Bridging the Gap between Field Experiments and Machine Learning: The EC H2020 B-GOOD Project as a Case Study towards Automated Predictive Health Monitoring of Honey Bee Colonies.

Honey bee colonies have great societal and economic importance. The main challenge that beekeepers face is keeping bee colonies healthy under ever-changing environmental conditions. In the past two decades, beekeepers that manage colonies of Western honey bees (Apis mellifera) have become increasingly concerned by the presence of parasites and pathogens affecting the bees, the reduction in pollen and nectar availability, and the colonies' exposure to pesticides, among others. Hence, beekeepers need to know the health condition of their colonies and how to keep them alive and thriving, which creates a need for a new holistic data collection method to harmonize the flow of information from various sources that can be linked at the colony level for different health determinants, such as bee colony, environmental, socioeconomic, and genetic statuses. For this purpose, we have developed and implemented the B-GOOD (Giving Beekeeping Guidance by computational-assisted Decision Making) project as a case study to categorize the colony's health condition and find a Health Status Index (HSI). Using a 3-tier setup guided by work plans and standardized protocols, we have collected data from inside the colonies (amount of brood, disease load, honey harvest, etc.) and from their environment (floral resource availability). Most of the project's data was automatically collected by the BEEP Base Sensor System. This continuous stream of data served as the basis to determine and validate an algorithm to calculate the HSI using machine learning. In this article, we share our insights on this holistic methodology and also highlight the importance of using a standardized data language to increase the compatibility between different current and future studies. We argue that the combined management of big data will be an essential building block in the development of targeted guidance for beekeepers and for the future of sustainable beekeeping.

The effect of COVID-19 confinement on the activity behaviour of red deer.

The COVID-19 pandemic has drastically affected people's social habits, especially those related to outdoor activities. We intended to understand the effects of the two national lockdowns in Portugal on the presence and activity of a wild population of red deer (Cervus elaphus) by analysing data from camera traps installed at Lousã mountain, in the central part of Portugal. The cameras were set between 2019 and 2021, and a total of 2434 individual contacts of red deer and 182 contacts of people were recorded. Results showed a higher human presence in the mountain area during the COVID-19 outbreak, especially during the first lockdown in 2020 (0.05 ± 0.17 individuals/day), compared to the same period of the year before the pandemic (0.02 ± 0.05 individuals/day), which resulted in an increase of people by 150%. The increase in human presence did not have a significant direct effect on the presence of red deer. Despite the low overlap of activity patterns between people and red deer, deer showed avoidance behaviour in the 24 h after the detection of human presence on camera traps, as well as an increase in daily activity during the 2020 lockdown, showing red deer's awareness of human visitation. These results showed that people's increased search for cultural services in wild environments during COVID-19 lockdowns, such as hiking and biking, seemed to influence the population of red deer, albeit momentarily.

Differences in the Diets of Female and Male Red Deer: The Meaning for Sexual Segregation.

Sexual segregation is a common phenomenon among animals, particularly dimorphic ones. Although widely addressed, the reasons and consequences of sexual segregation are still an important topic in need of better understanding. In this study, we mainly evaluate the diet composition and feeding behaviour of animals, which are related to the use of different habitats by the sexes, a special case of sexual segregation also termed habitat segregation. Sexually size dimorphic males and females often have different energetic and nutritional needs and, thus, different diets. We collected fresh faecal samples from wild Iberian red deer (Cervus elaphus L.) in Portugal. Samples were analysed in terms of diet composition and quality. As expected, both sexes differed in their diet composition, with males eating more arboreous species than females, but this difference was affected by sampling periods. Diet composition of both sexes had the biggest differences (and the lowest overlap) in spring, which corresponds to the end of gestation and beginning of birth. These differences might be a consequence of the sexual body size dimorphism characteristic of this species, as well as of different needs due to different reproductive costs. No differences regarding the quality of the excreted diet were observed. Our results may help to understand some patterns of sexual segregation observed in this red deer population. However, besides foraging ecology, other factors may also be contributing to sexual segregation in this Mediterranean population of red deer, and further studies focusing on sexual differences regarding feeding behaviour and digestibility are needed.

Hydrological changes caused by the construction of dams and reservoirs: The CECP analysis.

