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- 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.
- Projeto BEEHAPPY: sanidade da abelha melífera (Apis mellifera L.) dos Açores: comparação dos padrões epidemiológicos num laboratório natural únicoPublication . Pinto, M. Alice; Henriques, Dora; Rodrigues, Pedro João; Miranda, Joachim; Martín-Hernández, Raquel; Amaral, Joana S.O BEEHAPPY é um projeto financiado pela Fundação para a Ciência e a Tecnologia (FCT), é coordenado pelo CIMO e envolve uma equipa de 16 especialistas multidisciplinares provenientes de várias instituições nacionais (CIMO-Centro de Investigação de Montanha, REQUIMTE-Rede de Química e Tecnologia, CEDRI -Centro de Investigação em Digitalização e Robótica Inteligente) e estrangeiras (SLU-Swedish University of Agricultural Sciences, CIAPA-IRIAF- Centro de Investigación Apícola y Agroambiental- Instituto Regional de Investigación y Desarrollo Agralimentario y Forestal, INIA-Peru- Instituto Nacional de Innovación Agraria). O BEEHAPPY centra-se nas populações de abelha dos Açores e tem como principais objectivos (i) o estudo da interação entre a abelha e as suas principais doenças (viroses e Nosemose) e (ii) o desenvolvimento de ferramentas com vista a identificar as populações de abelha (ferramenta informática) e avaliar a autenticidade do mel dos Açores (ferramenta molecular), o qual apresenta um grande potencial comercial. Para se atingirem tais objetivos, irão ser executadas 10 tarefas dedicadas à abelha (Tarefas 1, 4, 5, 6, 7, 8, 9), patogénios (Tarefas 1, 2, 3) e mel (Tarefa 10; Figura 1).
- 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.
- Standard methods and good practices in Apis honey bee omics researchPublication . Techer, Maeva A.; Chakrabarti, Priyadarshini; Caesar, Lílian; Eynard, Sonia E.; Farrell, M. Catherine; Foster, Leonard J.; Gorrochategui-Ortega, June; Henriques, Dora; Li-Byarlay, Hongmei; Morré, Jeffrey T.; Newton, Irene L. G.; Parejo, Melanie; Pinto, M. Alice; Vignal, Alain; Zarraonaindia, Iratxe; McAfee, AlisonIn the past decades, COLOSS members have joined forces multiple times to develop and condense standard methods related to research on honey bees, their pests, pathogens, and colony products. This led to the publication of four open-access BEEBOOK volumes that have been utilized by researchers worldwide. Among the chapters, “Standard methods for molecular research in Apis mellifera,” written by Evans and collaborators in 2013, has been a cornerstone for the standardization of honey bee molecular studies. However, since sequencing technologies and analyzing algorithms have made tremendous progress, many described methods require updating. In parallel, other Apis species’ genomes have now been sequenced, thus opening new research avenues in a comparative framework. In this chapter, we add to the methods previously covered by Evans et al. in 2013 and provide updated methodology where necessary, including worked examples and bioinformatic analysis pipe-lines. We also cover topics which were not previously covered in depth, such as sequencing ancient samples, population genomics, proteomics, and sampling honey bee colony products for microbiome studies, among others. Our hope is for this to become a lasting resource for honey bee scientists as the field continues to advance.
- Development of LAMP Primers for the Detection of Pyrethroid Resistance Mutations in Varroa destructorPublication . Costa, Maíra; Yadró García, Carlos A.; Lopes, Ana; Bejaoui, Mohamed Khalil; Almeida, Jhennifer; Correia, Lucas; Sánchez, Sara; Li, Fernanda; Pinto, M. Alice; Henriques, DoraVarroa destructor is one of the main threats to Apis mellifera L., directly affecting colony health and contributing to their global decline. Control of this mite is traditionally achieved using acaricides, with pyrethroids such as tau-fuvalinate and fumethrin being the most used, acting on voltage-gated sodium channels (VGSC). However, the intensive use of these compounds by beekeepers has led to the emergence of resistance, associated with mutations at residues 918 and 925 of the VGSC gene [1]. Traditional methods for detecting these mutations, such as PCR, TaqMan and RT-PCR, are eective but require expensive laboratory equipment. In this context, Loop-mediated Isothermal Amplification (LAMP) is a promising alternative, offering rapid and cost-effective detection without the need for thermal cycling [2]. LAMP is based on the use of a set of four to six primers, including two inner primers (FIP and BIP), two outer primers (F3 and B3), and optionally two loop primers (LoopF and LoopB), which are introduced to accelerate the amplification reaction [3]. This study aimed to develop specific LAMP primers, using the NEB LAMP software, for the detection of the main mutations associated with Varroa destructor resistance to pyrethroids in Portugal. The predictive efficiency, specificity, and thermodynamic properties of the designed primers were assessed using BLAST, eLAMP, and OligoAnalyzer tools, considering qPCR parameters. This work successfully identified specific primer sets, including loop primers, for the detection of the mutation at position 925, which may be used in future experimental validations for rapid diagnostic applications.
