Effectiveness of permanent drift fences in reducing roadkill risk of amphibians.

Roads are an important source of human economic progress, but also a threat to wildlife populations and natural habitats. Roads are responsible for the direct mortality of hundreds of millions of animals worldwide, with special negative effects for amphibians. Since the middle of the twentieth century, various types of mitigation measures have been constructed to reduce the negative effects of roads. However, despite the large availability of potential solutions designed for this purpose, there is still a knowledge gap about their effectiveness for amphibians. This study analysed whether permanent concrete drift fences reduced the roadkill risk for amphibians. We applied a before-after-control-impact (BACI) design in two road segments with concrete drift fences for amphibians. We recorded amphibians on these road segments three years before and three years after the fence installation. We further tested whether the presence of these mitigation measures transferred the animals to sites adjacent to the drift fences, creating new potential mortality aggregation sites (fence-end effect). Our results show a significant reduction in the number of amphibians reaching the sites with the drift fences. We were, however, unable to demonstrate the potential movement route transference, as our results were inconclusive. Despite the increase in amphibian numbers at the control sites in the first year after fence installation, the following two years presented similar amphibian numbers as the pre-fence years. We recognise the importance of permanent drift fences in reducing the mortality of amphibian populations; however, we encourage future studies to include tunnel-crossing data as well, to truly unveil the roadkill reduction power of amphibian mitigation measures, while maintaining or increasing connectivity between roadside habitats.

Multispecies landscape functional connectivity enhances local bird species' diversity in a highly fragmented landscape.

Local species assemblages are likely the result of habitat and landscape filtering. However, there is still limited knowledge on how landscape functional connectivity complements habitat attributes in mediating local species assemblages in real-world fragmented landscapes. In this study, we set up a non-manipulative experimental design in a standard production forest to demonstrate how functional connectivity determines the spatial distribution of a bird community. We test single- and multispecies spatially explicit, landscape functional connectivity models framed within the circuit theory, considering also patch attributes describing habitat size and quality, to weight their effects on species occurrence and community assemblage. We found that single-species functional connectivity effects contributed positively for occurrence of each species. However, they rarely provided competing alternatives in predicting community parameters when compared to multispecies connectivity models. Incorporating multispecies connectivity showed more consistent effects for all community parameters, than single-species models, since the overlap between species' dispersal abilities in the landscape shows poor agreement. Habitat size and quality, though less important, were also determinant in explaining community parameters while possibly relating to the provision of suitable nesting and foraging conditions. Both habitat and landscape filters concur to govern community assembly, though likely influencing different processes: while landscape connectivity determines which species can reach a patch, habitat quality determines which species settle in the patch. Our results also suggest that surrogating multispecies connectivity from single species has potential to source bias by assuming species perceive landscape and its barriers similarly. Inference on this issue must be gathered from as much species as possible.

Factors influencing predator roadkills: The availability of prey in road verges.

Road mortality is the most noticeable effect of roads on wildlife. Road verges may provide important refuges for small mammals and rabbits, particularly when roads cross intensive agricultural or grazed areas. In these circumstances, the increasing use of verges by prey species may attract predators to road surroundings increasing the risk of roadkill. The aim of this study was to quantify the role of prey availability (small mammals and rabbits) on predator road casualties, taking into account road and surrounding landscape characteristics. We analyzed this effect on different predator species, such as, snakes, owls and mammal carnivores. The study took place in a 10 km stretch of a National Road (EN4) in southern Portugal. Relationships among predator mortality and explanatory variables (prey abundance, landscape characteristics, and road verge features) were evaluated using Multivariate Redundancy Analysis (RDA) followed by a variation partitioning. Our results show that, although landscape features explained most of the mortality variation, the prey availability was also very important. Roadkills of Montpellier snake and Egyptian mongoose are strongly associated with wild rabbit abundance on verges, while mortality of stone marten, barn owl and tawny owl is related with wood mouse abundance, Mediterranean forest (montado) density, and verge shrub density. Implications for verge management and implementation of mitigation actions are discussed. We suggest vegetation removal in verges to decrease shelter and food availability for prey, and/or the promotion of habitat for prey in areas distant from roads.

Accounting for Connectivity Uncertainties in Predicting Roadkills: a Comparative Approach between Path Selection Functions and Habitat Suitability Models.

