Suchergebnisse

We assembled the mitogenome of Apis mellifera siciliana, which was previously identified as African by the tRNA-leu-cox2 intergenic region. The mitogenome is 16,590 bp long. The gene content and organization are identical to other A. mellifera mitogenomes, containing 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. Phylogenetic analysis showed a close mitochondrial relationship between A. m. siciliana and other African subspecies such as Apis mellifera sahariensis, Apis mellifera intermissa, and Apis mellifera ruttneri. ; This work was supported by MEDIBEES - Monitoring the Mediterranean Honey Bee Subspecies and their Resilience to Climate Change for the Improvement of Sustainable Agro-Ecosystems; BEEHAPPY ([POCI-01-0145- FEDER-029871]; FCT and COMPETE/QREN/EU). MEDIBEES is part of the PRIMA program supported by the European Union. Fundac¸~ao para a Ciência e a Tecnologia (FCT) provided financial support by national funds (FCT/MCTES) to CIMO [UIDB/00690/2020]. Dora Henriques is supported by the project BEEHAPPY ([POCI-01-0145-FEDER-029871]; FCT and COMPETE/QREN/EU). ; info:eu-repo/semantics/publishedVersion

This work has received funding from the Programa Nacional Apícola 2020-2022 under the project "AUTENT+ Desenvolvimento de abordagens inovadoras com vista à valorização e exploração do potencial de mercado do mel Português". The authors are also grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020). D. Henriques is supported by the project BeeHappy (POCI-01-0145-FEDER-029871) funded by FEDER (Fundo Europeu de Desenvolvimento Regional) through the program COMPETE 2020—POCI (Programa Operacional para a Competividade e Internacionalização), and by Portuguese funds through FCT and A.R. Lopes by the PhD scholarship funded by the FCT (SFRH/BD/143627/2019). ; Honey is a natural product widely consumed around the globe, not only for its taste and nutritional value, but also for its health benefits. Being a product of high dietary relevance and increasing demand, it has also become a target of economically motivated adulteration. According to the 2014 European Parliament report on the food crisis, fraud in the food chain and the control thereof, honey is among the 10 food products most prone of being adulterated [1]. Up until now, honey authenticity was mainly focused on the issues of sugars addition and botanical and geographical origin. However, recently an increased attention has been paid to the entomological origin of honey. To this aim, different approaches have been proposed to differentiate honey produced by different Apis mellifera subspecies, including those from distinct mitochondrial (mt) DNA lineages [2]. This work aimed to develop a novel real-time PCR method coupled with HRM analysis that allows for the simultaneous differentiation of honeybee from maternal lineages A, M and C, for further application in honey authentication. In this sense, data previously obtained from the mitogenomes of a total of 112 honeybees of different lineages were considered for the development of new DNA markers. Considering the aim of further application in honey, new primer sets were designed to amplify short fragments that included different single nucleotide polymorphisms (SNPs) allowing for HRM application. Three primer sets were proposed, amsCOI-F/amsCOI-R targeting the Cytochrome oxidase I (COI) gene, amsND1-F-amsND1-R targeting the NADH-ubiquinone oxidoreductase chain 1 (ND1) gene and amsCox3-F/amsCox3-R targeting the Cytochrome oxidase subunit III (Cox3) gene. Each primer set was first tested using qualitative PCR using DNA extracted from honeybees of A, M and C mtDNA lineages. After optimizing the real-time PCR conditions, each primer set was tested using a series of mtDNA extracted from honeybees. While amsCOI-F/amsCOI-R allowed only for the separation of the honeybees in two clusters, with lineage C and M clustering together, both the amsCox3-F/amsCox3-R and amsND1-F/amsND1-R set of primers allowed to differentiate the three lineages in separate clusters, with high level of confidence. As future work, the methodology will be assayed in commercial honey samples. ; info:eu-repo/semantics/publishedVersion

This volume contains the contributions of numerous participants at the IUFRO Landscape Ecology Working Group International Conference, which took place in Bragança, Portugal, from 21 to 24 of September 2010. The conference was dedicated to the theme Forest Landscapes and Global Change - New Frontiers in Management, Conservation and Restoration. The 128 papers included in this book follow the structure and topics of the conference. Sections 1 to 8 include papers relative to presentations in 18 thematic oral and two poster sessions. Section 9 is devoted to a wide-range of landscape ecology fields covered in the 12 symposia of the conference. The Proceedings of the IUFRO Landscape Ecology Working Group International Conference register the growth of scientific interest in forest landscape patterns and processes, and the recognition of the role of landscape ecology in the advancement of science and management, particularly within the context of emerging physical, social and political drivers of change, which influence forest systems and the services they provide. We believe that these papers, together with the presentations and debate which took place during the IUFRO Landscape Ecology Working Group International Conference – Bragança 2010, will definitively contribute to the advancement of landscape ecology and science in general. For their additional effort and commitment, we thank all the participants in the conference for leaving this record of their work, thoughts and science.

