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Population size is one of the basic demographic parameters for species management and conservation. Among different estimation methods, spatially explicit capture–recapture (SCR) models allow the estimation of population density in a framework that has been greatly developed in recent years. The use of automated detection devices, such as camera traps, has impressively extended SCR studies for individually identifiable species. However, its application to unmarked/partially marked species remains challenging, and no specific method has been widely used. We fitted an SCR-integrated model (SCR-IM) to stone marten Martes foina data, a species for which only some individuals are individually recognizable by natural marks, and estimate population size based on integration of three submodels: (1) individual capture histories from live capture and transponder tagging; (2) detection/nondetection or “occupancy” data using camera traps in a bigger area to extend the geographic scope of capture–recapture data; and (3) telemetry data from a set of tagged individuals. We estimated a stone marten density of 0.352 (SD: 0.081) individuals/km2. We simulated four dilution scenarios of occupancy data to study the variation in the coefficient of variation in population size estimates. We also used simulations with similar characteristics as the stone marten case study, comparing the accuracy and precision obtained from SCR-IM and SCR, to understand how submodels' integration affects the posterior distributions of estimated parameters. Based on our simulations, we found that population size estimates using SCR-IM are more accurate and precise. In our stone marten case study, the SCR-IM density estimation increased the precision by 37% when compared to the standard SCR model as regards to the coefficient of variation. This model has high potential to be used for species in which individual recognition by natural markings is not possible, therefore limiting the need to rely on invasive sa

Sarcomas are a heterogeneous group of tumors in which the role of ERK5 is poorly studied. To clarify the role of this MAPK in sarcomatous pathology, we used a murine 3-methyl-cholanthrene (3MC)-induced sarcoma model. Our data show that 3MC induces pleomorphic sarcomas with muscle differentiation, showing an increased expression of ERK5. Indeed, this upregulation was also observed in human sarcomas of muscular origin, such as leiomyosarcoma or rhabdomyosarcoma. Moreover, in cell lines derived from these 3MC-induced tumors, abrogation of Mapk7 expression by using specific shRNAs decreased in vitro growth and colony-forming capacity and led to a marked loss of tumor growth in vivo. In fact, transcriptomic profiling in ERK5 abrogated cell lines by RNAseq showed a deregulated gene expression pattern for key biological processes such as angiogenesis, migration, motility, etc., correlating with a better prognostic in human pathology. Finally, among the various differentially expressed genes, Klf2 is a key mediator of the biological effects of ERK5 as indicated by its specific interference, demonstrating that the ERK5–KLF2 axis is an important determinant of sarcoma biology that should be further studied in human pathology.

Biodiversity is dramatically declining worldwide due to the complex processes that include interactions among threats. Studies investigating how multiple drivers (inter)act to influence spatio-temporal shifts in species distribution ranges, which are among the most anticipated changes in biodiversity in the future, are still scarce in the scientific literature, particularly at meaningful conservation planning scales. We used species occurrence data for eight terrestrial Mediterranean carnivores at a regional scale of southern Spain to: 1) estimate environmental favourability to each species during two periods (1960s and present); 2) predict current favourability assuming the same environment-species relationships as the ones observed in the 1960s (expected favourability); and 3) analyse single and interactive effects of human persecution, presence of protected areas, and prey richness on favourability changes (present vs. expected) for each carnivore species. We found that, with a few exceptions, environmentally favourable areas for carnivore occurrence were more widely distributed in the past than today and, for most species, favourability tended to increase inside protected areas, with low frequency of poisoning events and high prey richness. Notwithstanding, for rare species such as Iberian grey wolf (Canis lupus signatus) and Iberian lynx (Lynx pardinus), interactions among drivers were the most important predictors of favourability changes, underpinned by a compensatory role of protected areas in the face of continued human persecution. We emphasize the role of protected areas in abating the effects of biodiversity threats and claim that scientific studies based on analyses of single-effect drivers that omit driver interactions may misinform conservation planning and require revision.

Human–wildlife conflicts are a growing problem in Northern Hemisphere where wild ungulates are one of the taxonomic groups most frequently involved. To mitigate these conflicts, it is essential to develop preventive actions able to avoid encounters between wildlife and human (activities). We here employed photo-trapping to evaluate the behaviour of red deer (Cervus elaphus) when confronted with dissuasive portable deterrents that function on the basis of changing patterns of light and ultrasound. This was done by following a before/after experimental design, with two phases: (i) a test phase, with active deterrents, and (ii) a control phase, without deterrents. When deterrents were activated they achieved a 48.96% reduction in the frequency of use by red deer (up to 66.64% when it was assessed on a thinner Sect. 10 m wide from the line of deterrents) and produced a reduction of 67.71% in the frequency of deterrent-line crossings. However, a habituation effect was detected since the use by red deer of the treatment area increased as time since treatment. These results indicate that these portable devices are effective as regards dissuading deer, mainly on short time scales. The deterrents tested here could be suitable for use at focal points for short periods or in combination with other methods to improve their effectiveness in vulnerability points. This device could potentially be used to mitigate conflicts caused by wildlife species and in response to relevant and timely situations, such as vehicle collisions and damage to crops, among others.

[Background]: Identification of membrane proteins differentially expressed on tumor cells is a key step in drug development. The carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a cell adhesion protein belonging to the immunoglobulin superfamily. Here, we explore the prognostic role CEACAM6 expression on patient outcome in cancer. [Methods]: A systematic search for studies evaluating the association between tumor expression of CEACAM6 and overall survival (OS) and disease-free survival (DFS) was performed. Hazard ratios (HR) were pooled in a meta-analysis using generic inverse variance and random effect modeling. Subgroup analyses were conducted based on tumor type and method of HR extraction. [Results]: Sixteen studies met the inclusion criteria. CEACAM6 expression was associated with worse OS [HR = 1.96, 95% confidence interval (CI) = 1.51–2.53], and DFS (HR = 2.49, 95% CI = 2.01–3.07) with subgroup analysis showing no significant differences between disease site subgroups. [Conclusions]: High expression of CEACAM6 is associated with worse OS and DFS in different malignancies. CEACAM6 is a target for the future development of novel therapeutics.

Cyclin-dependent kinases (CDKs) are a broad family of proteins involved in the cell cycle and transcriptional regulation. In this article, we explore the antitumoral activity of a novel proteolysis-targeting chimera (PROTAC) compound against CDK9. Breast cancer cell lines from different subtypes were used. Transcriptomic mapping of CDKs in breast cancer demonstrated that the expression of CDK9 predicted a detrimental outcome in basal-like tumors (HR = 1.51, CI = 1.08–2.11, p = 0.015) and, particularly, in the luminal B subtype with HER2+ expression (HR = 1.82, CI = 1.17–2.82, p = 0.0069). The novel CDK9 PROTAC, THAL-SNS-032, displayed a profound inhibitory activity in MCF7, T47D, and BT474 cells, with less effect in SKBR3, HCC1569, HCC1954, MDA-MB-231, HS578T, and BT549 cells. The three cell lines with HER2 overexpression and no presence of ER, SKBR3, HCC1569, and HCC1954 displayed an EC50 three times higher compared to ER-positive and dual ER/HER2-positive cell lines. BT474-derived trastuzumab-resistant cell lines displayed a particular sensitivity to THAL-SNS-032. Western blot analyses showed that THAL-SNS-032 caused a decrease in CDK9 levels in BT474, BT474-RH, and BT474-TDM1R cells, and a significant increase in apoptosis. Experiments in animals demonstrated an inverse therapeutic index of THAL-SNS-032, with doses in the nontherapeutic and toxic range. The identified toxicity was mainly due to an on-target off-tumor effect of the compound in the gastrointestinal epithelium. In summary, the potent and efficient antitumoral properties of the CDK9 PROTAC THAL-SNS-032 opens the possibility of using this type of compound in breast cancer only if specifically delivered to cancer cells, particularly in ER/HER2-positive and HER2-resistant tumors.