Functional connectivity modeling is increasingly used to predict the best spatial location for over- or underpasses, to mitigate road barrier effects and wildlife roadkills. This tool requires estimation of resistance surfaces, ideally modeled with movement data, which are costly to obtain. An alternative is to use occurrence data within species distribution models to infer movement resistance, although this remains a controversial issue. This study aimed both to compare the performance of resistance surfaces derived from path versus occurrence data in identifying road-crossing locations of a forest carnivore and assess the influence of movement type (daily vs. dispersal) on this performance. Resistance surfaces were built for genet (Genetta genetta) in southern Portugal using path selection functions with telemetry data, and species distribution models with occurrence data. An independent roadkill dataset was used to evaluate the performance of each connectivity model in predicting roadkill locations. The results show that resistance surfaces derived from occurrence data are as suitable in predicting roadkills as path data for daily movements. When dispersal was simulated, the performance of both resistance surfaces was equally good at predicting roadkills. Moreover, contrary to our expectations, we found no significant differences in locations of roadkill predictions between models based on daily movements and models based on dispersal. Our results suggest that species distribution models are a cost-effective tool to build functional connectivity models for road mitigation plans when movement data are not available.

Sampling effects on the identification of roadkill hotspots: Implications for survey design.

Although locating wildlife roadkill hotspots is essential to mitigate road impacts, the influence of study design on hotspot identification remains uncertain. We evaluated how sampling frequency affects the accuracy of hotspot identification, using a dataset of vertebrate roadkills (n = 4427) recorded over a year of daily surveys along 37 km of roads. "True" hotspots were identified using this baseline dataset, as the 500-m segments where the number of road-killed vertebrates exceeded the upper 95% confidence limit of the mean, assuming a Poisson distribution of road-kills per segment. "Estimated" hotspots were identified likewise, using datasets representing progressively lower sampling frequencies, which were produced by extracting data from the baseline dataset at appropriate time intervals (1-30 days). Overall, 24.3% of segments were "true" hotspots, concentrating 40.4% of roadkills. For different groups, "true" hotspots accounted from 6.8% (bats) to 29.7% (small birds) of road segments, concentrating from <40% (frogs and toads, snakes) to >60% (lizards, lagomorphs, carnivores) of roadkills. Spatial congruence between "true" and "estimated" hotspots declined rapidly with increasing time interval between surveys, due primarily to increasing false negatives (i.e., missing "true" hotspots). There were also false positives (i.e., wrong "estimated" hotspots), particularly at low sampling frequencies. Spatial accuracy decay with increasing time interval between surveys was higher for smaller-bodied (amphibians, reptiles, small birds, small mammals) than for larger-bodied species (birds of prey, hedgehogs, lagomorphs, carnivores). Results suggest that widely used surveys at weekly or longer intervals may produce poor estimates of roadkill hotspots, particularly for small-bodied species. Surveying daily or at two-day intervals may be required to achieve high accuracy in hotspot identification for multiple species.

Using the dead to infer about the living: Amphibian roadkill spatiotemporal dynamics suggest local populations' reduction.

Roads represent one of the main sources of wildlife mortality, population decline, and isolation, especially for low-vagility animal groups. It is still not clearly understood how wildlife populations respond to these negative effects over space and time. Most studies on wildlife road mortality do not consider the spatial and temporal components simultaneously, or the imperfect roadkill detection, both of which could lead to inaccurate assumptions and unreliable mitigation actions. In this study, we applied a multi-season occupancy model to a 14-year amphibian mortality dataset collected along 120 km of roads, combined with freely available landscape and remote sensing metrics, to identify the spatiotemporal patterns of amphibian roadkill in a Mediterranean landscape in Southern Portugal. Our models showed an explicit general decrease in amphibian roadkill. The Iberian painted frog (Discoglossus galganoi) experienced roadkill declines over time of ∼70 %, while the spiny common toad (Bufo spinosus) and the fire salamander (Salamandra salamandra) had a loss of nearly 50 %, and the Southern marbled newt (Triturus pygmaeus) had 40 %. Despite the decreasing trend in roadkill, spatial patterns seem to be rather stable from year to year. Multi-season occupancy models, when combined with relevant landscape and remote sensing predictors, as well as long-term monitoring data, can describe dynamic changes in roadkill over space and time. These patterns are valuable tools for understanding roadkill patterns and drivers in Mediterranean landscapes, enabling the differentiation of road sections with varying roadkill over time. Ultimately, this information may contribute to the development of effective conservation measures.

