Comparing two in-house developed SNP assays for inferring population structure in the honey bee (Apis mellifera L.)

The 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 caninfo:eu-repo/semantics/publishedVersio

Padrões de diversidade mitocondrial da abelha melífera em Portugal continental

A distribuição natural da abelha melífera (Apis mellifera L.) abrange a África, a Europa e o Médio Oriente (Figura 1). Esta ampla área geográfica é ocupada por 30 subespécies (raças geográficas) de abelhas (Ruttner 1988; Engel 1999; Sheppard e Meixner 2003; Meixner et al. 2011) que têm sido agrupadas em quatro linhagens (Ruttner 1988), nomeadamente: a linhagem do Médio Oriente (O), a linhagem Africana (A), a linhagem da Europa oriental (C) e a linhagem da Europa ocidental (M). A Europa alberga uma importante componente dessa diversidade representada pela ocorrência de duas das quatro linhagens (C e M). A linhagem C agrupa cerca de uma dezena de subespécies, entre as quais se encontram as duas mais utilizadas pela apicultura à escala mundial: a italiana (A. m. ligustica) e a carniola (A. m. carnica). A linhagem M apesar de ocupar uma extensa área que vai desde o Sul da Península Ibérica até ao Sul da Escandinávia e desde o Reino Unido até à Rússia, agrupa apenas duas subespécies: a abelha negra (A. m. mellifera), a norte dos Pirenéus, e a abelha ibérica (A. m. iberiensis), na Península Ibérica.Fundação para a Ciência e Tecnologi

Análisis jurídico político de la ley de la profesión de maestro

En los últimos treinta años desde 1941 el Magisterio nacional se venía rigiendo por un mismo escalafón. En 1962 y no pasó de la retórica se reconoció la docencia como carrera profesional a todos los niveles del sistema educativo al ratificarse el Convenio sobre Unificación Básica de la Educación en Centroamérica y se estableció que el Escalafón Nacional del Magisterio sería “El conjunto de disposiciones dirigidas a establecer garantías profesionales y económicas para los docentes. En 1969, el 9 de septiembre, se da la ley de La Profesión de Maestro y hasta en 1971 se dicta un nuevo Escalafón. La Ley de la Profesión de Maestro es un producto nítido de la primera gran huelga de ANDES 21 de junio. Durante cincuenta y ocho días los maestros y el pueblo salvadoreño se habían lanzado a las calles para exigir un estatuto protector del maestro. Se produjo una serie de acontecimientos sin precedentes en la historia del país

Employment of whole genome resequencing to reveal the evolutionary history and to develop molecular tools for Western European honey bees (Apis mellifera subspecies)

