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.
Nosema ceranae is an intracellular parasite that infects honeybees’ gut altering the digestive functions; therefore, it has the potential of affecting the composition of the gut microbiome. In this work, individual bees of known age were sampled both in spring and autumn, and their digestive tracts were assessed for N. ceranae infection. Intestinal microbiome was assessed by sequencing the bacterial 16S rRNA gene in two different gut sections, the anterior section (AS; midgut and a half of ileum) and the posterior section (PS; second half of ileum and rectum). A preliminary analysis with a first batch of samples (n = 42) showed that AS samples had a higher potential to discriminate between infected and non-infected bees than PS samples. As a consequence, AS samples were selected for subsequent analyses. When analyzing the whole set of AS samples (n = 158) no changes in α- or β-diversity were observed between infected and non-infected bees. However, significant changes in the relative abundance of Proteobacteria and Firmicutes appeared when a subgroup of highly infected bees was compared to the group of non-infected bees. Seasonality and bees’ age had a significant impact in shaping the bacteriome structure and composition of the bees’ gut. Further research is needed to elucidate possible associations between the microbiome and N. ceranae infection in order to find efficient strategies for prevention of infections through modulation of bees’ microbiome.
The microsporidia Nosema ceranae is an intracellular parasite of honeybees’ midgut, highly prevalent in Apis mellifera colonies for which important epidemiological information is still unknown. Our research aimed at understanding how age and season influence the onset of infection in honeybees and its development in the colony environment. Adult worker honeybees of less than 24h were marked and introduced into 6 different colonies in assays carried out in spring and autumn. Bees of known age were individually analyzed by PCR for Nosema spp. infection and those resulting positive were studied to determine the load by Real Time-qPCR. The age of onset and development of infection in each season was studied on a total of 2401 bees and the probability and the load of infection for both periods was established with two statistical models. First N. ceranae infected honeybees were detected at day 5 post emergence (p.e.; spring) and at day 4 p.e. (autumn) and in-hive prevalence increased from that point onwards, reaching the highest mean infection on day 18 p.e. (spring). The probability of infection increased significantly with age in both periods although the age variable better correlated in spring. The N. ceranae load tended to increase with age in both periods, although the age-load relationship was clearer in spring than in autumn. Therefore, age and season play an important role on the probability and the development of N. ceranae infection in honeybees, bringing important information to understand how it spreads within a colony.
Summary 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 broad‐range 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.
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.
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