Bee viruses: routes of infection in Hymenoptera

Numerous studies have recently reported on the discovery of bee viruses in different arthropod species and their possible transmission routes, vastly increasing our understanding of these viruses and their distribution. Here, we review the current literature on the recent advances in understanding the transmission of viruses, both on the presence of bee viruses in Apis and non-Apis bee species and on the discovery of previously unknown bee viruses. The natural transmission of bee viruses will be discussed among different bee species and other insects. Finally, the research potential of in vivo (host organisms) and in vitro (cell lines) serial passages of bee viruses is discussed, from the perspective of the host-virus landscape changes and potential transmission routes for emerging bee virus infections

Genetic diversification of an invasive honey bee ectoparasite across sympatric and allopatric host populations

Invasive parasites are major threats to biodiversity. The honey bee ectoparasite, Varroa destructor, has shifted host and spread almost globally several decades ago. This pest is generally considered to be the main global threat to Western honey bees, Apis mellifera, although the damages it causes are not equivalent in all its new host’s populations. Due to the high virulence of this parasite and the viruses it vectors, beekeepers generally rely on acaricide treatments to keep their colonies alive. However, some populations of A. mellifera can survive without anthropogenic mite control, through the expression of diverse resistance and tolerance traits. Such surviving colonies are currently found throughout the globe, with the biggest populations being found in Sub- Saharan Africa and Latin America. Recently, genetic differences between mite populations infesting surviving and treated A. mellifera colonies in Europe were found, suggesting that adaptations of honey bees drive mite evolution. Yet, the prevalence of such co-evolutionary adaptations in other invasive populations of V. destructor remain unknown. Using the previous data from Europe and novel genetic data from V. destructor populations in South America and Africa, we here investigated whether mites display signs of adaptations to different host populations of diverse origins and undergoing differing management. Our results show that, contrary to the differences previously documented in Europe, mites infesting treated and untreated honey bee populations in Africa and South America are genetically similar. However, strong levels of genetic differentiation were found when comparing mites across continents, suggesting ongoing allopatric speciation despite a recent spread from genetically homogenous lineages. This study provides novel insights into the co-evolution of V. destructor and A. mellifera, and confirms that these species are ideal to investigate coevolution in newly established host- parasite systems.Agencia Nacional de Investigación e InnovaciónPrograma ECOS-Su

Beeheal: standardization of laboratory methods for sample processing, nucleic acids extraction and PCR for microsporidia and viruses analysis

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/publishedVersio

Influence of chronic exposure to thiamethoxam and chronic bee paralysis virus on winter honey bees.

Co-exposure to pesticides and viruses is likely to occur in honey bee colonies. Pesticides can be present in pollen, nectar, and persist in stored food (honey and bee bread), and viruses can be highly prevalent in honey bee colonies. Therefore, the present study describes the influence of chronic co-exposure to thiamethoxam and Chronic bee paralysis virus (CBPV) on bee survival, virus loads, expression level of immune and detoxication genes, and pesticide metabolism Experiments were performed on honey bees collected from a winter apiary with reduced viral contaminations. No synergistic effect of co-exposure was observed on bee survival, nor on the ability of bees to metabolise the pesticide into clothianidin. However, we found that co-exposure caused an increase in CBPV loads that reached the viral levels usually found in overt infections. The effect of co-exposure on CBPV replication was associated with down-regulation of vitellogenin and dorsal-1a gene transcription. Nevertheless, the observed effects might be different to those occurring in spring or summer bees, which are more likelyco-exposed to thiamethoxam and CBPV and exhibit a different physiology

Genetic diversification of an invasive honey bee ectoparasite across sympatric and allopatric host populations.

Invasive parasites are major threats to biodiversity. The honey bee ectoparasite, Varroa destructor, has shifted host and spread almost globally several decades ago. This pest is generally considered to be the main global threat to Western honey bees, Apis mellifera, although the damages it causes are not equivalent in all its new host's populations. Due to the high virulence of this parasite and the viruses it vectors, beekeepers generally rely on acaricide treatments to keep their colonies alive. However, some populations of A. mellifera can survive without anthropogenic mite control, through the expression of diverse resistance and tolerance traits. Such surviving colonies are currently found throughout the globe, with the biggest populations being found in Sub-Saharan Africa and Latin America. Recently, genetic differences between mite populations infesting surviving and treated A. mellifera colonies in Europe were found, suggesting that adaptations of honey bees drive mite evolution. Yet, the prevalence of such co-evolutionary adaptations in other invasive populations of V. destructor remain unknown. Using the previous data from Europe and novel genetic data from V. destructor populations in South America and Africa, we here investigated whether mites display signs of adaptations to different host populations of diverse origins and undergoing differing management. Our results show that, contrary to the differences previously documented in Europe, mites infesting treated and untreated honey bee populations in Africa and South America are genetically similar. However, strong levels of genetic differentiation were found when comparing mites across continents, suggesting ongoing allopatric speciation despite a recent spread from genetically homogenous lineages. This study provides novel insights into the co-evolution of V. destructor and A. mellifera, and confirms that these species are ideal to investigate coevolution in newly established host-parasite systems

