Percorrer por autor "Higes, Mariano"
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- 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.
- BeeHeal: promoting bee health for sustainable agriculturePublication . Martín-Hernández, Raquel; Lopes, Ana; Chevjanovsky, Nor; Dalmon, Anne; Higes, Mariano; Le Conte, Yves; Pinto, M. Alice; Reyes-Carreño, Maritza; Soroker, VictoriaDuring the last years there is an alarming increase in the collapse of honeybee colonies (Apis mellifera) where bee parasites and pathogens like Varroa destructor mites, the microsporidia Nosema ssp. and viruses have played the leading role. Colony decline might compromise not only food security but also present and future income to the growers. Hence, four institutions from Mediterranean area have joined forces to put toghether a project entitled, “BEEHEAL: Promoting bee health for sustainable agriculture”. BEEHEAL is a collaborative research between Centro de Investigación Apícola y Agroambiental de Marchamalo - CAR (Spain), Centre de recherche Provence-Alpes-Côte d’Azur Unité: Abeilles et Environnement - INRA (France), Agricultural Research Organization, The Volcani Center - ARO (Israel) and Mountain Research Center (CMO), Polytechnic Institute of Bragança (Portugal). The aim of this project is to determine the phenology and interaction of the microsporidia Nosema ceranae and viruses including acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV), Black queen cell virus (BQCV) Chronic bee paralysis virus (CBPV) and Deformed wing virus (DWV), in Spain, France, Portugal and Israel. The findings of this project, which involves an active and unique cooperation among partners representing Mediterranean countries which encompasses a wide range of environmental and beekeeping management conditions, will contribute to ameliorate the damage caused by the expansion of N. ceranae through a rational implementation of existing treatments to avoid emergence of synergistic pathogens that accelerate colony collapse compromising food security. This project started at 2017 and it will end in 2020. BEEHEAL is funded through the ARIMNet2 (2016) Call by the following funding agencies: INIA (Spain), ANR (France), MOARD (Israel), and FCT (Portugal). This presentation will detail the tasks that are ongoing in the BEEHEAL project.
- Beeheal: standardization of laboratory methods for sample processing, nucleic acids extraction and PCR for microsporidia and viruses analysisPublication . Martín-Hernández, Raquel; Benito, M.; Chejanovsky, Nor; Le Conte, Yves; Dalmon, Anne; Higes, Mariano; Pinto, M. Alice; Reyes-Carreño, Maritza; Soroker, VictoriaBEEHEAL is a project designed to determine the phenology and interaction of Nosema ceranae and viruses in four Mediterranean countries: Spain, France, Portugal and Israel, including some territories where Varroa destructor is not present (Azores and Ouessant islands). This will allow us to study and compare the interactions between pathogens in a wide range of hosts, beekeeping and climatic conditions. The honey bee samples collected along the year in the different countries will be analysed for pathogens in three laboratories. This requires a standardization of methods to compare the results in order to assign the effect of every variable in a reliable way. To that end, the participating laboratories have been working together to establish the sampling methodology, the conservation of the samples, the nucleic acids extraction and the PCR analysis. We analyzed the sample processing for nucleic acid extraction on TE buffer (with or without Proteinase K), CTAB buffer or commercial kits (Qiagen). The maceration of bees (either individually or in composite samples) in TE buffer and posterior incubation at 96ºC for 20 minutes showed a good sensibility level and good value for N. ceranae DNA extraction. This method also allowed the conservation of RNA at -80ºC for a month in the TE solution for later RNA extraction. A joint protocol for sample processing, DNA and RNA extraction and PCR analysis has been developed but adjusted to the particular conditions and equipment of each laboratory. The standardization of methods to be implemented by each participating laboratory will avoid the biases on conclusions based on the diverse methods applied.
- Climate change-induced stress in the honey bee Apis mellifera L.- a genetic reviewPublication . Sagastume, Soledad; Cilia, Giovanni; Henriques, Dora; Yadró Garcia, Carlos A.; Corona, Miguel; Higes, Mariano; Pinto, M. Alice; Nanetti, Antonio; Martín-Hernández, RaquelClimate change is a powerful driver of stress, as it reinforces hotter and drier environments. For bees, the most concerning aspects of these new environmental conditions are the resistance and resilience of bees to changes in temperature, humidity and ultraviolet radiation, as well as the negative effect on diversity of food resources which can lead in nutritional stress. The climatic vulnerability of various bee species and subspecies varies worldwide, as they experience varying levels of stress and display distinct behaviors, weaknesses, and lifespans. To understand these differences, it is crucial to consider both the genetics and epigenetics of bees, as these factors play a key role in their response, resistance, and adaptation to new stressors. This review provides a guide of genetic and epigenetic markers involved in the cellular response of Apis mellifera to most common stressors derived from climate change. Understanding how the various molecular mechanisms interact to restore homeostasis during the stress response is essential for designing future studies based on molecular markers.
