Alessandro Balestrieri

Interspecific den sharing: a study on European badger setts using camera traps

Many mammals, both potential competitors and preys, have been reported to use the complex burrow system of European badger Meles meles setts as shelter, mainly in northern Europe and during winter, when badgers are lethargic. Nonetheless, until recent times observations of den sharing have been largely restricted to anecdotal information, because of the mainly nocturnal activity of most sett occupants. Using camera-trapping, we investigated both the mammal fauna associated with 24 badger setts located in northern and central Italy, and seasonal variation in the composition of specific assemblages, without interfering with the occupants’ activity. Trapping effort was 1,605 camera trap-days from December 2010 to December 2013. Badgers (two to six individuals per sett) shared their setts with a total of eight mammal species: crested porcupine Hystrix cristata, Eastern cottontail Sylvilagus floridanus, red fox Vulpes vulpes, pine marten Martes martes, stone marten Martes foina, wood mouse Apodemus sp., brown rat Rattus norvegicus and coypu Myocastor coypus. Den sharing was observed throughout the year, with a significant reduction of sharing during winter, when badgers were probably induced to move to alternative setts to avoid breeding porcupines. Eastern cottontails used badger burrows permanently and, at least in one occasion, reared their pups inside, although they can be easily preyed upon by badgers. Badger sett sharing may have favoured both the recent northward expansion of crested porcupines and settling of introduced cottontails in agricultural habitats.

Distribution and habitat use by pine marten Martes martes in a riparian corridor crossing intensively cultivated lowlands

The location of pine marten records in northern Italy suggests that main rivers may play the role of natural corridors favouring this species’ colonisation of cultivated lowlands. We assessed the distribution and habitat use by the pine marten on a 35xa0km long stretch of the River Ticino. Surveys were carried out between October 2011 and June 2012 along linear transects in a 2xa0×xa02xa0km grid. Using the variation in marking intensity as an indicator of habitat use, habitat selection was assessed at two landscape levels—at transect-scale by the χ2 test with Bonferroni’s confidence intervals for the proportion of use, and at grid-scale by multiple linear regression. By a polymerase chain reaction–restriction fragment length polymorphism method, 91 faecal samples were assigned to the pine marten. Faeces were mainly located in wooded areas, while fields were avoided. At the grid-scale of analysis, marking intensity was positively related to the mean area of wooded patches and negatively to their mean perimeter-area ratio. This suggests that pine marten relative abundance may partially depend on the degree of fragmentation and structure of residual woods. The survey protocol allowed to assess the probability of detection. Occupancy models outlined that heterogeneity in detection probability may arise as a result of variation in marking intensity, i.e. the number of marking individuals. Our results suggest that the availability of both woodland corridors and wood patches are major factors shaping pine marten distribution in intensively cultivated plains and that non-invasive genetic surveys are a cost-effective method for future studies at a broader scale.

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.

Multilevel habitat preferences of Apodemus sylvaticus and Clethrionomys glareolus in an intensively cultivated agricultural landscape

Small mammals are considered good model species for assessing the global effects of habitat loss and fragmentation in agricultural landscapes. With the aim of investigating the composition and habitat preferences of the small mammal community in an intensively cultivated area, we sampled the three main “macro-habitats” (wood patches, poplar plantations and crops) available on the western plain of the River Po (North Italy). The influence of habitat variables on each species’ occurrence was assessed at three different scales of observation: “macro-habitat”, “micro-habitat” (i.e. vegetation layers at trap sites) and the landscape level (i.e. land cover in plots of 500 m radius centred on each trap line). Overall, 3344 trap-nights yielded 169 Apodemus sylvaticus, 28 Clethrionomys glareolus and two Sorex araneus. The first species was dominant in all macro-habitats, while the other two species were trapped only in residual wood patches. The thickness of the fine litter was the main micro-habitat factor affecting the occurrence of both C. glareolus and A. sylvaticus in woods, while sites with herbaceous vegetation cover were preferred by the latter species in poplar plantations. Intraclass correlation coefficients showed that sites with thick litter or high herb cover were neither exclusive nor predominant in wood patches and poplar plantations, respectively, suggesting that the choice for micro-habitat parameters is subordinate to that of macro-habitat characteristics, mainly tree and shrub cover. At the landscape scale, no habitat variable affected the occurrence of both species. At the macro-habitat level, the strong selection for wood patches by both wood mice and bank voles and the negligible occurrence of small mammals in cultivated plots pointed out the major role for conservation played by residual semi-natural habitats in agricultural landscapes.

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.