- Diversity patterns of P450 genes in 17 honey bee subspeciesPublication . Li, Fernanda; Yadró Garcia, Carlos A.; Rufino, José; Rosa-Fontana, Annelise; Verbinnen, Gilles; Graaf, Dirk C.; Smet, Lina de; Pinto, M. Alice; Henriques, DoraHoney bees (Apis mellifera) inhabit a vast geographical range, spanning diverse natural and agricultural ecosystems. They are exposed to different levels and types of natural (such as plant allelochemicals) and synthetic (such as pesticides) xenobiotics within this range. Several genes have been implicated in the resistance of insects to pesticides, including the P450 monooxygenases superfamily that contains 46 genes. Here, the sequences of P450 monooxygenases from >1500 individuals representing 17 subspecies of the four honey bee main lineages will be analyzed. The functional annotation and effects of each variant will then be predicted using SnpEff and the allele frequency and FST (fixation index) of each SNP per population and evolutionary lineages will be calculated. It is expected to have highly differentiated SNPs among the different subspecies/lineages.
- Bioinformatics pipeline to evaluate patterns of diversity in detoxification genes in Western Honey BeePublication . Barbosa, Daniela; Li, Fernanda; Bashir, Sana; Lopes, Ana; Yadró García, Carlos A.; Quaresma, Andreia; Rufino, José; Rosa-Fontana, Annelise; Verbinnen, Gilles; Graaf, Dirk C.; Smet, Lina de; 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.
- Genomic DNA extraction from honey bee (Apis mellifera) queen spermathecal contentPublication . Yadró, Carlos A.; Lopes, Ana Rita; Henriques, Dora; Musin, Eduard; Wegener, Jakob; Pinto, M. AliceGenetic analysis of the honey bee spermathecal content can be particularly useful to provide an estimate of the genetic diversity and purity of the surrounding populations. Here we compared the concentration and quality of DNA extracted from queen spermatheca using four commercial kits to determine the best method to obtain DNA suitable for single nucleotide polymorphism genotyping by next-generation sequencing. The four kits were tested with different adjustments in the lysis incubation time, use of RNA-carrier, elution conditions and number of re-elutions. Only the use of QIAamp DNA Microkit with 3 h of lysis incubation, the addition of RNA-carrier and multiple re-elutions produced a DNA concentration over the required threshold.
- Maternal diversity patterns of Ibero-Atlantic populations reveal further complexity of Iberian honeybeesPublication . Pinto, M. Alice; Henriques, Dora; Neto, Margarida; Guedes, Helena; Muñoz, Irene; Azevedo, João; De la Rúa, PilarDissecting complex diversity patterns typically exhibited by organisms of hybrid zones is among the greatest challenges in evolutionary biology. This has been a long-standing goal of numerous Iberian honeybee studies. Herein, we further contribute to this goal by a fine-scale maternal survey of the Portuguese honeybee populations. In addition to confirm a predicted African ancestry of Portuguese populations and negligible levels of C-lineage introgression, our fine-scale survey revealed unparalleled levels of African maternal diversity and a remarkable north–south cline formed by African haplotypes of distinct ancestry. This survey further highlights the distinctiveness of Apis mellifera iberiensis by showing the existence of highly contrasting diversity patterns between the Atlantic and Mediterranean populations. Our findings further support an ancient natural invasion of the Iberian Peninsula possibly by Apis mellifera intermissa and also by Apis mellifera sahariensis. Protecting this African legacy is of unquestionable importance, especially with Iberia becoming warmer and dryer.
- DNA-based methods as a powerful tool for the entomological authentication of honeyPublication . Honrado, Mónica; Quaresma, Andreia; Lopes, Ana; Pinto, M. Alice; Henriques, Dora; Amaral, Joana S.Honey is a food widely consumed worldwide and much appreciated for its nutritional, organoleptic and health properties. However, it is also considered one of the food products most prone to be adulterated in the EU. Up until now, honey authenticity addressed mainly the issue of sugars addition and botanical origin. Still, increased attention has recently been paid to honey entomological origin as it also relates to its geographical origin since honeybees carrying mitochondrial DNA (mtDNA) of distinct ancestries can be found across Europe. While in Portugal mtDNA of the autochthonous subspecies Apis mellifera iberiensis belongs to the African (A) lineage, in the northeastern part of Iberia African mitotypes are replaced by mitotypes of western European (M-lineage) ancestry. The native distribution of the M-lineage A. m. mellifera expands from the Pyrenees to Scandinavia and from the British Isles to the Ural Mountains while the C-lineage A. m. ligustica and A. m. carnica subspecies are naturally found in the Apennine and Balkan peninsulas, respectively [1]. Also, certain honeys holding the protected designation of origin (PDO) label should be produced by autochthonous A. mellifera subspecies, as mentioned in their EU geographical indications register.