We investigated the influence of the construction of cascade dams and reservoirs on the predictability and complexity of the streamflow of the São Francisco River, Brazil, by using complexity entropy causality plane (CECP) in its standard and weighted form. We analyzed daily streamflow time series recorded in three fluviometric stations: São Francisco (upstream of cascade dams), Juazeiro (downstream of Sobradinho dam), and Pão de Açúcar station (downstream of Sobradinho and Xingó dams). By comparing the values of CECP information quantifiers (permutation entropy and statistical complexity) for the periods before and after the construction of Sobradinho (1979) and Xingó (1994) dams, we found that the reservoirs' operations changed the temporal variability of streamflow series toward the less predictable regime as indicated by higher entropy (lower complexity) values. Weighted CECP provides some finer details in the predictability of streamflow due to the inclusion of amplitude information in the probability distribution of ordinal patterns. The time evolution of streamflow predictability was analyzed by applying CECP in 2 year sliding windows that revealed the influence of the Paulo Alfonso complex (located between Sobradinho and Xingó dams), construction of which started in the 1950s and was identified through the increased streamflow entropy in the downstream Pão de Açúcar station. The other streamflow alteration unrelated to the construction of the two largest dams was identified in the upstream unimpacted São Francisco station, as an increase in the entropy around 1960s, indicating that some natural factors could also play a role in the decreased predictability of streamflow dynamics.

Inventory of tiger- and ground-beetles (Coleoptera, Caraboidea, Cicindelidae and Carabidae) in two sampling seasons of the Gorongosa National Park, Mozambique.

Background: The Gorongosa National Park (Mozambique) is one of the most emblematic protected areas in Africa, well known for its vertebrate biodiversity and restoration ecology efforts following the Mozambican civil war in 1992. The invertebrate biodiversity of Gorongosa National Park is still poorly studied, although the scarce information available indicates the existence of a rich number of species, namely in the case of tiger- and ground-beetles (Coleoptera, Caraboidea). Moreover, the study of arthropod assemblages is key for designing conservation practices since they are potentially accurate biodiversity and ecological indicators. Hence, the diversity assessment of Caraboidea beetles using standardised methodologies is likely to provide a new insight for future conservation planning and help to quantify the effects of climate change in areas identified as vulnerable to anthropogenic pressures, such as the Gorongosa National Park. New information: We report the occurrence of five tiger beetles (Cicindelidae) and 93 ground-beetles (Carabidae) species/morphospecies in Gorongosa National Park from a field survey funded by the ECOASSESS project. Sampling was performed in the four main habitat types present in the Park (miombo tropical forest, mixed dry forest, transitional forest and grasslands) between 25 October and 25 November 2019. In this sampling window, the turnover of Caraboidea species from the dry season to the wet season was recorded for the first time. Twenty-eight species of ground-beetles are new records to Mozambique, including three new subgenera and three new genera. Additional information on species phenology and habitat preferences is also provided.

Goitered Gazelle Gazella subgutturosa Responded to Human Disturbance by Increasing Vigilance Rather than Changing the Group Size.

Nonlethal human disturbances have been confirmed to have a negative effect on wildlife in a variety of ways, including on behaviors. In many studies, the impact of human disturbances on gregarious species of wildlife is assessed through changes in their social organization and vigilance. In our study in the Kalamaili Nature Reserve, China, we wanted to estimate the impacts of differing levels of human disturbances on two different group types (mixed-sex and all-male) of goitered gazelle, Gazella subgutturosa, living in three functional zones (experimental, buffer, and core zones), which represent high, medium, and low human disturbance levels, respectively. In addition, we studied the time spent vigilant as a function of group size with different levels of human disturbances in the three zones. In general, mixed-sex gazelle groups were of similar sizes in the three different zones, while all-male groups slightly differed in their sizes between the experimental and buffer zones. This may indicate that human disturbances have varying effects on the different group types, with smaller-sized, single-sex groups being more significantly affected by human disturbances than larger-sized, mixed-sex groups. Goitered gazelle showed higher vigilance levels in the experimental zone than in the two other zones. A trend of decreasing vigilance varying linearly with group size was also found in the three zones, and the rate of decrease was higher in the experimental zone. Increased habitat fragmentation and human activities brought goitered gazelle and humans closer together in the core zone. Therefore, stopping mining activities and returning the reserve to a continuous habitat with fewer environmental disturbances is the best way to establish and protect a stable population of this endangered species of gazelle.