Mapping high-risk areas for Mycobacterium tuberculosis complex bacteria transmission: Linking host space use and environmental contamination.

In many Mediterranean ecosystems, animal tuberculosis (TB), caused by Mycobacterium bovis, an ecovar of Mycobacterium tuberculosis complex (MTBC), is maintained by multi-host communities. It is hypothesised that interspecies transmission is mainly indirect via shared contaminated environments. Therefore, identifying spatial areas where MTBC bacteria occur and quantifying space use by susceptible hosts might help predict the spatial likelihood of transmission across the landscape. Here, we aimed to evaluate the transmission risk of MTBC in a multi-host system involving wildlife (ungulates and carnivores) and cattle (Bos taurus). We collected eighty-nine samples from natural substrates (water, soil, and mud) at 38 sampling sites in a TB endemic area within a Mediterranean agroforestry system in Portugal. These samples were analysed by real-time PCR to detect MTBC DNA. Additionally, host-specific space use intensity maps were obtained through camera-trapping covering the same sampling sites. Results evidenced that a significant proportion of samples were positive for MTBC DNA (49 %), suggesting that the contamination is widespread in the area. Moreover, they showed that the probability of MTBC occurrence in the environment was significantly influenced by topographic features (i.e., slope), although other non-significant predictor related with soil conditions (SMI: soil moisture index) incorporated the MTBC contamination model. The integration of host space use intensity maps with the spatial detection of MTBC showed that the red deer (Cervus elaphus) and wild boar (Sus scrofa) exhibited the highest percentages of high-risk areas for MTBC transmission. Furthermore, when considering the co-occurrence of multiple hosts, transmission risk analyses revealed that 26.5 % of the study area represented high-risk conditions for MTBC transmission, mainly in forest areas.

Ecological filtering shapes the impacts of agricultural deforestation on biodiversity.

The biodiversity impacts of agricultural deforestation vary widely across regions. Previous efforts to explain this variation have focused exclusively on the landscape features and management regimes of agricultural systems, neglecting the potentially critical role of ecological filtering in shaping deforestation tolerance of extant species assemblages at large geographical scales via selection for functional traits. Here we provide a large-scale test of this role using a global database of species abundance ratios between matched agricultural and native forest sites that comprises 71 avian assemblages reported in 44 primary studies, and a companion database of 10 functional traits for all 2,647 species involved. Using meta-analytic, phylogenetic and multivariate methods, we show that beyond agricultural features, filtering by the extent of natural environmental variability and the severity of historical anthropogenic deforestation shapes the varying deforestation impacts across species assemblages. For assemblages under greater environmental variability-proxied by drier and more seasonal climates under a greater disturbance regime-and longer deforestation histories, filtering has attenuated the negative impacts of current deforestation by selecting for functional traits linked to stronger deforestation tolerance. Our study provides a previously largely missing piece of knowledge in understanding and managing the biodiversity consequences of deforestation by agricultural deforestation.

Side-effects of analgesic kyotorphin derivatives: advantages over clinical opioid drugs.

The adverse side-effects associated with opioid administration restrain their use as analgesic drugs and call for new solutions to treat pain. Two kyotorphin derivatives, kyotorphin-amide (KTP-NH2) and ibuprofen-KTP-NH2 (IbKTP-NH2) are promising alternatives to opioids: they trigger analgesia via an indirect opioid mechanism and are highly effective in several pain models following systemic delivery. In vivo side-effects of KTP-NH2 and IbKTP-NH2 are, however, unknown and were evaluated in the present study using male adult Wistar rats. For comparison purposes, morphine and tramadol, two clinically relevant opioids, were also studied. Results showed that KTP-derivatives do not cause constipation after systemic administration, in contrast to morphine. Also, no alterations were observed in blood pressure or in food and water intake, which were only affected by tramadol. A reduction in micturition was detected after KTP-NH2 or tramadol administrations. A moderate locomotion decline was detected after IbKTP-NH2-treatment. The side-effect profile of KTP-NH2 and IbKTP-NH2 support the existence of opioid-based mechanisms in their analgesic actions. The conjugation of a strong analgesic activity with the absence of the major side-effects associated to opioids highlights the potential of both KTP-NH2 and IbKTP-NH2 as advantageous alternatives over current opioids.