Single nucleotide polymorphisms (SNPs) have a great potential in genetic identification and introgression studies of honey bees (Apis mellifera). To achieve SNPs full power in genetic analysis, they must be screened in a rapid, accurate and cost-effective manner. Matrix-assisted laser desorption-ionization–time-of-flight (MALDI–TOF) mass spectrometry is a promising tool for the high-throughput screening of SNPs at an affordable cost in the post-genome sequencing era. In this study, a reduced SNP panel has been validated for Iberian honey bees (A. m. iberiensis) and a pooling strategy is presented for allele frequencies determination by using MALDI-TOF technology. The reduced SNP panel contains 127 most ancestry-informative loci design in the dark honey bee (A. m. mellifera). We demonstrate the utility of this methodology in samples of Iberian honey bees (A. m. iberiensis) selected for its pathogen tolerance and in genomic DNA pools. The described method reduces costs and time and enables genotyping of up to thousands of honey bee samples by taking advantage of the high-throughput MALDI-TOF technology. ; This research is funded through FEDER and INIA (E-RTA2014-00003-C03 and 01), Regional Government of Murcia (19908/GERM/2015, Fundación Séneca) and BeeHope project (138573-BiodivERsA/0002/2014). I. Muñoz acknowledges the support of a Saavedra Fajardo fellowship from the Fundación Séneca (20036/SF/16). ; info:eu-repo/semantics/publishedVersion

The expansion of Africanized honeybees from South America to the southwestern United States in <50 years is considered one of the most spectacular biological invasions yet documented. In the American tropics, it has been shown that during their expansion Africanized honeybees have low levels of introgressed alleles from resident European populations. In the United States, it has been speculated, but not shown, that Africanized honeybees would hybridize extensively with European honeybees. Here we report a continuous 11-year study investigating temporal changes in the genetic structure of a feral population from the southern United States undergoing Africanization. Our microsatellite data showed that (1) the process of Africanization involved both maternal and paternal bidirectional gene flow between European and Africanized honeybees and (2) the panmitic European population was replaced by panmitic mixtures of A. m. scutellata and European genes within 5 years after Africanization. The post-Africanization gene pool (1998–2001) was composed of a diverse array of recombinant classes with a substantial European genetic contribution (mean 25–37%). Therefore, the resulting feral honeybee population of south Texas was best viewed as a hybrid swarm.

The expansion of Africanized honeybees from South America to the southwestern United States in 50 years is considered one of the most spectacular biological invasions yet documented. In the American tropics, it has been shown that during their expansion Africanized honeybees have low levels of introgressed alleles from resident European populations. In the United States, it has been speculated, but not shown, that Africanized honeybees would hybridize extensively with European honeybees. Here we report a continuous 11-year study investigating temporal changes in the genetic structure of a feral population from the southern United States undergoing Africanization. Our microsatellite data showed that (1) the process of Africanization involved both maternal and paternal bidirectional gene flow between European and Africanized honeybees and (2) the panmitic European population was replaced by panmitic mixtures of A. m. scutellata and European genes within 5 years after Africanization. The post-Africanization gene pool (1998–2001) was composed of a diverse array of recombinant classes with a substantial European genetic contribution (mean 25–37%). Therefore, the resulting feral honeybee population of south Texas was best viewed as a hybrid swarm. ; PRODEP II - Medida 5/Acção 5.3

Unmanaged honey bee colonies of local ecotype surviving without human intervention are likely to form a valuable genetic resource for the sustainability of managed apiaries as well conservation of threatened subspecies. In Ireland, following the Isle of Wight disease (which devastated honey bee colonies at the beginning of the 20th century) and subsequent hybridisation with C lineage bees, there has been a general acceptance by government agencies, scientists, and many beekeepers that no Apis mellifera mellifera (Amm) colonies persisted in the wild. However, sporadic reports were received in 2014/2015 of the existence of unmanaged honey bee colonies. Given that Ireland's human population is low in density with only 32 persons per square km in some rural areas and only approximately 3000 registered beekeepers, many of whom are reported to not favour purchasing imported bees, it is feasible that honeybees could have naturally adapted to introduced pathogens such as Varroa destructor. We initiated an investigation into the state of unmanaged honey bee colonies and in 2016 we launched a nationwide request through press and social media seeking locations of unmanaged colonies which realised over 170 replies in a short time period. We found that unmanaged colonies have utilised a wide variety of both natural and artificial cavities and survived unaided for periods reported to be from three to over 20 years. Given the difficulty in confirming the authenticity of these timings the survival of individual colonies has been monitored since 2016. Sixty-two of the colonies were sampled and a combined approach using mitochondrial, microsatellite and single nucleotide polymorphism (SNP) genotyping has shown the majority to be pure Apis mellifera mellifera and forming an integral part of the previously described pure Amm population in Ireland. This data, along with survival records for >2 years, and details of surrounding habitat and health of the unmanaged colonies, will be presented. ; info:eu-repo/semantics/publishedVersion