Biogeographic Patterns of Arbuscular Mycorrhizal Fungal Communities Along a Land-Use Intensification Gradient in the Subtropical Atlantic Forest Biome.

Information concerning arbuscular mycorrhizal (AM) fungal geographical distribution in tropical and subtropical soils from the Atlantic Forest (a global hotspot of biodiversity) are scarce and often restricted to the evaluation of richness and abundance of AM fungal species at specific ecosystems or local landscapes. In this study, we hypothesized that AM fungal diversity and community composition in subtropical soils would display fundamental differences in their geographical patterns, shaped by spatial distance and land-use change, at local and regional scales. AM fungal community composition was examined by spore-based taxonomic analysis, using soil trap cultures. Acaulospora koskei and Glomus were found as generalists, regardless of mesoregions and land uses. Other Acaulospora species were also found generalists within mesoregions. Land-use change and intensification did not influence AM fungal composition, partially rejecting our first hypothesis. We then calculated the distance-decay of similarities among pairs of AM fungal communities and the distance-decay relationship within and over mesoregions. We also performed the Mantel test and redundancy analysis to discriminate the main environmental drivers of AM fungal diversity and composition turnover. Overall, we found significant distance-decays for all land uses. We also observed a distance-decay relationship within the mesoregion scale (< 104 km) and these changes were correlated mainly to soil type (not land use), with the secondary influence of both total organic carbon and clay contents. AM fungal species distribution presented significant distance-decays, regardless of land uses, which was indicative of dispersal limitation, a stochastic neutral process. Although, we found evidence that, coupled with dispersal limitation, niche differentiation also played a role in structuring AM fungal communities, driven by long-term historical contingencies, as represented by soil type, resulting from different soil origin and mineralogy across mesoregions.

Land-Use Effect on Olive Groves Pest Prays oleae and on Its Potential Biocontrol Agent Chrysoperla carnea.

Olive growing has been intensified through the simplification of agricultural landscapes. In order to rethink the environmental drawbacks of these practices, conservation biological control techniques have been examined. In this work, Prays oleae and its natural enemy Chrysoperla carnea were monitored to account for the effects of the amount and diversity of different land-uses. We found that C. carnea showed an attraction to areas with high abundances of P. oleae but this predator did not display any affection by the different land-uses. Inversely, P. oleae abundance was lower in diverse landscapes and higher in simplified ones. Importantly, higher abundances of C. carnea were related to lower infestation levels of P. oleae in the late part of the season. These results corroborate the attraction of C. carnea to the olive moth, highlighting the potential of C. carnea as a biological control agent of this pest, assert that the promotion of land-use diversity can reduce P. oleae and confirm that landscapes dominated by olive groves can promote this pest. The present study aims at contributing to the discussion about the management of agricultural ecosystems by providing farmers with sustainable alternatives that do not have harmful effects on the environment and public health.

Physiological Stress Reactions in Red Deer Induced by Hunting Activities.

Hunting activity is usually seen as a factor capable of causing an intense stress response in wildlife that may lead to short but also long-term stress. In the Lousã Mountain, Portugal, the population of red deer (Cervus elaphus) is the target of intensive seasonal hunting. We collected and measured cortisol (and its metabolites) in three tissues types (blood, feces and hair) from red deer hunted during two hunting seasons to evaluate the stress levels at different time windows. We also assessed the immunological and physical condition of the animals. We predicted that the hunting activity would act as a stressor inducing increased short and long-term stress levels in the population. Results showed an increase in hair cortisol levels during the months of harvesting. Surprisingly, the tendency for plasma cortisol levels was to decrease during the hunting season, which could be interpreted as habituation to hunting activity, or due to the hunting duration. Contrary to our predictions, fecal cortisol metabolites did not show any clear patterns across the months. Overall, our results suggest an influence of hunting activities on the physiological stress in red deer. In addition, hair seems to be useful to measure physiological stress, although more studies are required to fully understand its suitability as an indicator of long-term stress. Methodologically, our approach highlights the importance of simultaneously using different methods to assess short and long-term effects in studies on physiological stress reactions.