Tese de Doutoramento em Biologia Molecular e Ambiental (Especialidade em Evolução, Biodiversidade e Ecologia)The Western honey bee, Apis mellifera L., acts as a pollinator, thus playing a role in the ecosystem of paramount importance. However, the genetic integrity of many subspecies has been threatened by introgressive hybridization. In an attempt to reverse this trend, the main goals of this dissertation were (i) to reveal the genetic structure of one of the most complex and diverse subspecies in Europe, the Iberian honey bee (Apis mellifera iberiensis), and (ii) to develop molecular tools for the Iberian honey bee and its sister subspecies, the Dark honey bee (Apis mellifera mellifera), both belonging to the western European lineage M. These molecular tools can be employed for breeding and conservation programs in western and southern Europe. The Dark honey bee A. m. mellifera has been severely threatened by hybridization with subspecies of eastern European (C lineage) ancestry, such as A. m. ligustica and A. m. carnica. Using 113 haploid honey bees collected from eight countries and genotyped with 1183 SNPs with the GoldenGate® Assay, five panels containing 48, 96, 144, 192 and 384 ancestry informative SNPs (fitted to the plexes of GoldenGate® Assays) were designed to estimate admixture proportions of C-lineage into A. m. mellifera. All SNP panels were able to estimate Clineage admixture proportions highly concordant with those inferred from the 1183 SNP dataset (r≥0.997). The discontinuation of Illumina’s GoldenGate® Assay and the need of a standard method to examine the purity of honey bee populations in a wide geographical area were the motivation to design four multiplexed SNP assays to be genotyped using the iPLEX MassARRAY system, having as a baseline the 144-plex SNP panel. An accurate and cost-effective tool was provided with all genomic information for 117 SNPs for immediate application in genetic surveys and conservation management of A. m. mellifera. The performance of the assays was assessed against the data from 27 Whole-Genome (WG) sequences and using a set of individuals obtained from controlled crosses. In addition, sensitivity tests indicate that this genotyping system has the potential to detect C-lineage introgression at a low frequency (diluted as 1:20 in DNA pools or as 1 F1 hybrid: 7 A. m. mellifera individuals in tissue pools). In contrast with A. m. mellifera, A. m. iberiensis populations exhibit a preserved complex genetic variation pattern. Although neutral processes have played an important role in shaping the Iberian diversity pattern, selection should not be ignored. Therefore, the WGS data of 87 Iberian honey bees were scanned for selection signals using three methods (Samβada, LFMM and PCAdapt) and two datasets (genomic and environmental). Candidate SNPs detected by at least two methods were further examined using a haplotype-based method and protein modelling. Among the 830 SNPs exhibiting selection signals, 90.2% lie in non-coding regions, suggesting that regulatory changes are important in local adaptation. An enrichment of nonsynonymous SNPs was also found, three of them leading to amino acid replacements, within or in the close vicinity of a functionally important site of proteins with functions related to lipids and transmembrane transport. Using both genetic and environmental data, candidate genes putatively under climate-driven adaptation were identified. Interestingly, membrane-related and circadian clock genes, which allow the organism to sense and fine-tune with environmental oscillations, are among the strongest candidate genes. This is particularly important in the context of rapid global change, helping to understand the mechanisms used by organisms to adapt to varying environmental conditions. Likewise for A. m. mellifera, it is important for A. m. iberiensis to have a cost-effective molecular tool capable of accurately detecting C-derived introgression. Reduced assays of highly informative SNPs were developed from 176 WGs. In addition, both the effects of sample size and of sampling a geographically restricted area on the number of highly informative SNPs were tested. Results show that a bias is introduced when the sample size is small (N≤10) and when sampling only captures a fraction of a population’s genetic diversity. The designed assays can be readily used for monitoring populations not only in the native range of A. m. iberiensis but also in the introduced range. Another molecular marker widely used to assess the genetic diversity in honey bees is the mitochondrial intergenic tRNAleu-cox2 region. Using mitogenome data from 123 individuals representing seven subspecies, three lineages (A, M and C) and three African sub-lineages (AI, AII and AIII), it was tested whether the information provided by this region is reliable for historical inference. While the mitogenome analysis supports the three evolutionary lineages defined by the tRNAleu-cox2 intergenic region, it does not support the existence of the three African sub-lineages. Finally, different parts of the mitogenome provided distinct results, implying that the conclusions drawn from studies using only one locus need to be taken with caution. Overall, in this dissertation a set of accurate and reliable tools was developed to be used in the preservation of the genetic integrity of honey bee populations of M-lineage ancestry. Moreover, new insights into genetic basis of Iberian honey bee local adaptation were provided by WGS and environmental data.A abelha-europeia, Apis mellifera L., exerce um papel