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Percorrer CIMO - Resumos em Proceedings Não Indexados à WoS/Scopus por Domínios Científicos e Tecnológicos (FOS) "Ciências Naturais::Ciências da Terra e do Ambiente"
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- Adaptive genomic variation in honey bees from arid regionsPublication . Yadró Garcia, Carlos A.; Henriques, Dora; Haddad, N.; Obeidat, W. M.; AlShagour, B.; Muz, Mustafa Necati; Arab, A.; Eissa, A. A.; Hosri, C.; Lamghari, F.; Arruda, J.; Rufino, José; Martín Hernández, R.; Nanetti, A.; Pinto, M. AliceThe Middle East is home to a large number of Apis mellifera subspecies that have evolved under extreme aridity and desertic conditions. Therefore, they represent a unique opportunity to disentangle the genetic basis of adaptation to such challenging environmental conditions. We arranged a collection of 514 drone samples (haploid males) split into two latitudinal transects. Transect 1 comprised 342 samples: 66 A. m. lamarckii (Egypt), 197 A. m. syriaca (Jordan), 30 A. m. syriaca (Lebanon), 21 A. m. meda (southeastern Turkey), and 27 A. m. anatoliaca (Anatolia, Turkey). Transect 2 comprised 173 samples: 14 A. m. jemenitica (Oman), 9 A. m. jemenitica (UAE), 75 A. m. meda (Iran), and 75 A. m. caucasia (northeastern Turkey). For all samples, whole genomes were resequenced, and environmental and bioclimatic variables were retrieved from WorldClim. For each transect, we applied three genotype–environment association methods (Samβada, pRDA, and LFMM) and one outlier-detection approach (PCAdapt) to identify SNPs associated with environmental adaptation. SNPs were annotated, and the intersection of genes between the four methods in each transect was analysed for gene enrichment. In both transects, enriched terms included genes related to biological regulation through endocrine, neuroendocrine, and neurotransmitter pathways. The regulation of gene expression was also enriched, including several transcription factors, especially HOX genes. Transmembrane and ionic transporters also seem to play a central role in environmental adaptation in these subspecies. Finally, neurogenesis, synapse establishment, and neural system development were also enriched. When gene enrichment analysis was set to exclude electronic annotations (automatically assigned, non-curated GO terms), enriched terms included processes such as behaviour, learning or memory, cognition, and associative and olfactory learning and behaviour. Our findings point to a complex suite of regulatory and sensory 62 pathways that collectively shape the environmental adaptation of honey bee subspecies that have evolved in arid environments.
- Assessing pesticide impact on honeybee gut microbiota: a call for microbial diversity as an environmental risk assessment endpointPublication . Rosa-Fontana, Annelise; Aguado, Daniel; Martín Hernández, Raquel; Higes, Mariano; Henriques, Dora; Pinto, M. AliceA recent roadmap for integrating environmental microbiota into risk assessments under the European Food Safety Authority (EFSA) remit has been published. The honeybee gut microbiota has emerged as a promising avenue to protect bees against stressors. Honeybees exhibit a stable core microbiota, and dysbiosis may serve as an indicator of adverse conditions. We investigated the gut microbiota of newly emerged “Apis mellifera iberiensis” workers exposed to a single concentration of the insecticide flupyradifurone (FPF, 36 ppm). The control groups included pure syrup (negative control, NC) and syrup supplemented with 1% acetone (acetone control, AC). Laboratory trials followed official guidelines (OECD No. 245). The abdomen of each bee was separated from the thorax, and DNA extraction was performed individually. Full-length 16s rRNA amplicon metagenomic was sequenced through PacBio sequel II (HiFi/CCS mode). The Shannon diversity index was used to analyze honeybee gut microbiota composition across experimental groups. Our results revealed a significant increase in bacterial community diversity (Shannon index, P = 0.003) after ten days of chronic exposure to FPF. This effect was more pronounced when compared to the AC group (P = 0.003) than to the NC group (P = 0.03). These findings demonstrate that FPF disrupts the honeybee gut microbiota. This study represents the first characterization of honeybee gut microbiota strictly adhering to OECD guidelines without modifications or adaptations. Furthermore, we have provided new insights into pesticide risk assessment, highlighting an overlooked aspect of bee health assessment. We propose integrating this approach into pesticide risk assessments by using diversity indices as comparative parameters. Specifically, we advocate for the inclusion of honeybee gut microbiota dysbiosis as a sublethal effect in the initial screening phase of risk assessments (laboratory-based assays) and as a key parameter for evaluating pollinator health.
