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Analyzing the mutational load of driver mutations in melanoma could provide valuable information regarding its progression. We aimed at analyzing the heterogeneity of mutational load of BRAF V600E in biopsies of melanoma patients of different stages, and investigating its potential as a prognosis factor. Mutational load of BRAF V600E was analyzed by digital PCR in 78 biopsies of melanoma patients of different stages and 10 nevi. The BRAF V600E load was compared among biopsies of different stages. Results showed a great variability in the load of V600E (0%-81%). Interestingly, we observed a significant difference in the load of V600E between the early and late melanoma stages, in the sense of an inverse correlation between BRAF V600E mutational load and melanoma progression. In addition, a machine learning approach showed that the mutational load of BRAF V600E could be a good predictor of metastasis in stage II patients. Our results suggest that BRAF V600E is a promising biomarker of prognosis in stage II patients., Competing Interests: The authors have declared that no competing interests exist.

To assess the influence of food type on biomarkers, mussels (Mytilus galloprovincialis) were maintained under laboratory conditions and fed using 4 different microalgae diets ad libitum for 1 week: (a) Isochrysis galbana; (b) Tetraselmis chuii; (c) a mixture of I. galbana and T. chuii; and (d) a commercial food (Microalgae Composed Diet, Acuinuga). Different microalgae were shown to present different distribution and fate in the midgut. I. galbana (≈4 μm Ø) readily reached digestive cells to be intracellularly digested. T. chuii (≈10 μm Ø and hardly digestible) was retained in stomach and digestive ducts for long times and extracellularly digested. Based on these findings, it appeared likely that the presence of large amounts of microalgal enzymes and metabolites might interfere with biochemical determinations of mussel's biomarkers and/or that the diet-induced alterations of mussels' digestion could modulate lysosomal and tissue-level biomarkers. To test these hypotheses, a battery of common biochemical, cytological and tissue-level biomarkers were determined in the gills (including activities of pyruvate kinase, phosphoenolpyruvate carboxykinase and cytochrome c oxidase) and the digestive gland of the mussels (including protein, lipid, free glucose and glycogen total content, lysosomal structural changes and membrane stability, intracellular accumulation of neutral lipids and lipofuscins, changes in cell type composition and epithelial thinning, as well as altered tissue integrity). The type of food was concluded to be a major factor influencing biomarkers in short-term experiments though not all the microalgae affected biomarkers and their responsiveness in the same way. T. chuii seemed to alter the nutritional status, oxidative stress and digestion processes, thus interfering with a variety of biomarkers. On the other hand, the massive presence of I. galbana within digestive cells hampered the measurement of cytochemical biomarkers and rendered less reliable the results of biochemical biomarkers (as these could be attributed to both the mussel and the microalgae). Research to optimize dietary food type, composition, regime and rations for toxicological experimentation is urgently needed. Meanwhile, a detailed description of the food type and feeding conditions should be always provided when reporting aquatic toxicological experiments with mussels, as a necessary prerequisite to compare and interpret the biological responses elicited by pollutants., Competing Interests: The authors have declared that no competing interests exist.

Knowledge on the trophic interactions among predators and their prey is important in order to understand ecology and behaviour of animals. Traditionally studies on the diet composition of insectivorous bats have been based on the morphological identification of prey remains, but the accuracy of the results has been hampered due to methodological limitations. Lately, the DNA metabarcoding and High Throughput Sequencing (HTS) techniques have changed the scene since they allows prey identification to the species level, ultimately giving more precision to the results. Nevertheless, the use of one single primer set to amplify faecal DNA produces biases in the assessed dietary composition. Three horseshoe bats overlap extensively in their distribution range in Europe: Rhinolophus euryale, R. hipposideros and R. ferrumequinum. In order to achieve the deepest insight on their prey list we combined two different primers. Results showed that the used primers were complementary at the order and species levels, only 22 out of 135 prey species being amplified by both. The most frequent prey of R. hipposideros belonged to Diptera and Lepidoptera, to Lepidoptera in R. euryale, and Lepidoptera, Diptera and Coleoptera in R. ferrumequinum. The three bats show significant resource partitioning, since their trophic niche overlap is not higher than 34%. Our results confirm the importance of combining complementary primers to describe the diet of generalist insectivorous bats with amplicon metabarcoding techniques. Overall, each primer set showed a subset of the prey composition, with a small portion of the total prey being identified by both of them. Therefore, each primer presented a different picture of the niche overlap among the three horseshoe bats due to their taxonomic affinity., Competing Interests: The authors have declared that no competing interests exist.