Wing membrane and fur samples as reliable biological matrices to measure bioaccumulation of metals and metalloids in bats.

There is a growing conservation concern about the possible consequences of environmental contamination in the health of bat communities. Most studies on the effects of contaminants in bats have been focused on organic contaminants, and the consequences of bat exposure to metals and metalloids remain largely unknown. The aim of this study was to evaluate the suitability of external biological matrices (fur and wing membrane) for the assessment of exposure and bioaccumulation of metals in bats. The concentration of arsenic, cadmium, cobalt, chromium, copper, manganese, nickel, lead, selenium and zinc was measured in internal organs (liver, heart, brain), internal (bone) and external tissues (wing membrane, fur) collected from bat carcasses of four species (Hypsugo savii, Nyctalus leisleri, Pipistrellus pipistrellus, Pipistrellus pygmaeus) obtained in windfarm mortality searches. With the exception of zinc (P = 0.223), the results showed significant differences between the concentrations of metals in the analyzed tissues for all metals (P < 0.05). Significant differences were also found between organs/tissues (P < 0.001), metals (P < 0.001) and a significant interaction between organs/tissues and metals was found (P < 0.001). Despite these results, the patterns in terms of metal accumulation were similar for all samples. Depending on the metal, the organ/tissue that showed the highest concentrations varied, but fur and wing had the highest concentrations for most metals. The variability obtained in terms of metal concentrations in different tissues highlights the need to define standardized methods capable of being applied in monitoring bat populations worldwide. The results indicate that wing membrane and fur, biological matrices that may be collected from living bats, yield reliable results and may be useful for studies on bats ecotoxicology, coupled to a standardized protocol for large-scale investigation of metal accumulation.

Everolimus in combination with Imatinib overcomes resistance in Chronic myeloid leukaemia.

Although Imatinib and other tyrosine kinase inhibitors (TKIs) have excellent results, the appearance of resistance is a problem in chronic myeloid leukaemia (CML). PI3K/AKT/mTOR pathway is activated by BCR-ABL playing a crucial rule in CML. This study aimed to evaluate the therapeutic potential of Everolimus, in CML models sensitive and resistant to Imatinib. We used one CML cell line sensitive to Imatinib (K562) and two resistant (K562-RC and K56-RD). Cell lines were treated with Everolimus alone and in combination with Imatinib. Cell viability was analysed by resazurin assay. Cell death and cell cycle were analysed by flow cytometry. Additionally, we also studied peripheral blood samples obtained from 52 patients under TKI treatment. Everolimus reduced cell line viability in sensitive (IC50 = 20 µM) and resistant models (K562-RC, IC50 = 25 µM; K562-RD, IC50 = 30 µM). This drug induced cell death by apoptosis and cell cycle arrest in G0/G1 phase. Everolimus also reduced cell viability by increasing apoptosis of haematopoietic stem cells (CD34+ cells) with low cytotoxicity to lymphocytes. Everolimus at 25 µM increased apoptotic cells 18.7% in CD34+ cells and only 8% in lymphocytes. The response to Everolimus was influenced by TKI treatment, with a better response in samples from patients under 2nd and 3rd generation TKI and with less toxicity to lymphocytes. Our results reveal that Everolimus induce cell death in CML cells sensitive and resistant to Imatinib, with low cytotoxicity to normal cells, suggesting that Everolimus could be an alternative targeted therapeutic approach in CML patients, even in cases of Imatinib resistance.

Maternal 5mCpG Imprints at the PARD6G-AS1 and GCSAML Differentially Methylated Regions Are Decoupled From Parent-of-Origin Expression Effects in Multiple Human Tissues.

