Understanding the nexus of nutrition, the microbiome, and virus susceptibility in the honey bee
Irene Newton, Adam Dolezal, and others aim to understand how interactions between honey bees and their symbiont, Bombella apis, protect brood from environmental perturbations such as nutritional and viral stress. In prior research, the Newton lab has found that B. apis protects brood from nutritive stress. The Dolezal lab has found that both adult and larval nutrition impacts virus susceptibility in honey bee workers. Their new project will connect these two lines of inquiry, identifying links between B. apis colonization, nutritional supplementation, and virus susceptibility in both honey bee workers and queens.
Establishing a GEMS culture collection
Very few systems of study in biology include molecular level understanding of coevolution among multiple interactors since most studies still focus on pairwise interactions. An important aspect of GEMS is basic discovery – capturing natural variation in a diversity of microbial species within our single focal environment. Toward this end, Rachel Whitaker, Bill Metcalf, and many others are leading an effort that includes both targeted and general culturing strategies to capture rhizobia, bombella, pseudomonads, streptomyces, and fungal species plus many new bacteria from field sites at the W.K. Kellogg Biological Station. Culturing will be paired with culture-independent sampling of soil, leaf, and other environments and expanded in future years to capture microbial viruses and other elements.
Mutualism response to stress
Symbiosis with rhizobial bacteria is a strategy legumes employ to meet one stress - the challenge of nitrogen limitation. In previous work, this research team has documented a degradation of this symbiosis in the long-term nitrogen fertilization plots at the W. K. Kellogg Biological Station (KBS), where rhizobia from high N plots were less beneficial for host plants. However, nitrogen availability is only one dimension of the environmental context in which the clover-rhizobium symbiosis exists. How does a system so sensitively tuned to one stressor (e.g., nitrogen) respond when confronted with another major environmental stressor? Does the partnership collapse under the pressure of multiple and potentially conflicting constraints, or do the eco-evolutionary feedbacks inherent in the symbiosis contribute to rapid adaptation? To what extent are relationships with other partners (e.g., "symbionts of symbionts" and members of the plant microbiome) involved in the response? Tony Yannarell, Jen Lau, and others are investigating the constraints that may limit adaptation to multiple stressors (nitrogen, drought) using the clover-rhizobium symbionts from KBS plots. They hypothesize that relationships with a broad array of partners in the microbiome can help the clover-rhizobium symbioses adapt to multiple environmental stressors.
Mutualism feedbacks on pollination
The clover-rhizobium and bee-virus systems are linked through plant traits that have important implications for bee biology; therefore, we hypothesize that the evolution of less cooperative rhizobia in response to long-term nitrogen (N)-addition will affect interactions with pollinators because of changes in floral traits. Adam Dolezal, Jen Lau, Alex Harmon-Threatt, and others will test this hypothesis by quantifying the effects of both N and rhizobium evolution on plant traits and investigating how these traits affect bee visitation, behavior, and physiology. This starting point will facilitate future work that aims to identify how observed changes in plant traits affect bee immunity and health and also how rhizobium evolutionary effects on bee visitation feeds back to affect plant demography and might contribute to observed legume declines in high N environments in the field.
Recombination, HGT, and natural selection in bacterial evolutionary responses
It is clear that horizontal gene transfer (HGT), acting at various phylogenetic scales, is responsible for major evolutionary innovations in microbes. Detecting HGT, particularly at local/population genomic scales, is a difficult and potentially computationally- and statistically-challenging problem. We have yet to settle on a set of comprehensive and generalizable methods for detecting recombination and HGT in bacterial genomes so that these patterns can be combined with signatures of natural selection to address evolutionary responses in ecologically relevant traits in natural bacterial populations. In Rhizobium symbionts of legumes, for example, the region of highest differentiation between control and N-evolved strains (which have become less beneficial for plant hosts) appears to have been transmitted across a diverse set of chromosomal lineages; however, addressing this problem with rigor will require additional focused work and novel methods. Using existing datasets consisting of hundreds of rhizobial strains, GEMS postdoc Mario Ceron Romero is working with Katy Heath, Rachel Whitaker, and Tandy Warnow to test existing methodologies and develop novel pipelines to address these questions, which can be applied to Bombella and Pseudomonas populations and beyond as part of ongoing research in the Institute.