essencial no ecossistema como polinizador. No entanto, a sua integridade genética tem sido ameaçada por hibridação introgressiva. Na tentativa de contribuir para contrariar esta tendência esta dissertação tem os seguintes objetivos: (i) revelar a estrutura genética de uma das subespécies mais complexas da Europa, a abelha ibérica (Apis mellifera iberiensis) e (ii) desenvolver ferramentas moleculares para a abelha ibérica e para a abelha-negra (Apis mellifera mellifera), ambas pertencentes à linhagem Europeia Ocidental ou M. Estas ferramentas podem ser usadas tanto em programas de melhoramento como de conservação. A abelha-negra tem sido ameaçada pela hibridação com subespécies da Europa Oriental (linhagem C), como a A. m. ligustica e a A. m. carnica. Usando como base 113 abelhas haploides provenientes de oito países genotipadas com 1183 na GoldenGate® Assay, foram desenvolvidos cinco painéis com 48, 96, 144, 192 e 384 SNPs (adequados para a tecnologia GoldenGate® Assay) com o objetivo de estimar introgressão da linhagem C em A. m. mellifera. Todos os painéis tiveram estimativas de introgressão semelhantes à obtida quando os 1183 SNPs foram usados (r≥0,997). A descontinuação da GoldenGate® Assay e a necessidade de um método para monitorizar a introgressão numa vasta área geográfica motivaram o desenvolvimento de quatro painéis de SNPs apropriados para o sistema iPLEX MassARRAY, tendo como base o painel de 144 SNPs, anteriormente desenvolvido. No final foi divulgada uma ferramenta precisa e económica, juntamente com toda a informação genética dos 117 SNPs, para uma aplicação imediata em estudos genéticos e de conservação da A. m. mellifera. O desempenho deste painel foi avaliado por intermédio de 27 genomas e usando indivíduos obtidos por cruzamentos controlados. Adicionalmente foram efetuados testes de sensibilidade que demonstraram que esta técnica permite a deteção de alelos da linhagem C, mesmo quando em baixas frequências (diluído de 1:20 numa pool de DNA e na proporção 1 F1:7 A. m. mellifera numa pool de tecidos). Contrariamente à A. m. mellifera, as populações de A. m. iberiensis ainda não estão ameaçadas e têm uma estrutura genética complexa. Os processos neutrais tiveram um papel importante na moldagem da diversidade genética, no entanto o papel da seleção não pode ser ignorado. Por isso, foram procurados sinais de seleção usando um total de 87 genomas de abelhas ibéricas, três métodos (Samβada, LFMM e PCAdapt) e dois tipos de dados (genómicos e ambientais). Os SNPs detetados por pelo menos dois destes métodos foram analisados usando métodos haplótipicos e modelação de proteínas. Entre os 830 SNPs com sinais de seleção, 90,2% estão em regiões não codificantes, sugerindo que a regulação tem um papel crucial na adaptação local. Foi também encontrado um enriquecimento de SNPs não-sinónimos, três deles levam à substituição de aminoácidos situados dentro ou perto de locais funcionais de proteínas relacionadas com lípidos e transporte transmembranar. Usando dados genéticos ambientais foram identificados genes relacionados com adaptação a diferentes climas. Curiosamente, os candidatos mais fortes foram genes relacionados com a membrana e o relógio circadiano que permitem que o organismo detete e se adapte a variações ambientais. Estudos de adaptação local são especialmente importantes no contexto das mudanças climáticas, ajudando a perceber quais os mecanismos usados para a adaptação a diferentes condições ambientais. Ferramentas moleculares económicas e precisas para estimar a introgressão da linhagem C são tão importantes para a A. m. mellifera como para a A. m. iberiensis, por isso quatro painéis ultra-reduzidos foram desenvolvidos usando o genoma completo de 176 indivíduos. Adicionalmente, os efeitos do tamanho da amostra e da amostragem geograficamente confinada foram avaliados no número de SNPs fixos. Verificou-se que existe um enviesamento quando o tamanho da amostra é ≤10 e quando a amostragem representa uma pequena porção da diversidade genética. Os painéis ultra-reduzidos podem ser utilizados na monitorização da integridade genética não só na área nativa mas também em locais onde a abelha ibérica foi introduzida. Outro marcador muito usado na avaliação da diversidade genética das abelhas é a região intergénica tRNAleu-cox2 do DNA mitocondrial. Usando dados do mitogenoma de 123 indivíduos de sete subespécies diferentes, três linhagens (A, M e C) e três sublinhagens africanas (AI, AII e AIII), foi testado se esta região é fidedigna para inferência histórica. As análises mitogenómicas suportam as três linhagens evolutivas definidas por essa região intergénica mas não sustentam a existência das três sub-linhagens africanas. É também de salientar que diferentes partes do mitogenoma fornecem diferentes resultados, sugerindo que as conclusões retiradas de estudos que utilizem um só locus devem ser tomadas com precaução. Nesta dissertação foram desenvolvidas ferramentas precisas e fidedignas que podem ser usadas para a preservação da integridade genética das populações europeias de abelhas da linhagem M. Os dados de sequenciação e ambientais ajudaram na compreensão da base genética da adaptação local da abelha ibérica.A sua execução foi financiada pela Bolsa de Doutoramento SFRH/BD/84195/2012, atribuída pela Fundação para a Ciência e a Tecnologia (FCT). Contou ainda com os recursos disponibilizados pelo projecto BeeHope: 2013‐2014 BiodivERsA/FACCE‐JPI (joint call for research proposals, with the national funders FCT, Portugal, CNRS, France, and MEC, Spain) e do projeto COMPETE/QREN/EU (PTDC/BIABEC/099640/2008)

