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- 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.
- Distribution of pyrethroid and amitraz resistance in Varroa destructor mites from honey bee (Apis mellifera) colonies in PortugalPublication . Costa, Maíra; Yadró Garcia, Carlos A.; Rodrigues, Cláudia; Lopes, Ana Rita; Pérez-Pérez, Antonio; Martín Hernández, Raquel; Higes, Mariano; Pinto, M. Alice; Henriques, DoraHoney bees (Apis mellifera) are widely used for pollination and honey production worldwide. However, they face a serious threat from the ectoparasitic mite Varroa destructor. This parasite causes varroosis and transmits multiple viruses, such as the Deformed wing virus (DWVV), compromising the health and survival of colonies. While there are many acaricides commercially available to treat colonies against varroa, the recurrent use of certain molecules is leading to the development of varroa resistance. In Portugal, two classes of synthetic compounds are currently used: pyrethroids (fluvalinate and flumethrin) and formamidines (amitraz). However, the excessive and repeated use of acaricides has led to the development of resistance in mite populations. Pyrethroid resistance is linked to mutations in the Voltage-Gated Sodium Channel (VGSC) gene, with amino acid substitutions, at position 925 and 918. Amitraz resistance is associated with mutations in the Octopamine-like β-adrenergic receptor (Octβ2R). The amino acid changes include asparagine (N) to serine (S) at position 87 (N87S), observed in France; tyrosine (Y) to histidine (H) at position 215 (Y215H), in the USA; phenylalanine (F) to leucine (L) at position 290 (F290L), in Spain; and tyrosine (Y) to phenylalanine (F) at position 337 (Y337F), in Turkey. The distribution of resistance alleles has not yet been explored in Portugal. To fill this data gap, Varroa mites from honey bee colonies across various regions of the country were analyzed through DNA extraction, PCR with specific primers, and Sanger sequencing. The results confirmed the of widespread distribution of resistance alleles to pyrethroids at positions 925 and 918, like those observed in Spain. In contrast, preliminary results did not detect any amitraz resistance alleles, although the small sample size warrants caution in interpretation. This study represents an important step in understanding V. destructor resistance to chemical treatments in Portugal, providing essential data for monitoring acaricide resistance and supporting the development of more effective management strategies to help beekeepers better protect their colonies.
- Evolution of amitraz resistance in varroa destructor: historical assessment of the F290L mutation in iberian populationsPublication . Costa, Maíra; Pérez-Pérez, António; Santos, Ana Clara; Prado, Rafaela; Lopes, Doralinda; Lopes, Ana Rita; Yadró Garcia, Carlos A.; Martín-Hernández, Raquel; Higes, Mariano; Pinto, M. Alice; Henriques, DoraVarroa destructor is one of the main parasites of the honey bee (Apis mellifera), causing significant colony losses worldwide. The control of this mite often relies on acaricides, with amitraz being among the most widely used products in Portugal and Spain over the last decade. However, the continuous and persistent use of the same active substance exerts strong selective pressure on parasite populations, potentially favouring alleles associated with treatment resistance. Recently, a point mutation resulting in a phenylalanine-to-leucine substitution at position 290 (F290L) was identified in V. destructor populations from Spain, putatively associated with amitraz resistance. This mutation has been detected at high frequency in recent samples, suggesting resistance may have evolved due to sustained amitraz use. To assess whether the F290L mutation was already present before the widespread amitraz adoption, DNA extraction and sequencing analysis will be performed on mites collected since 2006, prior to the massive use of this treatment in Iberian beekeeping. This approach will clarify whether the allele frequency of F290L increased because of the selective pressure exerted by amitraz or if it already existed in high frequencies in earlier populations. This study will contribute to a better understanding of the evolution of resistance and to the development of more effective monitoring and management strategies to combat V. destructor.
