Stygobiont species show common, typical traits derived from their adaptation to subterranean life. Due to the general absence of light in cave environments, the majority of them are eyeless. Although the absence of eyes generally does not allow them to perceive luminous stimuli, some stygobionts still present phototaxis. Previous studies determined that different species of the eyeless amphipod crustaceans of the genus Niphargus are able to react to light; this has been interpreted as an adaptation to avoid dangerous surface habitats, even if recent studies suggest that this could also be an adaptation to exploit them when a situation is less dangerous (i.e., during the night). Niphargus thuringius is a stygobiont amphipod that can also be observed in spring environments despite possessing all the main morphological features of subterranean organisms, such as depigmentation and a lack of eyes. In the present study, we test how the species respond to light stimuli according to the light cycle and predation risk experienced during a conditioning period. We assessed the reactions to light stimuli of adult individuals of N. thuringius after 30 days of rearing in microcosms with different conditions of light occurrence (total darkness or a light/darkness daily cycle) and predation risk (without predators, with one predator, and with two predators). Both light stimuli during the test and rearing conditions affected the behavior of Niphargus thuringius. With light stimuli, individuals presented a strong photophobic response. Moreover, individuals reared in conditions of high predation risk preferred a more sheltered environment during behavioral tests than individuals reared in safe conditions. Our results add a new species to those of stygobiont amphipods known to display negative phototaxis, confirming that this pattern is widespread and conserved in the field. N. thuringius could be a good candidate model to perform further studies aiming to assess if differences occur between spring populations and populations present in deeper groundwater.

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.