Mitochondrial SNP markers to monitor evolutionary lineage ancestry in Apis mellifera mellifera conservation programs

The European dark honey bee, Apis mellifera mellifera , is threatened inmost of its native range, in part, due to introgressive hybridization with bees from the highly divergent C-lineage, mainly Apis mellifera carnica and Apis mellifera ligustica (De la Rúa et al. 2009; Pinto et al. 2014). Yet, the maintenance of locally adapted genetic diversity is critical for the population long-term survival and sustainability (De la Rúa et al. 2009; Meixner 2010). The growing awareness that genetic diversity is important for sustainable beekeeping led to implementation of different conservation and breeding programs throughout Europe, which are in need of reliable and costefficient molecular tools to accurately monitor Clineage introgression into A. m. mellifera (De la Rúa et al. 2009; Henriques et al. 2018a, b; Meixner 2010). The large mating flight distances and the polyandrous mating system make it challenging to preserve honey bee subspecies in an open conservation area where intruders can fly in (Neumann et al. 1999). It is therefore necessary to regularly control the genetic ancestry of new or superseded colonies.This work was financed by FEDER (Fundo Europeu de Desenvolvimento Regional) through the program COMPETE 2020–POCI (Programa Operacional para a Competitividade e Internacionalização) and by Portuguese funds through FCT (Fundação para a Ciência e a Tecnologia) in the framework of the project BeeHappy (POCI-01-0145-FEDER-029871). Melanie Parejo was supported by a mobility fellowship awarded from the Swiss National Science Foundation (SNSF).info:eu-repo/semantics/publishedVersio

The Welsh dark bee (Apis mellifera mellifera) is not extinct

Due to past and present imports of Apis mellifera ligustica (Italian bees), Apis mellifera carnica (Carniolan bees) and the English Buckfast bee (a hybrid strain) across its entire natural range, Apis mellifera mellifera is now threatened with extinction by genetic pollution through hybridization. Whilst the status of remnant A. m. mellifera populations is well documented on the European mainland, few studies have been undertaken to identify surviving populations on the British Isles. A few A. m. mellifera stocks are thought to persist in Scotland and the southwest of England and recently, Ireland might appear to contain multiple non-hybridized A. m. mellifera populations. 163 young worker bees, representing 121 colonies from across Wales, were genetically screened in an attempt to identify remnant A. m. mellifera stocks, as part of a conservation breeding program. Recent studies have demonstrated that honey bees of local origin have significantly higher survival chances than honey bees of non-local origin due to their adaptation to their local environment, suggesting that conservation of locally adapted honey bees is a logistical and practical possibility to develop sustainable apiculture. Within this survey, we made use of a custom-tailored SNP genotype assay to estimate the extent of C lineage introgression in the ncDNA as well as sequencing of the tRNAleucox2 intergenic region of the mtDNA to check for the ancestry of the tested coloniesinfo:eu-repo/semantics/publishedVersio

A note to transfer a generic database pseudocode for storing chronological data from research in apiaries

In honey bee research conducted in apiaries, a large amount of information is usually generated requiring a flexible database for storing and retrieving data. Here, we developed a generic database pseudocode, based on the abstraction of the apiary system, for data collected from the colonies through time.We thank J Chávez-Galarza for the fruitful discussions during the design of the database architecture. This research was funded through the 2013-2014 BiodivERsA/FACCE-JPI joint call for research proposals, with the national funders “Fundação para a Ciência e Tecnologia” (Portugal), “Agence Nationale de la Recherche” (France), and “Ministério de Economia y Competividade” (Spain).info:eu-repo/semantics/publishedVersio

Polymorphisms in cytochrome P450 versus cline distribution of evolutionary lineages in Apis mellifera iberiensis

Honey bees (Apis mellifera) are the most prominent and economically important pollinator species worldwide. However, the reported decline of its populations in several regions of the world over the last decades is of concern. The causes are manifold, including the spread of pathogens and parasites, malnutrition and habitat loss, climate change and xenobiotics, especially pesticides. Among the main mechanisms used by insects to cope with the adverse effects of xenobiotics is the metabolic resistance mediated mainly by three superfamilies of enzymes: the cytochrome P450 monooxygenases, the glutathione transferases and the carboxylesterases. We hypothesize that the genetic background influences the sensitivity to pesticides or detoxification capacity of different honey bee populations, ecotypes and subspecies. The Iberian Peninsula provides an interesting scenario to study the genetic variability of the cytochrome P450 genes given the co-occurrence of two clinally distributed evolutionary lineages, as a result of secondary contact. In this study, the genetic variability of six genes of the cytochrome P450 superfamily (CYP6AS3, CYP6AS4, CYP6AS5, CYP6AS7, CYP6AS12 and CYP6AS17) was analyzed in the Iberian honey bee (Apis mellifera iberiensis) to provide more information on the mechanisms of resistance to xenobiotics and to identify the genetic variation involved in local adaptation. Genomic signal of selective sweeps was detected in three genes, of which CYP6AS5 presents the highest number of point mutations under selection, being proposed as a candidate gene to perform gene expression studies. We discuss the correlation between the variability of P450 genes and the distribution of the evolutionary lineages in the Iberian Peninsula. The identification of polymorphisms in these genes promises to shed light on the relationship between diversity and xenobiotic tolerance of A. m. iberiensis.info:eu-repo/semantics/publishedVersio