A cross-sectional study was carried out to evaluate shared pathogens that can be transmitted by close or non-close contact at the domestic–wild ruminants’ interface. During summer–autumn 2015, a total of 138 cattle and 203 wild ruminants (red deer, Cervus elaphus, and fallow deer, Dama dama) were sampled in Doñana National Park (DNP, south-western Spain), a Mediterranean ecosystem well known for the interaction network occurring in the ungulate host community. Pestiviruses, bovine respiratory syncytial virus (BRSV; Bovine orthopneumovirus), bovine herpesvirus 1 (BoHV-1; Bovine alphaherpesvirus 1) and Mycobacterium tuberculosis complex (MTC) were assessed using serological, microbiological and molecular techniques. The overall seroprevalence against viruses in cattle was 2.2% for pestiviruses, 11.6% for BRSV and 27.5% for BoHV-1. No virus-specific antibodies were found in wildlife. MTC incidence in cattle was 15.9%, and MTC seroprevalence in wild ruminants was 14.3%. The same Mycobacterium bovis spoligotypes (SB1232, SB1230 and SB1610) were identified in cattle, red deer and fallow deer. The serological results for the selected respiratory viruses suggest epidemiological cycles only in cattle. Surveillance efforts in multi-host epidemiological scenarios are needed to better drive and prioritize control strategies for shared pathogens.

In recent decades, intensive techniques of livestock raising have flourished, which has largely replaced traditional farming practices such as transhumance. These changes may have affected scavengers’ behaviour and ecology, as extensive livestock is a key source of carrion. This study evaluates the spatial responses of avian scavengers to the seasonal movements of transhumant herds in south-eastern Spain. We surveyed the abundance of avian scavengers and ungulates, and analysed the factors affecting the space use by 30 GPS-tracked griffon vultures (Gyps fulvus). Griffons’ foraging activity increased in the pasturelands occupied by transhumant herds, which implied greater vulture abundance at the landscape level during the livestock season. In contrast, facultative scavengers were more abundant without transhumant livestock herds, and the abundance of wild ungulates did not change in relation to livestock presence. We conclude that fostering transhumance and other traditional farming systems, tothe detriment of farming intensification, could favour vulture conservation.

Camera trapping is a widely used tool in wildlife research and conservation, and a plethora of makes and models of camera traps have emerged. However, insufficient attention has been paid to testing their performance, particularly under field conditions. In this study, we have comparatively tested five of the most frequently used makes of camera trap (Bushnell, KeepGuard, Ltl Acorn, Reconyx and Scoutguard) to identify the key factors behind their probability of detection (i.e. the probability that the camera successfully capturing a usable photograph of an animal passing through the field of view) and trigger speed (i.e. the time delay between the instant at which a motion is detected, and the time at which the picture is taken). We used 45 cameras (nine devices of each make) with infrared flash in a field experiment in which a continuous remote video was used in parallel (as a gold-standard) to discover the animals that entered the camera trap detection zone. The period (day/night), distance between animals and cameras, model, species, deployment height and activation sensitivity were significantly related to the probability of detection. This probability was lower during the night than during the day. There was a greater probability of detecting a given species when the cameras were set at its shoulder height. The interaction between species and the distance between the animals and the cameras significantly affected the trigger speed, meaning that the closer the animals that entered the detection zone, the higher the trigger speed, with substantial differences among species. This was probably related by movement speed. In conclusion, this study shows differences in the performance of camera trap models and settings, signifying that caution is required when making direct comparisons among results obtained in different experiments, or when designing new ones. These results provide empirical guidelines for best practices in camera trapping and highlight the relevance

Studying textbooks is relevant due to the money annually invested (about half-billion dollars) in the Brazilian textbook program, known as PNLD. This work analyses the learning Physics strategies of 75 students belonging to the 1st High School year from a Brazilian school. The analysis was based on data collected by a questionnaire about study habits applied in 2019. The questionnaire includes 17 items considering their reading habits, the use of their Physics textbook, and study strategies. The objective was to uncover how and when students use the Physics textbook since the governmental policies have consolidated their presence in Brazilian schools. Recent research on the subject indicates on one hand that teachers use Physics textbooks, especially to prepare their classes, and that students usually use them sporadically and superficially. In general, school practices tend to transform Physics textbooks into simple exercise sources, which can generate distortions in the teaching and learning of this discipline. On the other hand, recent research about what features students’ value most in a textbook also shows us that they desire a material with correct concepts, summaries, and diagrams that allow easy identification of the key concepts, but also with themes and approaches that arouse interest and exemplify the content. The results we obtained corroborate the research on Physics textbooks of the last decade but bring new data such as the importance of the internet in students' study habits. The presence and importance of the internet in students' study routines cannot always be a diversification of Physics teaching or learning activities. On the contrary, this positioning can be defended by two arguments. The first one is based on our everyday school life experience considering that most teachers are unaware of the sites and web contents accessed by the students. The same can be said of video lessons available on the web. Admittedly, the use of the Internet, appli

Different educational levels are promoting a model based on the development and assessment of professional competences, proposed by The European Higher Education Area (EHEA), on the Bologna Declaration. This learning process, including assessment, needs to be designed and implemented according to the competences acquired by students to achieve learning results previously expected. To do this, it is necessary to design learning activities and to know the way they are assessed. Learning assessment needs to be aligned with learning activities, intending to provide students the possibility of accomplishing learning results and becoming more competitive. For this reason, people responsible for the education and learning process, need to plan activities and the way results are assessed. Assessment is a crucial step in the education and learning process. It is necessary to monitor and improve the whole process, as well as to assess learning performance for every student individually. The use of an assessment with training purposes promotes a greater student’s commitment and helps detect deviations in competence acquisition to be solved over time. The competency-based learning process has provoked such a transformation in the higher-education field that has attempted to satisfy current training requirements. In this context, at university, the use of rubrics is considered as an innovative educational tool, promoting competence assessment both for students and professors. Rubrics have become an important tool for competence assessment and customized feedback, them both being considered as a useful way to unify criteria for activity development and assessment, that is to say, uniting the assessment criteria. Rubrics permit to manage expectations for an activity or task, defining indicators that reflect the sides of the task subject to assessment, performance levels indicating the quality achieved and criteria to assign those performance levels and, using this quality, acquiri

Olea pollen concentrations have been studied in relation to the typology of air masses, pollen grain sources and marine nature during advections in a coastal enclave in the south-eastern Iberian Peninsula. Since Spain is the world's leading olive producer, and olive growing extends throughout the Mediterranean basin, this location is ideal for the study of long-distance transport events (LTD) during the main pollen season (MPS). The air masses were classified using the calculation of 48-h back trajectories at 250, 500 and 750 m above ground level using the HYSPLIT model. After that, the frequency of LDT events from Africa and Europe was found to be 8.7% of the MPS days. In contrast, regional air masses were found in 38.6% of the MPS days. This was reflected in pollen concentrations, with significantly higher concentrations (p-value <0.05) on days with regional air masses compared to days with European air masses. Regarding the source areas, the importance of nearby sources with intense olive cultivation was confirmed (i.e., Andalusia). This proximity was relevant beyond the attenuations observed when the advections acquired a marine nature as the air mass back trajectories moved over the sea (p-value <0.001). The review of air mass typologies, source areas and pollen concentrations resulted in establishing peak dates and the detection of LDT associated with these peak dates. Distortions in the typical path of each air mass explained alterations in pollen concentrations on consecutive days. The recirculation and loops of the air mass back trajectories varied the pollen load that every type of air mass could originally contain.

[EN] For more than a decade, the European Union has been implementing an ambitious energy policy focused on reducing CO2 emissions, increasing the use of renewable energy and improving energy efficiency. This paper investigates the factors that hinder the application of renewable energy technologies (RETs) in existing university buildings in Spain and Portugal. Following a qualitative methodology, 33 technicians working in the infrastructure management offices of 24 universities have been interviewed. The factors identified have been classified into economic-financial, administrative and legislative barriers, architectural, urban planning, technological, networking, social acceptance, institutional and others. It is concluded that there have not been sufficient economic incentives to carry out RETs projects in this type of building. Conditioning factors can act individually or jointly, generating a greater effect. Most participants consider that there are no social acceptance barriers. Knowledge of these determinants can facilitate actions that help implement this technology on university campuses in both countries.

Models@runtime (models at runtime) are based on computation reflection. Runtimemodels can be regarded as a reflexive layer causally connected with the underlying system. Hence,every change in the runtime model involves a change in the reflected system, and vice versa. Tothe best of our knowledge, there are no runtime models for Python applications. Therefore, wepropose a formal approach based on Petri Nets (PNs) to model, develop, and reconfigure Pythonapplications at runtime. This framework is supported by a tool whose architecture consists of twomodules connecting both the model and its execution. The proposed framework considers executionexceptions and allows users to monitor Python expressions at runtime. Additionally, the applicationbehavior can be reconfigured by applying Graph Rewriting Rules (GRRs). A case study usingService-Level Agreement (SLA) violations is presented to illustrate our approach.

