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- Comparative Analysis of DNA Extraction Methods for Individual Varroa destructorPublication . Costa, MaĆra; Lopes, Ana; YadrĆ³ Garcia, Carlos A.; Vitrio, Nathalia; GonƧalves, 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 Quantusā¢ Fluorometer apparatus, and NanoDropā¢. The manual extraction DNA concentration did not vary across incubation times and the concentration values varied between 0.240-0.545 ng/Ī¼l (Quantus), 0.72-4.49 ng/Ī¼l (spectrophotometer), and 0.0-1.47 ng/Ī¼l (NanoDrop). While extraction automatic approach yielded higher respectively 0.483-0.631 ng/Ī¼l, 20.33-9.0 ng/Ī¼l, and 5.3-6.8 ng/Ī¼l. 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.
- Projeto MITE- Varroa e vĆrus transmitidos: MonItorizaĆ§Ć£o de muTaƧƵes e dEsenvolvimento de ferramentas moleculares inovadorasPublication . Henriques, Dora; YadrĆ³ Garcia, Carlos A.; Lopes, Ana; Costa, MaĆra; Rufino, JosĆ©; MartĆn-HernĆ”ndez, Raquel; Higes, Mariano; Silva, Dinis; Pinto, M. AliceO Ć”caro ectoparasita varroa (Varroa destructor), que causa a doenƧa varroose, e alguns dos vĆrus transmitidos, como o vĆrus das asas deformadas (Deformed wing vĆrus ā DWV), sĆ£o apontados como umas das mais importantes ameaƧas para a abelha melĆfera a nĆvel global. O controle mais eficaz da varroa envolve o uso de acaricidas, sintĆ©ticos ou orgĆ¢nicos. No entanto, o uso intensivo dos acaricidas sintĆ©ticos tem levado ao desenvolvimento de resistĆŖncia da varroa ao tratamento em muitas regiƵes do mundo, o que tem causada uma maior perda de colĆ³nias. A base molecular de alguns dos mecanismos de resistĆŖncia da varroa Ć s molĆ©culas sintĆ©ticas mais usadas (os piretroides formamidinas) foi descrita recentemente. Esta informaĆ§Ć£o, quando associada a testes genĆ©ticos de fĆ”cil implementaĆ§Ć£o, permite a monitorizaĆ§Ć£o das populaƧƵes de varroa o que poderĆ” ajudar na luta integrada contra a varroose. Ao contrĆ”rio do que acontece com a varroa, para os vĆrus nĆ£o hĆ” nenhum tratamento disponĆvel. Em Portugal continental, nĆ£o Ć© conhecida a distribuiĆ§Ć£o e prevalĆŖncia dos vĆrus mais importantes das abelhas. No entanto, compreender a distribuiĆ§Ć£o e disseminaĆ§Ć£o das doenƧas Ć© essencial ao desenvolvimento de estratĆ©gias adequadas ao seu controlo e contenĆ§Ć£o. Genericamente, neste projeto pretende-se verificar se existem em Portugal populaƧƵes de varroa portadoras dos alelos que conferem resistĆŖncia aos piretrĆ³ides e ao amitraz, e em caso afirmativo estudar a sua distribuiĆ§Ć£o geogrĆ”fica. Pretende-se tambĆ©m modernizar o setor apĆcola ao desenvolver-se ferramentas moleculares inovadoras que possam ser facilmente usadas na luta integrada contra a varroose e concomitantemente na deteĆ§Ć£o dos vĆrus associados, como o DWV.
- Challenges in varroosis control: preliminary investigation of amitraz resistance in varroa destructor in PortugalPublication . Costa, MaĆra; Lopes, Ana; YadrĆ³ Garcia, Carlos A.; GonƧalves, Mariana Lousada; Coelho, Liliana; Pires, Sancia; Pinto, M. Alice; Henriques, DoraVarroosis is a disease caused by the ectoparasitic mite Varroa destructor, identified as one of the most significant global threats to the honey bee (Apis mellifera). The most effective control of this mite is through synthetic or organic acaricides. However, the excessive and repeated use of synthetic acaricides has led to the development of resistance. Amitraz is a synthetic pesticide commonly used in the control of V. destructor, but resistance to this compound has been observed. Previous studies observed a substitution of asparagine by serine at position 87 (N87S) of the OctĪ²2R gene, associated with amitraz resistance in France, and a substitution of tyrosine by histidine at position 215 (Y215H), associated with amitraz resistance in the USA. Building upon this knowledge, we aim to implement the first screening in Portugal for mutations associated with V. destructor resistance to amitraz. Unlike several European countries and the USA, Portugal lacks information regarding gene variation implicated in V. destructor resistance to amitraz, as well as allelic frequencies and their geographical distribution. To investigate the resistance mechanism, primers were developed to amplify the two known target regions of amitraz in V. destructor. DNA was extracted from individual female varroa mites using a commercial extraction kit, and the obtained DNA was PCR-amplified with the developed primers, followed by Sanger sequencing. With the knowledge obtained, we hope to assist beekeepers in selecting the most suitable acaricide to manage V. destructor in their apiaries and gain a deeper understanding of amitraz resistance in Portugal.
