Effects of predation risk on the sensory asymmetries and defensive strategies of Bufotes balearicus tadpoles.

Lateralization consists of the differential use of bilateral organs or limbs and is well described in many taxa and in several contexts. Common ecological frameworks where it can be observed are foraging and predatory ones, with benefits related to both visual and auditory lateralization such as faster response or increasing neural processing ability. Anuran amphibians are considered relevant models for investigating lateralization, due to their great ecological variety and the possibility of easily being raised under laboratory conditions. By adopting the "rotational preference test", we used Balearic green toad tadpoles to test the effects of behavioural defensive responses triggered by different predator types (native vs alien, i.e. dragonfly larvae Aeshna cyanea and adult red swamp crayfish Procambarus clarkii) and diets (fasted vs. tadpole-fed predators) on their lateralization. We recorded tadpoles' responses to five different chemical cues: clean water (control treatment), fasted dragonfly larvae and crayfish, and tadpole-fed dragonfly larvae and crayfish. Green toad tadpoles did not show a bias in a predominant direction, although lateralization occurred at the individual level, as shown by the intensity index (LA). Perceived predation risk was the highest in tadpoles exposed to the combined chemical cues of conspecific prey and native predators, which elicited both changes in the intensity of lateralization and a marked reduction in tadpoles' activity level. Our results suggest that contextual predation threat may induce very rapid changes in the expression of asymmetries at the individual level, and might play a role as part of the complex defensive strategies adopted by prey in the attempt to escape predators.

Reliable wolf-dog hybrid detection in Europe using a reduced SNP panel developed for non-invasively collected samples.

BACKGROUND: Understanding the processes that lead to hybridization of wolves and dogs is of scientific and management importance, particularly over large geographical scales, as wolves can disperse great distances. However, a method to efficiently detect hybrids in routine wolf monitoring is lacking. Microsatellites offer only limited resolution due to the low number of markers showing distinctive allele frequencies between wolves and dogs. Moreover, calibration across laboratories is time-consuming and costly. In this study, we selected a panel of 96 ancestry informative markers for wolves and dogs, derived from the Illumina CanineHD Whole-Genome BeadChip (174 K). We designed very short amplicons for genotyping on a microfluidic array, thus making the method suitable also for non-invasively collected samples. RESULTS: Genotypes based on 93 SNPs from wolves sampled throughout Europe, purebred and non-pedigree dogs, and suspected hybrids showed that the new panel accurately identifies parental individuals, first-generation hybrids and first-generation backcrosses to wolves, while second- and third-generation backcrosses to wolves were identified as advanced hybrids in almost all cases. Our results support the hybrid identity of suspect individuals and the non-hybrid status of individuals regarded as wolves. We also show the adequacy of these markers to assess hybridization at a European-wide scale and the importance of including samples from reference populations. CONCLUSIONS: We showed that the proposed SNP panel is an efficient tool for detecting hybrids up to the third-generation backcrosses to wolves across Europe. Notably, the proposed genotyping method is suitable for a variety of samples, including non-invasive and museum samples, making this panel useful for wolf-dog hybrid assessments and wolf monitoring at both continental and different temporal scales.

Contextual behavioural plasticity in Italian agile frog (Rana latastei) tadpoles exposed to native and alien predator cues.

Predation is a strong driver for the evolution of prey behaviour. To properly assess the actual risk of predation, anuran tadpoles mostly rely on water-borne chemical cues, and their ability to evaluate environmental information is even more crucial when potential predators consist of unknown alien species. Behavioural plasticity - that is, the capacity to express changes in behaviour in response to different environmental stimuli - is crucial to cope with predation risk. We explored the defensive behaviour of Italian agile frog (Rana latastei) tadpoles when exposed to the chemical cues of two predator species, one native (dragonfly larvae) and one alien (red swamp crayfish). Firstly, we observed whether a plastic life history trait (i.e. hatching time) might be affected by native predatory cues. Secondly, we recorded a suite of behavioural responses (activity level, lateralization and sinuosity) to each cue. For assessing lateralization and sinuosity, we developed a C++ code for the automatic analysis of digitally recorded tadpole tracks. Hatching time seemed not to be affected by the potential risk of predation, while both predator species and diet affected tadpoles' defensive behaviour. Tadpoles responded to a predator threat by two main defensive strategies: freezing and 'zig-zagging'. While the first behaviour had previously been reported, the analysis of individual trajectories indicated that tadpoles can also increase path complexity, probably to prevent predators from anticipating their location. We also recorded a decrease in lateralization intensity, which suggests that under predation risk, tadpoles tend to scrutinize the surrounding environment equally on both sides.

