Salamandra Larvae Research Articles

Detection of elusive fire salamander larvae (Salamandra salamandra) in streams via environmental DNA

Abstract In the face of the global biodiversity crisis, the monitoring of species richness and diversity is experiencing an increased demand entailing a raise in cost and time investment. The analysis of species-specific DNA fragments in environmental samples (eDNA) such as from water or soil, facilitate the molecular detection of species without the specific sampling of individuals. The invasive chytrid fungus Batrachochytrium salamandrivorans (Bsal) is infecting natural fire salamander populations (Salamandra salamandra) and causes chytridiomycosis resulting in infrequent regional extinctions of populations across Central Europe. With regard to the expanding distribution of Bsal over the last years, cost-effective monitoring of fire salamanders is important for the conservation of this species. Based on a real-time quantitative PCR (qPCR) assay, we developed a new protocol to detect S. salamandra larvae in streams via eDNA, using species-specific primers of the mitochondrial control region (D-loop). We tested the efficiency of qPCR primer sets for six combinations of DNA extraction kits coupled with subsequent PCR inhibitor removal kits for obtaining qPCR-detectable S. salamandra eDNA from water filters, that were taken both from natural streams and artificial water tanks in the laboratory as positive controls. We found that the DNeasy Blood & Tissue Kit in combination with the DNeasy PowerClean CleanUp Kit performed best for detecting salamander larvae from natural streams. Our experimental protocol paves the way for resource-saving approaches to monitor S. salamandra larvae, but also confirms the limits to this eDNA approach in that it requires optimized laboratory protocols.

Compassionate approaches for the conservation and protection of fire salamanders

The Near Eastern fire salamander, Salamandra infraimmaculata, is considered an endangered species in Israel and is near-threatened regionally. For 25 years, our laboratory has sought ethical sampling methods to protect individuals and populations of Salamandra. To “mark” individuals for estimating dispersal and population size, we use non-invasive individual-specific markings from photographs of larvae and adults. We demonstrated through mesocosm experiments (which are less mortality-driven than in nature) that exotic Gambusia affinis have extreme negative mortality effects on Salamandra larvae. From a compassionate conservation aspect, G. affinis should not be killed and placed in habitats where amphibians are not in danger and mosquitoes can be controlled. We identified breeding-site characteristics demonstrating that permanent breeding sites support larger adult populations than temporary breeding sites. For population genetics studies, we take minimal sized tail tips from adults (which have no adverse effects) for microsatellite data. For gene expression studies, rather than sacrifice entire bodies, we demonstrated that by taking only small larval tail tips, we could follow gene expression. We additionally demonstrated that tail tip removal does not affect survival, time to or size at metamorphosis. We documented high road kill rates at a specific breeding site. To prevent potential disease spread, we sterilize boots and sampling gear. We use results for implementing or recommending conservation of individuals and populations – e.g., identifying: movement corridors for breeding site dispersal; roadkill hotspots for under-road tunnels; suitable habitat for pool construction for more effective conservation; utilizing population genetics for recommending management units; information on demography and genetic diversity to identify hotspots for conservation; removal of Gambusia for amphibian protection.

Differences in chytridiomycosis infection costs between two amphibian species from Central Europe

Batrachochytrium dendrobatidis (Bd) causes the disease chytridiomycosis associated with amphibian declines. Response and costs of infection varies greatly between species. Bd can induce a stress response in amphibians resulting in elevated corticosterone (CORT). We exposed Bombina variegata and Hyla arborea tadpoles to Bd+ or Bd- Salamandra salamandra larvae and measured CORT release rates, Bd infection loads, and survival through metamorphosis. Tadpoles of both species exposed to Bd+ larvae had elevated CORT release rates compared to tadpoles exposed to Bd- larvae. Bombina variegata appear less resistant to infection than H. arborea, showing higher Bd loads and more infected individuals. Within species, we did not find differences in cost of infection on survival, however more B. variegata tadpoles reached metamorphosis than H. arborea. The differences in resistance may be species specific, owing to higher immunity defenses with H. arborea having higher overall CORT release rates, and differences in antimicrobial peptides, or to differences in Bd strain or other unexplored mechanisms.