Deep mitochondrial introgression and hybridization among ecologically divergent vole species.

The completion of speciation is typically difficult to ascertain in rapidly diverging taxa but the amount of hybridization and gene flow in sympatry or parapatry contains important information about the level of reproductive isolation achieved. Here, we examined the progress in speciation between the Mediterranean (Microtus duodecimcostatus) and the Lusitanian pine vole (M. lusitanicus), which are part of the most rapid radiation of species known in mammals. These two Iberian pine voles are classified as separate species because of differences in morphology and ecology, but relatively many ambiguous individuals can be found in sympatric conditions. Our phylogenetic analyses of rangewide data from the mitochondrial cytochrome b gene (mtDNA) demonstrated high levels of diversity and a basal separation in two parapatric lineages. However, mtDNA affiliation was at odds with morphological classification or geographical distribution of the taxa. In contrast, statistical analyses of microsatellites (nucDNA) showed two clear genetic clusters in allopatry and sympatry generally matching morphological classification. This cytonuclear discordance over a large geographic area suggests historical introgression of mtDNA from M. duodecimcostatus to M. lusitanicus. There was statistical evidence for at least two recent hybrids in the sympatry zone but gene flow is apparently low given clear-cut differences in nucDNA. Our results indicate a relatively advanced speciation process in these Iberian pine voles without fully established reproductive isolation. This situation enables use of combined population genomic and experimental approaches for the separation of patterns and mechanisms in the ongoing explosive diversification of these and other Arvicoline rodents in the future.

Modification of Bacterial Cellulose Biofilms with Xylan Polyelectrolytes.

The effect of the addition of two [4-butyltrimethylammonium]-xylan chloride polyelectrolytes (BTMAXs) on bacterial cellulose (BC) was evaluated. The first strategy was to add the polyelectrolytes to the culture medium together with a cell suspension of the bacterium. After one week of cultivation, the films were collected and purified. The second approach consisted of obtaining a purified and homogenized BC, to which the polyelectrolytes were added subsequently. The films were characterized in terms of tear and burst indexes, optical properties, surface free energy, static contact angle, Gurley porosity, SEM, X-ray diffraction and AFM. Although there are small differences in mechanical and optical properties between the nanocomposites and control films, the films obtained by BC synthesis in the presence of BTMAXs were remarkably less opaque, rougher, and had a much lower specular gloss. The surface free energy depends on the BTMAXs addition method. The crystallinity of the composites is lower than that of the control material, with a higher reduction of this parameter in the composites obtained by adding the BTMAXs to the culture medium. In view of these results, it can be concluded that BC-BTMAX composites are a promising new material, for example, for paper restoration.

Amidated and Ibuprofen-Conjugated Kyotorphins Promote Neuronal Rescue and Memory Recovery in Cerebral Hypoperfusion Dementia Model.

Chronic brain ischemia is a prominent risk factor for neurological dysfunction and progression for dementias, including Alzheimer's disease (AD). In rats, permanent bilateral common carotid artery occlusion (2VO) causes a progressive neurodegeneration in the hippocampus, learning deficits and memory loss as it occurs in AD. Kyotorphin (KTP) is an endogenous antinociceptive dipeptide whose role as neuromodulator/neuroprotector has been suggested. Recently, we designed two analgesic KTP-derivatives, KTP-amide (KTP-NH2) and KTP-NH2 linked to ibuprofen (IbKTP-NH2) to improve KTP brain targeting. This study investigated the effects of KTP-derivatives on cognitive/behavioral functions (motor/spatial memory/nociception) and hippocampal pathology of female rats in chronic cerebral hypoperfusion (2VO-rat model). 2VO-animals were treated with KTP-NH2 or IbKTP-NH2 for 7 days at weeks 2 and 5 post-surgery. After behavioral testing (week 6), coronal sections of hippocampus were H&E-stained or immunolabeled for the cellular markers GFAP (astrocytes) and NFL (neurons). Our findings show that KTP-derivatives, mainly IbKTP-NH2, enhanced cognitive impairment of 2VO-animals and prevented neuronal damage in hippocampal CA1 subfield, suggesting their potential usefulness for the treatment of dementia.