Righting behaviour in the European pond turtle (Emys orbicularis): relations between behavioural and morphological lateralization.

Lateralization represents a key property of many behavioural traits, with the right and left sides of the brain providing different and integrative functions. Common ecological contexts where lateralization can be observed are foraging and predatory ones, where both visual and auditory lateralization may provide advantages such as faster response and increasing neural processing capacity. This is crucial in selecting a safe refuge during a predatory attack and may strongly affect the outcome of predator-prey interactions. For animals like turtles, a critical condition may occur when they are overturned on their own shell, which causes difficulties in breathing and thermoregulation, making them more vulnerable to predators. Therefore, the ability to right is a critical adaptive component related to survival in aquatic turtles, which has been observed to be lateralized. However, an overlooked feature of behavioural lateralization is its possible relationship with asymmetry in external morphology. Here we investigated this topic in freshwater European pond turtles Emys orbicularis, looking at a possible relation between lateralization in righting behaviour response and asymmetry in the shape of turtles' plastron and carapace. Righting performance (total time needed to completely turn) appeared to depend on shell shape. We found that none of the morphometric variables was related to a lateralization index calculated as the first side from which turtles tried to right. However, a strong negative correlation between the asymmetry index of plastron and the turning direction emerged, with more symmetric animals tending to turn to the right side.

Discrimination of group numerousness under predation risk in anuran tadpoles.

For social animals, group size discrimination may play a major role in setting the trade-off between the costs and benefits of membership. Several anuran tadpoles show different degrees of social aggregation when exposed to the risk of predation. Despite the importance of aggregative behaviour as an anti-predatory response, the mechanism underlying tadpole choice of the group to join to has not been sufficiently investigated. To establish whether visual cues provide sufficient information to enable tadpoles to choose between aggregations differing in size, we explored the abilities of the larvae of two anuran species (green toad Bufotes balearicus and edible frog Pelophylax esculentus) to discriminate among four numerical combinations of conspecific tadpoles (1 vs. 4, 3 vs. 4, 4 vs. 6 and 4 vs. 8), either in the presence or absence of predatory cues. Our results suggest that in anuran larvae the capacity to discriminate between quantities is limited to small numbers (1 vs. 4 for B. balearicus and both 1 vs. 4 and 3 vs. 4 for P. esculentus). Predator-exposed toad tadpoles stayed longer close to the larger group, supporting aggregation as a major anti-predator behaviour in bufonids, while frog tadpoles showed a preference for the smaller groups, though in predator-free trials only, probably associated with lower intra-specific competition.

Long-term impact of agricultural practices on the diversity of small mammal communities: a case study based on owl pellets.

Small mammals have been seldom used as indicators of biodiversity responses to environmental changes, probably because their long-term population trend in a given area is not easy to monitor. To assess the impact of agricultural intensification in a protected area of northern Italy, we compared the composition of its small mammal communities, as assessed in 1994-1995 and 2015-2016 by the analysis of owl pellets (N = 265 and 302, respectively), which provides an effective and affordable method for assessing changes in the diversity and structure of small mammal assemblages over time. We recorded a sharp reduction in the frequency of occurrence of shrews (Sorex spp. and Crocidura spp.), which were replaced by generalist/anthropophilic rats (Rattus norvegicus) and house mice (Mus domesticus). Overall richness and diversity of the community varied only slightly, while trophic level and functional diversity indices clearly reflected the decline of the predator-level fraction of the community. We could reliably exclude both broad-scale land use- and climate changes as drivers of variation in the composition of small mammal communities and ascribe the decline of insectivores to changes in agricultural practices, namely the increase in cover of maize fields and spread of both herbicides and insecticides. Our results are consistent with the general opinion that crop specialization and increasing chemical inputs reduce the diversity and abundance of invertebrate prey, with bottom-up effects on higher trophic levels.

Context-dependent behavioural lateralization in the European pond turtle, Emys orbicularis (Testudines, Emydidae).