Cold case: The disappearance of Egypt bee virus, a fourth distinct master strain of deformed wing virus linked to honeybee mortality in 1970’s Egypt

In 1977, a sample of diseased adult honeybees (Apis mellifera) from Egypt was found to contain large amounts of a previously unknown virus, Egypt bee virus, which was subsequently shown to be serologically related to deformed wing virus (DWV). By sequencing the original isolate, we demonstrate that Egypt bee virus is in fact a fourth unique, major variant of DWV (DWV-D): more closely related to DWV-C than to either DWV-A or DWV-B. DWV-A and DWV-B are the most common DWV variants worldwide due to their close relationship and transmission by Varroa destructor. However, we could not find any trace of DWV-D in several hundred RNA sequencing libraries from a worldwide selection of honeybee, varroa and bumblebee samples. This means that DWV-D has either become extinct, been replaced by other DWV variants better adapted to varroa-mediated transmission, or persists only in a narrow geographic or host range, isolated from common bee and beekeeping trade routes

A SNP assay for assessing diversity in immune genes in the honey bee (Apis mellifera L.)

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/publishedVersio

Diversity and Global Distribution of Viruses of the Western Honey Bee, Apis mellifera

Funding: This research was permitted and funded by the COLOSS network (https://coloss.org/).Peer reviewedPublisher PD

Projeto BEEHEAL: promover a saúde da abelha para uma agricultura sustentável

O BEEHEAL, com o título original “Promoting bee health for sustainable agriculture”, é um projeto internacional colaborativo aprovado no âmbito da Ação ERA-Net ARIMNet2 (Coordination of Agricultural Research in the Mediterranean). O projeto é coordenado por Raquel Martín- Hernández, investigadora do “Centro de Investigación Apícola y Agroambiental de Marchamalo” (CAR), Espanha. Para além deste centro de investigação, representado por Raquel Martín-Hernández e Mariano Higes, o consórcio inclui mais três instituições, nomeadamente: o Centro de Investigação de Montanha (CIMO) do Instituto Politécnico de Bragança, representado por M. Alice Pinto e Ana Rita Lopes, o “Centre de Recherche Provence-Alpes-Côte d’Azur Unité: Abeilles et Environnement do “Institut National de la Recherche Agronomique” (INRA), França, representado por Yves Le conte, Anne Dalmon e Maritza Maritza Reyes-Carreno, e o “Volcani Center” da “Agricultural Research Organization” (ARO), Israel, representado por Nor Chevjanovsky e Victoria Soroker. As populações de abelha melífera (Apis mellifera L.) têm vindo a sofrer perdas acentuadas em todo o mundo. Estas perdas estão relacionado com vários factores, que podem atuar sozinhos ou em combinação, incluindo (i) propagação de parasitas e agentes patogénicos exóticos , como por exemplo o ácaro ectoparasita Varroa destructor, o qual serve de vetor de transmissão de vários vírus, e o fungo microsporídeo Nosema ceranae, (ii) exposição das colónias a agro-químicos, (iii) má nutrição, (iv) alterações climáticas, entre outros (vanEngelsdorp & Meixner, 2010; Potts et al., 2010).Ao Paulo Ventura pelo acompanhamento técnico feito ao apário no primeiro ano do projeto. O BEEHEAL é financiado por ARIMNet2 (2016) com os financiadores nacionais Instituto Nacional de Investigación y Teccnologia Agraria y alimentaria (INIA – Espanha), Agence Nationale de la recherche (ARN – France), Ministry off Agriculture & Rural Development, (MOARD – Israel) e Fundação para a Ciência e a Tecnologia (FCT – Portugal)info:eu-repo/semantics/publishedVersio

Virus Prevalence in Egg Samples Collected from Naturally Selected and Traditionally Managed Honey Bee Colonies across Europe

Monitoring virus infections can be an important selection tool in honey bee breeding. A recent study pointed towards an association between the virus-free status of eggs and an increased virus resistance to deformed wing virus (DWV) at the colony level. In this study, eggs from both naturally surviving and traditionally managed colonies from across Europe were screened for the prevalence of different viruses. Screenings were performed using the phenotyping protocol of the 'suppressed in ovo virus infection' trait but with qPCR instead of end-point PCR and a primer set that covers all DWV genotypes. Of the 213 screened samples, 109 were infected with DWV, 54 were infected with black queen cell virus (BQCV), 3 were infected with the sacbrood virus, and 2 were infected with the acute bee paralyses virus. It was demonstrated that incidences of the vertical transmission of DWV were more frequent in naturally surviving than in traditionally managed colonies, although the virus loads in the eggs remained the same. When comparing virus infections with queen age, older queens showed significantly lower infection loads of DWV in both traditionally managed and naturally surviving colonies, as well as reduced DWV infection frequencies in traditionally managed colonies. We determined that the detection frequencies of DWV and BQCV in honey bee eggs were lower in samples obtained in the spring than in those collected in the summer, indicating that vertical transmission may be lower in spring. Together, these patterns in vertical transmission show that honey bee queens have the potential to reduce the degree of vertical transmission over time