Carcass Persistence and Detectability: Reducing the Uncertainty Surrounding Wildlife-Vehicle Collision Surveys.

Carcass persistence time and detectability are two main sources of uncertainty on roadkill surveys. In this study, we evaluate the influence of these uncertainties on roadkill surveys and estimates. To estimate carcass persistence time, three observers (including the driver) surveyed 114km by car on a monthly basis for two years, searching for wildlife-vehicle collisions (WVC). Each survey consisted of five consecutive days. To estimate carcass detectability, we randomly selected stretches of 500m to be also surveyed on foot by two other observers (total 292 walked stretches, 146 km walked). We expected that body size of the carcass, road type, presence of scavengers and weather conditions to be the main drivers influencing the carcass persistence times, but their relative importance was unknown. We also expected detectability to be highly dependent on body size. Overall, we recorded low median persistence times (one day) and low detectability (<10%) for all vertebrates. The results indicate that body size and landscape cover (as a surrogate of scavengers' presence) are the major drivers of carcass persistence. Detectability was lower for animals with body mass less than 100g when compared to carcass with higher body mass. We estimated that our recorded mortality rates underestimated actual values of mortality by 2-10 fold. Although persistence times were similar to previous studies, the detectability rates here described are very different from previous studies. The results suggest that detectability is the main source of bias across WVC studies. Therefore, more than persistence times, studies should carefully account for differing detectability when comparing WVC studies.

Characterization of purified bacterial cellulose focused on its use on paper restoration.

Bacterial cellulose (BC) synthesized by Gluconacetobacter sucrofermentans CECT 7291 seems to be a good option for the restoration of degraded paper. In this work BC layers are cultivated and purified by two different methods: an alkaline treatment when the culture media contains ethanol and a thermal treatment if the media is free from ethanol. The main goal of these tests was the characterization of BC layers measured in terms of tear and burst indexes, optical properties, SEM, X-ray diffraction, FTIR, degree of polymerization, static and dynamic contact angles, and mercury intrusion porosimetry. The BC layers were also evaluated in the same terms after an aging treatment. Results showed that BC has got high crystallinity index, low internal porosity, good mechanical properties and high stability over time, especially when purified by the alkaline treatment. These features make BC an adequate candidate for degraded paper reinforcement.

Relative effects of road risk, habitat suitability, and connectivity on wildlife roadkills: the case of tawny owls (Strix aluco).

BACKGROUND: Despite its importance for reducing wildlife-vehicle collisions, there is still incomplete understanding of factors responsible for high road mortality. In particular, few empirical studies examined the idea that spatial variation in roadkills is influenced by a complex interplay between road-related factors, and species-specific habitat quality and landscape connectivity. METHODOLOGY/PRINCIPAL FINDINGS: In this study we addressed this issue, using a 7-year dataset of tawny owl (Strix aluco) roadkills recorded along 37 km of road in southern Portugal. We used a multi-species roadkill index as a surrogate of intrinsic road risk, and we used a Maxent distribution model to estimate habitat suitability. Landscape connectivity was estimated from least-cost paths between tawny owl territories, using habitat suitability as a resistance surface. We defined 10 alternative scenarios to compute connectivity, based on variation in potential movement patterns according to territory quality and dispersal distance thresholds. Hierarchical partitioning of a regression model indicated that independent variation in tawny owl roadkills was explained primarily by the roadkill index (70.5%) and, to a much lesser extent, by landscape connectivity (26.2%), while habitat suitability had minor effects (3.3%). Analysis of connectivity scenarios suggested that owl roadkills were primarily related to short range movements (<5 km) between high quality territories. Tawny owl roadkills were spatially autocorrelated, but the introduction of spatial filters in the regression model did not change the type and relative contribution of environmental variables. CONCLUSIONS: Overall, results suggest that road-related factors may have a dominant influence on roadkill patterns, particularly in areas like ours where habitat quality and landscape connectivity are globally high for the study species. Nevertheless, the study supported the view that functional connectivity should be incorporated whenever possible in roadkill models, as it may greatly increase their power to predict the location of roadkill hotspots.