Lateralization presents clear advantages in ecological contexts as the dominance of one brain side prevents the simultaneous activation of contrasting responses in organisms with laterally located eyes. This is crucial in selecting a safe refuge during a predatory attack and may strongly affect predator-prey interactions. We explored the possible presence of lateralization in the anti-predatory behaviour of European pond turtles, considering their escape facing a possible predatory attack. Thirty individuals (17 males, 13 females) were exposed to three different environmental situations of gradually increasing predatory threat: escape underwater from an unsafe shelter, diving into the water from a basking site and righting after being overturned. All turtles were tested 20 times for each of the three experiments (60 trials per individual; 1800 overall trials). We recorded multiple behavioural responses in the general context of predation risk. This was done in order to assess both the existence of lateralization and possible correlations among different behaviours as function of lateralization. The number of significant responses to the left side was always prevalent in each of the three simulated anti-predatory situations, suggesting the existence of a lateralized behaviour in this species. At the individual level, the differences we found in the three experiments could be related to different ecological contexts and consequent risk of predation. Our findings, among the few on chelonians, support the possible involvement of right hemisphere activity and, most importantly, reveal how the complexity of a general predatory context can affect the laterality of escape behaviour.

Continuous and discrete quantity discrimination in tortoises.

The ability to estimate quantity, which is crucially important in several aspects of animal behaviour (e.g. foraging), has been extensively investigated in most taxa, with the exception of reptiles. The few studies available, in lizards, report lack of spontaneous discrimination of quantity, which may suggest that reptiles could represent an exception in numerical abilities among vertebrates. We investigated the spontaneous ability of Hermann's tortoises ( Testudo hermanni) to select the larger quantity of food items. Tortoises were able to choose the larger food item when exposed to two options differing in size, but equal in numerousness (0.25, 0.50, 0.67 and 0.75 ratio) and when presented with two groups differing in numerousness, but equal in size (1 versus 4, 2 versus 4, 2 versus 3 and 3 versus 4 items). The tortoises succeeded in both size and numerousness discrimination, and their performance appeared to depend on the ratio of items to be discriminated (thus following Weber's Law). These findings in chelonians provide evidence of an ancient system for the extrapolation of numerical magnitudes from given sets of elements, shared among vertebrates.

Cutaneous carcinogenic risk evaluation in 375 patients treated with narrowband-UVB phototherapy: A 15-year experience from our Institute.

BACKGROUND: Narrowband-ultraviolet B (NB-UVB) is widely used for the treatment of several dermatological diseases. A cutaneous carcinogenic effect has been hypothesized, but not proved. METHODS: We retrospectively reviewed the data of patients treated with NB-UVB between January 1998 and December 2013 at the Dermatology Unit of our University Hospital, to evaluate the cutaneous carcinogenic risk of NB-UVB. RESULTS: In all, 375 patients were included, each receiving a mean follow-up of 6.9 years. Vitiligo and psoriasis were the most common diseases. In total, 19 non-melanoma skin cancers (NMSCs) were diagnosed in eight patients, after a mean latency of 5.2 years after the first radiation. No malignant melanoma (MM) was observed. The incidence rates of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) were 620.2/100 ̇000 p/y and 116.3/100 ̇000 p/y. NMSCs were more frequent in patients affected by psoriasis (P = .0232), with older age at the first radiation (mean = 68.8 years, P = .0001). CONCLUSION: Despite the small number of patients and limited follow-up, our data suggest that NB-UVB may trigger cutaneous carcinogenesis, mainly in patients at risk for NMSCs, increasing their personal risk for single and multiple neoplasms, usually superficial BCCs. MM risk does not seem to be enhanced.

Fear is the mother of invention: anuran embryos exposed to predator cues alter life-history traits, post-hatching behaviour and neuronal activity patterns.

