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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.
- Viral genetic landscape in the honey bee populations of the Azores archipelagoPublication . Lopes, Ana; Miranda, Joachim; Martín-Hernández, Raquel; Pinto, M. AliceHoney bee decline can be attributed to many stressors, including pathogens and parasites, such as varroa and its vectored viruses. The global honey bee trade poses an important risk of pathogen dissemination. The arrival of varroa in the Azores because of (illegal) queen importations is clear evidence of that. Varroa was Ƥrst detected on Pico in 2000, on Flores in 2001, and on Faial in 2008. The other six islands of the archipelago remain mite-free. Therefore, the Azores provide an interesting setting to study the impact of varroa in the viral landscapes, adding to previous studies. In July/August of 2014/2015 we sampled 402 colonies distributed across 8 islands. These were screened for DWV, BQCV and LSV by RT-qPCR and the positive samples were further examined by high-throughput sequencing (HTS) to uncover whether varroa has altered the viral genetic composition. Of the 8 islands, São Jorge and Terceira had no DWV-positive colonies and Pico and Flores showed the highest DWV prevalence. BQCV and LVS were detected on all the islands. The HST data indicated that DWV-A, -B, and -C variants were all present in the Azores, although with varying prevalences (DWV-A: 56%; DWV-C: 31.2%; DWV-B: 12.2%). The phylogenetic tree revealed a geographic pattern, in which most of the DWV variants from São Miguel and Santa Maria formed a clade with the DWV-C reference, whereas Pico, Faial, Flores, and Graciosa clustered with the DWV-A reference. Interestingly, the DWV-A sequences from Pico were more closely related with those from Faial than with those from Flores. If the DWV-A variant was introduced with varroa, then the illegal queen imports on Pico and Flores had independent origins, whereas the DWV-A variants on Faial might have originated from Pico. DWV-A and B have acquired a world-wide distribution in the wake of varroa, while variants DWV-C and DWV-D have become extremely rare, one plausible scenario is that DWV-C was one of the original genotypes in the Azores predating varroa and has been replaced by the variants A and B, due to their superior adaptation to varroamediated transmission. Also of interest is the distinct clade formed by the BQCV and LSV sequences of São Miguel and Santa Maria, as well as the independent cluster formed by Flores sequences supporting the previous scenario. All these Ƥndings will be discussed in this communication.
- Varroa destructor shapes the unique viral landscapes of the honey bee populations of the Azores archipelagoPublication . Lopes, Ana Rita; Low, Matthew; Martín-Hernández, Raquel; Miranda, Joachim; Pinto, M. AliceThe worldwide dispersal of the ectoparasitic mite Varroa destructor from its Asian origins has fundamentally transformed the relationship of the honey bee (Apis mellifera) with several of its viruses, via changes in transmission and/or host immunosuppression. The extent to which honey bee-virus relationships change after Varroa invasion is poorly understood for most viruses, in part because there are few places in the world with several geographically close but completely isolated honey bee populations that either have, or have not, been exposed long-term to Varroa, allowing for separate ecological, epidemiological, and adaptive relationships to develop between honey bees and their viruses, in relation to the mite's presence or absence. The Azores is one such place, as it contains islands with and without the mite. Here, we combined qPCR with meta-amplicon deep sequencing to uncover the relationship between Varroa presence, and the prevalence, load, diversity, and phylogeographic structure of eight honey bee viruses screened across the archipelago. Four viruses were not detected on any island (ABPV-Acute bee paralysis virus, KBV-Kashmir bee virus, IAPV-Israeli acute bee paralysis virus, BeeMLV-Bee macula-like virus); one (SBV-Sacbrood virus) was detected only on mite-infested islands; one (CBPV-Chronic bee paralysis virus) occurred on some islands, and two (BQCV-Black queen cell virus, LSV-Lake Sinai virus,) were present on every single island. This multi-virus screening builds upon a parallel survey of Deformed wing virus (DWV) strains that uncovered a remarkably heterogeneous viral landscape featuring Varroa-infested islands dominated by DWV-A and -B, Varroa-free islands na & iuml;ve to DWV, and a refuge of the rare DWV-C dominating the easternmost Varroa-free islands. While all four detected viruses investigated here were affected by Varroa for one or two parameters (usually prevalence and/or the Richness component of ASV diversity), the strongest effect was observed for the multi-strain LSV. Varroa unambiguously led to elevated prevalence, load, and diversity (Richness and Shannon Index) of LSV, with these results largely shaped by LSV-2, a major LSV strain. Unprecedented insights into the mite-virus relationship were further gained from implementing a phylogeographic approach. In addition to enabling the identification of a novel LSV strain that dominated the unique viral landscape of the easternmost islands, this approach, in combination with the recovered diversity patterns, strongly suggests that Varroa is driving the evolutionary change of LSV in the Azores. This study greatly advances the current understanding of the effect of Varroa on the epidemiology and adaptive evolution of these less-studied viruses, whose relationship with Varroa has thus far been poorly defined.
