Salamander Larvae Research Articles

Exposure to polystyrene nanoplastics causes immune damage, oxidative stress and intestinal flora disruption in salamander (Andrias davidianus) larvae

The toxic effects of nanoparticles have been increasingly investigated, but there has been limited research on amphibians, especially those of conservation value. This study examined the effects of different concentrations (0, 0.04, 0.2, 1, 5 mg/L) of polystyrene nanoplastics (PS-NPs, 80 nm) on the short-term exposure (7 d) of Andrias davidianus. Results demonstrated the concentration-dependent enrichment of PS-NPs in the intestine. Histological lesions displayed increased hepatic macrophages with cellular rupture, broken intestinal villi, decreased cuprocytes and crypt depression. Antioxidant- and inflammation-related enzyme activities were analysed, and it was found that hepatic and intestinal MDA content and CAT activity were highest in the N-1 group and SOD activity was highest in the N-0.2 group (p < 0.05). AKP activity continued to decline, and iNOS activity was highest in the N-0.2 group (p < 0.05). il-10, tgf-β, bcl-w and txnl1 were significantly downregulated in the N-0.2 group, while il-6 and il-8 were markedly upregulated in the N-0.2 group (p < 0.05). Exposing to PS-NPs decreased probiotic bacteria (Cetobacterium, Akkermansia) and increased pathogenic bacteria (Lachnoclostridium). Our results suggest that NPs exposure can have deleterious effects on salamanders, which predicts that NPs contamination may lead to continued amphibian declines. Therefore, we strongly recommend that attention be paid to amphibians, especially endangered species, in the field of NPs.

Salamander loss alters montane stream ecosystem functioning and structure through top‐down effects

AbstractAmphibians are among the most endangered taxa worldwide, but little is known about how their disappearance can alter the functioning and structure of freshwater ecosystems, where they live as larval stages. This is particularly true for urodeles, which often are key predators in these ecosystems. The fire salamander (Salamandra salamandra) is a common predator in European fresh waters, but the species is declining due to habitat loss and the infection by fungal pathogens. We studied the consequences of fire salamander loss from three montane streams, by comparing two key ecosystem processes (periphyton accrual and leaf litter decomposition) and the structure of three communities (periphytic algae, aquatic hyphomycetes and invertebrates) using stream enclosures with and without salamander larvae. Salamander loss did not cause changes in invertebrate abundance or community structure, except for one stream where abundance increased in the absence of salamander larvae. However, salamander loss led to lower periphyton accrual, changes in algal community structure and slower leaf litter decomposition, with no associated changes in fungal communities or microbial decomposition. The changes observed may have been caused by release of salamander predatory pressure on invertebrates, which could have promoted their grazing on periphyton, in contrast to their preference for leaf shredding in the presence of salamander. Our study demonstrates an important role of salamander larvae in montane streams through top‐down control of lower trophic levels and thus in regulating key stream ecosystem processes. Our results highlight the need for improving protection measures for amphibians to prevent these alterations on ecosystem structure and function.

Performance of different automatic photographic identification software for larvae and adults of the European fire salamander.

For many species, population sizes are unknown despite their importance for conservation. For population size estimation, capture-mark-recapture (CMR) studies are often used, which include the necessity to identify each individual, mostly through individual markings or genetic characters. Invasive marking techniques, however, can negatively affect the individual fitness. Alternatives are low-impact techniques such as the use of photos for individual identification, for species with stable distinctive phenotypic traits. For the individual identification of photos, a variety of different software, with different requirements, is available. The European fire salamander (Salamandra salamandra) is a species in which individuals, both at the larval stage and as adults, have individual specific patterns that allow for individual identification. In this study, we compared the performance of five different software for the use of photographic identification for the European fire salamander: Amphibian & Reptile Wildbook (ARW), AmphIdent, I3S pattern+, ManderMatcher and Wild-ID. While adults can be identified by all five software, European fire salamander larvae can currently only be identified by two of the five (ARW and Wild-ID). We used one dataset of European fire salamander larval pictures taken in the laboratory and tested this dataset in two of the five software (ARW and Wild-ID). We used another dataset of European fire salamander adult pictures taken in the field and tested this using all five software. We compared the requirements of all software on the pictures used and calculated the False Rejection Rate (FRR) and the Recognition Rate (RR). For the larval dataset (421 pictures) we found that the ARW and Wild-ID performed equally well for individual identification (99.6% and 100% Recognition Rate, respectively). For the adult dataset (377 pictures), we found the best False Rejection Rate in ManderMatcher and the highest Recognition Rate in the ARW. Additionally, the ARW is the only program that requires no image pre-processing. In times of amphibian declines, non-invasive photo identification software allowing capture-mark-recapture studies help to gain knowledge on population sizes, distribution, movement and demography of a population and can thus help to support species conservation.

