First data on the prevalence and distribution of pathogens in bumblebees (Bombus terrestris and Bombus pascuorum) from Spain

Bumblebees provide pollination services not only to wildflowers but also to economically important crops. In the context of the global decline of pollinators, there is an increasing interest in determining the pathogen diversity of bumblebee species. In this work, wild bumblebees of the species Bombus terrestris and Bombus pascuorum from northern and southern Spain were molecularly screened to detect and estimate prevalence of pathogens. One third of bumblebees were infected: while viruses only infected B. pascuorum, B. terrestris was infected by Apicystis bombi, Crithidia bombi and Nosema bombi. Ecological differences between host species might affect the success of the pathogens biological cycle and consequently infection prevalence. Furthermore, sex of the bumblebees (workers or males), sampling area (north or south) and altitude were important predictors of pathogen prevalence. Understanding how these factors affect pathogens distribution is essential for future conservation of bumblebee wild populations

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

Epidemiology of the Microsporidium Nosema ceranae in Four Mediterranean Countries

Nosema ceranae is a highly prevalent intracellular parasite of honey bees’ midgut worldwide. This Microsporidium was monitored during a long-term study to evaluate the infection at apiary and intra-colony levels in six apiaries in four Mediterranean countries (France, Israel, Portugal, and Spain). Parameters on colony strength, honey production, beekeeping management, and climate were also recorded. Except for São Miguel (Azores, Portugal), all apiaries were positive for N. ceranae, with the lowest prevalence in mainland France and the highest intra-colony infection in Israel. A negative correlation between intra-colony infection and colony strength was observed in Spain and mainland Portugal. In these two apiaries, the queen replacement also influenced the infection levels. The highest colony losses occurred in mainland France and Spain, although they did not correlate with the Nosema infection levels, as parasitism was low in France and high in Spain. These results suggest that both the effects and the level of N. ceranae infection depends on location and beekeeping conditions. Further studies on host-parasite coevolution, and perhaps the interactions with other pathogens and the role of honey bee genetics, could assist in understanding the difference between nosemosis disease and infection, to develop appropriate strategies for its control

Epidemiología de la nosemosis tipo C : influencia de factores bióticos y abióticos en el desarrollo de la infección

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Veterinaria, leída el 24-04-2023Las abejas melíferas son fundamentales para el mantenimiento de la biodiversidad en ecosistemas agrarios y naturales por su papel polinizador de cultivos y plantas silvestres, además de su interés por la elaboración de alimentos y otros productos de consumo humano. Estos insectos sociales son susceptibles de sufrir la infección de muchos patógenos que ponen en peligro la viabilidad de las colonias. Entre ellos, el microsporidio Nosema ceranae, agente causante de la nosemosis tipo C, es uno de los más prevalentes a nivel mundial. Sin embargo, a pesar de su amplia distribución y de las importantes repercusiones patológicas que tiene sobre las colonias de abejas, todavía se desconocen importantes aspectos de su epidemiología y, sobre todo, de su posible interacción con otros factores bióticos. Por este motivo, en la presente Tesis Doctoral se ha estudiado el papel de diversos factores involucrados en la infección utilizando colonias infectadas por N. ceranae de manera natural, con el fin de comparar el desarrollo de la infección a diferentes niveles (apiario, colonia y abejas individuales). Para ello, se ha realizado un estudio comparativo de la infección en seis colmenares distribuidos en cuatro países mediterráneos para analizar cómo varía la infección a lo largo del tiempo durante un período de veinte meses. Se comprobó que las condiciones de cada apiario y el manejo apícola influyen en el desarrollo de la infección afectando de forma significativa tanto el porcentaje de parasitación dentro de la colonia como la mortalidad de las colmenas. También se estudió el papel de las condiciones climáticas y, pese a que se intuye cierta influencia, no se ha podido establecer ninguna relación entre éstas y la parasitación del microsporidio dentro de la colonia. También cabe destacar que la mortalidad de las colonias registradas a lo largo del tiempo en los colmenares parece obedecer a un conjunto de factores y no solamente a la parasitación por N. ceranae...Honey bees are essential to maintain biodiversity in agricultural and natural ecosystems due to their role as pollinators of crops and wild plants, in addition to their interest in the production of food and other products for human consumption. These social insects are susceptible to infection by many pathogens that threaten the viability of colonies. Among them, the microsporidium Nosema ceranae, the causative agent of nosemosis type C, is one of the most prevalent worldwide. However, despite its wide distribution and the important pathological impact it has on honey bee colonies, important aspects of its epidemiology and especially its possible interaction with other biotic factors are still unknown.For this reason, in this PhD Thesis the role of several factors involved in the infection have been studied using colonies naturally infected by N. ceranae, in order to compare the development of infection at different levels (apiary, colony and individual honey bees).In order to do this, a comparative study of the infection was carried out in six apiaries distributed in four Mediterranean countries to analyse how the infection varies over time during a period of twenty months. It was found that the conditions of each apiary and the beekeeping management influence the development of the infection, significantly affecting both the percentage of parasitization within the colony and the mortality of the colonies. The role of climatic conditions were also studied and, although some influence is suspected, it has not been possible to establish any relationships between these and the levels of the microsporidium within the colony. It is also noteworthy that the mortality of the colonies recorded over time in the apiaries seems to be due to a combination of factors and not only by N. ceranae infection...Depto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaTRUEunpu

First data on the prevalence and distribution of pathogens in bumblebees (Bombus terrestris and Bombus pascuorum) from Spain

Bumblebees provide pollination services not only to wildflowers but also to economically important crops. In the context of the global decline of pollinators, there is an increasing interest in determining the pathogen diversity of bumblebee species. In this work, wild bumblebees of the species Bombus terrestris and Bombus pascuorum from northern and southern Spain were molecularly screened to detect and estimate prevalence of pathogens. One third of bumblebees were infected: while viruses only infected B. pascuorum, B. terrestris was infected by Apicystis bombi, Crithidia bombi and Nosema bombi. Ecological differences between host species might affect the success of the pathogens biological cycle and consequently infection prevalence. Furthermore, sex of the bumblebees (workers or males), sampling area (north or south) and altitude were important predictors of pathogen prevalence. Understanding how these factors affect pathogens distribution is essential for future conservation of bumblebee wild populations

Wide diversity of parasites in Bombus terrestris (Linnaeus, 1758) revealed by a high-throughput sequencing approach

Assessing the extent of parasite diversity requires the application of appropriate molecular tools, especially given the growing evidence of multiple parasite co-occurrence. Here, we compared the performance of a next-generation sequencing technology (Ion PGM ™ System) in 12 Bombus terrestris specimens that were PCR-identified as positive for trypanosomatids (Leishmaniinae) in a previous study. These bumblebees were also screened for the occurrence of Nosematidae and Neogregarinorida parasites using both classical protocols (either specific PCR amplification or amplification with broadrange primers plus Sanger sequencing) and Ion PGM sequencing. The latter revealed higher parasite diversity within individuals, especially among Leishmaniinae (which were present as a combination of Lotmaria passim, Crithidia mellificae and Crithidia bombi), and the occurrence of taxa never reported in these hosts: Crithidia acanthocephali and a novel neogregarinorida species. Furthermore, the complementary results produced by the different sets of primers highlighted the convenience of using multiple markers to minimize the chance of some target organisms going unnoticed. Altogether, the deep sequencing methodology offered a more comprehensive way to investigate parasite diversity than the usual identification methods and provided new insights whose importance for bumblebee health should be further analysed

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-stone species that has been severely affected by biological invasions. To fill the gap, here we identified single nucleotide polymorphisms (SNPs) in honey bee immune genes and then developed an assay to be genotyped using the high-sample-throughput iPLEX MassARRAY system that can be readily used for population surveys. The assay was constructed using SNPs detected in whole-genome scans of 123 individuals originating from a wide geographical area, representing seven A. mellifera subspecies and three evolutionary lineages (M-Western European, C- Eastern European, A- African). In this dataset, a total of 35,782 SNPs distributed through 180 genes were found. Only polymorphic SNPs (MAF>0.05) SNPs located in putatively functional regions were retained. Other filtering criteria inked to the MassARRAY® MALDI-TOF genotyping system were used and an assay with 107 SNPs was obtained. A total of 16 SNPs were discarded either due to inconsistent calls and/or misidentification of heterozygous positions. The final assay contains 91 quality-proved functional SNPs covering 89 innate immune genes. The 89 genes belong to several families and pathways, such as IMD, JAK-STAT, Toll and RNAi. This gene set also includes genes that have been found to be expressed in honey bees infected with the viruses SINV, IAPV, DWV and Nosema spp. This medium-density-SNP assay was applied to 156 samples from four countries (Portugal, Spain, France and Israel) and the admixture analysis clustered the samples according to their lineage and subspecies, suggesting that the immune SNPs can be also used to reconstruct population structure. This newly-developed SNP assay can be applied to monitoring diversity in immune genes, identifying the genetic basis of disease susceptibility, and even inferring genetic structure.info:eu-repo/semantics/publishedVersio

IN PRESS - Mitochondrial and nuclear diversity of colonies of varying origins: contrasting patterns inferred from the intergenic tRNAleu-cox2 region and immune SNPs

In this study, we gathered sequence data from the tRNAleu-cox2 intergenic mitochondrial (mtDNA) region concurrently with single nucleotide polymorphism (SNP) data from 91 loci of nuclear DNA (ncDNA). The data was obtained from 156 colonies sampled in six apiaries from four countries. The full dataset was analysed and discussed for genetic patterns with a focus on cytonuclear diversity and admixture levels.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 (ERANET) received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 618127. 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).info:eu-repo/semantics/publishedVersio