- Vespa velutina nigrithorax intracolonial microsatellite diversity: comparison between nests of the founder and leading edge populations in PortugalPublication . Costa, MaĆra; Quaresma, Andreia; Doblas-Bajo, MĆ³nica; Maamri, Sarra; Henriques, Dora; Pinto, M. AliceThe invasive yellow-legged hornet, Vespa velutina nigrithorax, is a voracious predator of honeybees which has been causing important economic losses to apiculture across Europe. It was first seen in 2004 in France, and it rapidly spread throughout the European countries. In 2011, V. v. nigrithorax was first reported in Portugal, in the region of Viana do Castelo, and since then it has been spreading throughout the country, with BraganƧa representing the edge of its distributional range in northeastern Portugal. The aim of this study was to compare the intra-colony genetic diversity of nests collected Viana do Castelo and BraganƧa. A total of 120 workers (sterile females) were sampled from two nests from Viana do Castelo and two from BraganƧa (30 worker per nest). Total DNA was extracted from the thorax of each individual using the NucleospinĀ® Tissue (Macherey-Nagel). The individuals were genotyped using 16 microsatellite loci divided into three multiplex combinations. The lengths of the fragments were determined using GeneMapper 3.7 (Applied Biosystems). Genetic diversity statistics, which included observed number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and unbiased expected heterozygosity (uHe) were estimated using GENALEX 6.5. This software was also implemented to assess genetic structure using a Principal Coordinate Analyses (PCoA). Our results show that both nests from both origins have low genetic diversity, with BraganƧa showing higher genetic diversity (Na=2; Ne= 1.600; Ho=0.427; He=0.330; uHe=0.333) than Viana do Castelo (Na=1.750; Ne=1.482; Ho=0.363; He=0.258; uHe=0.260). The genetic structure analysis shows that the two nest origins form two distinct groups. These results suggest that yellowlegged hornet population from the region of BraganƧa may be an expansion from multiple source populations.
- Distribution of Alleles Linked to Pyrethroid and Amitraz Resistance in Varroa destructor across PortugalPublication . Costa, MaĆra; YadrĆ³ Garcia, Carlos A.; Lopes, Ana; Quaresma, Andreia; Rodrigues, ClaĆŗdia; Pinto, M. Alice; Henriques, DoraVarroa destructor, an ectoparasitic mite of honey bees (Apis mellifera), is a significant threat to apiculture by causing varroosis and transmitting dangerous viruses such as the deformed wings virus. This mite can be controlled by the use of synthetic or organic acaricides. Currently, in Portugal, two classes of synthetic compounds are used: pyrethroids (fluvalinate and flumethrin), and formamidines (amitraz). However, excessive and repeated use of acaricides has led to resistance in mite populations. Resistance to pyrethroids is primarily associated with mutations in the voltage-gated sodium channel gene, and involves an amino acid change at position 925, where a leucine (L) is found in the wild type. At this position, three alleles have been described that confer Varroa resistance to pyrethroids, where leucine is replaced by valine (L925V), isoleucine (L925I), or methionine (L925M). A novel mutation at position 918 was described in a population in the Valencian Community in Spain, where the amino acid methionine, generally found at position 918, was replaced by leucine (M918L). For amitraz, resistance is linked to mutations in the OctĪ²2R gene, involving a substitution of asparagine by serine at position 87 (N87S), found in France, and a substitution of tyrosine by histidine at position 215 (Y215H), found in the USA. However, the distribution of these resistance-associated alleles remains unexplored in Portugal. To address this gap, we sampled Varroa mites from honey bee colonies from different locations in Portugal, and used a PCR assay with mutation-specific primers, followed by Sanger sequencing of the amplicons. The results confirmed pyrethroid resistance alleles at positions 925 and 918 in the Portuguese populations. In contrast, amitraz-resistance alleles remained undetected, although this result should be interpreted with caution as the sample size was reduced. These findings represent a step forward for understanding the status of Varroa resistance in Portugal, providing baseline data for the development of more effective, region-specific management strategies. Further research with larger sample sizes is essential to confirm these preliminary observations.