Stress response of fire salamander larvae differs between habitat types.

The larvae of the European fire salamander (Salamandra salamandra) can inhabit two different habitats: streams and ponds. Streams are characterized by lower predation risks and higher food availability. Thus, ponds are considered a less suitable habitat. To investigate the differential impacts of these two habitats on larval physiology, we measured the stress response of larvae. After successfully validating the measure of water-borne corticosterone release rates in fire salamander larvae, we measured the baseline and stress-induced corticosterone of 64 larvae from ponds and streams in the field. We found that larvae in ponds have a higher baseline and stress-induced corticosterone levels. Additionally, we performed a reciprocal transplant experiment (RTE) and tested whether larvae can adapt their stress responses to changing habitats. After two weeks, we did not find an increase in corticosterone levels when comparing stress-induced corticosterone values with baseline corticosterone values in larvae transferred into ponds, irrespective of their habitat of origin. However, larvae transferred into streams still exhibited an increase in the stress-induced corticosterone response in comparison with the baseline values. These results show that non-invasive hormone measurements can provide information on the habitat quality and potential adaptation and thus emphasize the potential for its use in conservation efforts.

Tonic immobility behaviour does not differ between fire salamander larvae from ponds and streams.

Tonic immobility is an antipredator defence in which the prey animal remains motionless after physical contact with the predator, pretending to be dead. This behaviour has been observed among a variety of taxa but has received only little attention in amphibian larvae. During our field studies with fire salamander larvae, we observed that larvae from different habitats display tonic immobility after handling. In our study site, we find larvae in pond and stream habitats, that differ in several aspects such as their stress response and their risk-taking behaviour, likely caused by the very different habitat conditions. We measured the time that the tonic immobility behaviour was displayed but found no difference between larvae from the two habitat types. Likewise, we also found no correlation between the size of the larvae and the duration of displaying the behaviour. In conclusion, we found that fire salamander larvae show tonic immobility, but found no evidence that the different habitat conditions influence the tonic immobility behaviour.

Bifenthrin induces changes in clinical poisoning symptoms, oxidative stress, DNA damage, histological characteristics, and transcriptome in Chinese giant salamander (Andrias davidianus) larvae

Bifenthrin (BF) is a broad-spectrum insecticide that has gained widespread use due to its high effectiveness. However, there is limited research on the potential toxic effects of bifenthrin pollution on amphibians. This study aimed to investigate the 50 % lethal concentration (LC50) and safety concentration of Chinese giant salamanders (CGS) exposed to BF (at 0, 6.25,12.5,25 and 50 μg/L BF) for 96 h. Subsequently, CGS were exposed to BF (at 0, 0.04, and 4 μg/L BF) for one week to investigate its toxic effects. Clinical poisoning symptoms, liver pathology, oxidative stress factors, DNA damage, and transcriptome differences were observed and analyzed. The results indicate that exposure to BF at 4 μg/L significantly decreased the adenosine-triphosphate (ATP), superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT) contents in the brain, liver, and kidney of CGS. Additionally, the study found that the malondialdehyde (MDA), reactive oxygen species (ROS), and 8-hydroxydeoxyguanosine (8-OHdG) contents were increased. The liver tissue exhibited significant inflammatory reactions and structural malformations. RNA-seq analysis of the liver showed that BF caused abnormal antioxidant indices of CGS. This affected molecular function genes such as catalytic activity, ATP-dependent activity, metabolic processes, signaling and immune system processes, behavior, and detoxification, which were significantly upregulated, resulting in the differential genes significantly enriched in the calcium signaling pathway, PPARα signaling pathway and NF-kB signaling pathway. The results suggest that BF induces the abnormal production of free radicals, which overwhelms the body's self-defense system, leading to varying degrees of oxidative stress. This can result in oxidative damage, DNA damage, abnormal lipid metabolism, autoimmune diseases, clinical poisoning symptoms, and tissue inflammation. This work provides a theoretical basis for the rational application of bifenthrin and environmental risk assessment, as well as scientific guidance for the conservation of amphibian populations.