- Origins, diversity, and adaptive evolution of DWV in the honey bees of the Azores: the impact of the invasive mite Varroa destructorPublication . Lopes, Ana Rita; Low, Matthew; Martín-Hernández, Raquel; Pinto, M. Alice; Miranda, JoachimDeformed wing virus (DWV) is a honey bee virus, whose emergence from relative obscurity is driven by the recent host-switch, adaptation, and global dispersal of the ectoparasitic mite Varroa destructor (a highly efficient vector of DWV) to reproduction on honey bees (Apis mellifera). Our study examines how varroa affects the continuing evolution of DWV, using the Azores archipelago, where varroa is present on only three out of the eight Islands, as a natural experimental system for comparing different evolutionary conditions and trajectories. We combined qPCR of 494 honey bee colonies sampled across the archipelago with amplicon deep sequencing to reveal how the DWV genetic landscape is altered by varroa. Two of the varroa-free Islands were also free of DWV, while a further two Islands were intriguingly dominated by the rare DWV-C major variant. The other four Islands, including the three varroa-infested Islands, were dominated by the common DWV-A and DWV-B variants. The varroa-infested Islands had, as expected, an elevated DWV prevalence relative to the uninfested Islands, but not elevated DWV loads, due the relatively high prevalence and loads of DWV-C on the varroa-free Islands. This establishes the Azores as a stable refuge for DWV-C and provides the most convincing evidence to date that at least some major strains of DWV may be capable of not just surviving, but actually thriving in honey bees in the absence of varroa-mediated transmission. We did not detect any change in DWV genetic diversity associated with island varroa status but did find a positive association of DWV diversity with virus load, irrespective of island varroa status.
- 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.
- 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.
- 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.
- Vírus: os inimigos invisíveis da abelha (Apis mellifera L.)Publication . Lopes, Ana; Henriques, Dora; Pinto, M. AliceNas últimas décadas, a abelha melífera (Apis mellifera L.), tem sido sujeita a várias pressões, tais como, alterações climáticas, perda e fragmentação de habitat, o uso intensivo de agroquímicos e pesticidas, entre outros, que afetam a sua nutrição e a sanidade comprometendo assim a sua a longevidade. Aliado a todos estes fatores, a introdução de predadores (e.g. Vespa velutina), parasitas (e.g. Varroa destructor) e patógenos (e.g. Nosema ceranae) de outros continentes e a interação entre eles com o hospedeiro tem culminado em enormes perdas de colónias a nível mundial. O ácaro ectoparasita Varroa destructor e o fungo Microsporidia Nosema spp. (fungos parasitas unicelulares e formadores de esporos) têm sido apontados como um os principais agentes causadores de mortalidade das colónias. Porém, nos últimos anos as viroses têm vindo a ocupar um lugar de destaque, especialmente devido à estreita associação da varroa ao vírus das asas deformadas (DWV). Os vírus são parasitas intracelulares obrigatórios, uma vez que só conseguem reproduzir-se no interior de uma célula do hospedeiro. Tipicamente medem menos de 0,2 µm, e por isso não podem ser visualizados no microscópio ótico, sendo frequentemente identificados a partir de sintomas específicos que provocam na abelha, como é o caso da deformação das asas causada pelo DWV. Até ao momento, foram isolados mais de 50 vírus na abelha (Beaurepaire et al., 2020) dos quais 24 foram descritos por Bill Baley e Brenda Ball entre os anos 60 e 80 no Reino Unido (de Miranda et al., 2013). A maioria dos vírus das abelhas têm como material genético ARN (ácido ribonucleico), sendo que apenas o vírus filamentoso da Apis mellifera (AmFV) e o vírus iridescente Apis (AIV) têm ADN (ácido de desoxirribonucleico; de Miranda et al., 2013).