- Maternal ancestry of honey bees on SĆ£o Miguel-Azores (Portugal)Publication . Costa, MaĆra; Lopes, Ana; Doblas-Bajo, MĆ³nica; Quaresma, Andreia; Barreira, Diogo; Henriques, Dora; Pinto, M. AliceThe Apis mellifera iberiensis was introduced to the Azores in the 16th century by Portuguese settlers likely from the northern part of mainland Portugal. Many years later, in the 1980s, occurred a new introduction using honey bee subspecies from C-lineage maternal ancestry. This left a C-lineage signature in the Azores, with some islands, such as Pico or Graciosa, showing high frequency of C-lineage maternal ancestry as revealed by the study conducted in 2014/2015 (Ferreira et al. 2020). Hence, this study aimed to evaluate the C-lineage maternal signature on SĆ£o Miguel, seven years later (samples collected in 2023). A total of 192 colonies were sampled across the island and the total DNA was extracted from a pool of fifteen right front legs, were removed and placed in a 2.0-mL screwcap tube containing two zirconia beads of 2 mm diameter. The DNA extracts were then amplified using PCR targeting the tRNAleu-cox2 region of the mitochondrial DNA, followed by Sanger sequencing. The results show a prevalence of 64% for lineage A, contrasted with the 70% recorded in 2014/2015. Additionally, lineage C had a prevalence of 20%, compared to 27% in 2014/2015. In the A-lineage, the most prevalent haplotype was A14, while in the C-lineage, it was the C2 haplotype. Consequently, it can be inferred even almost 45 years after the C-lineage ancestry introduction, there are still a considerable frequency of this foreign maternal origin.
- Vespa velutina: using microsatellites to compare the genetic diversity of founding nests in the leading-edge populations in PortugalPublication . Costa, MaĆra; Quaresma, Andreia; Doblas-Bajo, MĆ³nica; Henriques, Dora; Pinto, M. AliceThe invasive yellow-legged hornet (Vespa velutina nigrithorax) is a voracious predator of honeybees and other pollinators, its diffusion generates relevant environmental and socio-economic impacts. Native from China, it rapidly started spreading throughout Europe, after its first sight in France in 2004. V. v. nigrithorax was first reported in Portugal, in the region of Viana do Castelo, in 2011 and since then it has been spreading through the country, with BraganƧa representing the Northeastern edge of its distribution in Portugal. The aim of this study was to compare the genetic diversity between nests collected in Viana do Castelo and BraganƧa. Four nests were analysed, two from Viana do Castelo and two from BraganƧa. Each sample nest is represented by 30 workers making up a total of 120 individuals analysed for this study. Total DNA was extracted from the thorax of each individual using the NucleospinĀ® Tissue (Macherey-Nagel). The individuals were genotyped using 16 microsatellite loci divided into three multiplex combinations. The lengths of the fragments were determined using GeneMapper 3.7 (Applied Biosystems). Genetic diversity statistics, which included observed number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and unbiased expected heterozygosity (uHe) were estimated using GENALEX 6.5. This software was also implemented to assess genetic structure using a Principal Coordinate Analyses (PCoA). Our results show that both nests from both origins have low genetic diversity, with BraganƧa showing higher genetic diversity (Na=2; Ne= 1.600; Ho=0.427; He=0.330; uHe=0.333) than Viana do Castelo (Na=1.750; Ne=1.482; Ho=0.363; He=0.258; uHe=0.260). The genetic structure show that distinct groups might be in the origin of both nestsā location. These results suggest that the population of Vespa velutina from the region of BraganƧa may be an expansion from multiple source populations.
- Projeto MITE- Varroa e vĆrus transmitidos: monItorizaĆ§Ć£o de muTaƧƵes e dEsenvolvimento de ferramentas moleculares inovadorasPublication . Henriques, Dora; YadrĆ³ Garcia, Carlos A.; Lopes, Ana Rita; Rufino, JosĆ©; Costa, MaĆra; Coelho, Marta; MartĆn-HernĆ”ndez, Raquel; Higes, Mariano; FederaĆ§Ć£o FenapĆcola; Pinto, M. AliceA abelha melĆfera (Apis melĆfera L.) tem sido ameaƧada por diferentes fatores, tais como pesticidas, alteraƧƵes climĆ”ticas, mĆ” nutriĆ§Ć£o, espĆ©cies invasoras tais como o predador Vespa velutina, a bactĆ©ria causadora da loque americana Paenibacillus larvae, e principalmente, o Ć”caro parasita Varroa destructor (vulgarmente conhecido como varroa), que Ć© apontado como umas das mais importantes ameaƧas para a abelha melĆfera a nĆvel global.