Food and light availability induce plastic responses in fire salamander larvae from contrasting environments.

Phenotypic plasticity has been proposed as a mechanism facilitating the colonisation and adaptation to novel environments, such as caves. However, phenotypic plasticity in subterranean environments remains largely unexplored. Here, we test for plasticity in growth and development of fire salamander larvae (Salamandra salamandra) from subterranean and surface habitats, in response to contrasting food availability and light conditions. We hypothesized that: (i) low food availability and absence of light decrease larval growth and delay metamorphosis, (ii) light conditions mediate the effects of food availability on growth and time to metamorphosis, and (iii) larval response to contrasting light and food conditions is shaped by the habitat of origin. Our study showed that reduced food availability significantly delayed metamorphosis and slowed total length and body mass growth rates, while exposure to constant darkness slowed body mass growth rate. However, larvae slowed growth rates and increased time to metamorphosis without compromising size at metamorphosis. The effect of food availability on growth and time to metamorphosis did not change under different light conditions. Fire salamanders from subterranean and surface habitats responded differently only in relation to contrasting food availability conditions. Specifically, larvae from the surface habitat grew faster in high food conditions, while growth in larvae from the subterranean habitat was not influenced by food availability. Initial size also appeared to be an influential factor, since larger and heavier larvae grew slower, metamorphosed faster, and the size advantage was maintained in newly-metamorphosed juveniles. Overall, the results of our experiment suggest that plasticity and local adaptation favor the exploitation of aquatic subterranean habitats for breeding by fire salamanders, allowing successful development even under food shortage and day-length constraints, without compromising metamorphic size. Our findings have implications for conservation because they confirm that phenotypic plasticity plays a critical role in allowing fire salamanders to overcome altered environmental conditions.

Landscape of fear in freshwater ecotones: How predation risk and light conditions affect mesopredator activity and foraging in springs

Abstract Springs are environments that can provide general insights into factors favouring diversity in ecotones, but they are often neglected in freshwater studies. One of the challenging processes acting in ecotones is the landscape of fear (LOF), the space–time variation of perceived predation risk. Spring exploitation often involves species that are mesopredators in surface fresh water and that can become apex predators in ground water, as in the case of the fire salamander larvae (Salamandra salamandra). Here, we aim to determine whether the activity and foraging patterns of the fire salamander in springs are affected by LOF. We surveyed the night‐ and daytime abundance of fire salamander larvae in 15 springs to assess predator occurrence. We also reared 48 salamander larvae with and without non‐lethal exposure to predators within tanks simulating groundwater or surface freshwater light features. Before and after a month of rearing, we tested larva efficiency in catching prey when exposed to predator chemical cues, both in light and dark conditions. In the field, the number of active fire salamander larvae was significantly higher during the night. At night, the number of active larvae across the transition area between ground water and surface water was higher in plots closer to the surface. Testing and rearing conditions significantly affected larva behaviour, and prey capture was significantly more effective in light conditions. It was less successful in larvae reared with predator chemical cues and in the presence of predators. Moreover, larvae reared with predators under light conditions were slower than those raised with predators in dark conditions. Our results show that LOF can interact with extant environmental features and constitute a significant behavioural pressure for mesopredator species living in freshwater ecotones.

PREVALENCE OF RANAVIRUS IN SPOTTED SALAMANDER (AMBYSTOMA MACULATUM) LARVAE FROM CREATED VERNAL POOLS IN WEST VIRGINIA, USA.