Joint effects of rising temperature and the presence of introduced predatory fish on montane amphibian populations

AbstractAmphibian populations in montane habitats are often subjected to high thermal variability, which may exacerbate anthropogenic impacts such as the introduction of exotic species. Here, we present data from two experiments exploring the joint effects of rising temperatures and the presence of waterborne cues from an exotic predatory fish on the short‐ and long‐term antipredatory responses (i.e. activity and time to metamorphosis respectively) of Rana iberica and Salamandra salamandra larvae from two montane amphibian populations. We found some evidence of a cumulative effect of an increase in temperature and the presence of predators. Although predator recognition was not precluded at rising temperatures, we observed an increase in larval activity in warmer water, which might negatively affect survival by favoring prey detectability by predators. We also observed a strong quadratic effect of temperature and a joint effect of temperature and predators on larval development: at intermediate temperatures, larvae exposed to exotic trout cues had greatly accelerated metamorphosis. These results suggest that warmer conditions might be particularly harmful for larvae in montane wetlands enduring the presence of exotic predators.

Influence of fire salamander larvae on among-pool distribution of mosquito egg rafts: oviposition habitat selection or egg raft predation?

Many amphibian populations are in decline worldwide. Surprisingly, few studies have examined how such declines may benefit mosquitoes. Amphibian larvae may compete with and prey upon mosquito larvae, and may alter oviposition habitat selection (OHS) of mosquito adults. However, often overlooked, observed among-pool egg distributions attributed to OHS may additionally or alternatively be explained by egg predation. Temporary pools of mountainous areas of the Mediterranean serve as larval habitat for both the mosquito, Culiseta longiareolata, and the salamander, Salamandra infraimmaculata. We found Culiseta larvae and egg rafts to be highly vulnerable to predation by pre-metamorphosing Salamandra larvae, but not to metamorphosing ones. In outdoor mesocosm experiments, oviposition avoidance by Culiseta females in response to caged Salamandra was not demonstrated regardless of salamander developmental stage. Egg raft abundance was significantly reduced in free-roaming, pre-metamorphosing Salamandra but not by metamorphosing ones. Thus, Salamandra larvae may have little deterrence on Culiseta oviposition. Instead, fewer egg rafts are attributed largely to egg predation. This study highlights the importance of egg raft predation in addition to OHS when interpreting the influence of predators on prey egg distributions. It also highlights that a cost of declining amphibian populations is their reduced impacts on mosquito populations.

Deleterious effects by mosquitofish (Gambusia affinis) on the endangered fire salamander (Salamandra infraimmaculata)

AbstractInvasive fishes, via competition and predation, may be an important factor in endangering populations of amphibians and other aquatic fauna. The mosquitofish, Gambusia affinis, commonly stocked into amphibian breeding sites for mosquito control worldwide, has recently been found in some breeding sites of the endangered fire salamander Salamandra infraimmaculata in northern Israel. A comparison of Salamandra larvae in natural pools varying in Gambusia densities suggested that Gambusia negatively affects Salamandra by causing damage to its appendages, in particular, its tailfin (as manifested in a smaller tail:body ratio). Comparison of the same pool before and after mosquitofish introduction suggested strong negative effects on larval performance and number of emerging metamorphs. To explicitly test the hypothesis that Gambusia negatively affects Salamandra larval development, growth and survival, we conducted a replicated outdoor mesocosm experiment. In this experiment, we also tested how habitat structural complexity, in the form of radiating cords simulating artificial vegetation, might mediate damage caused by Gambusia. We used a two‐by‐two factorial design: the presence or absence of increased structural complexity crossed with the presence or absence of mosquitofish. After a short period, mosquitofish strongly reduced larval wet weight and survival, and increased body damage in the form of a reduced larval tail:body ratio and partially missing gills and limbs. No Salamandra larvae exposed to mosquitofish survived to metamorphosis in either habitat type. We conclude that Gambusia strongly and negatively affects Salamandra larvae and that the introduction of Gambusia into sites containing Salamandra is inconsistent with the goal of preserving this endangered urodele.

Xanthophore migration from the dermis to the epidermis and dermal remodeling during Salamandra salamandra salamandra (L.) larval development.

During larval development of Salamandra salamandra salamandra chromatophores organize to form the definitive pigment pattern constituted by a black background with yellow patches that are characterized by epidermal xanthophores and dermal iridophores. Simultaneously the dermis undergoes remodeling from the larval stage to that typical of the adult. In the present study we ultrastucturally and immunocytochemically examined skin fragments of S. s. salamandra larvae and juveniles in order to investigate the modalities of xanthophore migration and differentiation in the context of dermal remodeling from the larval to adult stage. Semithin and thin sections showed that the dermis in newly born larvae consists of a compact connective tissue (basement lamella), to which fibroblasts and xanthophores adhere, and of a loose deep collagen layer. As larval development proceeds, fibroblasts and xanthophores invade the basement lamella, skin glands develop and the adult dermis forms. At metamorphosis, xanthophores reach the epidermis crossing through the basal lamina. We examined immunocytochemically the expression of signal molecules, such as fibronectin, vitronectin, beta1-integrin, chondroitin sulfate, E-cadherin, N-cadherin and plasminogen activator, which are known to be involved in regulating morphogenetic events. Their role in dermal remodeling and in pigment pattern formation is discussed.