- Note: Cytonuclear patterns of a honey bee population from the Azores show a stable population at the nuclear but not at the mitochondrial DNA levelPublication . Henriques, Dora; Lopes, Ana Rita; Costa, MaĆra; Quaresma, Andreia; Doblas-Bajo, MĆ³nica; Pinto, M. AliceThe Azores archipelago has been the stage for multiple introductions of Apis mellifera from varying origins, which have led to widespread admixture and the existence of phenotypically and genotypically heterogeneous populations. This is evident on the SĆ£o Miguel Island, where the historically introduced black phenotype of A. m. iberiensis (lineage M) co-exists with the contemporaneously introduced yellow phenotype of C-lineage ancestry. Interestingly, the cytonuclear markers used herein revealed that C-lineage ancestry is residual at the nuclear level for both the black (5.82 Ā± 1.66%) and yellow (5.91 Ā± 1.85%) phenotypes, although this is more pronounced at the mitochondrial level (27.27% for black and 14.74% for yellow). While the C-lineage contribution has remained stable at the nuclear level for over 20 years, there has been a recent decrease in the proportion of C-derived mitotypes.
- An unparalleled survey of honey bee genetic diversity in Europe ā insights from wing shape, nuclear whole genome, and mitochondrial DNA dataPublication . Pinto, M. Alice; Li, Fernanda; Lopes, Ana Rita; Taliadoros, Demetris; Costa, MaĆra; YadrĆ³ Garcia, Carlos A.; Cunha, Larissa; Henriques, Dora; Martin Hernandez, Giselle; Albo, Alexandre; BlažytÄ-ÄereÅ”kienÄ, Laima; Brodschneider, Robert; Brusbardis, Valters; Carreck, Norman; Charistos, Leonidas; Chlebo, Robert; Cillia, Giovanni; Coffey, Mary F.; Dahle, Bjorn; Danneels, Ellen; Dobrescu, Constantin; Filipi, Janja; Gajda, Anna; Gratzer, Kristina; Groeneveld, Linn; Hatjina, Fani; Johannesen, Jes; Kolasa, Michal; Kƶrmendy-RĆ”cz, JĆ”nos; KovaÄiÄ, Marin; Kristiansen, Preben; Dupleix-Marchal, Anna; Martikkala, Maritta; McCormack, Grace; MartĆn-HernĆ”ndez, Raquel; Nanetti, Antonio; Pavlov, Borce; Pietropaoli, Marco; Poirot, Benjamin; Radev, Zheko; Raudmets, Aivar; RenĆ©-Douarre, Vincent; Roessink, Ivo; Å kerl, Maja Ivana SmodiÅ”; Soland, Gabriele; Titera, Dalibor; Van der Steen, Jozef; Varnava, Andri; VejsnƦs, Flemming; Graaf, Dirk C. de; Webster, MathewEurope is home to 10 subspecies of Apis mellifera, classified into four main lineages sensu Ruttner: M (western and northern European), C (southeastern European), A (African), and O (Middle Eastern). However, large-scale movements of honey bee genetic materialādriven by transhumance and, more importantly, queen tradingāhas intensified gene flow, particularly from the C-lineage subspecies A. m. carnica and A. m. ligustica, threatening the genetic integrity of several subspecies and blurring the genetic boundaries between subspecies. Here, we analyzed over 1,200 recently collected samples from apiaries in 33 countries using a multi-marker approach: maternally inherited mitochondrial DNA (tRNAleu-cox2 intergenic region), nuclear genome-wide single nucleotide polymorphisms (SNPs), and wing shape (DeepWingsĀ©) to provide the most updated and extensive account of honey bee genetic variation in Europe. Our results reveal a strong dominance of C-lineage ancestry across Europe, with noteworthy introgression signals in island subspecies and A. m. mellifera populations outside conservation apiaries. Notably, all three markers consistently captured these broad introgression patterns. This unprecedented survey highlights a worrying trend of genetic homogenization in European honey bee populations. Moreover, the widespread prevalence of C-lineage genetic introgression underscores the need for conservation efforts to preserve the diversity shaped by evolution, which is crucial for adaptation to growing environmental challenges.