Following the European Union (EU) legislation, honey should be produced by the western honey bee, Apis mellifera. Across Europe, 10 different A. mellifera subspecies can be found, comprising 3 different lineages (A, M and C) based on mtDNA [1]. In general, honey bees occupy allopatric geographical ranges according to their evolutionary lineages, allowing to establish an entomological origin for honey produced in different EU countries. Additionally, several honeys with protected designation of origin (PDO) detail the subspecies traditionally used in their production [2]. While numerous works focused on the botanical and/or geographical authenticity of honey, only a few have attempted its entomological authentication. For that purpose, DNA-based methods have been considered as the most suitable tools since they allow the unequivocal species identification. So far, only few works described the use of DNAbased methods to establish the entomological origin of honey [3,4] and those were focused on different species of honey bees, including Meliponini and/or Trigonini stingless bees. To our knowledge, this is the first attempt to distinguish among different European honey bee subspecies commonly used in honey production, with further application to honey authentication. In this work, DNA markers were developed for the differentiation of A. mellifera subspecies DNA in honey. For this purpose, individuals of A. m. iberiensis lineage A (n=22) from Portugal and Spain (n=5), A. m. iberiensis lineage M from Spain (n=7), A. m. mellifera lineage M from France, Netherlands, Scotland and Norway (n=7), A. m. ligustica lineage C from Italy (n=4), A. m. carnica lineage C from Croacia and Serbia (n=4) and commercial Buckfast lineage C bees (n=10) were tested. Different sets of primers were designed targeting the cytochrome oxidase I gene. The specificity and sensitivity of the designed primers were assayed by qualitative polymerase chain reaction (PCR). Species-specific primers successfully allowed the identification of A. m. iberiensis lineage A by end-point PCR. The use of real-time PCR coupled with High Resolution Melting analysis allowed the separation of A. mellifera honey bee subspecies in different clusters according to their lineages. The developed methodologies were applied to the analysis of authentic honey samples from Portugal (produced by A. m. iberiensis lineage A), Spain (produced by A. m. iberiensis lineage M), and Italy (produced by A. m. ligustica lineage C), allowing its successful entomological origin identification. ; This work has been supported by FCT (Fundação para a Ciência e Tecnologia) through project UID/QUI/50006/2013 – POCI/01/0145/FEDER/007265 with financial support from FCT/ MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 and by the project NORTE-01-0145-FEDER-000011. S. Soares and J. Costa are grateful to FCT grants (SFRH/ BPD/102404/2014 and SFRH/BD/75091/2010) financed by POPH-QREN (subsidized by FSE and MCTES). ; info:eu-repo/semantics/publishedVersion

The arrival to the United States of the Africanized honey bee, a hybrid between European subspecies and the African subspecies Apis mellifera scutellata, is a remarkable model for the study of biological invasions. This immigration has created an opportunity to study the dynamics of secondary contact of honey bee subspecies from African and European lineages in a feral population in South Texas. An 11-year survey of this population (1991-2001) showed that mitochondrial haplotype frequencies changed drastically over time from a resident population of eastern and western European maternal ancestry, to a population dominated by the African haplotype. A subsequent study of the nuclear genome showed that the Africanization process included bidirectional gene flow between European and Africanized honey bees, giving rise to a new panmictic mixture of A. m. scutellata- and European-derived genes. In this study, we examined gene flow patterns in the same population 23 years after the first hybridization event occurred. We found 28 active colonies inhabiting 92 tree cavities surveyed in a 5.14 km(2) area, resulting in a colony density of 5.4 colonies/km(2). Of these 28 colonies, 25 were of A. m. scutellata maternal ancestry, and three were of western European maternal ancestry. No colonies of eastern European maternal ancestry were detected, although they were present in the earlier samples. Nuclear DNA revealed little change in the introgression of A. m. scutellata-derived genes into the population compared to previous surveys. Our results suggest this feral population remains an admixed swarm with continued low levels of European ancestry and a greater presence of African-derived mitochondrial genetic composition.