Closed-form exact solutions for periodic motions of a nonlinear oscillator, which contains a quadratic mixed-parity restoring force, are derived analytically. This oscillator is characterised by two real parameters, which are the coefficients of the linear and the quadratic terms, and has single-well potential. All possible combinations of positive and negative values of these coefficients providing periodic motions are considered, and two families of exact solutions are obtained in terms of Jacobi elliptic functions. The periods are given in terms of the complete elliptic integral of the first kind, the behaviour of these periods as a function of the initial amplitude is analysed, and the exact solutions for certain values of these parameters are plotted.

En este artículo se presenta un estudio comparativo entre El valiente justiciero o El ricohombre de Alcalá (1657) de Agustín Moreto y la refundición de Dionisio Solís (1811), un dramaturgo del siglo XIX. Esta refundición no ha sido estudiada anteriormente, salvo un breve análisis estructural de la pieza. De forma que ofreceremos un estudio comparativo entre la obra barroca y la refundición homónima, con el objetivo de establecer las semejanzas y diferencias entre ambas e intentar justificar los cambios introducidos por el refundidor.

Arthropod-borne flaviviruses, such as Zika virus (ZIKV), Usutu virus (USUV), and West Nile virus (WNV), are a growing cause of human illness and death around the world. Presently, no licensed antivirals to control them are available and, therefore, search for broad-spectrum antivirals, including host-directed compounds, is essential. The PI3K/Akt pathway controls essential cellular functions involved in cell metabolism and proliferation. Moreover, Akt has been found to participate in modulating replication in different viruses including the flaviviruses. In this work we studied the interaction of flavivirus NS5 polymerases with the cellular kinase Akt. In vitro NS5 phosphorylation experiments with Akt showed that flavivirus NS5 polymerases are phosphorylated and co-immunoprecipitate by Akt. Polymerase activity assays of Ala- and Glu-generated mutants for the Akt-phosphorylated residues also indicate that Glu mutants of ZIKV and USUV NS5s present a reduced primer-extension activity that was not observed in WNV mutants. Furthermore, treatment with Akt inhibitors (MK-2206, honokiol and ipatasertib) reduced USUV and ZIKV titers in cell culture but, except for honokiol, not WNV. All these findings suggest an important role for Akt in flavivirus replication although with specific differences among viruses and encourage further investigations to examine the PI3K/Akt/mTOR pathway as an antiviral potential target.

[Background]: Although the anti-HER2 antibody trastuzumab augments patient survival in HER2+ breast cancer, a relevant number of patients progress to this treatment. In this context, novel drug combinations are needed to increase its antitumor activity. In this work, we have evaluated the efficacy of proteolysis targeting chimera (PROTAC) compounds based on BET inhibitors (BETi) to augment the activity of trastuzumab in HER2+ breast cancer models., [Methods]: BT474 and SKBR3 HER2+ breast cancer cell lines were used. The effects of trastuzumab and the BET-PROTAC MZ1 either alone or in combination, were evaluated using MTT proliferation assays, three-dimensional invasion and adhesion cultures, flow cytometry, qPCR and Western blot. In vivo studies were carried out in a xenografted model in mice. Finally, a Clariom_S_Human transcriptomic array was applied to identify deregulated genes after treatments., [Results]: MZ1 induced a higher antiproliferative effect compared to the BETi JQ1. The combination of MZ1 and -trastuzumab significantly decreased cell proliferation, the formation of three-dimensional structures and cellular invasion compared to either of the drugs alone. Evaluation of apoptosis resulted in an increase of cell death following treatment with the combination, and biochemical studies displayed modifications of apoptosis and DNA damage components. In vivo administration of agents alone or combined, to tumors orthotopically xenografted in mice, resulted in a decrease of the tumor volume only after MZ1-Trastuzumab combination treatment. Results from a transcriptomic array indicated a series of newly described transcription factors including HOXB7, MEIS2, TCERG1, and DNAJC2, that were associated to poor outcome in HER2+ breast cancer subtype and downregulated by the MZ1-trastuzumab combination., [Conclusions]: We describe an active novel combination that includes the BET-PROTAC MZ1 and trastuzumab, in HER2+ tumors. Further studies should be performed to confirm these findings and pave the way for their future clinical development.

Small-conductance calcium-activated potassium (SK) channels are crucial for learning and memory. However, many aspects of their spatial organization in neurons are still unknown. In this study, we have taken a novel approach to answering these questions combining a pre-embedding immunogold labeling with an automated dual-beam electron microscope that integrates focused ion beam milling and scanning electron microscopy (FIB/SEM) to gather 3D map ultrastructural and biomolecular information simultaneously. Using this new approach, we evaluated the number and variability in the density of extrasynaptic SK2 channels in 3D reconstructions from six dendritic segments of excitatory neurons and six inhibitory neurons present in the stratum radiatum of the CA1 region of the mouse. SK2 immunoparticles were observed throughout the surface of hippocampal neurons, either scattered or clustered, as well as at intracellular sites. Quantitative volumetric evaluations revealed that the extrasynaptic SK2 channel density in spines was seven times higher than in dendritic shafts and thirty-five times higher than in interneurons. Spines showed a heterogeneous population of SK2 expression, some spines having a high SK2 content, others having a low content and others lacking SK2 channels. SK2 immunonegative spines were significantly smaller than those immunopositive. These results show that SK2 channel density differs between excitatory and inhibitory neurons and demonstrates a large variability in the density of SK2 channels in spines. Furthermore, we demonstrated that SK2 expression was associated with excitatory synapses, but not with inhibitory synapses in CA1 pyramidal cells. Consequently, regulation of excitability and synaptic plasticity by SK2 channels is expected to be neuron class- and target-specific. These data show that immunogold FIB/SEM represent a new powerful EM tool to correlate structure and function of ion channels with nanoscale resolution.

Basal-like breast cancer is an incurable disease with limited therapeutic options, mainly due to the frequent development of anti-cancer drug resistance. Therefore, identification of druggable targets to improve current therapies and overcome these resistances is a major goal. Targeting DNA repair mechanisms has reached the clinical setting and several strategies, like the inhibition of the CHK1 kinase, are currently in clinical development. Here, using a panel of basal-like cancer cell lines, we explored the synergistic interactions of CHK1 inhibitors (rabusertib and SAR020106) with approved therapies in breast cancer and evaluated their potential to overcome resistance. We identified a synergistic action of these inhibitors with agents that produce DNA damage, like platinum compounds, gemcitabine, and the PARP inhibitor olaparib. Our results demonstrated that the combination of rabusertib with these chemotherapies also has a synergistic impact on tumor initiation, invasion capabilities, and apoptosis in vitro. We also revealed a biochemical effect on DNA damage and caspase-dependent apoptosis pathways through the phosphorylation of H2AX, the degradation of full-length PARP, and the increase of caspases 3 and 8 activity. This agent also demonstrated synergistic activity in a platinum-resistant cell line, inducing an increase in cell death in response to cisplatin only when combined with rabusertib, while no toxic effect was found on non-tumorigenic breast tissue-derived cell lines. Lastly, the combination of CHK1 inhibitor with cisplatin and gemcitabine resulted in more activity than single or double combinations, leading to a higher apoptotic effect. In conclusion, in our study we identify therapeutic options for the clinical development of CHK1 inhibitors, and confirm that the inhibition of this kinase can overcome acquired resistance to cisplatin.

Aging is a continuous, universal, and irreversible process that determines progressive loss of adaptability. The liver is a critical organ that supports digestion, metabolism, immunity, detoxification, vitamin storage, and hormone signaling. Nevertheless, the relationship between aging and the development of liver diseases remains elusive. In fact, although prolonged fasting in adult rodents and humans delays the onset of the disease and increases longevity, whether prolonged fasting could exert adverse effects in old organisms remains incompletely understood. In this work, we aimed to characterize the oxidative stress and nuclear proteome in the liver of 3-month- and 24-month-old male Wistar rats upon 36 h of fasting and its adaptation in response to 30 min of refeeding. To this end, we analyzed the hepatic lipid peroxidation levels (TBARS) and the expression levels of genes associated with fat metabolism and oxidative stress during aging. In addition, to gain a better insight into the molecular and cellular processes that characterize the liver of old rats, the hepatic nuclear proteome was also evaluated by isobaric tag quantitation (iTRAQ) mass spectrometry-based proteomics. In old rats, aging combined with prolonged fasting had great impact on lipid peroxidation in the liver that was associated with a marked downregulation of antioxidant genes (Sod2, Fmo3, and Cyp2C11) compared to young rats. Besides, our proteomic study revealed that RNA splicing is the hepatic nuclear biological process markedly affected by aging and this modification persists upon refeeding. Our results suggest that aged-induced changes in the nuclear proteome could affect processes associated with the adaptative response to refeeding after prolonged fasting, such as those involved in the defense against oxidative stress.