RISK OF PREDATION AND HATCHING OF RESTING EGGS IN THE OSTRACOD HETEROCYPRIS INCONGRUENS

Most crustaceans inhabiting temporary pools have resting stages that can remain dormant for many years. Only a fraction of the resting stages hatch each time the pool floods, as a safeguard against unpredictable events resulting in complete reproductive failure. There is likely to be a selective advantage in the ability of resting stages to respond to signals indicating likely environmental conditions and to adjust their hatching fraction in response. We examine whether such an ability exists in the resting stages (diapausing eggs) of the ostracod Heterocypris incongruens, which inhabits temporary freshwater pools in northern Israel. The active stages are subject in some seasons to predation by larvae of the fire salamander (Salamandra salamandra L.). We predicted that a lower fraction of H. incongruens eggs would hatch in water in which S. salamandra larvae had been kept than in water that had not contained S. salamandra larvae. We found no such effect. We discuss possible reasons for the absence of the expected response. A lower proportion of H. incongruens eggs hatched in water with higher conductivity. This may reduce the risk of active stages being killed by desiccation before being able to complete their life cycle

Cytochemical and immunocytochemical investigations on epidermal mitochondria-rich cells in Salamandra salamandra salamandra (L.) larvae

In the present study we set out to investigate the expression of E-cadherin, N-cadherin, β 1-integrin, fibronectin and vitronectin in the mitochondria-rich cells (MRC) of the skin of Salamandra salamandra salamandra. Moreover MRC were stained with five lectins ( Triticum vulgaris; Dolichos biflorus; Glycine max; Arachis hypogaea and Canavalia ensiformis). Larval MRC expressed both adhesion molecules and extracellular matrix glycoproteins and bound all lectins tested. Juvenile MRC did not react with the antisera utilized, but they stained with the lectins. Both the lectins and the regulatory molecules proved to be good cytochemical markers for distinguishing morphologically differentiated MRC during the larval life of Salamandra salamandra salamandra. The adhesion molecules and matrix glycoproteins are of great utility for maintaining the correct tissue architecture. In Salamandra salamandra salamandra larvae these molecules may be crucial for stability and for the correct localization and fate of all skin elements, including specialized cells such as larval MRC.

Non-consumptive effects of larval Salamandra on crustacean prey: can eggs detect predators?

Predators affect prey populations not only by prey consumption but also in nonconsumptive ways including modifying prey behavior. I tested the effects of fire salamander larvae (Salamandra infraimmaculata) on populations of co-occurring crustacean species in artificial outdoor pools. I also tested whether these effects were due entirely to prey consumption by Salamandra larvae or alternatively to some nonconsumptive effect. The soil (containing crustacean eggs) added to the artificial pools was collected from a dried-out temporary pool that is inhabited by Salamandra during the early part of the hydroperiod. I randomly assigned the pools to one of three treatments: control, free Salamandra, or caged Salamandra. Free salamander larvae could roam the entire pool and prey upon crustaceans. Caged salamander larvae were placed within a cage with having 250-μm mesh windows. They could not prey upon the crustaceans but could, for example, influence them by chemical cues. Densities of the three dominant crustacean species (Arctodiaptomus similis, Ceriodaphnia quadrangula and Cyzicus sp.) were drastically reduced in both salamander treatments compared to the control. Crustacean densities, however, were not significantly different between the two salamander treatments. One plausible explanation is that crustacean eggs can detect the presence of this predator via chemical cues and delay hatching.

Cannibalistic phenomena in salamandra salamandra larvae in certain water bodies and under experimental conditions

Salamandra salamandra larvae originating from a single batch (layed by one female) were raised at different temperatures and on ion-rich or distilled water. Temperature, but not the presence or absence of ions affected growth. Larvae originating from one batch or from several batches born on the same day, were kept without food for long periods of several months in order to observe cannibalistic predation under such conditions. This phenomenon is known in natural populations of salamanders inhabitating either rain pools in rocks or permanent springs poor in aquatic life. It was found that cannibalism increased under experimental conditions proportionally to the increase in size between the larvae.