Digging into the genomic past of Swiss honey bees by whole-genome sequencing museum specimens

Historical specimens in museum collections provide opportunities to gain insights into the genomic past. For the Western honey bee, Apis mellifera L., this is particularly important because its populations are currently under threat worldwide and have experienced many changes in management and environment over the last century. Using Swiss Apis mellifera mellifera as a case study, our research provides important insights into the genetic diversity of native honey bees prior to the industrial-scale introductions and trade of non-native stocks during the 20th century-the onset of intensive commercial breeding and the decline of wild honey bees following the arrival of Varroa destructor. We sequenced whole-genomes of 22 honey bees from the Natural History Museum in Bern collected in Switzerland, including the oldest A. mellifera sample ever sequenced. We identify both, a historic and a recent migrant, natural or human-mediated, which corroborates with the population history of honey bees in Switzerland. Contrary to what we expected, we find no evidence for a significant genetic bottleneck in Swiss honey bees, and find that genetic diversity is not only maintained, but even slightly increased, most probably due to modern apicultural practices. Finally, we identify signals of selection between historic and modern honey bee populations associated with genes enriched in functions linked to xenobiotics, suggesting a possible selective pressure from the increasing use and diversity of chemicals used in agriculture and apiculture over the last century.info:eu-repo/semantics/publishedVersio

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

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

AmelHap: Leveraging drone whole-genome sequence data to create a honey bee HapMap

Honey bee, Apis mellifera, drones are typically haploid, developing from an unfertilized egg, inheriting only their queen’s alleles and none from the many drones she mated with. Thus the ordered combination or ‘phase’ of alleles is known, making drones a valuable haplotype resource. We collated whole-genome sequence data for 1,407 drones, including 45 newly sequenced Scottish drones, collectively representing 19 countries, 8 subspecies and various hybrids. Following alignment to Amel_HAv3.1, variant calling and quality filtering, we retained 17.4 M high quality variants across 1,328 samples with a genotyping rate of 98.7%. We demonstrate the utility of this haplotype resource, AmelHap, for genotype imputation, returning >95% concordance when up to 61% of data is missing in haploids and up to 12% of data is missing in diploids. AmelHap will serve as a useful resource for the community for imputation from low-depth sequencing or SNP chip data, accurate phasing of diploids for association studies, and as a comprehensive reference panel for population genetic and evolutionary analyses.For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising from this submission. This work was supported by a grant from the CB Dennis British Beekeepers’ Research Trust awarded to MB and DW, and through strategic investment funding to the Roslin Institute from the Biotechnology and Biological Sciences Research Council (BBS/E/D/30002276). MP was supported by a Basque Government grant (IT1233-19)

Developing reduced SNP assays from whole-genome sequence data to estimate C-lineage introgression in the Iberian honeybee (Apis mellifera iberiensis)

The honeybee has been subject to a growing number of threats. In Western Europe one such threat is large-scale introductions of commercial strains (C-lineage), which is leading to introgressive hybridization and even the local extinction of native populations (M-lineage). Here, we developed reduced assays of highly informative SNPs from 176 whole genomes to estimate C-lineage introgression in ;M-lineage subspecies Apis mellifera iberiensis. We started by evaluating the effects of sample size and sampling a geographically restricted area on the number of highly informative SNPs. We demonstrated that a bias in the number of fixed SNPs (FST=1) is introduced when the sample size is small (N≤10) and when sampling only captures a small fraction of a population’s genetic diversity. These results underscore the importance of having a representative sample when developing reliable reduced SNP assays for organisms with complex genetic patterns. We used a training dataset to design four independent SNP assays selected from pairwise FST between the Iberian and C-lineage honeybees. The designed assays, which were validated in holdout and simulated hybrid datasets, proved to be highly accurate and can be readily used for monitoring populations not only in the native range of A. m. iberiensis in Iberia but also in the introduced range in the Balearic islands, Macaronesia, and South America, in a time- and cost-effective manner. While our approach used the Iberian honeybee as model system, it has a high value in a wide range of scenarios for the monitoring and conservation of potentially hybridized domestic and wildlife populations.info:eu-repo/semantics/publishedVersio

Population genetic diversity and dynamics of the honey bee brood pathogen Melissococcus plutonius in a region with high prevalence.

European foulbrood (EFB) is a honey bee brood disease caused by the bacterium Melissococcus plutonius. Large-scale EFB outbreaks have been reported in several countries in recent decades, which entail costly sanitation measures of affected apiaries to restrict the spread of this contagious pathogen. To mitigate its impact, a better understanding of the population dynamics of the etiological agent is required. We here used multi-locus sequence typing (MLST) to infer the genetic diversity and geographical distribution of 160M. plutonius isolates collected from EFB symptomatic honey bee colonies seven years apart. Isolates belonged to three clonal complexes (CCs) known worldwide and to 12 sequence types (STs), of which five were novel. Phylogenetic and clustering analyses showed that some of these novel sequence types have likely evolved locally during a period of outbreak, but most disappeared again. We further screened the isolates for melissotoxin A (mtxA), a putative virulence gene. The prevalence of STs in which mtxA was frequent increased over time, suggesting that this gene promotes spread. Despite the increased frequency of this gene in the population, the total number of cases decreased, which could be due to stricter control measures implemented before the second sampling period. Our results provide a better understanding of M. plutonius population dynamics and help identify knowledge gaps that limit efficient control of this emerging disease.This research was funded by the Swiss Federal Food Safety and Veterinary Office grant number 1.12.15, the University of Lausanne and Agroscope. EGH was funded by a BBSRC CASE studentship in partnership with Bee Disease Insurance and the National Bee Unit. GEB was funded jointly by a grant from BBSRC, Defra, NERC, the Scottish Government and the Wellcome Trust, under the Insect Pollinator Initiative (BB/I000801/1)