Herbivorous arthropods cause immense damage in crop production annually. Consumption of these pests by insectivorous animals is of significant importance to counteract their adverse effects. Insectivorous bats are considered amongst the most voracious predators of arthropods, some of which are known crop pests. In vineyard-dominated Mediterranean agroecosystems, several crops are damaged by the attack of insect pests. In this study we aimed 1) to explore the diet and pest consumption of the lesser horseshoe bat Rhinolophus hipposideros and 2) analyse whether the composition of pest species in its diet changes throughout the season. We employed a dual-primer DNA metabarcoding analysis of DNA extracted from faeces collected in three bat colonies of a wine region in Southwestern Europe during the whole active period of most pest species. Overall, 395 arthropod prey species belonging to 11 orders were detected; lepidopterans and dipterans were the most diverse orders in terms of species. Altogether, 55 pest species were identified, 25 of which are known to cause significant agricultural damage and 8 are regarded as pests affecting grapevines. The composition of pest species in faeces changed significantly with the season, thus suggesting several periods should be sampled to assess the pest consumption by bats. As a whole, the results imply that R. hipposideros acts as a suppressor of a wide array of agricultural pests in Mediterranean agroecosystems. Therefore, management measures favouring the growth of R. hipposideros populations should be considered., Competing Interests: The authors have declared that no competing interests exist.