Birds have historically suffered adverse effects by toxic elements, such as As, Pb, Hg, and Cd. However, reports on exposure to a wide range of elements, including rare earth elements and other minor elements of emerging concern, and the potential consequences for wildlife are still scarce. This study evaluates blood concentrations of 50 elements and their related effects on lutein and vitamin levels in the Eurasian blackbird (Turdus merula) and wild rock pigeon (Columba livia), inhabiting different scenarios of contaminant exposure. Blood concentrations of As, Cd, and Pb (and Mn in T. merula) were increased in both species captured in the mining area, compared to the control site. T. merula also showed increased As, Cd, and Pb concentrations in blood in the agricultural–urban area, as compared to the control area, together with the highest Hg levels, which could be related to agricultural practices and industrial activities. Decreases of 33 and 38% in the plasma retinol levels in T. merula inhabiting the mining and the agricultural–urban areas, respectively, as compared to the control site, were associated with increased Pb, As, and Cd exposure. This could be du

Methods for estimating population densities of unmarked species using camera traps are still under development. One such method is called ‘random encounter model (REM)’ and, to our knowledge, has never been used to estimate densities of mountain-dwelling ungulates. In this study, we tested the REM method to estimate the density of Balkan chamois (Rupicapra r. balcanica) in a Mediterranean habitat, Mt. Biokovo. To meet the assumptions of REM, we systematically placed 25 camera traps throughout the known range of the population (approximately 65 km2) at the intersections of 2-km grid cells. Prior to data collection, population density was estimated by visual counts on sample plots in August 2020. Cameras were operational between July 2020 and September 2020 and active throughout the 24-h period. All parameters for REM (i.e. average movement speed, angle and radius) were estimated using exclusively camera trap data. We obtained 279 independent events of chamois from 2503 camera trap days. The density estimate obtained by REM resulted to be 20.65 ± 5.27 ind. km−2, slightly higher than the reference value obtained by visual counts: 17.33 ± 0.98 ind. km−2. Other parameters required to calculate density were speed (1.62 km·day−1 ± 0.21), detection radius (5.56 m ± 0.21) and detection angle (1.16 + 0.05 radians). Therefore, REM has shown comparable results to visual counts and may have potential for estimating density of mountain ungulates, especially in rugged and inaccessible mountainous areas with low detectability where other approaches are inadequate or impossible.

Recently, the survival of Mycobacterium bovis on livestock mineral blocks has been confirmed, but little is known about its implication in the transmission of animal tuberculosis (TB) under field conditions. The objective of this study was to describe the shared use of mineral supplements in four extensive beef cattle farms from a high TB prevalence area in South Central Spain, to identify the main factors explaining their use, and characterize its potential role for the transmission of Mycobacterium tuberculosis Complex (MTC). This is relevant to design control measures at the wildlife-livestock interface. Animal activity was monitored by camera-trapping at 12 mineral supplementation points during spring and fall. Additionally, swabs were periodically taken from the mineral substrates and analyzed by PCR searching for MTC DNA. Cattle, pig, goat, sheep, wild boar, and red deer were all recorded licking on mineral supplementation points. Livestock species were the main users and presented a diurnal use pattern. Wild ungulates presented a nocturnal-crepuscular use pattern, with scarce overlapping with livestock. Wild boar presence was positively related to cattle presence at mineral supplementation points, whereas red deer presence was higher in supplemental points closer to forested areas and in farms without hunting pressure. We recorded 266 indirect wildlife-livestock interactions (i.e., two consecutive visits that occurred within 78 h), all of them derived from 21 unique wildlife visits. All the analyzed swabs resulted negative to MTC DNA. Comparing to other environmental sources of MTC in our study area, mainly water ponds, this research evidenced that mineral blocks are less attractive to wildlife. However, the potential for interspecific transmission of MTC or other pathogens cannot be discarded. The risk for interaction at mineral supplementation points and further transmission can be prevented by implementing specific measures in the context of integral biose

1.Population density estimations are essential for wildlife management and conservation. Camera traps have become a promising cost-effective tool, for which several methods have been described to estimate population density when individuals are unrecognizable (i.e. unmarked populations). However, comparative tests of their applicability and performance are scarce., 2.Here, we have compared three methods based on camera traps to estimate population density without individual recognition: Random Encounter Model (REM), Random Encounter and Staying Time (REST) and Distance Sampling with camera traps (CT-DS). Comparisons were carried out in terms of consistency with one another, precision and cost-effectiveness. We considered six natural populations with a wide range of densities, and three species with different behavioural traits (red deer Cervus elaphus, wild boar Sus scrofa and red fox Vulpes vulpes). In three of these populations, we obtained independent density estimates as a reference., 3. The densities estimated ranged from 0.23 individuals/km2 (fox) to 34.87 individuals/km2 (red deer). We did not find significant differences in terms of density values estimated by the three methods in five out of six populations, but REM has a tendency to generate higher average density values than REST and CT-DS. Regarding the independents’ densities, REM results were not significantly different in any population, and REST and CT-DS were significantly different in one population. The precision obtained was not significantly different between methods, with average coefficients of variation of 0.28 (REST), 0.36 (REM) and 0.42 (CT-DS). The REST method required the lowest human effort., 4. Synthesis and applications. Our results show that all of the methods examined can work well, with each having particular strengths and weaknesses. Broadly, Random Encounter and Staying Time (REST) could be recommended in scenarios of high abundance, Distance Sampling with camera traps (CT-DS) in those of low abundance while Random Encounter Model (REM) can be recommended when camera trap performance is not optimal, as it can be applied with less risk of bias. This broadens the applicability of camera trapping for estimating densities of unmarked populations using information exclusively obtained from camera traps. This strengthens the case for scientifically based camera trapping as a cost-effective method to provide reference estimates for wildlife managers, including within multi-species monitoring programmes.

We found severe acute respiratory syndrome coronavirus 2 RNA in 6 (8.4%) of 71 ferrets in central Spain and isolated and sequenced virus from 1 oral and 1 rectal swab specimen. Natural infection occurs in kept ferrets when virus circulation among humans is high. However, small ferret collections probably cannot maintain virus circulation.

Bluetongue is a vector-borne disease affecting domestic and wild ruminants, with a major socioeconomic impact. Endemic circulation of the bluetongue virus serotype 4 (BTV-4) and BTV-1 have occurred in Spain since 2004 and 2007, respectively. However, epidemiological studies have seldom been approached from a long-term perspective in wild ruminants. A total of 881 deer (red deer and fallow deer) were necropsied from 2005 to 2018 as part of the DNP health-monitoring program. Serum samples were tested for antibodies against BTV with the aims of assessing the temporal trend and to evaluate the modulating factors: individual, populational, environmental, and stochastic. Red deer displayed statistically significant higher seroprevalences of BTV (SBT; 78.6 ± 3.8%) than fallow deer (53.1 ± 4.7%). The detection of BTV-1 and BTV-4 by the serum neutralization test in calves suggested the circulation of both serotypes over the study period. For red deer, wet years together with high densities could provide suitable conditions for vector borne BTV transmission. Moreover, proximity to high suitability habitat for Culicoides, permanent pasturelands, was associated with higher SBT. The differences in the ecology and behaviour of deer species influencing the exposure to the vectors could determine the differences found in the SBT patterns. This study evidences the role that deer species may play in the maintenance of BTV, however, elucidating the epidemiological role of host in different contexts as well as the consequences of climate change on the competent vector populations and its potential effect on the dynamics of BTV infection in hosts communities deserve further research.