Ranavirosis is a disease of high concern for amphibians due to widespread documentation of its lethal and sublethal impacts and its high transmission potential across populations and species. We investigated whether spotted salamander (Ambystoma maculatum) ranavirus prevalence and viral load were associated with habitat characteristics, genetic diversity, corticosterone levels, and body size. In 2015 and 2016, we sampled 34 recently created vernal pools in the Monongahela National Forest, West Virginia, USA. We collected tail clippings from 1,128 spotted salamander larvae and waterborne hormone samples from 436 of those larvae, along with eight environmental characteristics of the pools. Over the 2-yr period, we detected ranavirus in 62% of pools, with prevalence ranging from 0% to 63% (mean, 7.68%). Spotted salamander size was positively correlated with ranavirus presence and viral load; however, we did not find associations between ranavirus prevalence or viral load and habitat characteristics, spotted salamander genetic diversity, relatedness, effective number of breeders, or corticosterone levels. The widespread occurrence of ranavirus in the vernal pools illustrates the potential for rapid natural introduction of the pathogen to created wetlands. Managers could consider monitoring local distributions of ranavirus before creation of new vernal pools to guide strategic placement of the wetlands to minimize occurrence and prevalence of this pathogen.

Habitat connectivity supports the local abundance of fire salamanders (Salamandra salamandra) but also the spread of Batrachochytrium salamandrivorans

ContextHabitat connectivity can stabilise animal populations by facilitating immigration and genetic exchange, but it increases the risk of infectious diseases being spread by hosts. Chytridiomycosis caused by Batrachochytrium salamandrivorans (Bsal) threatens European salamander diversity. The extent to which the connectivity of populations of fire salamanders (Salamandra salamandra) contributes to the spread of Bsal remains unclear.ObjectivesWe analysed the impact of habitat connectivity of fire salamanders on the spread of Bsal. Moreover, we show how local salamander abundance is associated with habitat connectivity over a five-year period.MethodsWe developed fire salamander habitat suitability models (HSMs) for the Eifel area (Germany), currently considered the core of the range of Bsal in Europe. Habitat models were used to calculate pairwise resistance between salamander occurrences to test whether Bsal presence and salamander abundance were associated with habitat connectivity.ResultsFire salamanders are widely distributed in the Eifel. Solid bedrock and topographic positioning were important predictors of stream suitability as breeding habitats, while deciduous forests and grassland cover determined overall fire salamander habitat suitability along with breeding habitat suitability. Bsal-positive salamander occurrences were better-connected than Bsal-negative or untested occurrences. Nevertheless, fire salamander larvae were more abundant in well-connected sites.ConclusionThe connection of salamander populations by suitable habitat seems to support local salamander abundance while facilitating the spread of Bsal. In situ conservation measures counteracting host species connectivity to interrupt Bsal transmission pathways must be implemented with caution, as they may weaken the demographic advantages of connectivity.

Encouraging news for in situ conservation: Translocation of salamander larvae has limited impacts on their skin microbiota.

The key role of symbiotic skin bacteria communities in amphibian resistance to emerging pathogens is well recognized, but factors leading to their dysbiosis are not fully understood. In particular, the potential effects of population translocations on the composition and diversity of hosts' skin microbiota have received little attention, although such transfers are widely carried out as a strategy for amphibian conservation. To characterize the potential reorganization of the microbiota over such a sudden environmental change, we conducted a common-garden experiment simulating reciprocal translocations of yellow-spotted salamander larvae across three lakes. We sequenced skin microbiota samples collected before and 15 days after the transfer. Using a database of antifungal isolates, we identified symbionts with known function against the pathogen Batrachochytrium dendrobatidis, a major driver of amphibian declines. Our results indicate an important reorganization of bacterial assemblages throughout ontogeny, with strong changes in composition, diversity and structure of the skin microbiota in both control and translocated individuals over the 15 days of monitoring. Unexpectedly, the diversity and community structure of the microbiota were not significantly affected by the translocation event, thus suggesting a strong resilience of skin bacterial communities to environmental change-at least across the time-window studied here. A few phylotypes were more abundant in the microbiota of translocated larvae, but no differences were found among pathogen-inhibiting symbionts. Taken together, our results support amphibian translocations as a promising strategy for this endangered animal class, with limited impact on their skin microbiota.

