Recipient Organization
The University of North Carolina at Greensboro
1400 Spring Garden Street
Greensboro,NC 27412
Performing Department
Biology
Non Technical Summary
Honeybees are responsible for pollinating crops that make up at least one-third of our diet, and their value to global agricultural production reaches many billions of dollars per year. Since 2006, the world's honeybee colonies have undergone elevated mortality, and annual colony losses continue to rise. The reasons for this phenomenon, commonly called colony collapse disorder, are not fully understood, but several possible causes have been suggested, such as pesticides, genetically modified crops, habitat fragmentation, climate change, and various diseases and parasites. However, no single stressor has been shown to account for the declines.Gut microbial communities influence animal health in many ways including synthesis of vitamins, balance of metabolism, defense against pathogens, behavior, and developmental and immune responses. Perturbed gut communities can be detrimental to host health. Honeybees are exposed to a number of chemicals, such as pesticides, herbicides, and antibiotics, which are potent sources of disturbance to microbial communities. In view of growing evidence for the importance of the gut microbial communities in animal health and the largely unexplained decline of honeybee colonies, the effects of chemical exposure on the honeybee gut microbiome are of major interest.The work proposed here will provide a full description of how exposure to pesticides, herbicides, and antibiotics affects the native gut microbial communities of honeybees. This project will also address how disruption of the healthy gut community affects susceptibility to invasion by opportunistic pathogens. These findings will provide insights on the importance of resident gut microbes in honeybee health and will be particularly relevant to beekeepers by revealing what types of perturbations of gut communities increase the risk for pathogen infection. Thus, the results from this project will provide insight into the decline of honeybee populations.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
The proposed research will investigate the effects of chemically induced perturbation on the native gut microbiota of honeybee. These perturbations can have negative impacts on bee health, for example increased susceptibility to pathogen infection. I will determine the effects of antibiotics, pesticides, and herbicides on the native gut microbial community structure, and will evaluate whether recovery of the microbiota can be achieved after perturbation. Furthermore, it will address how disruption of the healthy community affects susceptibility to invasion by non-resident species, particularly opportunistic pathogens. To our knowledge, this is the first study to look at the effects of antibiotics, pesticides, and herbicides on the native honeybee gut microbial community structure.The long-term goal of this proposal is to understand the effects of perturbation on host- associated microbial communities. The honeybee, an agriculturally and economically important pollinator, will be used as a model system to address the following supporting objectives:Identify the impact of perturbation on the population dynamics of the gut microbiomeDetermine the ability of gut microbial community to recover after perturbationDetermine how perturbation of the gut microbiome promotes invasion by opportunistic pathogens.Findings of this project will provide insights into the importance of resident gut microbes for honeybee health.
Project Methods
Key to the proposed work is an experimental approach that will enable identifying fine-scale community changes that cannot be detected using 16S rRNA sequence profiling. As opposed to the highly conserved 16S rRNA, protein-coding genes evolve much faster. For example, bacterial strains differing by only 1% in 16S sequence can differ by 10-20% in single copy, universal protein-coding genes. This is directly observed in the bee bacteria. Thus, an approach based on protein-coding genes can reveal patterns of strain diversity and fine-scale differences in the community structure. This approach echoes an older method known as multilocus sequence typing (MLST), which is used to classify strains within species. Here, the critical difference is that it will be applied to obtain comprehensive characterizations of entire uncultured communities, and will exploit high- throughput sequencing technologies. Using the complete genomes from the core species of the bee gut microbiome, I have defined marker genes for each species. These markers were selected based on their universal presence in strains and their ability to capture maximal diversity. Using these markers I will be able to track strain level diversity much more accurately than is possible with 16S rRNA profiling. Moreover, traditional rRNA 16S profiling will also be applied in order to define the overall composition of the community (i.e. the presence of the major phyla).Objective 1. Identify the impact of perturbation on population dynamics of the gut microbiomeThe Raymann lab maintains five hives on the UNCG North campus and has established detailed protocols for experimentally manipulating gut microbiota of honeybees. NEWs will be collected from a removable frame kept overnight in an incubator. After emerging, the bees will be marked and released back into the colony so they can naturally acquire their microbiota. After one week, I will collect marked bees and subject them to i) antibiotics (tylosin) ii) imidacloprid iii) glyphosate and iv) combinations of the three treatments. Controls will be fed sterile sugar syrup. Cohorts of bees will be dissected before and after chemical treatment. DNA will be extracted from the gut using established techniques, and Illumina-based amplicon profiling of 16S rRNA and defined protein-coding gene markers will be performed. I will also estimate the total community size pre- and post-treatment by quantifying 16S rDNA copies using qPCR (adjusting for number of rRNA operons per genome). All DNA sequence data will be analyzed using bioinformatic approaches including sequence comparison, estimation of gene polymorphism, identification of operational taxonomic units (OTUs), and phylogenetic and taxon-based analysis of diversity within and between samples. Pilot experiments based on Illumina sequencing of the V4 region of the 16S rRNA gene have confirmed that treatment with tetracycline has a significant impact on the community structure of the microbiota.Objective 2: Determine the ability of the gut microbial community to recover after perturbationI will perform controlled experiments to test if different environmental factors play a role in the recovery of the gut microbial community after chemical perturbation (described in Objective 1). These experiments will involve allowing the bees to recover after chemical treatment in solitary cup cages, in cup cages containing normal, untreated workers taken from the hive, and in intact hives outdoors. The recovery groups maintained in cup cages will be fed with a sterile diet of sugar syrup. The bees will be allowed to recover for two weeks, and survival rates will be recorded daily. Cohorts of bees will be sampled at several time points during the experiment, and Illumina-based amplicon profiling will be done as described above.Objective 3. Determine how perturbation of the gut community promotes invasion by opportunistic pathogensSeveral opportunistic pathogens of the honeybee have been isolated in the lab and can be easily cultured. Newly emerged workers will be marked and released into the hive so they can naturally acquire their microbiota. After one week, I will collect marked bees and subject them to different chemical treatments (as described above). I will then expose the bees to various opportunistic pathogens of bees, e.g. Serratia marcescens, Escherichia coli, and Hafnia alvei. The colonization of the pathogen in the gut will be determined by plating the guts on LB agar plates (native gut members will not grow on this medium). Bees will be sampled (dissected) and survival rates will be monitored at multiple time points throughout treatment. DNA will be extracted from the gut using established techniques, and Illumina-based amplicon profiling of 16S rRNA and defined protein-coding gene markers will be performed to determine pre- and post-infection community composition. The relative abundance of the opportunistic pathogen will be compared to the entire community using qPCR. All DNA sequence data will be analyzed using the bioinformatics approaches described above.Progress towards completion of the project objectives will be based on each aim in the proposed timeline (2 years), which includes completing treatment, recovery, and infection experiments, data collection and analysis, and preparing manuscripts.