[EN] Pollinators of most of the plants cultivated in the Mediterranean regions of Europe are still unknown. We provide new data and we review previously available information on bees (Apoidea) associated with three economically important crops in Spain: melon (Cucumis melo L.), watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) and almond (Prunus dulcis (Mill.) D.A.Webb). We found that, overall, 98 bee species spanning four families visit flowers of the studied crop species, and 46 additional species were sampled within the crops with pan-traps. The bee assemblages visiting melon include 7–33 species, with moderate to high importance of small Lasioglossum (Halictidae) and of honey bee (Apis mellifera Linnaeus, 1758: Apidae), and in one case of small Ceratina: Apidae. Watermelon flowers are visited by 11–14 species of bees, with highest abundance of honey bees or of small Lasioglossum, depending on the locality. Bees collected on almond trees included 12–37 species, with the honey bee and medium-size to large Andrena (Andrenidae) and Osmia (Megachilidae) being predominant. The new samples expanded the geographical distributions of nearly one- fourth of the collected species. Diversity estimators slightly varied even within fields of the same crop, and a cluster analysis suggested both a certain overlap between melon and watermelon and a role of geographical distance on similarity among bee assemblages, though these patterns were much clearer using presence data rather than abundances. Below- ground-nesting and solitary species were more frequently collected than above-ground-nesting and eusocial species, but for melon and watermelon the highest abundances were recorded for eusocial species. Almost exclusively polylectic species visited the flowers of the three studied crops. The results of this study could help in planning conservation actions to maintain this important diversity of potential pollinators in Spanish areas covered by these crops., [FR] En Europe, dans la région méditerranéenne, les pollinisateurs de la plupart des plantes cultivées demeurent encore inconnus. Nous apportons ici de nouvelles données et analysons celles déjà acquises sur les abeilles (Apoidea) associées à trois cultures économiquement importantes en Espagne : le melon (Cucumis melo L.), la pastèque (Citrullus lanatus (Thunb.) Matsum. & Nakai) et l’amandier (Prunus dulcis (Mill.) D.A. Webb). Au total, 98 espèces de quatre familles d’abeilles ont visité les fleurs de ces trois plantes, tandis que 46 autres espèces ont été capturées à l’aide de pièges colorés dans leurs champs. Les fleurs de melon sont visitées par 7 à 33 espèces d’abeilles, parmi lesquelles dominent plus ou moins fortement de petits Lasioglossum (Halictidae) et l’abeille domestique (Apis mellifera Linnaeus, 1758 : Apidae) (et, dans un cas, de petites cératines : Apidae). Les fleurs de pastèque sont visitées par 11 à 14 espèces d’abeilles, dont les plus abondantes sont, selon le site, l’abeille mellifère ou de petits Lasioglossum. Les abeilles récoltées sur les amandiers représentent 12 à 37 espèces, parmi lesquelles prédominent l’abeille mellifère, des andrènes (Andrenidae) de taille moyenne à grande et des osmies (Megachilidae). Nos échantillonnages ont permis d’élargir la répartition géographique de près d’un quart des espèces collectées. Les estimateurs de diversité montrent de légères variations y compris entre champs d’une même culture, tandis que l’analyse typologique suggère que les assemblages d’abeilles des cultures de melon et de pastèque se chevauchent partiellement mais aussi que la similarité de ces assemblages dépend de la distance géographique, les résultats des modèles étant beaucoup plus nets à partir des données de présence que des données d’abondance. Les espèces solitaires et terricoles ont été plus fréquemment capturées que les espèces eusociales ou celles nichant au-dessus du sol mais, mais dans les cultures de melon et de pastèque, ce sont l

Current results do not provide conclusive evidence on the effect of BCG vaccination on COVID‐19 alone or in combination with other factors. To address this limitation, in this study we used a citizen science initiative on the COVID‐19 pandemic to collect data worldwide during 2 October 2020–30 October 2020 (1,233 individuals) in a structured way for analysing factors and characteristics of affected individuals in relation to BCG vaccination. For the first time, the results of our study suggested that vaccination with BCG may increase the risk for COVID‐19 at certain age, particularly in individuals vaccinated at childhood. Childhood BCG vaccination increased the likelihood of being diagnosed with COVID‐19 fivefold in COVID‐19 low‐incidence countries and threefold in high‐incidence countries. A reasonable explanation for this effect is the activation of certain innate immunity mechanisms associated with inflammatory reactions. These factors should be considered when analysing the risks associated with this global pandemic.

Understanding the dynamics of a wildlife population in relation to hunting strategies is essential to achieve sustainable management. We used monitoring data over 25 years from two red deer (Cervus elaphus) populations with different management (with and without supplemental feeding) in South Central Spain to: (i) characterise the density dependence of population dynamics under contrasted management, and (ii) provide the basis for sustainable extraction by considering the theoretical maximum sustainable yield (MSYt) as the reference. The red deer population displayed a typical management reactive culling approach (‘saw-tooth-like’ curves), with occasional strong annual harvests but not occurring on a regular basis. Interestingly, we found reduced population growth at high densities in both populations, indicating that density-mediated factors determined population growth even when artificial feeding was provided. However, no effects of sex not age class of the extracted population on the population growth rate were determined. The total number of animals hunted was only slightly above those predicted by MSYt (i.e. K50%) in both populations, despite high densities close to theoretical K, being consistent throughout the study period. The extraction rates (30.3 and 34.0%, for supplemented and unsupplemented populations, respectively) were 13.3% and 10.2% lower compared to the MSYt situation in the unsupplemented and supplemented populations, respectively. Long term population monitoring data provided feasible and suitable baseline values to optimise the sustainable exploitation of red deer populations in the Mediterranean ecosystem under these contrasting management scenarios. Adaptive management, involving objective-driven decision making informed by data on red deer population dynamic, can contribute (i) to maximising the total extraction over the long term while (ii) reducing the ecological impact of high population densities.

Insects are highly involved in accidental introductions in non-native areas. Potential distribution modelling is routinely used to predict the dynamics of such range expansions, giving insights on which areas are climatically suitable for establishment. However, even in areas where climatic conditions are unsuitable, colonization may be still possible in sub-areas with particular, human activity-driven microclimates, such as cities. We used as a model species the Asian mud-dauber wasp Sceliphron curvatum (Hymenoptera: Sphecidae), which arrived in Europe 40 years ago, to show that cities may be used by alien species to enlarge their distribution into climatically non-optimal areas. By using an average consensus from six different models, we predicted that, based on climate, S. curvatum would find the highest suitability in most part of Mediterranean basin, which are characterized by high summer temperatures and reduced climatic oscillations. The species is indeed often observed in such areas, but also in Central Europe, where suitability is overall lower. At such latitudes, however, the wasp was more often found in cities (which have the highest suitability) than in peri-urban and rural areas, possibly according to the urban ‘heat island’ effect. In Southern Europe, where climate is overall more favourable for the species, suitability tended to be more similar in both urban and rural environments, and urban detections were indeed rarer. The inclusion of population density in the model improved the suitability of Northern areas in an expected urbanization-driven jeopardized pattern. Hence, S. curvatum would be able to colonize in the future at least some climatically unsuitable Northern areas, reaching up to 70° latitude, by using cities as the main sites for establishment.

High‐density populations of large ungulates are now widespread. However, the perception of overabundance only appears when it produces a problem for humans, such as a loss of plant diversity, damage to agricultural crops and forestry, ungulate‐vehicle collisions, a nuisance to humans, disease transmission to livestock or changes in habitat for other species. The admissible level of density depends on the ecological and socio‐economic context in which the population is located, and defining this level is important, in order to determine management strategies and actions., We describe the main contexts in which ungulate overabundance occurs in Europe, record the causes of overabundance and evaluate which set of indicators of ecological change is the most appropriate for monitoring and diagnosing overabundance in each scenario., Our review of 318 published papers revealed six contexts of wild ungulate overabundance in Europe (protected areas, hunting areas, forestry, arable farming, livestock farming and [peri]urban areas). In addition to population abundance, four sets of indicators of environmental change could be used to monitor overabundance within these contexts (impacts on habitats, impact on animal performance, increments in diseases and parasite loads, and increments in nuisance to humans)., Nine species of ungulate were found to be overabundant. Red deer Cervus elaphus was the species most likely to be overabundant in the contexts of protected areas (detailed in 27% of papers on that context) and hunting areas (38%); roe deer Capreolus capreolus in forestry (28%); wild boar Sus scrofa in arable farming (60%), livestock farming (29%) and (peri)urban areas (38%). Our evidence shows that the diagnosis and monitoring of ungulate population overabundance via indicators of ecological change, and the management actions required to control these undesirable situations, are strongly context‐dependent.