Molecular techniques allow non-invasive dietary studies from faeces, providing an invaluable tool to unveil ecological requirements of endangered or elusive species. They contribute to progress on important issues such as genomics, population genetics, dietary studies or reproductive analyses, essential knowledge for conservation biology. Nevertheless, these techniques require general methods to be tailored to the specific research objectives, as well as to substrate- and species-specific constraints. In this pilot study we test a range of available primers to optimise diet analysis from metabarcoding of faeces of a generalist aquatic insectivore, the endangered Pyrenean desman (Galemys pyrenaicus, É. Geoffroy Saint-Hilaire, 1811, Talpidae), as a step to improve the knowledge of the conservation biology of this species. Twenty-four faeces were collected in the field, DNA was extracted from them, and fragments of the standard barcode region (COI) were PCR amplified by using five primer sets (Brandon-Mong, Gillet, Leray, Meusnier and Zeale). PCR outputs were sequenced on the Illumina MiSeq platform, sequences were processed, clustered into OTUs (Operational Taxonomic Units) using UPARSE algorithm and BLASTed against the NCBI database. Although all primer sets successfully amplified their target fragments, they differed considerably in the amounts of sequence reads, rough OTUs, and taxonomically assigned OTUs. Primer sets consistently identified a few abundant prey taxa, probably representing the staple food of the Pyrenean desman. However, they differed in the less common prey groups. Overall, the combination of Gillet and Zeale primer sets were most cost-effective to identify the widest taxonomic range of prey as well as the desman itself, which could be further improved stepwise by adding sequentially the outputs of Leray, Brandon-Mong and Meusnier primers. These results are relevant for the conservation biology of this endangered species as they allow a better characterization of its food and habitat requirements., Competing Interests: We have the following interests. Jorge González-Esteban is employed by DESMA Estudios Ambientales. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Visual attractiveness and rarity often results in large carnivores being adopted as flagship species for stimulating conservation awareness. Their hunting behaviour and prey selection can affect the population dynamics of their prey, which in turn affects the population dynamics of these large carnivores. Therefore, our understanding of their trophic ecology and foraging strategies is important for predicting their population dynamics and consequently for developing effective conservation programs. Here we concentrate on an endangered species of carnivores, the snow leopard, in the Himalayas. Most previous studies on snow leopard diet lack information on prey availability and/or did not genetically check, whether the identification of snow leopard scats is correct, as their scats are similar to those of other carnivores. We studied the prey of snow leopard in three Himalayan regions in Nepal (Sagarmatha National Park (SNP), Lower Mustang (LM) and Upper Manang (UM) in the Annapurna Conservation Area, during winter and summer in 2014-2016. We collected 268 scats along 139.3 km linear transects, of which 122 were genetically confirmed to belong to snow leopard. Their diet was identified by comparing hairs in scats with our reference collection of the hairs of potential prey. We determined prey availability using 32-48 camera-traps and 4,567 trap nights. In the SNP, the most frequent prey in snow leopard faeces was the Himalayan tahr in both winter and summer. In LM and UM, its main prey was blue sheep in winter, but yak and goat in summer. In terms of relative biomass consumed, yak was the main prey everywhere in both seasons. Snow leopard preferred large prey and avoided small prey in summer but not in winter, with regional differences. It preferred domestic to wild prey only in winter, and in SNP. Unlike most other studies carried out in the same area, our study uses genetic methods for identifying the source of the scat. Studies solely based on visual identification of samples may be strongly biased. Diet studies based on frequency of occurrence of prey tend to overestimate the importance of small prey, which may be consumed more often, but contribute less energy than large prey. However, even assessments based on prey biomass are unlikely to be accurate as we do not know whether the actual size of the prey consumed corresponds to the average size used to calculate the biomass eaten. For example, large adults may be too difficult to catch and therefore mostly young animals are consumed, whose weight is much lower. We show that snow leopard consumes a diverse range of prey, which varies both regionally and seasonally. We conclude that in order to conserve snow leopards it is also necessary to conserve its main wild species of prey, which will reduce the incidence of losses of livestock., Competing Interests: The authors have declared that no competing interests exist.

A thorough search for bat herpesviruses was carried out in oropharyngeal samples taken from most of the bat species present in the Iberian Peninsula from the Vespertilionidae, Miniopteridae, Molossidae and Rhinolophidae families, in addition to a colony of captive fruit bats from the Pteropodidae family. By using two degenerate consensus PCR methods targeting two conserved genes, distinct and previously unrecognized bat-hosted herpesviruses were identified for the most of the tested species. All together a total of 42 potentially novel bat herpesviruses were partially characterized. Thirty-two of them were tentatively assigned to the Betaherpesvirinae subfamily while the remaining 10 were allocated into the Gammaherpesvirinae subfamily. Significant diversity was observed among the novel sequences when compared with type herpesvirus species of the ICTV-approved genera. The inferred phylogenetic relationships showed that most of the betaherpesviruses sequences fell into a well-supported unique monophyletic clade and support the recognition of a new betaherpesvirus genus. This clade is subdivided into three major clades, corresponding to the families of bats studied. This supports the hypothesis of a species-specific parallel evolution process between the potentially new betaherpesviruses and their bat hosts. Interestingly, two of the betaherpesviruses' sequences detected in rhinolophid bats clustered together apart from the rest, closely related to viruses that belong to the Roseolovirus genus. This suggests a putative third roseolo lineage. On the contrary, no phylogenetic structure was detected among several potentially novel bat-hosted gammaherpesviruses found in the study. Remarkably, all of the possible novel bat herpesviruses described in this study are linked to a unique bat species., Competing Interests: The authors have declared that no competing interests exist.