Occurrence of fumonisins in feed for swine and horses.

BACKGROUND: Fumonisin B1 (FB1), fumonisin B2 (FB2), and overall mycotoxins feed contamination may cause several effects on crops production and animal health. The contamination occurred predominantly in corn and corn-based foods and feeds. AIMS: This survey intends to provide the occurrence of fumonisins in swine and equine mixed feeds in Portugal, making an overview from 2007 to 2010. METHODS: A total of 363 samples were analyzed, 258 from swine feed and 105 from horse feed with HPLC method. The detection limit was 50 µg/kg for FB1 and 100 µg/kg for FB2. RESULTS: The overall results were 13% of FB1 occurrence from 2007 to 2010. FB1 was detected in about 17.0% of swine feed samples, being more frequent in 2010 (32.9%). In this year (2010) levels ranged between 66.7 and 3815.5 µg/kg. FB2 occurred only in 2010 in swine feed (6 samples, ranging between 104.0 to 467.2 µg/kg) and in horse feed (1 sample). CONCLUSIONS: This represents an increase in occurrence through the analyzed years, but this may not be a threat to animal health, once the values were below the recommended guidance values from European Commission.

How long do the dead survive on the road? Carcass persistence probability and implications for road-kill monitoring surveys.

BACKGROUND: Road mortality is probably the best-known and visible impact of roads upon wildlife. Although several factors influence road-kill counts, carcass persistence time is considered the most important determinant underlying underestimates of road mortality. The present study aims to describe and model carcass persistence variability on the road for different taxonomic groups under different environmental conditions throughout the year; and also to assess the effect of sampling frequency on the relative variation in road-kill estimates registered within a survey. METHODOLOGY/PRINCIPAL FINDINGS: Daily surveys of road-killed vertebrates were conducted over one year along four road sections with different traffic volumes. Survival analysis was then used to i) describe carcass persistence timings for overall and for specific animal groups; ii) assess optimal sampling designs according to research objectives; and iii) model the influence of road, animal and weather factors on carcass persistence probabilities. Most animal carcasses persisted on the road for the first day only, with some groups disappearing at very high rates. The advisable periodicity of road monitoring that minimizes bias in road mortality estimates is daily monitoring for bats (in the morning) and lizards (in the afternoon), daily monitoring for toads, small birds, small mammals, snakes, salamanders, and lagomorphs; 1 day-interval (alternate days) for large birds, birds of prey, hedgehogs, and freshwater turtles; and 2 day-interval for carnivores. Multiple factors influenced the persistence probabilities of vertebrate carcasses on the road. Overall, the persistence was much lower for small animals, on roads with lower traffic volumes, for carcasses located on road lanes, and during humid conditions and high temperatures during the wet season and dry seasons, respectively. CONCLUSION/SIGNIFICANCE: The guidance given here on monitoring frequencies is particularly relevant to provide conservation and transportation agencies with accurate numbers of road-kills, realistic mitigation measures, and detailed designs for road monitoring programs.

Using presence signs to discriminate between similar species.

The Lusitanian and the Mediterranean pine voles (Microtus lusitanicus Gerbe, 1879 and Microtus duodecimcostatus de Selys-Longchamps, 1839) are fossorial sister species and have an allopatric pattern of distribution in Portugal, which includes a potential sympatry area in the centre of the country. The present study aimed to determine the validity of using presence signs in the field for discrimination of the two species in an area of sympatry (Northern Alentejo) and the characteristics that achieve the best classification accuracy. A total of 175 trapping plots were sampled across the study area. Prior to the set up of traps, ten presence signs were randomly selected for measurements of four variables: proportion of soil mounds, mean diameter of mounds, proportion of burrow openings and mean diameter of burrow openings. On the basis of a classification tree analysis, results showed that presence signs can be used to discriminate plots inhabited by one or the other species in the studied sympatry area. The characteristic that most accurately enables species identification is the proportion of burrow openings: for every ten presence signs found in a plot, if more than eight have an opening, then it is inhabited by M. lusitanicus (i.e. mostly burrow openings with few or no mounds present); if eight or fewer have an opening, M. duodecimcostatus is present (i.e. mostly mounds with few or no burrow openings).