, elastic membrane upon which many tiny animals are adapted to live and move. We show that it may be equally important to the minute animals living beneath it by examining air-breathing mechanics in five species (three families) of anuran (frog) tadpoles. Air-breathing is essential for survival and development in most tadpoles, yet we found that all tadpoles at small body sizes were unable to break through the water's surface to access air. Nevertheless, by 3 days post-hatch and only 3 mm body length, all began to breathe air and fill the lungs. High-speed macrovideography revealed that surface tension was circumvented by a novel behaviour we call ‘bubble-sucking’: mouth attachment to the water's undersurface, the surface drawn into the mouth by suction, a bubble ‘pinched off’ within the mouth, then compressed and forced into the lungs. Growing tadpoles transitioned to air-breathing via typical surface breaching. Salamander larvae and pulmonate snails were also discovered to ‘bubble-suck’, and two insects used other means of circumvention, suggesting that surface tension may have a broader impact on animal phenotypes than hitherto appreciated.