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- Development of LAMP Primers for the Detection of Pyrethroid Resistance Mutations in Varroa destructorPublication . Costa, MaĆra; YadrĆ³ GarcĆa, Carlos A.; Lopes, Ana; Bejaoui, Mohamed Khalil; Almeida, Jhennifer; Correia, Lucas; SĆ”nchez, 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 ešæææective 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.
- 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.
- IdentificaĆ§Ć£o de bacteriĆ³fagos com potencial no controlo da tuberculose da oliveiraPublication . Costa, MaĆra; Baptista, Paula; CameirĆ£o, Cristina Sofia Lopes; Nava, Gilmar AntĆ“nioPseudomonas savastanoi pv. savastanoi (Pss) Ć© a bactĆ©ria patogĆ©nica causal da doenƧa tuberculose-da-oliveira, sendo uma das principais responsĆ”veis pelas perdas significativas na produĆ§Ć£o e qualidade do olival em todo o mundo especialmente em paĆses do MediterrĆ¢neo. O seu ataque na oliveira ocorre na parte aĆ©rea, formando nĆ³dulos visĆveis no tronco, caule, folhas e frutos, nĆ£o existindo um tratamento especĆfico e eficaz quando estabelecida no olival. Ocasionando a necessidade no desenvolvimento de ferramentas inovadoras, seguras e eficazes para o seu controlo. Assim, neste trabalho pretendeu-se identificar bacteriĆ³fagos com potencial de serem usados contra a tuberculose. Devido Ć elevada especificidade dos fagos, procedeu-se numa primeira fase ao estudo da comunidade bacteriana endofĆtica associada a tumores e a ramos de oliveira sem sintomas com o intuito de constituir uma coleĆ§Ć£o de estirpes de Pss que serĆ” Ćŗtil no processo de seleĆ§Ć£o de bacteriĆ³fagos. O isolamento em cultura seguida pela identificaĆ§Ć£o dos isolados bacterianos atravĆ©s da sequenciaĆ§Ć£o da regiĆ£o 16S do rDNA permitiu identificar um total de 69 taxa, sendo Pantoea e Erwinia os gĆ©neros mais abundantes em caules assintomĆ”ticos, tendo destaque nos sintomĆ”ticos os gĆ©neros Pantoea e Curtobacterium. O mĆ©todo por maceraĆ§Ć£o gerou uma maior diversidade taxonĆ³mica que no mĆ©todo de explante, sendo nos caules sintomĆ”ticos que verificou maior diversidade e abundĆ¢ncia bacteriana relativamente aos caules assintomĆ”ticos. O isolamento de bacteriĆ³fagos em amostras de solo de olival permitiu identificar, pela primeira vez, um total de nove bacteriĆ³fagos com capacidade de causar a lise de Pss. A sua aĆ§Ć£o na luta contra a tuberculose deverĆ” ser avaliada no futuro com ensaios in planta. Quando avaliada a eficiĆŖncia dos bacteriĆ³fagos contra a Pss em fatias de cenoura biolĆ³gica, nĆ£o se obteve diferenƧas significativas, no entanto os resultados obtidos sugerem que os bacteriĆ³fagos podem ser considerados possĆveis agentes de biocontrolo contra a multiplicaĆ§Ć£o do patĆ³geno, necessitando, porĆ©m, de estudos complementares.
- 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.