According to the European Union legislation, honey is the natural sweet substance produced by Apis mellifera, also known as European honeybee. However, in other regions of the world, honey is traditionally obtained from other bee species. Among those, A. cerana (also known as Asian honeybee) is also of economic importance since it is used in apiculture. Due to the decline of the wild populations of the A. cerana in some countries, such as Japan and parts of China, there is an increasingly interest in preserving the native Asian honeybee, being its honey increasingly valued. Owing to the growing demand for this traditional product, the honey produced by A. cerana attains a much higher market value compared to that of A. mellifera, thus being prone to adulteration. So far, only a few protein-based methods have been proposed to assess honey entomological origin [1], which in fact is related to its geographical origin since bee species generally occupy different geographical ranges according to their evolutionary lineages [2]. In this work, DNA methods were developed for the specific identification of A. mellifera and A. cerana DNA in honey. For this purpose, bees of A. cerana from Thailand, China and Vietnam and honeybees of 4 different subspecies of A. mellifera (iberiensis, mellifera, ligustica and carnica) from EU countries were used. Different sets of primers were designed targeting the tRNAleu - COII intergenic region and the 16S rRNA gene. For both cases, the specificity and sensitivity of the designed primers were assayed by qualitative polymerase chain reaction (PCR). DNA was extracted from honey samples as previously described [3]. PCR with primers targeting the tRNAleu - COII intergenic region allowed the specific detection of A. cerana. The applicability of the proposed new PCR method was assayed with authentic A. cerana and A. mellifera honey samples, which enabled the identification of A. cerana honey. PCR targeting the 16S rRNA gene successfully amplified both honeybee species, but without being able to differentiate them. However, the use of real-time PCR with 16S rRNA primers coupled with High Resolution Melting (HRM) analysis allowed the differentiation of both species in distinct clusters (Fig. 1). The developed new HRM methodology was further applied to the analysis of authentic honey samples from Vietnam (produced from A. cerana and A. mellifera honeybees) and from Portugal (produced from A. mellifera honeybees), as well as commercial samples of honey labelled as produced in the EU, allowing its successful entomological origin identification [4]. Both developed techniques proved their effectiveness for establishing the entomological origin of honey and can be considered as useful tools for authentication/control purposes. ; This work was supported by FCT (Fundação para a Ciência e Tecnologia) through project UID/QUI/50006/2013 - POCI/01/0145/ FEDER/007265 with financial support from FCT/MEC through notional funds and eo-financed by FEDER, under the Partnership Agreement PT2020, and project NORTE- 01- 0145- FEDER- 0 00011. S. Soores, L. Grozino and J. Costa ore grateful to FCT grants (SFRH/BPD/102404/2014, SFRH/BD/132462/2017 and SFRH/ BD/75091/2010) financed by POPH-QREN (subsidized by FSE and MCTES). ; info:eu-repo/semantics/publishedVersion

Honey is the natural sweet substance produced by honey bees. According to the European Union legislation, it should be produced by the westem honey bee, Apis mellifera. However, in Ásia, honey is traditionally obtained from other bee species, mainly the eastera honey bee Apis cerana. So far, only a few protein-based methods have been proposed to assess honey entomological origin[ ], which in fact is related to its geographical origin since bee species generally occupy different geographical ranges according to their evolutionary lineages [ ]. In this work, DNA markers were developed for the specifíc identification ofA. mellifera and A. cerana in honey. For this purpose, bees of A. cerana from Thailand, China and Vietnam and honey bees of 4 different subspecies of A. mellifera (iberiensis, mellifera, ligustíca, carnica) from EU countries were used. Different sets ofprimers were designed targeting the 16S rRNA gene and the tRNAleu - COII intergenic region. The specificity and sensitivity of the designed primers were assayed by qualitative polymerase chain reaction (PCR). Primers targeting the intergenic region successfully differentiated A. cerana from A. mellifera. Positive amplifications were obtained for ali the bees with 16S rRNA primers. However, the use of real-time PCR coupled with High Resolution Melting analysis allowed the separation of the two honey bee species in different clusters. The developed methodologies were applied to the analysis ofauthentic honey samples from Vietnam (produced from A. cerana and A. mellifera bees) and from Portugal allowing its successful entomological origin identification. ; To the project UID/QUI/50006/2013 - POCI/01/0145/FEDER/007265 for financiai support from FCT/MEC through national fünds and co-fmanced by FEDER. S. Soares and J. Costa are gratefül to FCT grants (SFRH/BPD/102404/2014 and SFRH/BD/75091/2010) fmancedby POPH-QREN (subsidized by FSE and MCTES). ; info:eu-repo/semantics/publishedVersion