Spatial organisation of European badger (Meles meles) in northern Italy as assessed by camera-trapping

Italy is amongst the European countries for which data on badger Meles meles numbers and variation in population size are still too few to assess its conservation status. With the aim of estimating badger density in northern Italy, between February 2013 and March 2014, we extensively surveyed an isolated hilly area for badger setts and assessed group size by monitoring a sample of setts by two sessions of camera-trapping. The size and shape of each home range were assessed from main sett coordinates using the Dirichlet tessellation method. Then, assuming that badger home ranges tend to be delimited by hard boundaries or geographical features, the polygon shapes were successively modified by hand to draw more realistic home ranges. The overall trapping effort was 754 camera trap-days. The number of badger individuals sharing the same sett/entrances was recorded, distinguishing individuals based on the time and hole of emergence and, possibly, tail patterns. Groups consisted of two to four adult individuals, averaging 2.75 badgers/group. Camera-trapping proved to be a cost-effective and time-saving method. Depending on the method used to draw home ranges, in the study area, sett density was 0.34–0.51 sett/km2, whilst badger density was 0.93–1.4 adult individuals/km2. Both sett and badger densities were rather high with respect to most available data for continental Europe. As badger mean group size seems to be rather constant throughout both hilly and plain areas of the River Po basin, sett density may be used as an accurate estimator of badger density throughout this wide area.

Long-term monitoring by roadkill counts of mammal populations living in intensively cultivated landscapes

Elusive behaviour and financial constraints hamper the long-term monitoring of mammal population, particularly in intensively cultivated and urbanised landscapes, where most survey methodologies cannot be applied effectively. Provided that there is a direct relationship between each species’ density and frequency of road casualties, roadkill counts may represent a cost-effective alternative method to collect abundance data over long periods. We quantified the numbers of casualties of mammal species from 2001 to 2016 along two routes (65xa0km) crossing the heavily altered central River Po plain (N Italy). Each route was surveyed by car 10 times per month, covering 123,987xa0km and recording 15,589 road-kills from 15 species (15.3 roadkills/100xa0km/year). Most widespread mammals previously reported for the study area were recorded. Variation in each species’ roadkill numbers throughout the study period was consistent with available information on their distribution and abundance and the consistency of the patterns outlined on the two roads supported the hypothesis that the frequency of roadkills was related to each species’ density. Seasonal fluctuations in roadkill records could be related to either their reproductive cycles or dispersal patterns. For meso- and large species, the relationship between the occurrence of casualties and a set of 13 habitat variables was assessed by Logistic Regression Analysis. Based on our results, we believe that roadkill counts should be implemented to outline species’ population trends wherever high road density fragments wildlife habitats, and may represent a powerful citizen science-based method to collect large amounts of data over long periods.

Pine marten vs. stone marten in agricultural lowlands: a landscape-scale, genetic survey

We applied molecular analysis methods to faecal samples to determine both the overall level of occupancy for pine marten (Martes martes) and current stone marten (Martes foina) distribution in the western Po plain. Surveys were carried out in a 10xa0×xa010-km grid, applying a hybrid sampling design. The specific identification of faecal samples was accomplished either by a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method or by amplifying and sequencing a 330-bp mtDNA fragment of the control region (D-loop). Identification success was 93.7xa0% by the PCR-RFLP and 71.7xa0% by DNA sequencing. Overall, we collected 47 pine marten records and 24 stone marten records. Thirty-six squares (81.8xa0%) were found to be positive for at least one marten species, the distribution range of the two species scarcely overlapping. The pine marten was shown to be widespread in lowland areas on the north bank of the River Po, which is probably acting as a barrier to its expansion. In this area, stone marten records were few, while it is was widespread on the south bank of the river. Pine marten expansion may have forced the stone marten to restrict itself to less suitable agricultural and urban areas. Nonetheless, we cannot exclude that stone marten range and/or numbers may being declining as a consequence of pine marten expansion. Six pine marten samples belonged to the Central-Northern European (CNE) phylogroup. The relatively high percentage of CNE martens is consistent with the hypothesis of an ongoing expansion of Alpine and trans-Alpine pine marten populations.

Behavioural and life history responses to predation risk by common frog tadpoles exposed to two predators during ontogeny

The presence of predators can induce changes in both the morphology and behaviour of Anuran larvae, affecting both their size and developmental stage at metamorphosis and, consequently, the fitness of adult individuals. Tadpoles have been shown to be capable of finely tuning their defensive responses according to the actual risk perceived, which is expected to vary according to the prey-to-predator size ratio. In this study, we exposed common frog (Rana temporaria) tadpoles (Gosner stages 28–30), for a period of 2xa0weeks, to the non-lethal presence of dragonfly larvae (Anax imperator) and backswimmers (Notonecta glauca). In such a narrow window of time, we expected behavioural responses to be similar for both predators and exposure to predation risk to have negligible effects on tadpole development and weight. Overall, tadpoles increased hiding behaviour and were less active when predators were present in the experimental mesocosms, but behavioural responses were constrained to the early phase of the ontogeny and were no longer used when tadpoles reached a threshold size. Developmental rate slightly slowed down for predator treatments in comparison to controls, possibly as a consequence of energetic investment in unrecorded morphological defences. Although variation in laboratory conditions and protocols makes it hard to compare the results of different experiments, our results contribute to verify the consistency of behavioural responses in Anuran larvae.

Towards Extinction and Back: Decline and Recovery of Otter Populations in Italy

In the second half of the twentieth century, European otter populations crashed in large areas of the Italian peninsula. As a consequence, at the end of the century the Italian otter population consisted of five isolated nuclei, the extinction of which had been foretold to occur within a few decades.