A hallmark of imprinted genes in mammals is the occurrence of parent-of-origin-dependent asymmetry of DNA cytosine methylation (5mC) of alleles at CpG islands (CGIs) in their promoter regions. This 5mCpG asymmetry between the parental alleles creates allele-specific imprinted differentially methylated regions (iDMRs). iDMRs are often coupled to the transcriptional repression of the methylated allele and the activation of the unmethylated allele in a tissue-specific, developmental-stage-specific and/or isoform-specific fashion. iDMRs function as regulatory platforms, built through the recruitment of chemical modifications to histones to achieve differential, parent-of-origin-dependent chromatin segmentation states. Here, we used a comparative computational data mining approach to identify 125 novel constitutive candidate iDMRs that integrate the maximal number of allele-specific methylation region records overlapping CGIs in human methylomes. Twenty-nine candidate iDMRs display gametic 5mCpG asymmetry, and another 96 are candidate secondary iDMRs. We established the maternal origin of the 5mCpG imprints of one gametic (PARD6G-AS1) and one secondary (GCSAML) iDMRs. We also found a constitutively hemimethylated, nonimprinted domain at the PWWP2AP1 promoter CGI with oocyte-derived methylation asymmetry. Given that the 5mCpG level at the iDMRs is not a sufficient criterion to predict active or silent locus states and that iDMRs can regulate genes from a distance of more than 1 Mb, we used RNA-Seq experiments from the Genotype-Tissue Expression project and public archives to assess the transcriptional expression profiles of SNPs across 4.6 Mb spans around the novel maternal iDMRs. We showed that PARD6G-AS1 and GCSAML are expressed biallelically in multiple tissues. We found evidence of tissue-specific monoallelic expression of ZNF124 and OR2L13, located 363 kb upstream and 419 kb downstream, respectively, of the GCSAML iDMR. We hypothesize that the GCSAML iDMR regulates the tissue-specific, monoallelic expression of ZNF124 but not of OR2L13. We annotated the non-coding epigenomic marks in the two maternal iDMRs using data from the Roadmap Epigenomics project and showed that the PARD6G-AS1 and GCSAML iDMRs achieve contrasting activation and repression chromatin segmentations. Lastly, we found that the maternal 5mCpG imprints are perturbed in several hematopoietic cancers. We conclude that the maternal 5mCpG imprints at PARD6G-AS1 and GCSAML iDMRs are decoupled from parent-of-origin transcriptional expression effects in multiple tissues.

Trisomy 21 Alters DNA Methylation in Parent-of-Origin-Dependent and -Independent Manners.

The supernumerary chromosome 21 in Down syndrome differentially affects the methylation statuses at CpG dinucleotide sites and creates genome-wide transcriptional dysregulation of parental alleles, ultimately causing diverse pathologies. At present, it is unknown whether those effects are dependent or independent of the parental origin of the nondisjoined chromosome 21. Linkage analysis is a standard method for the determination of the parental origin of this aneuploidy, although it is inadequate in cases with deficiency of samples from the progenitors. Here, we assessed the reliability of the epigenetic 5mCpG imprints resulting in the maternally (oocyte)-derived allele methylation at a differentially methylated region (DMR) of the candidate imprinted WRB gene for asserting the parental origin of chromosome 21. We developed a methylation-sensitive restriction enzyme-specific PCR assay, based on the WRB DMR, across single nucleotide polymorphisms (SNPs) to examine the methylation statuses in the parental alleles. In genomic DNA from blood cells of either disomic or trisomic subjects, the maternal alleles were consistently methylated, while the paternal alleles were unmethylated. However, the supernumerary chromosome 21 did alter the methylation patterns at the RUNX1 (chromosome 21) and TMEM131 (chromosome 2) CpG sites in a parent-of-origin-independent manner. To evaluate the 5mCpG imprints, we conducted a computational comparative epigenomic analysis of transcriptome RNA sequencing (RNA-Seq) and histone modification expression patterns. We found allele fractions consistent with the transcriptional biallelic expression of WRB and ten neighboring genes, despite the similarities in the confluence of both a 17-histone modification activation backbone module and a 5-histone modification repressive module between the WRB DMR and the DMRs of six imprinted genes. We concluded that the maternally inherited 5mCpG imprints at the WRB DMR are uncoupled from the parental allele expression of WRB and ten neighboring genes in several tissues and that trisomy 21 alters DNA methylation in parent-of-origin-dependent and -independent manners.

The importance of lizards and small mammals as reservoirs for Borrelia lusitaniae in Portugal.