Environmental Drivers of Amphibian Breeding Phenology across Multiple Sites

A mechanistic understanding of phenology, the seasonal timing of life history events, is important for understanding species’ interactions and the potential responses of ecological communities to a rapidly changing climate. We present analysis of a seven-year dataset on the breeding phenology of wood frogs (Rana sylvatica), tiger salamanders (Ambystoma tigrinum), blue-spotted salamanders (Ambystoma laterale), and associated unisexual Ambystoma salamanders from six wetlands in Southeast Michigan, USA. We assess whether the ordinal date of breeding migrations varies among species, sexes, and individual wetlands, and we describe the specific environmental conditions associated with breeding migrations for each species/sex. Breeding date was significantly affected by species/sex identity, year, wetland, and the interactions between species/sex and year as well as wetland and year. There was a great deal of variation among years, with breeding occurring nearly synchronously among groups in some years but widely spaced between groups in other years. Specific environmental triggers for movement varied for each species and sex and changed as the breeding season progressed. In general, salamanders responded to longer temperature lags (more warmer days in a row) than wood frogs, whereas wood frogs required longer precipitation lags (more rainy days in a row) than salamanders. Wood frogs were more likely to migrate around the time of a new moon, whereas in contrast, Ambystoma salamander migration was not associated with a moon phase. Ordinal day was an important factor in all models, suggesting that these amphibians require a latency period or similar mechanism to avoid breeding too early in the year, even when weather conditions appear favorable. Male wood frogs migrated earlier than female wood frogs, and male blue-spotted salamanders migrated earlier than female A. laterale and associated unisexual females. Larger unisexual salamanders migrated earlier than smaller individuals. Differences in species’ responses to environmental cues led to wood frogs and A. laterale breeding later than tiger salamanders in colder years but not in warmer years. This suggests that, as the climate warms, wood frog and A. laterale larvae may experience less predation from tiger salamander larvae due to reduced size differences when they breed simultaneously. Our study is one of few to describe the proximate drivers of amphibian breeding migrations across multiple species, wetlands, and years, and it can inform models predicting how climate change may shift ecological interactions among pond-breeding amphibian species.

Pool Age and Local Habitat Are Associated with Effective Number of Breeders in Spotted Salamander Populations Colonizing Created Vernal Pools

Population genetics can reveal whether colonization of created habitats has been successful and inform future strategies for habitat creation. We used genetic analysis to investigate spotted salamander (Ambystoma maculatum) colonization of created vernal pools and explored the impact of habitat characteristics on the genetic diversity and connectivity of the pools. Our first objective was to examine genetic structure, differentiation, diversity, and potential for a founder effect. Our second objective was to determine if habitat characteristics were associated with effective number of breeders, relatedness, or genetic diversity. We sampled spotted salamander larvae in 31 created vernal pools (1–5 years old) in Monongahela National Forest (WV) in May and June 2015 and 2016. The youngest pools exhibited genetic differentiation, a founder effect, and low effective number of breeders. Effective number of breeders was positively associated with pool age, vegetation cover, pool diameter, and sample size. Vegetation cover was also negatively associated with relatedness. Genetic diversity did not have strong environmental predictors. Our results indicated the effective number of breeders increased and genetic differentiation decreased within 4–5 years of pool creation, a sign of rapid colonization and potential population establishment. Our research also showed that higher vegetative cover within the pool and larger pool diameters could impact habitat quality and should be incorporated into future pool creation.