A molecular tool to detect genetic introgression from Spermatheca content

The genetic integrity of Apis mellifera mellifera is threatened by introgression in many places of its native distribution, after recurrent importations of commercial queens typically belonging to the divergent C-lineage. A growing interest in keeping and protecting A. m. mellifera has motivated the development of conservation programs in many places of Europe. As part of the conservation efforts, isolated mating stations are set to avoid unwanted crosses, but these are not always effective as matings with unwanted drones are frequently reported. An interesting method to monitor the degree of isolation of mating stations could be through genetic analysis of the queen spermatheca contents. While this method implies that queens selected for monitoring are sacrificed, it can be a powerful way of assessing the effectiveness of mating stations because it would allow easy detection of unwanted alleles. Here, we developed an SNP-based tool suited to the analysis of DNA extracted from spermatheca or from pooled DNA of varying sources. To that end, we first designed an SNP panel from whole-genome sequence data generated from 228 drones, of which 148 belonged to the M-lineage (117 A. m. iberiensis and 31 A. m. mellifera) and 80 to the C-lineage (46 A. m. carnica and 34 A. m. ligustica). A total of 5,007 highly differentiated SNPs was found. Based on different criteria, 130 SNPs were selected to be included in the genotyping tool. This tool is based on the NEBNext Direct Genotyping Solution that allows high-throughput, sequence-based target genotyping of single-individual or pooled DNA. To assess the tool’s sensitivity and accuracy, 142 samples (DNA extracted from spermatheca and tissue, as well as known DNA mixtures) were genotyped. After removing the problematic SNPs, 81 were retained and these were able to provide an estimate of the pool introgression level with great accuracy. This tool represents a significant advance in the genetic analysis of honey bee colonies with a variety of applications, including breeding and conservation of A. m. mellifera.info:eu-repo/semantics/publishedVersio

Developing reduced SNP assays from whole-genome sequence data to estimate introgression in an organism with complex genetic patterns, the Iberian honeybee (Apis mellifera iberiensis)

The most important managed pollinator, the honeybee (Apis mellifera L.), has been subject to a growing number of threats. In western Europe, one such threat is large-scale introductions of commercial strains (C-lineage ancestry), which is leading to introgressive hybridization and even the local extinction of native honeybee populations (M-lineage ancestry). Here, we developed reduced assays of highly informative SNPs from 176 whole genomes to estimate C-lineage introgression in the most diverse and evolutionarily complex subspecies in Europe, the Iberian honeybee (Apis mellifera iberiensis). We started by evaluating the effects of sample size and sampling a geographically restricted area on the number of highly informative SNPs. We demonstrated that a bias in the number of fixed SNPs (FST = 1) is introduced when the sample size is small (N ≤ 10) and when sampling only captures a small fraction of a population’s genetic diversity. These results underscore the importance of having a representative sample when developing reliable reduced SNP assays for organisms with complex genetic patterns. We used a training data set to design four independent SNP assays selected from pairwise FST between the Iberian and C-lineage honeybees. The designed assays, which were validated in holdout and simulated hybrid data sets, proved to be highly accurate not only in the native range of A. m. iberiensis in Iberia but also in the introduced range in the Balearic islands, Macaronesia and South America, in a time-and cost-effective manner. While our approach used the Iberian honeybee as model system, it has a high value in a wide range of scenarios for the monitoring and conservation of potentially hybridized domestic and wildlife populations

A short exposure to a semi-natural habitat alleviates the honey bee hive microbial imbalance caused by agricultural stress

Honeybee health and the species' gut microbiota are interconnected. Also noteworthy are the multiple niches present within hives, each with distinct microbiotas and all coexisting, which we termed "apibiome". External stressors (e.g. anthropization) can compromise microbial balance and bee resilience. We hypothesised that (1) the bacterial communities of hives located in areas with different degrees of anthropization differ in composition, and (2) due to interactions between the multiple microbiomes within the apibiome, changes in the community of a niche would impact the bacteria present in other hive sections. We characterised the bacterial consortia of different niches (bee gut, bee bread, hive entrance and internal hive air) of 43 hives from 3 different environments (agricultural, semi-natural and natural) through 16S rRNA amplicon sequencing. Agricultural samples presented lower community evenness, depletion of beneficial bacteria, and increased recruitment of stress related pathways (predicted via PICRUSt2). The taxonomic and functional composition of gut and hive entrance followed an environmental gradient. Arsenophonus emerged as a possible indicator of anthropization, gradually decreasing in abundance from agriculture to the natural environment in multiple niches. Importantly, after 16 days of exposure to a semi-natural landscape hives showed intermediate profiles, suggesting alleviation of microbial dysbiosis through reduction of anthropization.This work was funded by the Dept. of Economic Development and Competitiveness of the Basque Government (Gobierno Vasco/Eusko Jaurlaritza), R&D&I grants for the agricultural, food and fishing sectors of the Basque Autonomous Community (37-2017-00044), and the Research Group IT1233-19 of the Basque University System. JG was supported by the Department of Agriculture, Fisheries and Food of the Basque Government (Gobierno Vasco/Eusko Jaurlaritza) through a subsidy programme of training aid and support. These funding bodies provided the financial support to the research, but did not participate in the design of the study, analysis and interpretation of data, and writing of the manuscript