Behavioral plasticity is a key feature allowing animals to broaden their dietary niche when novel food resources become available, and long-fingered bats provide an appropriate model system to study the underpinnings of behavioral plasticity, since although generally being an insectivorous species, some individuals have been reported to catch fish. Aiming to get insight into the origin of fishing behavior in long-fingered bats, we studied in the field the differences in sensorial and mechanical reactions to insect-like (stationary) and fish-like (temporary) prey stimuli between well-known piscivorous and strictly insectivorous individuals. Both piscivorous and insectivorous individuals exhibited a qualitatively similar reaction to temporary target stimuli (longer and deeper dips and terminal echolocation phase skewed towards buzz I compared to stationary stimuli). Nevertheless, the quantitative differences observed in the sensorial and mechanical features (the intensity of the shift was significantly greater in piscivorous than in insectivorous individuals) show that piscivorous individuals have honed their capture technique likely enhancing the fishing success. Thus, our results suggest that the fishing technique was developed from a primary reaction shared by all long-fingered bats. All individuals seem to be mechanically and sensorially adapted to detect and capture fish, although under appropriate environmental conditions, they would further improve their technique by experience and/or social learning., Competing Interests: The authors have declared that no competing interests exist.

Mountain environments, characterized by high levels of endemism, are at risk of experiencing significant biodiversity loss due to current trends in global warming. While many acknowledge their importance and vulnerability, these ecosystems still remain poorly studied, particularly for taxa that are difficult to sample such as bats. Aiming to estimate the amount of cryptic diversity among bats of a Neotropical montane cloud forest in Talamanca Range-south-east Central America-, we performed a 15-night sampling campaign, which resulted in 90 captured bats belonging to 8 species. We sequenced their mitochondrial cytochrome c oxidase subunit I (COI) and screened their inter- and intraspecific genetic variation. Phylogenetic relations with conspecifics and closely related species from other geographic regions were established using Maximum Likelihood and Bayesian inference methods, as well as median-joining haplotype networks. Mitochondrial lineages highly divergent from hitherto characterized populations (> 9% COI dissimilarity) were found in Myotis oxyotus and Hylonycteris underwoodi. Sturnira burtonlimi and M. keaysi also showed distinct mitochondrial structure with sibling species and/or populations. These results suggest that mountains in the region hold a high degree of endemicity potential that has previously been ignored in bats. They also warn of the high extinction risk montane bats may be facing due to climatic change, particularly in isolated mountain systems like Talamanca Range., Competing Interests: The authors have declared that no competing interests exist.

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.

The estimation of maturity and sex of fish stocks in European waters is a requirement of the EU Data Collection Framework as part of the policy to improve fisheries management. On the other hand, research on fish biology is increasingly focused in molecular approaches, researchers needing correct identification of fish sex and reproductive stage without necessarily having in house the histological know-how necessary for the task. Taking advantage of the differential gene transcription occurring during fish sex differentiation and gametogenesis, the utility of 5S ribosomal RNA (5S rRNA) and General transcription factor IIIA (gtf3a) in the molecular identification of sex and gametogenic stage was tested in different economically-relevant fish species from the Bay of Biscay. Gonads of 9 fish species (, Atlantic, Atlantic-chub and horse mackerel, blue whiting, bogue, European anchovy, hake and pilchard and megrim), collected from local commercial fishing vessels were histologically sexed and 5S and 18S rRNA concentrations were quantified by capillary electrophoresis to calculate a 5S/18S rRNA index. Degenerate primers permitted cloning and sequencing of gtf3a fragments in 7 of the studied species. 5S rRNA and gtf3a transcript levels, together with 5S/18S rRNA index, distinguished clearly ovaries from testis in all of the studied species. The values were always higher in females than in males. 5S/18S rRNA index values in females were always highest when fish were captured in early phases of ovary development whilst, in later vitellogenic stages, the values decreased significantly. In megrim and European anchovy, where gonads in different oogenesis stages were obtained, the 5S/18S rRNA index identified clearly gametogenic stage. This approach, to the sexing and the quantitative non-subjective identification of the maturity stage of female fish, could have multiple applications in the study of fish stock dynamics, fish reproduction and fecundity and fish biology in general.

