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.

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.

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.

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.

Microplastic contamination in terrestrial ecosystems: A study using barn owl (Tyto alba) pellets.

Microplastics (MPs) are recognised as an emerging environmental problem that needs to be carefully monitored. So far, MPs have been widely recorded in marine and freshwater ecosystems. Still, few studies have focused on MP occurrence in terrestrial ecosystems, although soils are suspected to be one of the main MP reservoirs. To test a non-invasive method for assessing MP contamination in terrestrial ecosystems, we analysed the pellets of a top terrestrial predator, the barn owl (Tyto alba). Sixty pellets were collected from three agricultural areas (20 pellets each) and analysed to assess both barn owl diet and MP content. Thirty-four MPs were confirmed by micro-Fourier Transform Infrared Spectroscopy (µ-FTIR) analysis in 33% of the pellets (min-max 1-5 MPs per pellet). Most of the detected items were microfibres (88.2%). Polyethylene terephthalate, polyacrylonitrile and polyamide were the most abundant polymers. One of the three sites was significantly less contaminated. In the two sites with the highest MP occurrences, barn owl diet was characterised by predation on synanthropic rodents, particularly brown rats (Rattus norvegicus), which may indicate habitat degradation and increased exposure to MPs. Analyses also suggest that Savi's pine vole (Microtus savii) is the prey least at risk of MP contamination, probably due to its strictly herbivorous diet. We argue that the analysis of barn owl pellets may represent a cost-effective method for monitoring MP contamination in terrestrial ecosystems.

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.

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.

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.

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.

Predation on Amphibians May Enhance Eurasian Otter Recovery in Southern Italy.

Mediterranean freshwaters undergo extreme seasonal variation in water flow, which, exacerbated by water withdrawal for agriculture or hydroelectric purposes, may affect fish communities and thus prey availability for semi-aquatic predators, such as Eurasian otter Lutra lutra. To investigate the role played by food availability on the ongoing recovery of an otter population at the southernmost limit of its Italian range, we assessed otter diet by the analysis of 357 spraints collected from 2014 to 2017 on eight rivers, and both fish and amphibian availability by, respectively, electrofishing and visual encounter surveys. Fish and amphibians formed the bulk of otter diet, the latter resource contributing as much as fish to otter diet in spring. Use by otters of both fish and amphibians depended only fish availability, suggesting that amphibians constituted an alternative resource to be exploited in conditions of fish shortage. Accordingly, electrofishing showed that fish biomass may barely be sufficient to sustain the current otter population. ATR-FT-IR spectroscopy allowed to point out for the first time the occurrence of amphibian eggs in otter spraints, although the co-occurrence of anuran bones did not allow to discriminate between direct and passive predation. Overall results indicate that the expansion or even survival of this small otter population may depend on the effective management of water resources and reinforcement of fish assemblages.

XMRV and Public Health: The Retroviral Genome Is Not a Suitable Template for Diagnostic PCR, and Its Association with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Appears Unreliable.

A few years ago, a highly significant association between the xenotropic murine leukemia virus-related virus (XMRV) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), a complex debilitating disease of poorly understood etiology and no definite treatment, was reported in Science, raising concern for public welfare. Successively, the failure to reproduce these findings, and the suspect that the diagnostic PCR was vitiated by laboratory contaminations, led to the retraction of the paper. Notwithstanding, XMRV continued to be the subject of researches and public debates. Occasional positivity in humans was also detected recently, even if the data always appeared elusive and non-reproducible. In this study, we discuss the current status of this controversial association and propose that a major role in the unreliability of the results was played by the XMRV genomic composition in itself. In this regard, we present bioinformatic analyses that show: (i) aspecific, spurious annealings of the available primers in multiple homologous sites of the human genome; (ii) strict homologies between whole XMRV genome and interspersed repetitive elements widespread in mammalian genomes. To further detail this scenario, we screen several human and mammalian samples by using both published and newly designed primers. The experimental data confirm that available primers are far from being selective and specific. In conclusion, the occurrence of highly conserved, repeated DNA sequences in the XMRV genome deeply undermines the reliability of diagnostic PCRs by leading to artifactual and spurious amplifications. Together with all the other evidences, this makes the association between the XMRV retrovirus and CFS totally unreliable.

Modelling the Distribution of Forest-Dependent Species in Human-Dominated Landscapes: Patterns for the Pine Marten in Intensively Cultivated Lowlands.