Empirical comparison of microsatellite and SNP markers to estimate introgression in Apis mellifera mellifera

The genetic identity of the dark European honey bee, Apis mellifera mellifera is currently under pressure throughout most of its native range due to large scale commercial trade and replacement with honey bees of mainly Eastern European ancestry (C-lineage: Apis mellifera carnica and Apis mellifera ligustica). To counteract this process, numerous conservation efforts for the protection of native honey bees are sprouting across Europe. For the management of such protected areas and conservation breeding purposes, honey bee subspecies have been routinely identified through wing morphology and through DNA-hybrid tests using microsatellite markers. Currently, new methods are evolving including rapid innovations in single-nucleotide polymorphism (SNP) array technology and high-throughput sequencing. Here, we aim to quantify potential marker-specific biases of hybrid tests and give recommendations for applications in honey bee conservation management. Using an empirical dataset, we first assessed the accuracy of a recently developed reduced SNP panel to estimate C-lineage introgression in A. m. mellifera compared to whole-genome sequence (WGS) data. Using another independent data set, we estimated the differences in admixture proportions between the currently applied hybrid test based on microsatellites and the novel SNP test. We demonstrate that the SNP-based test which contains highly ancestry-informative markers is very efficient to estimate genome-wide ancestry. Furthermore, we report discrepancies between microsatellite and SNP-based admixture proportions. For conservation management, we, therefore, recommend the implementation of SNP-based hybrid tests to maintain high genetic variation within the breeding population, while minimizing influence of introduced honey bees.This work was supported by the Swiss Federal Office for Agriculture FOAG, the Fondation Sur-la-Croix, Basel, and by the 2013–2014 BiodivERsA/FACCE-JPI joint call for research proposals, with the national funders “Fundac¸ão para a Ciência e Tecnologia” (Portugal), “Agence Nationale de la Recherche” (France), and “Ministerio de Economía y Competividad” (Spain).info:eu-repo/semantics/publishedVersio

Formação de Assistentes Operacionais com funções de ação educativa, suas atitudes e práticas educativas: um contributo para a educação inclusiva

Nos últimos anos, em Portugal assiste-se a um aumento de crianças com Necessidades Educativas Especiais (NEE) a frequentar o ensino regular. Nesta perspetiva a inclusão de crianças com NEE no ensino regular transformou-se numa mudança complexa porque, tendo como objetivo melhorar a vidas das crianças, fez com que os estilos de vida e comportamentos de indivíduos se alterassem. Neste sentido, este estudo tem como objetivo de compreender as perspetivas das Assistentes Operacionais com funções de Ação Educativa em relação à sua formação, às suas atitudes face à inclusão de crianças com NEE nas salas do Jardim-de-infância. A investigação é de natureza qualitativa, optou-se por utilizar como método de recolha de dados a entrevista. Os participantes deste estudo foram Educadoras de Infância e Assistentes Operacionais com funções de Ação Educativa de uma Instituição Particular de Solidariedade Social. Os resultados mostram que as Assistentes Operacionais com funções de Ação Educativa desempenham as suas funções de forma insegura devido à falta de formação profissional. Revelam ainda, uma preocupação face à educação inclusiva com crianças com NEE.In the last years, in Portugal, the number of children with NEE attending the regular teaching has increased. In this perspective, the inclusion of children with NEE in the regular teaching, being the goal to improve children’s life, caused a complex change in the lifestyle and behavior of individuals. In this sense, this study has the purpose to understand the perspectives of the Educative Action Assistants concerning their learning, their attitudes before the inclusion of children with NEE in the classrooms of Kindergartens. The investigation has qualitative nature, and the interview has been the method of collecting data. The participants of this study are Infancy Teachers and Educative Action Assistants of a Private Institution of Social Solidarity. The results show that the Educative Action Assistants perform their tasks in an insecure way due to the short of professional learning. This study reveals also a concern before inclusive education with children with NEE