We analysed the whole-genome transcriptional profile of 6 cell lines of dark melanocytes (DM) and 6 of light melanocytes (LM) at basal conditions and after ultraviolet-B (UVB) radiation at different time points to investigate the mechanisms by which melanocytes protect human skin from the damaging effects of UVB. Further, we assessed the effect of different keratinocyte-conditioned media (KCM+ and KCM-) on melanocytes. Our results suggest that an interaction between ribosomal proteins and the P53 signaling pathway may occur in response to UVB in both DM and LM. We also observed that DM and LM show differentially expressed genes after irradiation, in particular at the first 6h after UVB. These are mainly associated with inflammatory reactions, cell survival or melanoma. Furthermore, the culture with KCM+ compared with KCM- had a noticeable effect on LM. This effect includes the activation of various signaling pathways such as the mTOR pathway, involved in the regulation of cell metabolism, growth, proliferation and survival. Finally, the comparison of the transcriptional profiles between LM and DM under basal conditions, and the application of natural selection tests in human populations allowed us to support the significant evolutionary role of MIF and ATP6V0B in the pigmentary phenotype.

The stone marten is a widely distributed mustelid in the Palaearctic region that exhibits variable habitat preferences in different parts of its range. The species is a Holocene immigrant from southwest Asia which, according to fossil remains, followed the expansion of the Neolithic farming cultures into Europe and possibly colonized the Iberian Peninsula during the Early Neolithic (ca. 7,000 years BP). However, the population genetic structure and historical biogeography of this generalist carnivore remains essentially unknown. In this study we have combined mitochondrial DNA (mtDNA) sequencing (621 bp) and microsatellite genotyping (23 polymorphic markers) to infer the population genetic structure of the stone marten within the Iberian Peninsula. The mtDNA data revealed low haplotype and nucleotide diversities and a lack of phylogeographic structure, most likely due to a recent colonization of the Iberian Peninsula by a few mtDNA lineages during the Early Neolithic. The microsatellite data set was analysed with a) spatial and non-spatial Bayesian individual-based clustering (IBC) approaches (STRUCTURE, TESS, BAPS and GENELAND), and b) multivariate methods [discriminant analysis of principal components (DAPC) and spatial principal component analysis (sPCA)]. Additionally, because isolation by distance (IBD) is a common spatial genetic pattern in mobile and continuously distributed species and it may represent a challenge to the performance of the above methods, the microsatellite data set was tested for its presence. Overall, the genetic structure of the stone marten in the Iberian Peninsula was characterized by a NE-SW spatial pattern of IBD, and this may explain the observed disagreement between clustering solutions obtained by the different IBC methods. However, there was significant indication for contemporary genetic structuring, albeit weak, into at least three different subpopulations. The detected subdivision could be attributed to the influence of the rivers Ebro, Tagus and Guadiana, suggesting that main watercourses in the Iberian Peninsula may act as semi-permeable barriers to gene flow in stone martens. To our knowledge, this is the first phylogeographic and population genetic study of the species at a broad regional scale. We also wanted to make the case for the importance and benefits of using and comparing multiple different clustering and multivariate methods in spatial genetic analyses of mobile and continuously distributed species.

In the present study we have investigated the population genetic structure of albacore (Thunnus alalunga, Bonnaterre 1788) and assessed the loss of genetic diversity, likely due to overfishing, of albacore population in the North Atlantic Ocean. For this purpose, 1,331 individuals from 26 worldwide locations were analyzed by genotyping 75 novel nuclear SNPs. Our results indicated the existence of four genetically homogeneous populations delimited within the Mediterranean Sea, the Atlantic Ocean, the Indian Ocean and the Pacific Ocean. Current definition of stocks allows the sustainable management of albacore since no stock includes more than one genetic entity. In addition, short- and long-term effective population sizes were estimated for the North Atlantic Ocean albacore population, and results showed no historical decline for this population. Therefore, the genetic diversity and, consequently, the adaptive potential of this population have not been significantly affected by overfishing.