Neurophysiological modifications associated to phenotypic plasticity in response to predators are largely unexplored, and there is a gap of knowledge on how the information encoded in predator cues is processed by prey sensory systems. To explore these issues, we exposed Rana dalmatina embryos to dragonfly chemical cues (kairomones) up to hatching. At different times after hatching (up to 40 days), we recorded morphology and anti-predator behaviour of tadpoles from control and kairomone-treated embryo groups as well as their neural olfactory responses, by recording the activity of their mitral neurons before and after exposure to a kairomone solution. Treated embryos hatched later and hatchlings were smaller than control siblings. In addition, the tadpoles from the treated group showed a stronger anti-predator response than controls at 10 days (but not at 30 days) post-hatching, though the intensity of the contextual response to the kairomone stimulus did not differ between the two groups. Baseline neuronal activity at 30 days post-hatching, as assessed by the frequency of spontaneous excitatory postsynaptic events and by the firing rate of mitral cells, was higher among tadpoles from the treated versus the control embryo groups. At the same time, neuronal activity showed a stronger increase among tadpoles from the treated versus the control group after a local kairomone perfusion. Hence, a different contextual plasticity between treatments at the neuronal level was not mirrored by the anti-predator behavioural response. In conclusion, our experiments demonstrate ontogenetic plasticity in tadpole neuronal activity after embryonic exposure to predator cues, corroborating the evidence that early-life experience contributes to shaping the phenotype at later life stages.

Isocline analysis of competition predicts stable coexistence of two amphibians.

We investigated the interaction between larvae of two anuran amphibian species (Rana temporaria and Bufo bufo) to test models of two-species competition. The study had a response surface experimental design with four replicates, each consisting of 24 density combinations. Larval performance-and, by assumption, change in population size-was defined by a linear combination of survival, growth, and development. We fit six competition models from the literature and discovered that density dependence was strongly non-linear, with the highest support for the Hassel-Comins model. Rana temporaria was competitively superior to B. bufo; the impacts of both species on growth and development were about five- to tenfold greater than those on survival. Isocline analysis predicted a stable configuration, which agrees with the observation that these two species are syntopic in nature. The results of this study confirm competition theory by identifying a model structure that agrees with data and making predictions that are broadly supportive of the observations.

Impacts, Potential Benefits and Eradication Feasibility of Aquatic Alien Species in an Integral Natural State Reserve.

Riverine wetlands are stepping-stone environments for the protection of local biodiversity, but they are particularly vulnerable to biological invasions. In order to take action against biological invasions, it is crucial to assess the impacts of alien species. However, it is also important to assess the potential benefits on ecosystem services that alien species could have. Once it has been verified that negative impacts are higher than potential benefits, it is important to propose feasible actions to contrast them. In this study, we assessed eight freshwater alien species recorded in an integral protected wetland using the Invasive Species Effects Assessment Tool (INSEAT) to quantify their negative impacts and potential benefits on ecosystem services. Moreover, for each species, we evaluated the feasibility of the main eradication techniques currently proposed in the literature using the Non-Native Risk Management scheme (NNRM), with the final aim of suggesting effective actions for their management. The INSEAT results indicated that all the assessed species had more impacts than benefits while NNRM provided useful indications on the best practical conservation actions to use for reducing the density, and therefore, the negative impacts on ecosystem services and the local biodiversity of the assessed alien species.

Predation cues induce predator specific changes in olfactory neurons encoding defensive responses in agile frog tadpoles.

Although behavioural defensive responses have been recorded several times in both laboratory and natural habitats, their neural mechanisms have seldom been investigated. To explore how chemical, water-borne cues are conveyed to the forebrain and instruct behavioural responses in anuran larvae, we conditioned newly hatched agile frog tadpoles using predator olfactory cues, specifically either native odonate larvae or alien crayfish kairomones. We expected chronic treatments to influence the basal neuronal activity of the tadpoles' mitral cells and alter their sensory neuronal connections, thereby impacting information processing. Subsequently, these neurons were acutely perfused, and their responses were compared with the defensive behaviour of tadpoles previously conditioned and exposed to the same cues. Tadpoles conditioned with odonate cues differed in both passive and active cell properties compared to those exposed to water (controls) or crayfish cues. The observed upregulation of membrane conductance and increase in both the number of active synapses and receptor density at the postsynaptic site are believed to have enhanced their responsiveness to external stimuli. Odonate cues also affected the resting membrane potential and firing rate of mitral cells during electrophysiological patch-clamp recordings, suggesting a rearrangement of the repertoire of voltage-dependent conductances expressed in cell membranes. These recorded neural changes may modulate the induction of an action potential and transmission of information. Furthermore, the recording of neural activity indicated that the lack of defensive responses towards non-native predators is due to the non-recognition of their olfactory cues.