- Assessment of the bioaccumulation potential of pharmaceuticals in periphyton in river systems of northeastern Portugal: methodological design and environmental perspectivesPublication . Voznakova, Alena; Geraldes, Ana Maria; Canle, MoisésPeriphyton is a complex biological matrix that plays a key role in aquatic ecosystems, not only as the basis of the food web, but also as a potential bioindicator of pollution. In the context of monitoring emerging pollutants, the study of periphyton provides complementary information to that obtained from water and sediment, especially regarding bioaccumulation processes and associated ecological risks. Its ability to retain and accumulate contaminants over time makes it a valuable tool for assessing long-term exposure and environmental quality. This study was carried out in the Douro river basin, in the region of Bragança (northeastern Portugal), where periphyton samples were collected from 14 sites during three seasonal campaigns throughout 2024. Samples were obtained by scraping hard substrates in river and reservoir systems, then preserved under cold conditions and freeze-dried for laboratory analysis. In addition to the development of a specific solid-phase extraction (SPE) and cleanup protocol for the detection of pharmaceutical compounds using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS/MS), periphyton samples were characterized to better understand their structure and composition as an environmental matrix. Periphyton characterization revealed consistent patterns, with dry weight generally between ~4 and 90 g/m² and chlorophyll content ranging from ~0.02% to 5%. The assessment of pharmaceutical contamination is still in progress, as methodological improvements are being applied due to the lack of reliable results with existing approaches. The methodological approach adopted will be presented, along with reflections on technical challenges and its potential contribution to integrated environmental assessment. Preliminary findings may also be included.
- Benchmarking lamp primer design platforms for pyrethroid resistance SNP detection in varroa destructorPublication . Bejaoui, Mohamed; Costa, Maíra; Pinto, M. Alice; Henriques, DoraVarroa destructor, an ectoparasitic mite of Apis mellifera, is a driver of colony and pollination service declines. Widely used pyrethroid acaricides targeting the mite voltage-gated sodium channel (VGSC) have selected for resistance mutations at codons 918 and 925 in Domain II. Genotyping 100 mites from 35 apiaries at the locus revealed ~40% pyrethroid-resistant haplotypes, with the double-resistant M918L/L925V variant at 43%, establishing the Portuguese baseline. PCR-based assays at this locus are robust but laboratory-bound, whereas loop-mediated amplification (LAMP) offers an isothermal, rapid, low-cost alternative for field resistance surveillance. Using this baseline, we benchmarked mutation - focused LAMP primer sets with PrimerExplorer V5, NEB LAMP Designer, and LAMP Designer v1.16 under shared, explicitly defined thermodynamic constraints. We constrained outer primers and inner-primer Tm to 55–70 °C, loops to ≈62–65 °C, GC to 40–70%, F2–B2 span to 120–180 bp, and filtered candidates using ΔG thresholds for hairpins and dimers. PrimerExplorer V5 provided flexible control over primer geometry around codons and loop placement, but required a loop-primer step and external specificity checks. NEB LAMP Designer rapidly generated optimised sets with conservative end-stability and self-dimer filtering, although limited parameter tuning and absence of BLAST restricted mutation-centred optimization. LAMP Designer v1.16 delivered an exhaustive thermodynamic assessment of hairpins, self-dimers, and cross-dimers together with BLAST-supported specificity and evaluation, at the cost of a steeper learning curve and reliance on proprietary software. In NEB LAMP Designer and LAMP Designer v1.16, targeted codons were embedded in distinct inner-primer segments (918 in F2, F1c; 925 in F1c, B1c, respectively), whereas PrimerExplorer V5 was used to design assays on codon 925 alone, given that M918L never occurred without L925V. Collectively, these comparisons show how software architecture and Tm/ΔG criteria shape LAMP primer solutions for resistance SNPs and inform the design of field-deployable diagnostics for evolutionary surveillance.