Silver nanoparticles (Ag NPs) are increasingly used in many products and are expected to end up in the aquatic environment. Mussels have been proposed as marine model species to evaluate NP toxicity in vitro. The objective of this work was to assess the mechanisms of toxicity of Ag NPs on mussel hemocytes and gill cells, in comparison to ionic and bulk Ag. Firstly, cytotoxicity of commercial and maltose stabilized Ag NPs was screened in parallel with the ionic and bulk forms at a wide range of concentrations in isolated mussel cells using cell viability assays. Toxicity of maltose alone was also tested. LC50 values were calculated and the most toxic Ag NPs tested were selected for a second step where sublethal concentrations of each Ag form were tested using a wide array of mechanistic tests in both cell types. Maltose-stabilized Ag NPs showed size-dependent cytotoxicity, smaller (20 nm) NPs being more toxic than larger (40 and 100 nm) NPs. Maltose alone provoked minor effects on cell viability. Ionic Ag was the most cytotoxic Ag form tested whereas bulk Ag showed similar cytotoxicity to the commercial Ag NPs. Main mechanisms of action of Ag NPs involved oxidative stress and genotoxicity in the two cell types, activation of lysosomal AcP activity, disruption of actin cytoskeleton and stimulation of phagocytosis in hemocytes and increase of MXR transport activity and inhibition of Na-K-ATPase in gill cells. Similar effects were observed after exposure to ionic and bulk Ag in the two cell types, although generally effects were more marked for the ionic form. In conclusion, results suggest that most observed responses were due at least in part to dissolved Ag.

Coherent ecological networks (EN) composed of core areas linked by ecological corridors are being developed worldwide with the goal of promoting landscape connectivity and biodiversity conservation. However, empirical assessment of the performance of EN designs is critical to evaluate the utility of these networks to mitigate effects of habitat loss and fragmentation. Landscape genetics provides a particularly valuable framework to address the question of functional connectivity by providing a direct means to investigate the effects of landscape structure on gene flow. The goals of this study are (1) to evaluate the landscape features that drive gene flow of an EN target species (European pine marten), and (2) evaluate the optimality of a regional EN design in providing connectivity for this species within the Basque Country (North Spain). Using partial Mantel tests in a reciprocal causal modeling framework we competed 59 alternative models, including isolation by distance and the regional EN. Our analysis indicated that the regional EN was among the most supported resistance models for the pine marten, but was not the best supported model. Gene flow of pine marten in northern Spain is facilitated by natural vegetation, and is resisted by anthropogenic landcover types and roads. Our results suggest that the regional EN design being implemented in the Basque Country will effectively facilitate gene flow of forest dwelling species at regional scale.

We aimed to study the selective pressures interacting on SLC45A2 to investigate the interplay between selection and susceptibility to disease. Thus, we enrolled 500 volunteers from a geographically limited population (Basques from the North of Spain) and by resequencing the whole coding region and intron 5 of the 34 most and the 34 least pigmented individuals according to the reflectance distribution, we observed that the polymorphism Leu374Phe (L374F, rs16891982) was statistically associated with skin color variability within this sample. In particular, allele 374F was significantly more frequent among the individuals with lighter skin. Further genotyping an independent set of 558 individuals of a geographically wider population with known ancestry in the Spanish population also revealed that the frequency of L374F was significantly correlated with the incident UV radiation intensity. Selection tests suggest that allele 374F is being positively selected in South Europeans, thus indicating that depigmentation is an adaptive process. Interestingly, by genotyping 119 melanoma samples, we show that this variant is also associated with an increased susceptibility to melanoma in our populations. The ultimate driving force for this adaptation is unknown, but it is compatible with the vitamin D hypothesis. This shows that molecular evolution analysis can be used as a useful technology to predict phenotypic and biomedical consequences in humans.