Anti-predator behavioral responses of Italian agile frog tadpoles (Rana latastei) exposed to microplastics.

Microplastics (MPs) are nowadays abundant, persistent, and ubiquitous in the environment, representing a new threat for terrestrial, marine, and freshwater ecosystems. Although anuran populations and species are globally declining, the effect of MP exposure on this taxon has been poorly investigated. With the aim of assessing the effects of microplastic exposure on the defensive responses of Italian agile frog (Rana latastei) tadpoles, we exposed them to three different concentrations (1, 7, and 50 mg L-1) of a mixture of plastic polymers (HPDE, PVC, PS, and PES) for 2 weeks. Then, we measured the total distance covered by individual tadpoles before and after exposure to tadpole-fed dragonfly larvae (Aeshna cyanea) cues. As expected, predation risk sharply lowered the total distance travelled by tadpoles; however, MP concentration did not affect their defensive performances. We also collected data on tadpole development, activity, and mortality. In contrast with previous experiments, neither tadpole growth nor mortality varied with MP concentration. Our results indicate that the intensity of MP effects on growth and development may depend on tadpole size, with large tadpoles being less susceptible to the negative effects of MP exposure.

Assessment of genetic diversity, population structure and wolf-dog hybridisation in the Eastern Romanian Carpathian wolf population.

The Carpathian Mountains have been constantly inhabited by grey wolves and present one of the largest distribution areas in Europe, comprising between 2300 and 2700 individuals in Romania. To date, however, relatively little is known about the Romanian wolf population. We aimed to provide a first assessment of genetic diversity, population structure and wolf-dog hybridisation based on 444 mostly non-invasively collected samples in the Eastern Romanian Carpathians. Pack reconstruction and analysis of population genetic parameters were performed with mitochondrial DNA control-region sequencing and microsatellite genotyping. We found relatively high levels of genetic diversity, which is similar to values found in previous studies on Carpathian wolves from Poland and Slovakia, as well as to the long-lasting Dinaric-Balkan wolf population. We found no significant population structure in our study region, suggesting effective dispersal and admixture. Analysis of wolf-dog hybridisation using a Single Nucleotide Polymorphism panel optimised for hybrid detection revealed low rates of admixture between wolves and domestic dogs. Our results provide evidence for the existence of a genetically viable wolf population in the Romanian Carpathians. The genetic data obtained in this study may serve as valuable baseline information for the elaboration of monitoring standards and management plans for wolves in Romania.

Multimodal Cues Do Not Improve Predator Recognition in Green Toad Tadpoles.

The anti-predator behaviour of green toad (Bufotes balearicus) tadpoles was investigated by exposing them to only the visual or chemical cues, or a combination of both, of a native predator, southern hawker Aeshna cyanea. We collected green toad egg strings in the field and tadpoles did not receive any predatory stimulus before the onset of the experiment. To manipulate chemical and visual cues independently, dragonfly larvae were caged inside a transparent plastic container, while chemical cues (odour of tadpole-fed dragonfly larvae) were injected into the surrounding arena. An empty container and water were used, respectively, as controls. The behaviour of individually tested tadpoles was videorecorded for 40 min, of which 20 were before their exposure to stimuli. Five second-distance frames were compared to assess both tadpole activity and position within the arena with respect to the visual stimulus. The tadpole level of activity strongly decreased after exposure to either chemical cues alone or in combination with visual cues, while visual cues alone apparently did not elicit any defensive response. The position of tadpoles inside the arena was not affected by visual cues, suggesting that green toad tadpoles mainly rely on olfactory cues to assess the level of predation risk.

Bacterial-Assisted Extraction of Bioactive Compounds from Cauliflower.

The market for nutraceutical molecules is growing at an impressive pace in all Western countries. A convenient source of bioactive compounds is found in vegetable waste products, and their re-use for the recovery of healthy biomolecules would increase the sustainability of the food production system. However, safe, cheap, and sustainable technologies should be applied for the recovery of these beneficial molecules, avoiding the use of toxic organic solvents or expensive equipment. The soil bacterium Bacillus subtilis is naturally endowed with several enzymes targeting complex vegetable polymers. In this work, a raw bacterial culture supernatant was used to assist in the extraction of bioactives using isothermal pressurization cycles. Besides a wild-type Bacillus subtilis strain, a new strain showing increased secretion of cellulases and xylanases, pivotal enzymes for the digestion of the plant cell wall, was also used. Results indicate that the recovery of compounds correlates with the amount of cellulolytic enzymes applied, demonstrating that the pretreatment with non-purified culture broth effectively promotes the release of bioactives from the vegetable matrix. Therefore, this approach is a valid and sustainable procedure for the recovery of bioactive compounds from food waste.