In recent years, the "forest-specialist" pine marten Martes martes has been reported to also occur also in largely fragmented, lowland landscapes of north-western Italy. The colonization of such an apparently unsuitable area provided the opportunity for investigating pine marten ecological requirements and predicting its potential south- and eastwards expansion. We collected available pine marten occurrence data in the flood plain of the River Po (N Italy) and relate them to 11 environmental variables by developing nine Species Distribution Models. To account for inter-model variability we used average ensemble predictions (EP). EP predicted a total of 482 suitable patches (8.31% of the total study area) for the pine marten. The main factors driving pine marten occurrence in the western River Po plain were the distance from watercourses and the distance from woods. EP suggested that the pine marten may further expand in the western lowland, whilst the negligible residual wood cover of large areas in the central and eastern plain makes the habitat unsuitable for the pine marten, except for some riparian corridors and the pine wood patches bordering the Adriatic coast. Based on our results, conservation strategies should seek to preserve remnant forest patches and enhance the functional connectivity provided by riparian corridors.

Detectability of the Eurasian otter by standard surveys: an approach using marking intensity to estimate false negative rates.

False negative detections may bias the surveys for rare species and reduce the reliability of models based on the proportion of occupied patches. We assessed the detectability of the Eurasian otter Lutra lutra through the standard survey method by analysing the detection history of 28 sampling stretches surveyed monthly between March 2001 and January 2003. Each survey negative for otter spraints was considered as a false negative if the otter had been recorded in the previous and/or following month (respectively, cFN and FN). Otter marking intensity (MI) (MI=N° of spraints per kilometre) was calculated and assumed to represent an index of its relative abundance. Spraints were found in 81.7% of all surveys. Yearly MI ranged from 1.02 to 101.4 spraints per kilometre. In 2002, mean MI was significantly lower than in the previous year, while no clear seasonal trend could be outlined. The minimum number of surveys required to establish the occurrence of the otter, as estimated by a probability model, was 2.6 and was inversely related to MI. For a sub-sample of 18 sampling stretches, the relation between the frequency of both cFN and FN and five variables of potential interest for otters was tested by means of stepwise linear multiple regressions, yielding two highly significant models, which both included only MI as the explanatory variable. The frequency of both FN and cFN was correlated to MI and the resulting equations used to assess the percentage of surveys positive for otters in both years. After the correction for non-detections, otter site occupancy did not vary between the 2 years, except for one river when applying the more conservative estimate of false negatives (cFN). Multiple visits and the assessing of MI should become standard components of otter surveys. This approach has broad applicability and may be applied to assess the large-scale distribution of other rare or elusive mammalian carnivores.

Exploitation of food resources by the Eurasian badger (Meles meles) at the altitudinal limit of its alpine range (NW Italy).

Food availability has been suggested to be the main factor shaping the altitudinal limits of species distributions. We analyzed the badger (Meles meles) diet in the western Italian Alps and, particularly, at the altitudinal limit of its range, with the main aim of highlighting any reduction in earthworm availability with altitude which could act as a limiting factor for badgers. Earthworms were by far the main food resource of badgers, followed by fruit. The two-month importance of these two items in badger diet was inversely correlated. Earthworm consumption was negatively correlated with air temperature. The seasonal pattern of earthworm use by badgers seemed to be influenced by the reproduction and estivation times of some epigeic species, rather than by climatic conditions per se. The eating of fruit by badgers was at least in part independent from the availability of earthworms. The badgers' efficacy in preying upon earthworms also in adverse conditions and their reliance on a wide variety of food resources suggest that worm availability might play a minor role in shaping the altitudinal limit of the species on the Alps.

Red fox (Vulpes vulpes) cannibalistic behaviour and the prevalence of Trichinella britovi in NW Italian Alps.

Food habits of the red fox (Vulpes vulpes) were studied in the Aosta Valley region (NW Italian Alps) and were related to the prevalence of Trichinella infection in the red fox itself and in two Mustelid species (the stone marten (Martes foina) and the badger (Meles meles)). The search of Trichinella by the automatic digestion of muscles samples led us to determine a prevalence of 3.5+/-1.2% in red foxes, 7.9+/-4.3% in stone martens and 1.9+/-1.8% in badgers, with no significant differences among the species. All larvae were identified as Trichinella britovi. The fox diet was assessed through the analysis of both faeces (n=180) and the stomach contents of road-killed animals (n=109). Our results confirmed the opportunistic feeding behaviour of the red fox, which is able to use various trophic resources, both of animal and vegetal origin: e.g. wild and cultivated fruits (F%=47.1; V%=67.3), rodents (F%=22.8; V%=64.8) and carrion (F%=15.6; V%=78.6) formed the bulk of the fox's diet. The frequency of occurrence of potential events of cannibalism was 1.0%, even if the complete absence of undigested remains, other than hairs, suggested the possibility of confusing cannibalism with coat-cleaning. We suggest that intra-specific necrophagy could not represent the unique way of transmission of the nematode in natural conditions.