Crimean–Congo haemorrhagic fever virus (CCHFV) is the causative agent of the severe tick‐borne, often fatal, zoonotic Crimean–Congo haemorrhagic fever (CCHF), which is widely distributed worldwide. The CCHFV transmission to humans occurs through tick bite, crushing of engorged ticks or contact with infected host blood. Previously, CCHFV genotype Africa III was reported in Spain. Given the emergence of CCHF and the role of ticks in pathogen maintenance and transmission, we investigated the presence and genotype identity of the virus in tick species parasitizing abundant wild host species in south‐western Spain. A total of 613 adult ticks were collected from hunter‐harvested wild ungulates in twenty locations throughout south‐western Spain. Ticks were identified, nucleic acids were extracted, RNA was analysed by a nested RT‐PCR targeting CCHFV S segment, and the amplicons were sequenced. According to the 212‐bp sequence amplified, the presence of CCHFV human genotype Europe V was detected in Hyalomma lusitanicum and Dermacentor marginatus ticks collected from red deer, fallow deer and Eurasian wild boar in different locations from south‐western Spain. Genotype Africa IV was also detected in a H. lusitanicum tick collected from a red deer. The detection of CCHFV in different tick species collected from various wild hosts and localities provided strong evidence of widespread CCHFV presence in the region, suggesting that the circulation of the virus in Spain requires more attention. Additionally, the identification of the CCHFV genotype Europe V in ticks suggested that its introduction in Spain was probably from Eastern Europe.

Oxidative stress has become a major concern in the field of spermatology, and one of the possible solutions to this acute problem would be the use of antioxidant protection; however, more studies are required in this field, as highly contradictory results regarding the addition of antioxidants have been obtained. Vitamin E is a powerful biological antioxidant, but its low stability and high hydrophobicity limit its application in spermatology, making the use of organic solvents necessary, which renders spermatozoa practically motionless. Keeping this in mind, we propose the use of hydrogels (HVEs) and nanoemulsions (NVEs), alone or in combination, as carriers for the controlled release of vitamin E, thus, improving its solubility and stability and preventing oxidative stress in sperm cells. Cryopreserved sperm from six stags was thawed and extended to 30 × 106 sperm/mL in Bovine Gamete Medium (BGM). Once aliquoted, the samples were incubated as follows: control, free vitamin E (1 mM), NVEs (9 mM), HVEs (1 mM), and the combination of HVEs and NVEs (H + N), with or without induced oxidative stress (100 µM Fe2+/ascorbate). The different treatments were analyzed after 0, 2, 5, and 24 h of incubation at 37 °C. Motility (CASA®), viability (YO-PRO-1/IP), mitochondrial membrane potential (Mitotracker Deep Red 633), lipid peroxidation (C11 BODIPY 581/591), intracellular reactive oxygen species production (CM-H2DCFDA), and DNA status (SCSA®) were assessed. Our results show that the deleterious effects of exogenous oxidative stress were prevented by the vitamin E-loaded carriers proposed, while the kinematic sperm parameters (p ˂ 0.05) and sperm viability were always preserved. Moreover, the vitamin E formulations maintained and preserved mitochondrial activity, prevented sperm lipid peroxidation, and decreased reactive oxygen species (ROS) production (p ˂ 0.05) under oxidative stress conditions. Vitamin E formulations were significantly different as regards the free vitamin E

Camera-trapping methods have been used to monitor movement and behavioural ecology parameters of wildlife. However, when considering movement behaviours to estimate DR is mandatory to include in the formulation the speed ratio, otherwise DR results will be biased. For instance, some wildlife populations present movement patterns characteristic of each behaviour (e.g. foraging or displacement between habitat patches), and further research is needed to integrate the behaviours in the estimation of movement parameters. In this respect, the day range (average daily distance travelled by an individual, DR) is a model parameter that relies on movement and behaviour. This study aims to provide a step forward concerning the use of camera-trapping in movement and behavioural ecology. We describe a machine learning procedure to differentiate movement behaviours from camera-trap data, and revisit the approach to consider different behaviours in the estimation of DR. Second, working within a simulated framework we tested the performance of three approaches to estimate DR: DROB (i.e. estimating DR without behavioural identification), DRTB (i.e. estimating DR by identifying behaviours manually and weighting each behaviour on the basis of the encounter rate obtained) and DRRB (i.e. estimating DR based on the classification of movement behaviours by a machine learning procedure and the ratio between speeds). Finally, we evaluated these approaches for 24 wild mammal species with different behavioural and ecological traits. The machine learning procedure to differentiate behaviours showed high accuracy (mean = 0.97). The DROB approach generated accurate results in scenarios with a speed-ratio (fast relative to slow behaviours) lower than 10, and for scenarios in which the animals spend most of the activity period on the slow behaviour. However, when considering movement behaviours to estimate DR is mandatory to include in the formulation the speed ratio, otherwise the DR results will

The heterogeneous nature of ejaculates highlights the relevance of studying the behavior of different sperm subpopulations. Changes in sperm motility and the increase in tyrosine phosphorylation are key events that usually occur during capacitation and can be modified by the cryopreservation process. However, the relationship between both events remains poorly defined throughout capacitation in the different sperm subpopulations. Fresh and frozen-thawed spermatozoa were incubated in capacitating (CAP) and non-capacitating (NC) media up to 240 min. Sperm kinematics, tyrosine phosphorylation and mitochondrial activity were measured by the CASA system and imaging flow cytometry. Four motile sperm subpopulations (SP) were identified in fresh and frozen-thawed ram semen after the cluster analysis. Incubation under CAP conditions over time led to greater changes in the percentage of spermatozoa included in each subpopulation compared to NC conditions, being different between fresh and frozen-thawed spermatozoa. The SP1, characterized by slow spermatozoa, progressively increased after 15 min in frozen-thawed samples incubated in both media but not in fresh ones. The SP4, characterized by fast and non-linear spermatozoa, showed a marked increase during CAP, but not under NC conditions, occurring more rapidly in frozen-thawed spermatozoa. This subpopulation (SP4) was also the only one positively and strongly correlated with mitochondrial activity and all phosphorylated sperm regions during capacitation, either in fresh or frozen-thawed samples. Our results indicated that in vitro capacitation induced significant changes in the distribution of motile sperm subpopulations, being affected by cryopreservation. Notwithstanding, the subpopulation which probably represents hyperactivated-like spermatozoa (SP4) also increased in frozen-thawed samples, occurring faster and simultaneously to the increment of mitochondrial activity and tyrosine phosphorylation of different sperm region

Crimean-Congo haemorrhagic fever virus (CCHFV) continues to cause new human cases in Iberia while its spatial distribution and ecological determinants remain unknown. The virus remains active in a silent tick-animal cycle to which animals contribute maintaining the tick populations and the virus itself. Wild ungulates, in particular red deer, are essential hosts for Hyalomma ticks in Iberia, which are the principal competent vector of CCHFV. Red deer could be an excellent model to understand the ecological determinants of CCHFV as well as to predict infection risks for humans because it is large, gregarious, abundant and the principal host for Hyalomma lusitanicum. We designed a cross-sectional study, analysed the presence of CCHFV antibodies in 1444 deer from 82 populations, and statistically modelled exposure risk with host and environmental predictors. The best-fitted statistical model was projected for peninsular Spain to map infection risks. Fifty out of 82 deer populations were seropositive, with individual population prevalence as high as 88%. The highest prevalence of exposure to CCHFV occurred in the southwest of the Iberian Peninsula. Climate and ungulate abundance were the most influential predictors of the risk of exposure to the virus. The highest risk regions were those where H. lusitanicum is most abundant. Eight of the nine primary human cases occurred in or bordering these regions, demonstrating that the model predicts human infection risk accurately. A recent human case of CCHF occurred in northwestern Spain, a region that the model predicted as low risk, pointing out that it needs improvement to capture all determinants of the CCHFV infection risk. In this study, we have been able to identify the main ecological determinants of CCHFV, and we have also managed to create an accurate model to assess the risk of CCHFV infection.

El orden de los quirópteros es uno de los grupos de mamíferos más abundantes, diversos y extendidos geográficamente por todo el mundo. Treinta y una especies pertenecientes a este orden habitan en la Península Ibérica. La diversidad de hábitats que pueden ocupar, combinada con la capacidad de vuelo, alta longevidad, la dispersión a largas distancias, formación de colonias y adaptación a ambientes antrópicos pueden facilitar el mantenimiento y la dispersión de patógenos zoonóticos potencialmente de relevancia para la salud humana. Además, son capaces de albergar grandes cargas de ectoparásitos altamente especializados como las garrapatas, entre otros artrópodos, que pueden ser portadores de patógenos zoonóticos y probablemente actúen como vectores en las poblaciones de quirópteros. Sin embargo, el papel de los murciélagos como reservorios naturales de patógenos y los factores que determinan los patrones epidemiológicos y riesgos de transmisión con otros grupos de especies es poco conocido. En este estudio se tomaron y se analizaron muestras de sangre de veinticinco especies de quirópteros de cuatro regiones de España. El estudio se centró en la detección, identificación y caracterización molecular de patógenos pertenecientes a los órdenes bacterianos Rickettsiales y Piroplasmida. Dentro de estos grupos, un gran número de especies de Rickettsia spp., Anaplasma spp., Babesia spp. y Theileria spp. son patógenos transmitidos por garrapatas que causan enfermedades relevantes para los seres humanos y los animales silvestres y domésticos. Como base para los factores determinantes de los patrones epidemiológicos encontrados (también multi-patógeno), se analizó la relación entre la prevalencia y diversidad de estos patógenos.