Differential effects of microplastic exposure on anuran tadpoles: A still underrated threat to amphibian conservation?

Microplastics (MPs) have been reported to threaten a wide variety of terrestrial, marine, and freshwater organisms. However, knowledge about the effects of MPs on anuran amphibians, one of the most threatened taxa worldwide, is still limited. To assess the effects of MPs on the growth and survival of the Italian agile frog (Rana latastei) and green toad (Bufotes balearicus), we exposed tadpoles to three different concentrations (1, 7, and 50 mg L-1) of an environmental relevant mixture of microplastics (HPDE, PVC, PS and PES), recording data on their activity level, weight and mortality rates. While the effects of MPs on green toad tadpoles were negligible, Italian agile frog tadpoles were severely affected both in terms of growth and activity level, with high mortality rates even at the lowest MP density (1 mg L-1). Our results suggest that MP contamination of freshwater habitats may contribute to the ongoing decline of anuran amphibians.

Lizard colour plasticity tracks background seasonal changes.

Environmental heterogeneity on a spatial and temporal scale fosters an organism's capacity to plastically alter coloration. Predation risk might favour the evolution of phenotypic plasticity in colour patterns, as individuals who change colour throughout the year may be able to improve their fitness. Here we explored the change in dorsal pigmentation of the Italian wall lizard (Podarcis siculus campestris) at three time points (March, July, October) during a period of activity in a Mediterranean natural area in southern Italy. Following a preliminary investigation conducted in 2018, during 2019 we captured 135 lizards and took a picture of their ventral scales to check for possible recapture over the sessions. Lizard dorsal pictures were collected in the field with the support of a reference chart to quantitatively estimate chromatic variables (hue, saturation and value). At the same time, pictures of the environmental background were collected. Our findings suggest that lizards are capable of altering dorsal coloration during seasonal change. They vary from green at the onset of spring, to brownish in the middle of summer and to a greyish colour in October. This modification closely followed environmental background colour variation and enhanced lizard crypsis during each season.

Exposure during embryonic development to Roundup® Power 2.0 affects lateralization, level of activity and growth, but not defensive behaviour of marsh frog tadpoles.

As glyphosate-based herbicides, sold under the commercial name Roundup®, represent the most used herbicides in the world, contamination of the freshwater environment by glyphosate has become a widespread issue. In Italy, glyphosate was detected in half of the surface waters monitoring sites and its concentrations were higher than environmental quality standards in 24.5% of them. It can last from days to months in water, leading to exposure for aquatic organisms and specifically to amphibians' larvae that develop in shallow water bodies with proven effects to development and behaviour. In this study, we tested the effects of a 96 h exposure during embryonic development of marsh frog's tadpoles to three ecologically relevant Roundup® Power 2.0 concentrations. As expected, given the low concentrations tested, no mortality was observed. Morphological measurements highlighted a reduction in the total length in tadpoles exposed to 7.6 mg a.e./L, while an increase was observed at lower concentrations of 0.7 and 3.1 mg a.e./L compared to control group. Tadpoles raised in 7.6 mg a.e./L also showed a smaller tail membrane than those raised in the control solution. Regarding behaviour, we tested tadpoles in two different sessions (Gosner stages 25 and 28/29) for lateralization, antipredator response and basal activity. Lower intensity of lateralization was detected in tadpoles raised at the highest Roundup® concentration in the first session of observation, while no significant difference among treatments was observed in the second one. In both sessions, effects of Roundup® Power 2.0 embryonic exposure on antipredator response, measured as the proportional change in activity after the injection of tadpole-fed predator (Anax imperator) cue, were not detected. Tadpoles exposed during embryonic development to Roundup® exhibited lower basal activity than the control group, with the strongest reduction for the 7.6 mg a.e./L treatment. Our results reinforce the concern of Roundup® contamination impact on amphibians.