BEEHEAL 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. ; This work has been developed under the BEEHEAL project. BEEHEAL is funded through the ARIMNet2 2016 Call by the following funding agencies: INIA (Spain), MOARD (Israel), ANR (France), and FCT (Portugal). ARIMNet2 (ERA-NET) has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 618127. ; info:eu-repo/semantics/publishedVersion

Nosema ceranae is a hot topic in honey bee health as reflected by numerous papers published every year. This review presents an update of the knowledge generated in the last 12 years in the field of N. ceranae research, addressing the routes of transmission, population structure and genetic diversity. This includes description of how the infection modifies the honey bee's metabolism, the immune response and other vital functions. The effects on individual honey bees will have a direct impact on the colony by leading to losses in the adult's population. The absence of clear clinical signs could keep the infection unnoticed by the beekeeper for long periods. The influence of the environmental conditions, beekeeping practices, bee genetics and the interaction with pesticides and other pathogens will have a direct influence on the prognosis of the disease. This review is approached from the point of view of the Mediterranean countries where the professional beekeeping has a high representation and where this pathogen is reported as an important threat. ; This work has been developed under the BEEHEAL project. BEEHEAL is funded through the ARIMNet2 2016 Call by the following funding agencies: INIA (Spain), MOARD (Israel), ANR (France) and FCT (Portugal). ARIMNet2 (ERA-NET) has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 618127. We also thank Dr. Tamara Gomez Moracho for the Nosema lifecycle design. ; info:eu-repo/semantics/publishedVersion

With a growing number of parasites and pathogens experiencing large-scale range expansions, monitoring diversity in immune genes of host populations has never been so important because it can inform on the adaptive potential to resist the invaders. Population surveys of immune genes are becoming common in many organisms, yet they are missing in the honey bee (Apis mellifera L.), a key managed pollinator species that has been severely affected by biological invasions. To fill the gap, here we identified single nucleotide polymorphisms (SNPs) in a wide range of honey bee immune genes and developed a medium-density assay targeting a subset of these genes. Using a discovery panel of 123 whole-genomes, representing seven A. mellifera subspecies and three evolutionary lineages, 180 immune genes were scanned for SNPs in exons, introns (< 4 bp from exons), 3' and 5´UTR, and < 1 kb upstream of the transcription start site. After application of multiple filtering criteria and validation, the final medium-density assay combines 91 quality-proved functional SNPs marking 89 innate immune genes and these can be readily typed using the high-sample-throughput iPLEX MassARRAY system. This medium-density-SNP assay was applied to 156 samples from four countries and the admixture analysis clustered the samples according to their lineage and subspecies, suggesting that honey bee ancestry can be delineated from functional variation. In addition to allowing analysis of immunogenetic variation, this newly-developed SNP assay can be used for inferring genetic structure and admixture in the honey bee. ; We are deeply indebted to Frank Aguiar, Luís Silva, Edgardo Melo, João Martins, João Melo, Manuel Moura, Manuel Viveiros, and Ricardo Sousa from "Direção Regional da Agricultura e Desenvolvimento Rural dos Açores" (Portugal), and to Laura Garreau, Laurent Maugis, Pascale Sauvage and Jacques Kermagoret, from "Association Conservatoire de l'Abeille Noir Bretonne" (France), for sampling the apiaries in São Miguel, Santa Maria, and Ouessant islands. Genotyping was outsourced to the Epigenetics and Genotyping laboratory, Central Unit for Research in Medicine (UCIM), University of Valencia, Spain. Data analyses were performed using computational resources at the Research Centre in Digitalization and Intelligent Robotics (CeDRI), Instituto Politécnico de Bragança. Ana Rita Lopes is supported by a PhD scholarship (SFRH/BD/143627/2019) from the Foundation for Science and Technology (FCT), Portugal. FCT provided financial support by national funds (FCT/MCTES) to CIMO (UIDB/00690/2020).This research was funded through the projects BEEHAPPY (POCI-01-0145- FEDER-029871, FCT and COMPETE/QREN/EU) and BEEHEAL. BEEHEAL was funded by the ARIMNet2 2016 Call by the following agencies: INIA (Spain), MOARD (Israel), ANR (France) and FCT (Portugal). ARIMNet2 (ERA-NET) received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 618127. ; info:eu-repo/semantics/publishedVersion