- Bioinformatics pipeline to evaluate patterns of diversity in detoxification genes in the Western honey bee (Apis mellifera)Publication . Barbosa, Daniela; Li, Fernanda; Bashir, Sana; Lopes, Ana Rita; Yadró Garcia, Carlos A.; Quaresma, Andreia; Rufino, José; Rosa-Fontana, Annelise; Verbinnen, Gilles; de Graaf, Dirk C.; De Smet, Lina; Taliadoros, Demetris; Webster, Matthew; Pinto, M. Alice; Henriques, DoraThe Western honey bee, Apis mellifera, displays significant genetic diversity in detoxification genes, which is pivotal for environmental adaptation and resilience. Herein, we developed a bioinformatics pipeline to investigate patterns of diversity in these genes, focusing on single nucleotide polymorphisms (SNPs) across A. mellifera populations, with variant annotation performed using both snpEff and the Variant Effect Predictor (VEP). Our pipeline integrates GATK, VCFtools, PLINK, bcftools, snpEff, and VEP to process genomic data systematically. Regions of interest were defined in a BED file for variant filtering. Using GATK, SNPs were extracted from a VCF file and conversion to PLINK format for population genetics analyses. Variants were filtered by minor allele frequency (MAF) and population differentiation (FST index) to identify SNPs with considerable. Variants were annotated with snpEff and VEP to predict functional impacts, enabling a comparative analysis of their annotation consistency and depth. Custom scripts were developed to map SNPs to detoxification genes, quantify SNP density, and integrated gene descriptions and lineage data. The resulting data were visualized using a combination of and generate different graphs using ggplot2 and chromoMap for chromossomal maps. Quality control steps were applied through the pipeline ensuring data reliability. Our findings reveal distinct SNP patterns in detoxification genes, highlighting candidate SNPs associated with A. mellifera subspecies-specific adaptations. The comparison of snpEff and VEP annotations provides insights into their strengths and limitations, which can help optimize software selection for genomic studies. This pipeline offers a reproducible framework for studying genetic diversity in A. mellifera that is adaptable to other species, advancing conservation and evolutionary genomics.
- Computational analysis to predict the impact of non-synonymous mutations in a protein: an example with the Odorant Receptor 49bPublication . Bashir, Sana; Shiraishi, Carlos S.H.; Yadró Garcia, Carlos A.; Henriques, Dora; Pinto, M. Alice; Abreu, Rui M.V.Non-synonymous mutations lead to amino acid substitutions within proteins, potentially affecting protein structure and function. While some mutations have a minimal impact on protein structure and function, others can alter their stability, conformation, and biological activity. Therefore, predicting how mutations affect proteins is important when uncovering the genetic basis of honey bee response to selective pressures. In a recent whole-genome scan for signatures of selection in Apis mellifera syriaca, we found a non-synonymous mutation under selection in the Odorant Receptor 49b (Or49b) gene. According to the literature, this gene plays a role in honeybee foraging, communication, and environmental adaptation. In an attempt to understand the biological impact at the molecular level of the discovered mutation, we assembled a pipeline that integrates bioinformatics and protein modelling techniques to evaluate the structural and functional consequences of a mutation in a protein. Protein structures were generated from a FASTA file using AlphaFold3, converted from mmCIF to PDB format, and visualized in PyMOL, where mutations were introduced. Functional site predictions were performed using Proteins Plus, and molecular dynamics simulations were conducted in YASARA to assess stability and conformational changes. Our findings suggest that the mutation under selection in the OR49b gene, which replaces tyrosine with histidine, alters protein dynamics by modifying its energy landscape and stability. At the end of the molecular dynamics simulation, the total potential energy of the wild-type protein was calculated as -384,675 kcal/mol. In contrast, this value increased to -287,075 kcal/mol for the mutant protein. This difference suggests that the mutation may affect the OR49b conformation, flexibility, and eventually its biological function. Whether this change affects honey bee olfactory perception remains uncertain. However, given that the mutation is under selection, it is plausible that the alternative amino acid variants confer an adaptive advantage in different environments. From a practical perspective, understanding how mutations affect biological function can ultimately assist honey bee breeders in selecting colonies for breeding programs, enhancing resilience and adaptability.