[ES]: La fiebre hemorrágica de Crimea Congo (FHCC) es la enfermedad vírica transmitida por garrapatas (Orden Ixodida) más extendida mundialmente. Está causada por un Orthonairovirus, cuyo ciclo de vida incluye garrapatas (principalmente Hyalomma), como vectores y reservorios, y animales como amplificadores del agente infeccioso u hospedadores/dispersores de los vectores. Los humanos son los únicos que padecen la enfermedad. Es posible que, siendo la abundancia de garrapatas variable en espacio y tiempo, la transmisión del virus de la fiebre hemorrágica de Crimea Congo (VFHCC) también esté sujeta a esta variación espaciotemporal y, por ello, ser un proceso dinámico. España, y más concretamente el Parque Nacional de Doñana (PND), es una zona con gran riesgo de entrada del virus debido a las condiciones adecuadas que presenta (ambiente, amplia presencia de garrapatas, gran variedad de mamíferos silvestres y zona esencial de parada de millones de aves migratorias), y por tanto ideal para la realización de este estudio, donde se buscó comprender la dinámica espaciotemporal del VFHCC y se trató de estimar la relación entre la abundancia de garrapatas vectores y el riesgo de exposición/infección al virus. El 60% de los ungulados silvestres analizados entre 2006 y 2019 resultaron seropositivos al VFHCC, mientras que el 12,8% de las garrapatas alimentadas analizadas entre 2015 y 2019 resultaron positivas a la PCR, detectándose tanto genotipo III (africano) como V (europeo), y observándose una variación temporal entre las muestras positivas. Se encontró además un efecto potencial de la abundancia predicha de Hyalomma lusitanicum sobre el riesgo de exposición al virus de los ungulados silvestres. Sin embargo, no se pudo confirmar tal efecto sobre la prevalencia de infección al VFHCC en garrapatas alimentadas., [EN]: Crimean-Congo hemorrhagic fever (CCHF) is the most widespread viral disease transmitted by ticks (Order Ixodida). It is caused by an Orthonairovirus, which life cycle includes ticks (mainly Hyalomma), as vectors and reservoirs, and animals as amplifiers of the infectious agent or as hosts/dispersers of the vectors. Humans are the only ones who suffer from the disease. Since tick abundance varies in space and time, it is possible that the transmission of CrimeanCongo hemorrhagic fever virus (CCHFV) also follows this spatiotemporal variation, and, therefore, it is a dynamic process. Spain, and more specifically Doñana National Park (PND), is considered and area under high risk of CCHFV circulation due to the favourable conditions it displays (environment, wide presence of ticks, a wide variety of wild mammals, and being an essential stopover for millions of migrating birds), and therefore ideal for the realization of this study. We tried to understand the spatiotemporal dynamics of the virus and we attempted to estimate the relationship between the abundance of the tick vectors and the risk of exposure/infection to the virus, all within the PND. The 60% of the wild ungulates analyzed between 2006 and 2019 were seropositive to CCFHV, while 12,8% of the fed ticks analyzed between 2015 and 2019 were PCR positive, detecting both genotype III (African) and genotype V (European), and observing a temporal variation among the positive samples. An effect of the predicted abundance of Hyalomma lusitanicum over the risk of exposure of the wild ungulates to the virus was also found, however, an effect over the prevalence of infection to VFHCC in fed ticks could not be confirmed.

This study describes chemical, physical, microbiological and technological characteristics of red deer milk and the effect of lactation on these parameters in order to know their potential aptitude to elaborate dairy products. During 18 weeks, milk from five hinds was monitored for composition, bacteriology, somatic cell count (SCC), physical properties and rennet coagulation. Mean values (g/100 g) for fat, protein, lactose and dry matter were 10.4, 7.1, 4.3 and 24.2, respectively, and for urea, 265 mg/100 mL. Except for lactose, a significant increase in these components was observed (p < 0.01) as lactation progressed. The average values for bacteriology and SCC were 5.3 log cfu/mL and 4.7 log cells/mL, respectively. Regarding physical properties, conductivity (mean: 2.8 ms/cm), viscosity (3.1 Cp), coordinates L* (89.9) and a* (−3.1) and milk fat globule diameter (D4,3: 6.1 µm) increased along with lactation while density (1.038 g/mL) decreased (p < 0.01). The pH (6.7), acidity (22.9° Dornic), coordinate b* (8.4) and ethanol stability (66.6% v/v) were stable during the study period. The stage of lactation also has a significant impact on milk coagulation properties and mean curd yield was 3.29 g/10 mL. These results suggest that red deer milk could be a potential innovative source of milk for the dairy industry.

[EN]: Ticks are obligate hematophagous arthropod ectoparasites involved in the transmission of a wide number of tick-borne pathogens (TBPs) causing diseases affecting human and animal health worldwide. Vaccines have improved the control of tick infestations and are consideredas the most effective and environmentally friendly approach for tick control and prevention of tick-borne diseases (TBDs). Regarding vaccine development, omics technologies advance the study of physiological and molecular function of tick genes and proteins, improving the identification and the characterization of candidate protective antigens, its functions and interacting proteins, thus contributing to the development of more efficacious and wide-targeted vaccines. This doctoral thesis addresses the use of omics approaches to the study of pathogen infection, host immune response in the tick Ixodes scapularis and human cells and vaccine development., [ES]: Las garrapatas son ectoparásitos artrópodos hematófagos obligados, involucrados en la transmisión de un amplio número de patógenos responsables de enfermedades que afectan a la salud humana y animal por todo el mundo. Las vacunas han mejorado el control de las infestaciones de garrapatas y son consideradas el enfoque más efectivo y respetuoso con el medio ambiente para el control de las garrapatas y la prevención de las enfermedades que transmiten. En referencia al desarrollo de vacunas, las tecnologías ómicas han permitido avanzar en el estudio de la función fisiológica y molecular de los genes y proteínas de las garrapatas, mejorando la identificación y caracterización de posibles antígenos protectores, sus funciones y proteínas interactuantes, contribuyendo así al desarrollo de vacunas más eficaces y de amplio alcance. Esta tesis doctoral aborda el uso de enfoques ómicos para el estudio de la infección producida por patógenos, la respuesta inmune del hospedador, en células de garrapata Ixodes scapularis y células humanas, y el desarrollo de vacunas.

[EN]: A better understanding of population density (i.e. the number of individuals per unit area) is essential for wildlife conservation and management. Despite the fact that a wide variety of methods with which to estimate population density have already been described and broadly used, there are still relevant gaps. In the last few decades, the use of remotely activated cameras (camera traps) has been established as an effective sampling tool when compared with alternative methods. Camera trapping could, therefore, be considered a reliable tool with which to monitor those situations in which classical methods have relevant limitations. It could, for example, be used with species whose behaviour is elusive and which have low detectability (as is the case of most mammals), or populations in which the animals can be identified individually by the spot patterns on their bodies. However, there is lack of information regarding those species for which it is not possible to identify individual animals (i.e. unmarked species). Some authors that have applied camera trapping originally considered relative abundance indexes in order to monitor unmarked populations. These indices were based on encounter rates (i.e. the number of animals detected per sampling unit) observed in camera trapping studies. Methods with which to estimate the population density of unmarked populations were later described, the first of which was the random encounter model (REM). The REM models the random encounters between moving animals and static cameras in order to estimate population density. The REM does this by employing three basic parameters: i) encounter rate, ii) detection zone (area in which the cameras effectively detect animals), and iii) day range (average daily distance travelled by each individual in the population). When this thesis was first started, it was broadly discussed that the application of the REM was limited by the difficulties involved in estimating the parameters required, [ES]: Mejorar el conocimiento sobre la densidad de población (es decir, el número de individuos por unidad de superficie) es fundamental para la gestión y conservación de la fauna silvestre. A pesar de que se han descrito y utilizado una gran variedad de métodos para calcular la densidad poblacional de la fauna silvestre, hoy en día todavía existen brechas importantes. En los últimos años, la técnica del fototrampeo (uso de cámaras activadas automáticamente) se ha establecido como una herramienta efectiva en comparación con otros métodos. Por tanto, el fototrampeo puede ser considerado una técnica fiable en aquellos casos en los que los métodos clásicos presentan limitaciones relevantes. Por ejemplo, al monitorizar especies con comportamiento esquivo y baja detectabilidad (como la mayor parte de los mamíferos), o poblaciones en las que los animales pueden ser reconocibles en base al patrón de manchas de su cuerpo. Sin embargo, existe una ausencia de información en especies en las que no es posible distinguir individuos en base al patrón de manchas de su cuerpo (comúnmente conocidas como “especies no marcadas”). Inicialmente, algunos autores consideraron índices relativos de abundancia para monitorizar estas poblaciones no marcadas. Estos índices estaban basados en la tasa de encuentro, es decir, el número de animales detectados por unidad de muestreo, obtenida en estudios de fototrampeo. Más tarde se describieron métodos que permitían calcular la densidad poblacional de especies no marcadas. El primero de estos métodos fue el modelo de encuentro aleatorio (REM por sus siglas en ingles). El REM modeliza los encuentros aleatorios entre animales móviles y cámaras trampa estáticas para finalmente estimar densidad poblacional. Para ello, el REM se basa en tres parámetros clave: i) la tasada de encuentro, ii) la zona de detección (área en la que las cámaras detectan a los animales), y iii) el rango diario (distancia media recorrida por cada individuo en la población). En