English

Abstract Freshwater crayfish rank among the most imperilled groups and play several critical roles in freshwater ecosystems, yet survey efforts remain limited throughout much of Canada and North America. Inadequate survey and monitoring efforts render our ecosystems vulnerable to the introduction and spread of harmful invasive species which can be difficult or impossible to control following establishment. During surveys targeting salamander larvae across Pelee Island, Ontario we incidentally captured crayfish and identified them to species. Three species were detected including two native species known only from collections in the 1960s (Creaserinus fodiens, Faxonius immunis) as well as the non-native White River Crayfish (Procambarus acutus) which has not been documented previously in Canada. Our oldest record of P. acutus is from 2015, and the species currently appears to be widespread and well established on Pelee Island and has also recently been recorded in Six Mile Lake on the Canadian Shield. We provide preliminary data on the aquatic habitats where each species was found on Pelee Island and strongly encourage additional monitoring efforts combined with targeted education efforts to prevent the spread of P. acutus to the Ontario mainland.

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

AbstractPrey organisms need to detect predators and respond with adequate antipredator behaviours. To recognize predators, chemosensory cues play a pivotal role, particularly in aquatic ecosystems, where sight may be restricted. However, it is less known whether the ability to use these cues, enabling appropriate antipredator behaviour, varies between subpopulations occupying different habitats with different predation risks. We examined antipredator behaviour of wild‐caught larval fire salamanders (Salamandra salamandra) from two different habitats, ponds and streams. Ponds and streams are inhabited by habitat‐specific predators, such as alpine newts (Ichthyosaura alpestris), which only occur in ponds. We confronted larvae from both habitats with either tap water (control) or tap water that had contained alpine newts (newt treatment) and investigated potential behavioural differences in activity and shelter emergence. In the activity test, pond larvae, but not stream larvae, were significantly less active when faced with chemical cues (potentially kairomones) from newts than those faced with a control stimulus, but stream larvae generally exhibited significantly lower activity than pond larvae. In the shelter‐emergence test, larvae from both habitats spent significantly less time outside a shelter in the newt treatment than in control water. This suggests that larval fire salamanders, independent of their habitat, recognize chemical cues from potential predators and alter their behaviour accordingly. Larvae of both habitats were able to detect and use chemical cues released from potential predators, but they differed slightly in their response to these chemical cues. This finding reflects varying antipredator strategies in response to the habitat‐specific differences.

Interspecific interactions between coexisting Siberian and Ezo salamander larvae examined by stable isotope analyses

Interspecific interactions between coexisting Siberian and Ezo salamander larvae examined by stable isotope analyses

Growth-Promotion Effects of Dissolved Amino Acids in Three Species of Hynobius Salamander Hatchlings.

It has been suggested that aquatic vertebrates may be able to meet their energy requirements by using the amino acids dissolved in environmental water. If this ability can be applied to aquatic organisms generally, then conventional ecological theories related to food web interactions should be revisited, as this would likely bring about significant advances in applications. Here, we prepared two 1 mM amino acid (phenylalanine and glycine) solutions in environmental water and conducted laboratory experiments to demonstrate the utilization of dissolved amino acids by hatchlings of three salamander species (Ezo: Hynobius retardatus, Tohoku: Hynobius lichenatus, and Japanese black: Hynobius nigrescens). Compared to controls (no amino acids in environmental water), the growth rate for Ezo salamanders was higher when larvae were reared in phenylalanine solution, while that for Japanese black salamanders was higher in glycine and phenylalanine solutions. Amino acids in environmental water had no effect on the growth of Tohoku salamanders. However, when growth was divided into early (days 1 to 5) and late (days 5 to 7) developmental stages, growth in early-developmental stage individuals was improved by phenylalanine treatment, even in Tohoku salamanders. The results showed that the growth of salamander larvae was improved when salamanders were reared in environmental water with high amino acid concentrations. Although aquatic bacteria may not have been removed completely from the environmental water, no other eukaryotes were present. Our results suggest an overlooked nutrient pathway in which aquatic vertebrates take up dissolved amino acids without mediation by other eukaryotes.

Host Identity Matters-Up to a Point: The Community Context of Batrachochytrium dendrobatidis Transmission.