- A Computational Approach to Study Non-Synonymous MutationsPublication . Bashir, Sana; Li, Fernanda; Shiraishi, Carlos S.H.; Yadró Garcia, Carlos A.; Barbosa, Daniela; Abreu, Rui M.V.; Rufino, José; Pinto, M. Alice; Henriques, DoraNon-synonymous SNPs (Single Nucleotide Polymorphisms) result in amino acid substitutions within proteins. While some may have minimal impact on protein structure and function, others can significantly alter stability, conformation, and biological activity. Therefore, it is crucial to predict how SNP-induced changes affect protein structure and function. Here, we developed a novel pipeline that integrates bioinformatics and molecular modeling techniques to evaluate the structural and functional consequences of a non-synonymous SNP in a protein. Initially, Protein Plus was used to predict the potential active sites, which will help to determine whether a mutation is located within the functional binding regions. Identification of active sites is necessary to prioritize mutations that may alter protein structure and function. PolyPhen-2 and I-Mutant were used to evaluate the functional impact of mutations and the thermodynamic stability of the proteins. Next, to visualize the structural changes, AlphaFold3 was used to generate highconfidence 3D models for wild and mutant proteins. Finally, molecular dynamics (MD) simulations were conducted using YASARA to explore the dynamic behavior and stability of both mutant and wild-type proteins. MD simulations provide valuable insight into structural flexibility, potential conformational shifts, and the overall impact of the mutation on protein function. This computational pipeline provides a detailed framework to evaluate the structural and energetic consequences of non-synonymous mutations.
- Computational Insights into the Impact of NonSynonymous Mutations in the ABC Transporter Gene of Apis melliferaPublication . Bashir, Sana; Li, Fernanda; Shiraishi, Carlos S.H.; Younes, Sarra Bin; Yadró Garcia, Carlos A.; Barbosa, Daniela; Abreu, Rui M.V.; Rufino, José; Pinto, M. Alice; Henriques, DoraNon-synonymous mutations lead to amino acid substitutions that can affect a protein’s structure and function, thereby influencing biological processes such as detoxification. ATP-binding cassette (ABC) transporters (an important superfamily of detoxification genes) play a critical role in the efflux of toxic compounds in honey bees (Apis mellifera), essential for their survival in environments exposed to natural and synthetic xenobiotics. In this study, non-synonymous mutations in 21 ABC transporter genes were identified. Then, computational tools were employed to investigate the structural and functional consequences of a non-synonymous mutation in the protein encoded by the ABC transporter gene LOC411997. Protein structures were generated from a FASTA file using AlphaFold3, converted from mmCIF to PDB format, and visualised in PyMOL, where mutations were introduced. Functional site predictions were performed using Proteins Plus, and molecular dynamics simulations were conducted in YASARA to assess stability and conformational changes. Our findings suggest that a mutation that replaces threonine with isoleucine alters the protein dynamics by modifying its energy landscape and stability. The total potential energy of the wild-type protein was calculated as -3,640,842 kcal/mol. In contrast, this value increased to -3,443,695 kcal/mol for the mutant protein. This difference suggests that the mutation may affect conformation, flexibility, and biological function of the protein encoded by the ABC transporter gene LOC411997. Understanding these conformational changes at the molecular level will contribute to strategies for improving honey bee’s resilience and conservation.