The intensification of agriculture in olive groves, especially the modification or elimination of spontaneous vegetation, alters the relationships in arthropod communities and reduces their interactions and ecosystem services. This study was carried out in nine olive groves in which there was either a planted cover crop, spontaneous cover crop or bare ground. The interactions of ground-dwelling, canopy and flying arthropods in trophic webs were calculated for each olive grove soil management regime at the family level taking into consideration their different functional traits: feeding guilds, specific agricultural traits and trophic level. Olive groves with spontaneous cover had trophic webs with a higher number of plausible links between arthropod families and a more balanced distribution of specimens among trophic levels compared to those with planted cover and bare ground. There was a similar number of arthropod families consisting of both pests and their natural enemies in the planted cover regime, while olive groves with bare ground had simpler trophic webs. The complexity of plausible trophic links was greater in olive groves with spontaneous plant cover despite the similar values for family richness in the three-olive grove soil management regimes. Qualitative values (such as functional traits) were more diverse in agroecosystems with spontaneous plant cover in which there were more sources of food.

The knowledge regarding the spatial ecology of red deer (Cervus elaphus) in different environments is crucial if effective management actions are to be designed. However, this knowledge continues to be scarce in the complex contexts of mixed land use and management circumstances. This study describes the spatial ecology of red deer monitored using GPS collars in Mediterranean ecosystems of South-Central Spain, considering the effect of individual and seasonal (food shortage period, rut, hunting season and food abundance period) factors on different land use and management scenarios, namely protected areas, mixed farms and fenced hunting estates. Our results showed less activity (ACT), a shorter daily range (DR) and a smaller home range (HR) during the food shortage period: ACT: 0.38 ± (SD) 0.12; DR: 3010.9 ± 727.3 m; and weekly HR: 122.2 ± 59.6 ha. With regard to land use, individuals were less ACT and had a smaller DR on fenced hunting estates (ACT: 0.24 ± 0.12; DR: 1946.3 ± 706.7 m) than in protected areas (ACT: 0.59 ± 0.12; DR: 4071.4 ± 1068.2 m) or on mixed farms (ACT: 0.57 ± 0.29; DR: 5431.1 ± 1939.5 m) in all the periods studied. Red deer selected land cover with forage and shelter when foraging and resting, respectively. When drive hunt events occurred (mixed farms and fenced hunting estates), the deer were more prone to select safer habitats (scrublands) and avoid open areas (crops or grasslands) than were their counterparts in protected areas. The patterns observed can be explained by sexual and seasonal differences as regards requirements, the response to disturbances and, interestingly, population management. Our results provide useful information with which to design scientifically-based species adaptive management in response to relevant and timely situations in Europe, such as the potential transmission of shared infections, vehicle collisions, and damage to crops and ecosystems.

The hepatitis E virus (HEV) is an emerging zoonotic pathogen whose main reservoir is suids. Most of the ecological and epidemiological aspects of its sylvatic cycle remain unknown. Thus, in this work, we study the drivers of HEV exposure in the wild boar population of Doñana National Park (DNP, southwest Spain) operating in the medium and long-term (2005–2018). Anti-HEV antibodies are widely distributed throughout the wild boar (46.7 ± 3.8%, 327 out of 700 sampled), showing a statistically significant age-increasing pattern. The temporal pattern displayed important interannual fluctuations. This could be mediated by marked variations in the population control of the wild boar, and subsequent changes in abundance rates, and its interplay with climatic conditions; as wet years together with a low abundance of wild boar led to the lowest seroprevalence. The fact that seroprevalence is high during conditions of high abundance, and not affected by rainfall level, is probably due to the increased interactions among the animals, and possibly, the subsequent higher environmental contamination with HEV particles. The proximity to the marshland (the main water body of the study area) is associated with a higher risk of testing positive, which is probably mediated by the preferential use of this area during the dry season and the favourable environmental conditions for the survival of HEV particles. A deeper understanding of the epidemiology of HEV in host communities deserves future research concerning other susceptible species. Most importantly, wild boar population control remains a challenge at the international level, and an increase of shared pathogen-related conflicts associated with this species is expected, as exemplified by HEV. Therefore, surveillance of wild boar diseases, including integrated population monitoring and sustainable population control programmes, will be essential to control the associated risks.

Animal tuberculosis (TB), which is caused by the Mycobacterium tuberculosis complex (MTC), is a zoonotic disease of global concern, and has a wide variety of wild and domestic reservoirs that can establish complex epidemiological systems. Of all the strategies employed to control TB, reducing the risks of interaction at the wildlife-livestock interface is a cornerstone. However, detailed protocols with which to assess and implement farm-specific preventive actions that can be employed against interactions with wildlife are lacking for extensive production systems. We describe an On-farm Wildlife Risk Mitigation Protocol that is applicable to beef cattle farming in Mediterranean environments in order to control the wildlife-livestock interaction and MTC transmission through the use of Farm-specific Action Plans (FsAP). We assessed the implementation and verification of FsAP in terms of its practical feasibility and acceptability by farmers (n=55 farms). Of the potential risk points, waterers (41.3 %) and waterholes (24.4 %) were the most common. Waterholes and springs were identified as the points with the greatest risks. Actions related to water management were essential on most farms (99 % of the high-risk points), as were those regarding wildlife management (36.4 % of the farms provided wild boar or cervids with supplementary food for hunting purposes). Overall, 75 % of the farmers adopted the plans to some extent, with an average of 31.8 % of actions implemented, but with high variability depending on the type of actions proposed. Farmers prioritised low-cost measures. Our results, in their entirety, indicate that the adoption of this On-farm Wildlife Risk Mitigation Protocol is practical and feasible in Mediterranean ecosystems, and can be easily transferred to professionals and adapted to other bioregions or epidemiological systems. The subsequent evaluation of FsAPs in terms of efficacy and cost-effectiveness, along with increasing their acceptance by farmers

Rhipicephalus spp. (Acari: Ixodidae) ticks are obligate hematophagous arthropods, which constitute a model for the study of vector-host interactions. The chemical composition or elementome of salivary glands (SG) and cement provides information relevant for the study of protein-based complex multifunctional tissues with a key role in tick biology. In this study, we characterized the elementome of cement cones in Rhipicephalus sanguineus collected from naturally infested dogs and in SG and cement of R. bursa collected from experimentally infested rabbits at different feeding stages. The elementome was characterized using scanning electron microscopy (SEM) combined with energy dispersive X-ray spectroscopy (EDS). The results showed the identification of up to 14 chemical elements in the cement, and suggested tick/host-driven differences in the cement elementome between tick species and between SG and cement within the same species. By still unknown mechanisms, ticks may regulate cement elementome during feeding to affect various biological processes. Although these analyses are preliminary, the results suggested that N is a key component of the cement elementome with a likely origin in SG/salivary proteins (i.e., Glycine (C2H5NO2)-rich superfamily member proteins; GRPs) and other tick/host-derived components (i.e. NAPDH). Future research should be focused on tick elementome and its functional implications to better understand cement structure and function.