AbstractThe level of detail on host communities needed to understand multihost parasite invasions is an unresolved issue in disease ecology. Coarse community metrics that ignore functional differences between hosts, such as host species richness, can be good predictors of invasion outcomes. Yet if host species vary in the extent to which they maintain and transmit infections, then explicitly accounting for those differences may be important. Through controlled mesocosm experiments and modeling, we show that interspecific differences between host species are important for community-wide infection dynamics of the multihost fungal parasite of amphibians (Batrachochytrium dendrobatidis [Bd]), but only up to a point. The most abundant host species in our system, fire salamander larvae (Salamandra salamandra), did not maintain or transmit infections. Rather, two less abundant "auxiliary" host species, Iberian tree frog (Hyla molleri) and spiny toad (Bufo spinosus) larvae, maintained and transmitted Bd. Frogs had the highest mean rates of Bd shedding, giving them the highest contributions to the basic reproduction number, R0. Toad contributions to R0 were substantial, however, and when examining community-level patterns of infection and transmission, the effects of frogs and toads were similar. Specifying more than just host species richness to distinguish salamanders from auxiliary host species was critical for predicting community-level Bd prevalence and transmission. Distinguishing frogs from toads, however, did not improve predictions. These findings demonstrate limitations to the importance of host species identities in multihost infection dynamics. Host species that exhibit different functional traits, such as susceptibility and infectiousness, may play similar epidemiological roles in the broader community.

The roles of landscape of fear and light in allowing the exploitation of spring habitats by subterranean amphipods: an experimental and field approach

Border habitats such as interfaces and ecotones promise research targets from an evolutionary and zoological point of view. Springs are typical ecotones that border two strongly distinct environments: surface and underground. They are exploited by both subterranean and surface species for which they may provide specific environmental pressures promoting phenotypic plasticity and local adaptations. The aim of this study is to understand how the landscape of fear (LOF) and physical constraints, like light occurrence, affect springs' exploitation by both a subterranean (Niphargus thuringius) and a surface crustacean amphipod species (Echinogammarus stammeri). From March to May 2021, we surveyed 15 springs, divided into 25 plots according to their distance to the border, and both day and night, we recorded amphipods activity and LOF levels for them. In a subterranean laboratory, we also reared 80 N. thuringius and 80 E. stammeri in safe and risky conditions with constant darkness and diel light variation assessing their activity and survival for 30 days. Risky conditions were represented by meso-predators (four fire salamander larvae) alone or with a top-predator (a dragonfly larva of the species Cordulegaster boltonii). While in field conditions, the activity of N. thuringius seemed negatively affected by the number of active predators, in laboratory experiments, the main role was played by the light treatment; activity was significantly higher in constant darkness conditions. E. stammeri activity in the field was higher in surface plots, while in laboratory conditions was affected by LOF. Predation risk negatively affected the survival of both amphipods. Our findings reveal that while light conditions seem to shape activity patterns of stygobionts strongly, predators have a lower effect on activity, even though predators have negative effects on survival. Moreover, physical constraints, such as light exposure, can affect antipredator responses of subterranean organisms, thus representing selective pressures for the exploitation of surface environments.

How Trophic Conditions Affect Development of Fire Salamander (Salamandra salamandra) Larvae: Two Extreme Cases

Reduced trophic resources can pose relevant constraints to the development of freshwater animals with complex life cycles. For amphibians, food deprived environments, such as high-altitude ponds and springs and groundwaters are frequently used for breeding. The aim of this study is to outline trophic conditions leading to extreme cases of delayed larval development and increased size at metamorphosis of a European widespread amphibian, the fire salamander (Salamandra salamandra). We collected 150 fire salamander larvae, split them in two groups, one with high and one with low trophic resource availability. We then observed the effects of nutritional conditions on larval development recording time to metamorphosis and average day growth. Moreover, in the field, we surveyed larvae growth and size at metamorphosis in two artificial subterranean sites with low prey availability. Trophic conditions strongly affected larval development and under low food treatment time to metamorphosis reached up to 416 days. In the subterranean environments we observed a similar pattern, with larvae requiring more than one year to attain metamorphosis but reaching unexpected large sizes. Environmental trophic conditions experienced during early stages can induce strong delay in metamorphosis of the fire salamander; this plasticity makes fire salamander larvae optimal models for comparative studies and cross-environment experiments.