- Conservation status of the honey bee subspecies native to the Mediterranean islandsPublication . Henriques, Dora; Yadró Garcia, Carlos A.; Yadró García, Carlos A.; Mangion, Marion Zammit; Galea, Thomas; Cilia, Giovannni; Nanetti, Antonio; Muz, Mustafa Necati; Muz, Dilek; Varnava, Andri; Hatjina, Fani; Charistos, Leonidas; Rufino, José; Martín Hernández, Raquel; Pinto, M. AliceThe Mediterranean islands have been a stage for honey bee diversification, hosting four of the 31 recognized subspecies: Apis mellifera siciliana in Sicily, A. m. ruttneri in Malta, A. m. cypria in Cyprus, and A. m. adami in Crete. However, owing to small population sizes, island subspecies are particularly vulnerable to introgressive hybridization when exotic queens or colonies are introduced into their geographically isolated territories. While previous genetic surveys—typically based on mitochondrial and/or microsatellite markers—have provided valuable insights, the current conservation status of these subspecies remains uncertain. Here, we sequenced the whole genomes of 327 samples collected from Sicily (n=97), Malta (n=79), Cyprus (n=37), and Crete (n=36), along with probable source populations (A. m. intermissa, A. m. sahariensis, A. m. media, A. m. anatoliaca, A. m. macedonica, A. m. cecropia, A. m. ligustica, A. m. carnica, A. m. caucasia), to provide a comprehensive and up-to-date assessment of the ancestry and diversity patterns of these insular subspecies. Whole-genome data were analysed using the software Admixture and additional analytical tools to explore ancestry and introgression patterns. For A. m. siciliana, 32 out of 97 samples (32.99%) exhibited high purity levels based on a threshold of 0.9. The situation in Malta was more concerning, with only 11 out of 79 samples (13.92%) classified as pure A. m. ruttneri. The most severe admixture was observed in Crete, where just 2 out of 36 samples (5.56%) were identified as pure A. m. adami, highlighting extensive hybridization with mainland subspecies. Meanwhile, A. m. cypria appears to be the best-preserved Mediterranean subspecies, with genetic integrity largely maintained except in the northwestern population, which exhibited shared ancestry with mainland A. m. anatoliaca. Overall, our findings highlight the urgent need for conservation efforts across these islands. While the native subspecies remain threatened by hybridization, their preservation might still be possible, provided that further importations of non-native colonies are effectively prevented.
- Contributos para a conservação dos ecossistemas costeiros e estuarinos: mitigação do impacto do consumo de água em piscinas e em instalações sanitárias.Publication . Geraldes, Ana Maria; Albuquerque, António; Silva, FloraOs processos ecológicos que ocorrem nos ecossistemas costeiros e estuarinos dependem fortemente do afluxo de água doce proveniente das bacias de hidrográficas. É este afluxo que influencia parâmetros com a entrada de sedimentos, a disponibilidade de nutrie ntes e a salinidade. No entanto, esta dinâmica tem sofrido profundas alterações, colocando em risco os serviços que estes ecossistemas proporcionam. Uma das causas destas alterações é o aumento constante do consumo de água para os mais diversos fins, causando uma rápida depleção dos recursos hídricos nas bacias hidrográficas. Urge assim, promover medidas de eficiência hídrica que reduzam as taxas de depleção dos recursos hídricos que atualmente se verificam. As piscinas públicas e residenciais têm um elevad o impacto no consumo de água nos espaços urbanos. Por outro lado, nas residências e nos edifícios públicos e comerciais são as instalações sanitárias que têm um maior peso no consumo de água potável. O objetivo desta apresentação é analisar, com base em casos de estudo, de que forma é possível reduzir os impactos que piscinas e instalações sanitárias têm no consumo de água. No caso das piscinas propõe-se que a água utilizada na lavagem dos filtros seja, após um tratamento relativamente simples, reutilizada para rega de jardins e outros fins não potáveis. Para mitigar os consumos de água nas instalações sanitárias é proposta a utilização de águas pluviais que são recolhidas em reservatórios que podem ser instalados nos respetivos edifícios.
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