- Colonisation patterns of Nosema ceranae in the Azores ArchipelagoPublication . Lopes, Ana Rita; Martín-Hernández, Raquel; Higes, Mariano; Segura, Sara K.; Henriques, Dora; Pinto, M. AliceNosema ceranae is a highly prevalent pathogen of Apis mellifera, which is distributed worldwide. However, there may still exist isolated areas that remain free of N. ceranae. Herein, we used molecular tools to survey the Azores to detect N. ceranae and unravel its colonisation patterns. To that end, we sampled 474 colonies from eight islands in 2014/2015 and 91 from four islands in 2020. The findings revealed that N. ceranae was not only present but also the dominant species in the Azores. In 2014/2015, N. apis was rare and N. ceranae prevalence varied between 2.7% in São Jorge and 50.7% in Pico. In 2020, N. ceranae prevalence increased significantly (p < 0.001) in Terceira and São Jorge also showing higher infection levels. The spatiotemporal patterns suggest that N. ceranae colonised the archipelago recently, and it rapidly spread across other islands, where at least two independent introductions might have occurred. Flores and Santa Maria have escaped the N. ceranae invasion, and it is remarkable that Santa Maria is also free of Varroa destructor, which makes it one of the last places in Europe where the honey bee remains naive to these two major biotic stressors.
- Distribuión de Nosema ceranae en abejas de las Azores y MadeiraPublication . Martín-Hernández, Raquel; Higes, Mariano; Sanchez, Carmen; Neves, Cátia J.; Henriques, Dora; Chávez-Galarza, Julio; Muñoz, Irene; Azevedo, João; De la Rúa, Pilar; Pinto, M. AliceLa abeja de la miel (Apis mellifera) es considerada como el insecto polinizador más importante. Actualmente está sufriendo un gran impacto en sus poblaciones por el efecto de patógenos comos los microsporidios del género Nosema entre otros factores. Las islas atlánticas como las Azores e Madeira constituyen un laboratorio natural idóneo para estudiar la influencia del manejo apicola en la dispersión de estes patógenos.
- 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.
- Epidemiology of the microsporidium nosema ceranae in four mediterranean countriesPublication . Jabal-Uriel, Clara; Barrios, Laura; Bonjour-Dalmon, Anne; Caspi-Yona, Shiran; Chejanovsky, Nor; Erez, Tal; Henriques, Dora; Higes, Mariano; Le Conte, Yves; Lopes, Ana; Meana, Aranzazu; Alice Pinto, M.; Teixeira, Amílcar; Reyes-Carreño, Maritza; Soroker, Victoria; Martín-Hernández, RaquelNosema ceranae is a highly prevalent intracellular parasite of honey bees’ midgut worldwide. This Microsporidium was monitored during a long-term study to evaluate the infection at apiary and intra-colony levels in six apiaries in four Mediterranean countries (France, Israel, Portugal, and Spain). Parameters on colony strength, honey production, beekeeping management, and climate were also recorded. Except for São Miguel (Azores, Portugal), all apiaries were positive for N. ceranae, with the lowest prevalence in mainland France and the highest intra-colony infection in Israel. A negative correlation between intra-colony infection and colony strength was observed in Spain and mainland Portugal. In these two apiaries, the queen replacement also influenced the infection levels. The highest colony losses occurred in mainland France and Spain, although they did not correlate with the Nosema infection levels, as parasitism was low in France and high in Spain. These results suggest that both the effects and the level of N. ceranae infection depends on location and beekeeping conditions. Further studies on host-parasite coevolution, and perhaps the interactions with other pathogens and the role of honey bee genetics, could assist in understanding the difference between nosemosis disease and infection, to develop appropriate strategies for its control.
- 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.
- First detection of Nosema ceranae in honey bees (Apis mellifera L.) of the Macaronesian archipelago of MadeiraPublication . Lopes, Ana; Martín-Hernández, Raquel; Higes, Mariano; Segura, Sara K.; Henriques, Dora; Pinto, M. AliceThe Microsporidia Nosema ceranae is an invasive pathogen affecting honey bee health, particularly in warm climates. In this study, N. ceranae was detected for the first time in honey bees (Apis mellifera L.) of the Madeira archipelago, indicating that this pathogen is now spreading across the entire Macaronesia. Nosema apis was not detected, and the high prevalence (67.7%) of N. ceranae indicates its dominance over N. apis.