Borrelia lusitaniae is a pathogen frequent in the Mediterranean area. Apart from lizards, evidence for birds and small mammals as competent reservoirs for this genospecies has been occasional. We collected questing ticks, skin biopsies and Ixodes sp. ticks feeding on lizards, birds and small mammals in a B. burgdorferi s.l. (sensu lato) enzootic area to assess their importance in the maintenance of B. lusitaniae. Borrelia lusitaniae was the most prevalent genospecies in questing ticks and was commonly found in larvae feeding on Psammodromus algirus. One biopsy infected with B. lusitaniae was collected from the tail of one Podarcis hispanica, which suggests systemic infection. Ixodes ricinus larvae feeding on Apodemus sylvaticus were infected with B. lusitaniae but with a lower prevalence. Our results reinforce the importance of lizards as reservoirs for B. lusitaniae, suggesting that P. algirus, in particular, acts as main reservoir for B. lusitaniae in Portugal.

5meCpG epigenetic marks neighboring a primate-conserved core promoter short tandem repeat indicate X-chromosome inactivation.

X-chromosome inactivation (XCI) is the epigenetic transcriptional silencing of an X-chromosome during the early stages of embryonic development in female eutherian mammals. XCI assures monoallelic expression in each cell and compensation for dosage-sensitive X-linked genes between females (XX) and males (XY). DNA methylation at the carbon-5 position of the cytosine pyrimidine ring in the context of a CpG dinucleotide sequence (5meCpG) in promoter regions is a key epigenetic marker for transcriptional gene silencing. Using computational analysis, we revealed an extragenic tandem GAAA repeat 230-bp from the landmark CpG island of the human X-linked retinitis pigmentosa 2 RP2 promoter whose 5meCpG status correlates with XCI. We used this RP2 onshore tandem GAAA repeat to develop an allele-specific 5meCpG-based PCR assay that is highly concordant with the human androgen receptor (AR) exonic tandem CAG repeat-based standard HUMARA assay in discriminating active (Xa) from inactive (Xi) X-chromosomes. The RP2 onshore tandem GAAA repeat contains neutral features that are lacking in the AR disease-linked tandem CAG repeat, is highly polymorphic (heterozygosity rates approximately 0.8) and shows minimal variation in the Xa/Xi ratio. The combined informativeness of RP2/AR is approximately 0.97, and this assay excels at determining the 5meCpG status of alleles at the Xp (RP2) and Xq (AR) chromosome arms in a single reaction. These findings are relevant and directly translatable to nonhuman primate models of XCI in which the AR CAG-repeat is monomorphic. We conducted the RP2 onshore tandem GAAA repeat assay in the naturally occurring chimeric New World monkey marmoset (Callitrichidae) and found it to be informative. The RP2 onshore tandem GAAA repeat will facilitate studies on the variable phenotypic expression of dominant and recessive X-linked diseases, epigenetic changes in twins, the physiology of aging hematopoiesis, the pathogenesis of age-related hematopoietic malignancies and the clonality of cancers in human and nonhuman primates.

A novel in-frame deletion affecting the BAR domain of OPHN1 in a family with intellectual disability and hippocampal alterations.

Oligophrenin-1 (OPHN1) is one of at least seven genes located on chromosome X that take part in Rho GTPase-dependent signaling pathways involved in X-linked intellectual disability (XLID). Mutations in OPHN1 were primarily described as an exclusive cause of non-syndromic XLID, but the re-evaluation of the affected individuals using brain imaging displayed fronto-temporal atrophy and cerebellar hypoplasia as neuroanatomical marks. In this study, we describe clinical, genetic and neuroimaging data of a three generation Brazilian XLID family co-segregating a novel intragenic deletion in OPHN1. This deletion results in an in-frame loss of exon 7 at transcription level (c.781_891del; r.487_597del), which is predicted to abolish 37 amino acids from the highly conserved N-terminal BAR domain of OPHN1. cDNA expression analysis demonstrated that the mutant OPHN1 transcript is stable and no abnormal splicing was observed. Features shared by the affected males of this family include neonatal hypotonia, strabismus, prominent root of the nose, deep set eyes, hyperactivity and instability/intolerance to frustration. Cranial MRI scans showed large lateral ventricles, vermis hypoplasia and cystic dilatation of the cisterna magna in all affected males. Interestingly, hippocampal alterations that have not been reported in patients with loss-of-function OPHN1 mutations were found in three affected individuals, suggesting an important function for the BAR domain in the hippocampus. This is the first description of an in-frame deletion within the BAR domain of OPHN1 and could provide new insights into the role of this domain in relation to brain and cognitive development or function.