- Resistance of varroa destructor to pyrethroids: a preliminary analysis in PortugalPublication . Costa, MaĆra; Lopes, Ana; YadrĆ³ Garcia, Carlos A.; Coelho, Marta; MartĆn-HernĆ”ndez, Raquel; Higes, Mariano; Pires, Sancia; Pinto, M. Alice; Henriques, DoraInfestations of Varroa destructor are closely associated with colony collapse in the honey bee (Apis mellifera). Currently, three classes of acaricidal compounds are utilized to combat these infestations: pyrethroids (fluvalinate and flumethrin), organophosphates (coumaphos) and formamidines (amitraz). The first cases of V. destructor resistance to pyrethroids were reported in Italy, followed by numerous instances of resistant populations worldwide. The molecular mechanism of V. destructor resistance to pyrethroids is well understood and primarily linked to an amino acid change at position 925, where a leucine (L) is typically found. At this position, three alleles have been described that confer resistance to pyrethroids, where leucine is substituted by valine (L925V), isoleucine (L925I), or methionine (L925M). A novel mutation at position 918 was described in a population from Spain, where the amino acid methionine, normally found at position 918, was replaced by leucine (M918L). In Portugal, gene variation associated with pyrethroid resistance remains unknown. To investigate this mechanism in V. destructor populations from Portuguese apiaries, DNA was extracted from individual mites. This DNA was PCR-amplified using the primers 273-IF_VD (5ā²-AAGCCGCCATTGTTACCAGA-3ā²) and 1973-IR_VD (5ā- CTGTTGTTACCGTGGAGCA-3ā²) and subjected to Sanger sequencing. The expected outcome is that mutations are found in Portugal at both positions 925 and 918, due to its geographical proximity to Spain. These results will contribute to the Portuguese beekeeping sector by facilitating the development of strategies for handling V. destructor resistance.
- 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.
- An unprecedented large-scale survey of honey bee mitochondrial diversity in Europe: c-lineage dominance and the need for conservation effortsPublication . Li, Fernanda; Costa, MaĆra; Lopes, Ana Rita; GonƧalves, Telma; Henriques, Dora; Quaresma, Andreia; YadrĆ³ Garcia, Carlos A.; Albo, Alexandre; BlažytÄ-ÄereÅ”kienÄ, Laima; Brodschneider, Robert; Brusbardis, Valters; Carreck, Norman L.; Charistos, Leonidas; Chlebo, Robert; Coffey, Mary F.; Dahle, BjĆørn; Danneels, Ellen; Dobrescu, Constantin; Dupleix-Marchal, Anna; Filipi, Janja; Gajda, Anna; Gratzer, Kristina; Groeneveld, Linn Fenna; Hatjina, Fani; Johannesen, Jes; Kolasa, Michal; Kƶrmendy-RĆ”cz, JĆ”nos; KovaÄiÄ, Marin; Kristiansen, Preben; Martikkala, Maritta; McCormack, Grace P.; MartĆn-HernĆ”ndez, Raquel; Pavlov, Borce; Pietropaoli, Marco; Poirot, Benjamin; Radev, Zheko; Raudmets, Aivar; RenĆ©-Douarre, Vincent; Roessink, Ivo; Å kerl, Maja Ivana SmodiÅ”; Soland-Reckeweg, Gabriele; Titera, Dalibor; Steen, Jozef van der; Varnava, Andri; VejsnƦs, Flemming; Webster, Matthew T.; Fedoriak, Mariia M.; Zarochentseva, Oksana; Graaf, Dirk C.; Pinto, M. AliceEurope is home to ten Apis mellifera subspecies, which belong to three mitochondrial lineages: the Western European (M), Eastern European (C), and African (A). However, the long-standing human-mediated movement of queens, primarily of C-lineage ancestry, has threatened the genetic integrity of many of these native subspecies through introgression and replacement. This has led to the establishment of conservation programs to recover the native lines in some European countries. The maternally-inherited mitochondrial DNA (mtDNA), particularly the highly polymorphic intergenic region tRNAleu-cox2, has been the marker of choice for assessing honey bee variation and introgression at large geographical scales. Herein, we will show the results of the tRNAleu-cox2 variation obtained from over 1200 colonies sampled across the range of the ten subspecies and covering 33 European countries. These revealed that apart from a few countries (Portugal, Spain, and Ireland) and isolated protected populations, European populations are predominantly dominated by C-lineage haplotypes, and many native subspecies exhibit a signature of C-derived introgression. In conclusion, this unprecedented survey of honey bee diversity across Europe underscores the concerning dominance of C-lineage genetic variation, highlighting the urgent need for strategic conservation efforts to preserve the native genetic diversity of Apis mellifera.
- 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.