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

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

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

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

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

Next-generation sequencing as a promising approach for assessing the entomological origin of honey

Honey is a food widely consumed worldwide and much appreciated for its nutritional and organoleptic properties as well as for its beneficial health effects. However, honey is also considered one of the foods most prone to be adulterated either by the admixing of honey with lower quality, by the addition of sugars, or by mislabeling of botanical and geographical origins, among other possible frauds.1 Therefore, typically, honey authentication has focused mainly on the development of techniques targeting these types of frauds. Recently, increased attention has been paid to honey’s entomological origin since it also relates with geographical origin whose label non-compliances are difficult to detect. Moreover, in the current context where native honeybees are increasingly threatened by introgression, due to the use of exotic queens, preservation of honeybee subspecies in their native ranges, to which they are better adapted, is perceived as of high importance. In this sense, valorisation of the honey produced by native subspecies has been suggested as a possible approach to generate higher income for beekeepers, contributing to the development of rural regions and of sustainable beekeeping based on conservation strategiesThe authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020), to Fenapícola and Capemel for supplying the Portuguese honeys, to Dr. Antonio Nanetti (CREA-AA) for the Italian honeys and to António Pajuelo (Pajuelo Consultores Apícolas S.L.) for the Spanish honeys. D. Henriques is supported by the project BeeHappy (POCI-01-0145-FEDER-029871) funded by FEDER, COMPETE 2020-POCI and FCT and A. Quaresma by the PhD scholarship funded by the FCT (DFA/BD/5155/2020)info:eu-repo/semantics/publishedVersio

Análisis de introgresión en Apis mellifera iberiensis y Apis mellifera mellifera usando polimorfismos de nucleótidos simples (SNPs)

Diferentes estudios han agrupado las subespecies de A. mellifera en cuatro linajes evolutivos basados sobre marcadores morfométricos, ecológicos, microsatélites y mtDNA: Africano (A), Medio Oriente (O), Este y Centro de Europa (C), Norte y Oeste de Europa (M). El linaje M está representado por las subespecies A. m. iberiensis y A. m. mellifera, cuya distribución es la Península Ibérica para la primera y desde los Pirineos hacia el Norte de Europa para la segunda. Durante las últimas décadas, la introducción masiva de subespecies del linaje C por apicultores ha ocasionado un fuerte flujo génico y más aún al casi completo remplazamiento de A. m. mellifera, como ha sido reportado para Alemania. Por tanto, el análisis de niveles de introgresión en programas de crianza y conservación es de vital importancia para evitar la perdida de diversidad genética y sustitución de especies nativas. Este estudio busca identificar los niveles de introgresión de subespecies del linaje C en las subespecies pertenecientes al linaje M a través de un análisis amplio del genoma usando SNPs. Para 711 individuos correspondiente a A. m. iberiensis y 88 individuos A. m. mellifera fueron genotipados 1536 SNPs. Las subespecies de linaje C A. m. ligustica y A. m. carnica fueron usados como poblaciones de referencia. Los niveles de introgresión fueron evaluados usando un método de agrupamiento Bayesiano implementado en el software STRUCTURE. Nuestros resultados indicaron que la introgresión en A. m .iberiensis no es significante, a diferencia en A. m. mellifera que presentó de 8% a 30% de introgresión. Considerando que muchas de las muestras de A. m. mellifera son provenientes de poblaciones integradas en programas de conservación en el Norte de Europa, este resultado evidencia el profundo contraste entre las dos subespecies del linaje M con respecto a su estado de conservación