Blowflies are insects of forensic interest as they may indicate characteristics of the environment where a body has been laying prior to the discovery. In order to estimate changes in community related to landscape and to assess if blowfly species can be used as indicators of the landscape where a corpse has been decaying, we studied the blowfly community and how it is affected by landscape in a 7,000 km2 region during a whole year. Using baited traps deployed monthly we collected 28,507 individuals of 10 calliphorid species, 7 of them well represented and distributed in the study area. Multiple Analysis of Variance found changes in abundance between seasons in the 7 analyzed species, and changes related to land use in 4 of them (Calliphora vomitoria, Lucilia ampullacea, L. caesar and L. illustris). Generalised Linear Model analyses of abundance of these species compared with landscape descriptors at different scales found only a clear significant relationship between summer abundance of C. vomitoria and distance to urban areas and degree of urbanisation. This relationship explained more deviance when considering the landscape composition at larger geographical scales (up to 2,500 m around sampling site). For the other species, no clear relationship between land uses and abundance was found, and therefore observed changes in their abundance patterns could be the result of other variables, probably small changes in temperature. Our results suggest that blowfly community composition cannot be used to infer in what kind of landscape a corpse has decayed, at least in highly fragmented habitats, the only exception being the summer abundance of C. vomitoria.

The long-fingered bat Myotis capaccinii is a European trawling bat reported to feed on fish in several Mediterranean locations, but the ecological circumstances of this behavior have not yet been studied. To elucidate the importance of fishing in this bat's diet, we evaluated the frequency and seasonal variation of fish remains in 3,000 fecal pellets collected from M. capaccinii at a nursery roost in Dénia (Eastern Iberian Peninsula) in 2008, 2009, and 2010. Fish consumption occurred evenly throughout the year. All otoliths found in feces were identified as belonging to the surface-feeding fish Gambusia holbrooki. Measuring otoliths, we estimated that the mean size of consumed fish was significantly smaller than the mean measured for available fish, suggesting that the long-fingered bat's relatively small body may constrain its handling of larger prey. Of note, one bat had eaten 15 fish, showing that fish may be a locally or seasonally important trophic resource for this species. By capturing 15 bats and radio-tracking the four with the most fish remains in their droppings, we also identified fishing areas, including a single fishing ground comprising several ponds within a golf course. Ponds hold a high density of G. holbrooki, suggesting that the amount of fish at the water surface may be the principal factor triggering fishing. The observed six-fold increase in percentage of consumed fish across the study period may be related to recent pond-building in the area. We discuss whether this quick behavioral response is a novel feature of M. capaccinii or an intrinsic feature that has erupted and faded locally along the species' history.

Molecular analysis of diet overcomes the considerable limitations of traditional techniques for identifying prey remains in bat faeces. We collected faeces from individual Mountain Long-eared Bats Plecotus macrobullaris trapped using mist nets during the summers of 2009 and 2010 in the Pyrenees. We analysed their diet using DNA mini-barcodes to identify prey species. In addition, we inferred some basic features of the bat's foraging ecology that had not yet been addressed. P. macrobullaris fed almost exclusively on moths (97.8%). As prey we detected one dipteran genus (Tipulidae) and 29 moth taxa: 28 were identified at species level (23 Noctuidae, 1 Crambidae, 1 Geometridae, 1 Pyralidae, 1 Sphingidae, 1 Tortricidae), and one at genus level (Rhyacia sp., Noctuidae). Known ecological information about the prey species allowed us to determine that bats had foraged at elevations between 1,500 and 2,500 m amsl (above mean sea level), mostly in subalpine meadows, followed by other open habitats such as orophilous grasslands and alpine meadows. No forest prey species were identified in the diet. As 96.4% of identified prey species were tympanate moths and no evidence of gleaning behaviour was revealed, we suggest P. macrobullaris probably forages by aerial hawking using faint echolocation pulses to avoid detection by hearing moths. As we could identify 87.8% of the analysed sequences (64.1% of the MOTUs, Molecular Operational Taxonomic Units) at species level, we conclude that DNA mini-barcodes are a very useful tool to analyse the diet of moth-specialist bats.