- Comparing two in-house developed SNP assays for inferring population structure in the honey bee (Apis mellifera L.)Publication . Henriques, Dora; Lopes, Ana; Pinto, M. AliceThe honey bee, Apis mellifera L., is under pressure globally due to several factors, one of them is the large-scale introduction of foreign queens and/or colonies which act as vectors of pathogens, and also threaten the genetic integrity of native populations. Different molecular tools have been developed to monitor the genetic integrity of the populations. SNPs (Single Nucleotide Polymorphism) have been preferred because are easily transferred between laboratories, have a low genotyping error, provide high-quality data, and are suitable for automation. Here, we compared the genotyping results obtained with two medium-density-SNP assays previously developed. One of assays was designed from 88 whole genomes of Apis mellifera iberiensis and 44 C-lineage individuals (the main ancestry of commercial bees) using fixed SNPs (FST=1) distributed in the 16 honey bee chromosomes. The other assay was designed from variation in immune genes using a discovery panel of 123 whole genomes, representing seven subspecies (A. m. iberiensis, A. m. mellifera, A. m. intermissa, A. m. sahariensis, A. m. ligustica, A. m. carnica, A. m. siciliana and three lineages (A, M and C). All the samples are from the native range of each subspecies and they were taken from inside the hives, placed in absolute ethanol and stored at -20ºC until DNA extraction. The tools were compared using 473 samples from the Azores, which harbour a genetically complex honey bee population. The samples were genotyped using the iPLEX MassARRAY® MALDI-TOF system. The membership proportions of each individual (Qvalue) were calculated using ADMIXTURE considering two genetic groups (K=2), with 10,000 iterations in 20 independent runs. Our results show that both assays provide similar Q-values, with a Pearson’s correlation of 0.89. Only 9.5% of the samples have an absolute Q-value difference > 0.10. The choice of the best SNP assay depends on the subspecies and the aim of the project. While the immune assays can be applied in different subspecies the other assay was specifically designed for A. m. iberiensis. Furthermore, if there is disease data available, the immune assay can
- Comparative Analysis of DNA Extraction Methods for Individual Varroa destructorPublication . Costa, Maíra; Lopes, Ana; Yadró Garcia, Carlos A.; Vitrio, Nathalia; Gonçalves, Telma; Pinto, M. Alice; Henriques, DoraThe ectoparasitic mite Varroa destructor is one of the major honey bee threats and it is associated to population worldwide decline. Genetic analyses using the mtDNA of V. destructor are fundamental for establishing the taxonomy and distribution of the mites. Consequently, low-quality DNA can lead to inaccurate or inconsistent data, making genetic interpretation more challenging. In this study, we compared the concentration and quality of DNA extracted from individual female V. destructor using two different commercial kits, aiming to identify the optimal method for obtaining high-quality DNA. Total DNA was extracted from mites using both an automated and manual extracted method. In addition, manual kit extraction tested three incubation procedure (1h, 5h, and overnight). DNA concentration was quantified using three different instruments: the SpectroStarVR Nano LVis Plate spectrophotometer, the Quantus™ Fluorometer apparatus, and NanoDrop™. The manual extraction DNA concentration did not vary across incubation times and the concentration values varied between 0.240-0.545 ng/μl (Quantus), 0.72-4.49 ng/μl (spectrophotometer), and 0.0-1.47 ng/μl (NanoDrop). While extraction automatic approach yielded higher respectively 0.483-0.631 ng/μl, 20.33-9.0 ng/μl, and 5.3-6.8 ng/μl. In conclusion, the automated kit extraction seems to be the best extraction method since it produced the higher-concentration DNA using only one individual mite.