- Improving soil health through nature-based solutions: the effects of olive-pomace-based compostsPublication . Royer, Ana Caroline; Lado, Marcos; Fonseca, Felícia; Hernandez Hernandez, Zulimar; Figueiredo, Tomás deLoss of soil organic matter (SOM) is a major issue in Mediterranean regions, especially in NE Portugal, where about 70% of soils are classified as Leptosols. On the other hand, olive groves dominate the region's farmland, and the olive oil industry generates large quantities of by-products. Two-phase olive pomace (OP) has a high organic load and is phytotoxic due to its high phenolic content. Composting OP with other agri-residues offers a nature-based solution that addresses untreated OP disposal challenges while recycling nutrients and supporting the circular economy. This study evaluates the effects of olive pomace-based composts (OPC) on soil physical and chemical properties. OPC was produced by composting OP with sheep manure and almond shells. In a pot trial, 3 OPCs produced with different %OP (OPC44, OPC31 and OPC25) and a commercial organic corrective (HMC) were incorporated in the 0-5cm layer of an eutric Leptosol (2.1% OM) at three doses (10, 20, 40 t.ha-1) + control. Pots were seeded with Italian ryegrass (Lolium multiflorum) and watered to keep soil moisture above 70% field capacity. After 138 days trial, soil properties were analysed for SOM and its physical fractionation, total C and N, extractable K, P Olsen, pH, effective cation exchange capacity (CEC), bulk density, porosity, field capacity, and aggregate stability. Multivariate analysis showed that compost dose had a stronger influence on soil properties than compost type. Linear regression revealed that SOM, total C, extractable K, pH and effective CEC increased proportionally with dose. The highest dose led to SOM and total C increases of 13.3 and 8.4 g·kg⁻¹, respectively. Compared to HMC, OPCs were better K sources. Organic amendment decreased bulk density (by 8-15%) and increased soil porosity, field capacity, and improved aggregate stability by ~20% (particularly with OPC31 and OPC44). OPC application also enhanced both physical and chemical protection of SOM. C content was highest in macroaggregates, stored as unprotected and coarse particulate OM. Higher doses — especially of OPC31 and OPC44 — promoted larger C accumulation in strongly physically and chemically protected pools, as compared to control and HMC. OPCs application increased mineral-associated OM, likely due to their colloidal nature, supporting long-term SOM stabilization. This study highlights the potential of OPC to improve SOM content, fertility, and soil structure in degraded Mediterranean soils. Composting OP provides a sustainable approach to valorise agri-waste, enhance soil functions, and support climate-smart agriculture.
- Incense honey (Pittosporum undulatum) in the Azores: Botanical authentication by using real-time PCR approachPublication . Lopes, Ana Rita; Moura, Monica B.M.V.; Grazina, Liliana; Costa, Joana; Amaral, Joana S.; Pinto, M. Alice; Mafra, IsabelHoney is a widely consumed food and much appreciated for its nutritional, organoleptic, and health properties. The honey produced in the Azores archipelago can be categorised as incense honey or multifloral honey. Incense honey classification should account with >30% from Pittosporum undulatum pollen grains and it is generally perceived as a unique and high-quality product and, consequently, susceptible to be adulterated through incorrect labelling or admixing with low-cost and low-quality honeys. Therefore, assessing the authenticity of such highly appreciated honey is a key issue for its valorisation. In this work, a real-time PCR method targeting the ITS region was proposed for the first time to detect P. undulatum species. To that end, the DNA extracted from the leaves of P. undulatum from other endemic species (e.g. Eucaliptus spp., Acacia spp., Trifolium spp., Castanea sativa Mill., Hydrangea macrophylla, Rhododendro indicum, Hedychium gardnerianum, Pericallis malvifolia) of the Azores and mainland Portugal was used to test the cross-reactivity of the ITS primers by qualitative PCR, revealing full specificity for P. undulatum. Posteriorly, a realtime PCR approach was proposed, exhibiting high analytical performance (PCR efficiency= 97.4 ± 6.4%, R͖= 0.991 ± 0.003) and a limit of quantification of 0.01 pg of incense DNA. The method was successfully applied to the 22 honey samples, from which incense was detected in all 9 monofloral incense honeys and in 5 out of 10 multifloral samples from the Azores. Generally, the quantitative results for incense DNA were in good agreement with the melissopalynological data, showing that all samples, except two, were according to their labelled statements. Herein, a new, simple, cost-effective and reliable molecular approach was proposed to authenticate and valorise the Azores honey.
- Landscape-scale genomic responses of the western honey bee (Apis mellifera) to pesticide pressurePublication . Lima, Daniela; Li, Fernanda; Yadró Garcia, Carlos A.; Taliadoros, Demetris; Webster, Matthew; Rufino, José; Roessink, I.; Buddendorf, Bas; Van der Steen, Jozef; Murcia-Morales, María; Fernández-Alba, Amadeo R.; Graaf, Dirk C. de; Lopes, Ana Rita; Pinto, M. Alice; Henriques, DoraWidespread pesticide use associated with intensive agriculture has been proven to impact the Western honey bee (Apis mellifera). However, despite evidence of declines in survival, development, foraging efficiency, and overall colony health, the genomic underpinning of this abiotic stressor is largely unknown. This study involved 102 whole-genome sequences from honey bee colonies from across Europe, sampled under the Better-B project, to search for genetic correlations to pesticide exposure using Genomic-Environment Association (GEA) analyses. The environmental exposure data were collected within the framework of the INSIGNIA-EU project, which used APIStrips (in-hive pesticide-adsorbing strips) to quantify pesticide residues brought into the hive by foraging in many of the sampled colonies. The APISTRIP data were complemented by modelled exposure grids from PEST-CHEMGRIDS and agriculture zones from the CORINE Land Cover. Employing three complementary GEA approaches-SAMBADA (spatial analysis tool), LFMM (latent factor mixed models), and RDA (Redundancy Analysis)—we identified SNPs in the bee’s genome significantly correlated with agricultural pressure and pesticide use. Notably, several genes with known roles in detoxification and stress response, including venom carboxylesterase6 and CYP336A1, were highlighted. These variants point to molecular pathways targeted by agrochemical toxicity, offering insights into the consequences of pesticide use. Such findings may, in the future and upon further validation, help refine risk assessment frameworks, support the development of resilience-oriented breeding programs, and promote sustainable apicultural practices.
- Occurrence of aflatoxins in food commodities produced and consumed in Angola and MozambiquePublication . Matusse, Cláudio ; Tolentino, Aritson D.S.; Lucamba, Zelda; Afonso, Sandra; Venâncio, Armando; Bila, João; Macuamule, Custódia; Rodrigues, PaulaAflatoxins, the mycotoxins produced by molds from the genus Aspergillus sect. Flavi that infest food and feed commodities, are a pressing issue in most African countries and their presence has led to a surge in health problems and food insecurity. The lack of effective monitoring and control in the Portuguese-Speaking African Countries (PALOP) has resulted in the underestimation of these mycotoxins. !e present work aimed to determine the occurrence of total aflatoxins (AFs) in several agricultural products – corn, beans, cassava flour, peanuts and rice – produced and consumed in the provinces of Cuanza Sul, Angola, and of Chongoene and Gaza, Mozambique. During 2023 and 2024, 236 samples (96 from Angola and 140 from Mozambique) were collected from local markets and analyzed for AFs using the lateral flow strip AgraStrip® Pro WATEX® (Romer) method. For Mozambique, 44% of all samples were positive for AFs (median=4.9 µg/kg). The highest incidence and contamination levels were found in corn, with all samples contaminated, ranging from the LOD to as high as 9200 µg/kg (median=26.9 µg/kg). Of these, 63% were contaminated above the Maximum Tolerable Limit (MTL) established by the Codex Alimentarius (15 µg/kg).
- Old and new climate change adaptation strategies by Montesinho mountain pastoralists, PortugalPublication . Aleixo-Pais, Isa G.; Castro, José; Frazão-Moreira, Amélia; Castro, João PauloMountain pastoralists are among the communities most affected by climate change, rendering empowerment of mountain communities and supportive policies essential.
- Plantas de pimento dulce (Capsicum annuum L.) em poscesacha como fuente de ingredientes antioxidantes y antidiabéticosPublication . Añibarro-Ortega, Mikel; López, Víctor; Dias, Maria Inês; Mandim, Filipa; Barros, Lillian; Pinela, JoséEl estrés oxidativo desempeña un papel fundamental en el desarrollo de enfermedades crónicas y metabólicas como la diabetes, enfermedades cardiovasculares y neurodegenerativas, obesidad y cáncer. Esto se debe a la producción excesiva de especies reactivas de oxígeno que dañan los lípidos, proteínas y ADN de los organismos, comprometiendo procesos celulares vitales [1]. Los antioxidantes naturales, como los polifenoles, pueden prevenir estos daños y hoy en día existe un creciente interés por el uso de extractos ricos en estos compuestos como ingredientes funcionales naturales. En el caso del cultivo de pimiento dulce (Capsicum annuum L.), tras la cosecha se genera una grande cantidad de biomasa vegetal infravalorada, que podría contener metabolitos secundarios de alto valor comercial. Así, el objetivo de este estudio fue valorizar estos subproductos agrícolas en línea con los objetivos de la Agenda 2030 de Desarrollo Sostenible de las Naciones Unidas. Para ello, los subproductos de la planta fueron empleados para preparar extractos hidroetanólicos [2], los cuales se caracterizaron con respecto a su perfil de compuestos fenólicos mediante HPLC-DAD-ESI/MSn y sus propiedades antioxidante, antidiabética, anti-obesidad, antiinflamatoria y citotóxica con base en ensayos celulares y enzimáticos in vitro. El análisis cromatográfico reveló un perfil fenólico con predominio de ácidos fenólicos, como el ácido clorogénico, y flavonas O-glicosiladas, concretamente luteolina y apigenina. Aunque los extractos presentaron cierta actividad en todos los ensayos realizados, donde más se destacaron fue en la actividad antioxidante, antidiabética y citotóxica en ciertas líneas celulares tumorales. Como conclusión, se puede afirmar que los subproductos de plantas de pimiento dulce son una prometedora fuente de compuestos bioactivos de alto valor añadido. Estos pueden tener propiedades beneficiosas para la salud del consumidor al ser incorporados en productos alimentarios. Además, fomentar esta práctica de circularidad será importante para la producción sostenible de ingredientes alimentarios naturales.
- Remoção de estrogénios por adsorção: Produção de adsorventes a partir de resíduos de cortiça e estudo cinéticoPublication . Exposto, Bruno Marques; Queiroz, Ana; Ribeiro, António E.; Brito, PauloOs poluentes emergentes são uma preocupação ambiental crescente no setor industrial. Estes compostos são detetados em concentrações cada vez superiores na natureza devido à sua persistência.1 Muitos poluentes emergentes são também considerados micropoluentes devido à sua perigosidade mesmo em concentrações muito baixas.