Eco-evolutionary forces maintaining genetic variation in Pseudomonas syringae
Explaining the abundance of genetic diversity in nature is a core goal of evolutionary biologists. It is known from other host species that tremendous diversity, both in terms of core genomes and in terms of effector repertoires, can be maintained in populations of the plant pathogen Pseudomonas syringae. Joy Bergelson, together with Mercedes Pascual and Rachel Whitaker, are working toward surveying and explaining pathogen effector populations in Pseudomonas associated with a single, homogenous population of clover at the W. K Kellogg Biological Station. The project goal is to ask whether clover harbors diverse populations of Pseudomonas syringae, with an eye towards understanding if horizontal gene transfer is a mechanism for maintaining this variation. Whole genome sequencing will enable the quantification of genomic diversity, including effectors, CRISPRs and other elements that are horizontally transferred. These explorations will provide a basis for formulating theory on plant-microbial interactions and the structure of genomic diversity that emerges from selection on genomic components subject to horizontal gene transfer. Studies of the eco-evolutionary dynamics and diversity patterns arising from such recognition in other host-pathogen systems, including CRISPR-induced immune diversification between microbial hosts and their viruses, are already ongoing. As part of GEMS, there is an opportunity to advance theory that encompasses a variety of systems where host recognition underlies vast genetic diversity of symbionts and pathogens.
Coevolutionary interactions between clover hosts and rhizobium symbionts
Nitrogen (N) addition is predicted to destabilize the legume-rhizobium mutualism. Lau and Heath have previously found data to support this theoretical prediction for the rhizobium partner. The potential role of evolution in the legume host in response to N-addition, or how N-addition will alter the coevolutionary feedbacks between leguminous hosts and their rhizobium symbionts, remain unaddressed. Jen Lau together with Katy Heath, Joy Bergelson, Carla Caceres and others will use approaches from quantitative genetics to investigate the potential for host evolutionary responses to N and to evolutionary changes in their rhizobium partners. Their initial project will identify the extent of plant genetic variation in traits underlying symbiotic interactions with rhizobia and predict evolutionary responses of plants to both N and rhizobium genetic variation. The goal is to begin to better understand the responses of the plant component of this series of nested mutualisms and the potential for coevolution between the plant and rhizobium partners. These results and generated plant lineages will lead to further experiments assessing how plant evolution feeds back to influence interactions with other community members and rhizobium evolution.
Evolutionary ecology of symbiosis using microbial mark-recapture
Over the past few decades, experimental procedures have been developed and refined for the study of microbial evolution under controlled laboratory conditions. Whether such information from test-tube studies pertains to the evolution of microorganisms and their hosts in nature is uncertain owing to the challenges associated with tracking genotypes and populations in the wild. One promising and recent development involves the insertion of barcodes into the genomes of microorganisms, which can then be retrieved through amplicon sequencing technology. Jay Lennon has generated proof-of-concept data in bacterial populations (i.e., Bacillus) with the goal of quantifying fitness costs associated with phage resistance and the production of dormant resting stages (i.e., endospores). Lennon, together with Jen Lau, Katy Heath, Rachel Whitaker, and Tony Yannarell will transfer these genetic and informatic tools to study the evolutionary ecology of Rhizobium leguminosarum, both during its interactions with clover hosts and in the soil environment. The project goal is to quantitatively track genotypes in nested symbionts to understand trade-offs and evolution and constraints on mutualisms involving plants and microbes. The proposed work addresses long-standing limitations that preclude a full understanding of the legume-rhizobium symbiosis, including challenges associated with quantifying fitness and evolutionary trajectories of bacteria in soil and plant life-stages. Molecular based mark-recapture approaches will synergize with other GEMS-related research that seeks to quantitatively understand the evolution of microbial populations (e.g., Pseudomonas, Streptomycetes, Bombella) embedded in complex communities.
Development of experimental tools for dissecting virus-honey bee interactions
Efforts to dissect honey bee-virus interactions are hampered by the absence of critical experimental tools. These limitations include (a) an inability to generate or study pure populations of virus due to the presence of persistent viral infection within currently existing honey bee cell culture systems, and (b) a lack of tools for genetic manipulation of viruses. GEMS researchers Chris Brooke, Adam Dolezal, and others will work to develop the cell culture systems and viral reverse genetics tools that will be critical for subsequent efforts to dissect the interplay between viruses, honey bee and alternative hosts, honey bee colonies, and the surrounding ecosystem. Altogether, the development of these critical experimental tools will enable a variety of future studies that will allow us to understand the molecular bases of broader within-host, colony-level, and ecosystem level dynamics.
Symbiosis genes, HGT, and mutualistic partner quality
Previous genomic work comparing the more beneficial rhizobium symbionts from ambient nitrogen environments to the less beneficial rhizobia from high nitrogen environments showed that genetic variation at the symbiosis gene region of the pSym symbiosis plasmid differentiated high and low-quality populations. The specific loci in this region are canonical symbiosis genes, including regulators and enzymes required for nitrogen fixation, but the effects of specific mutations on symbiosis phenotypes have not been determined, despite the ability to manipulate Rhizobium in the laboratory. In addition, the phylogenetic evidence to date suggests HGT of this region of the plasmid, across diverse chromosomal backgrounds, making this an even more interesting candidate genetic region for functional validation to link selection and HGT. Cari Vanderpool, Katy Heath, Rachel Whitaker, Amy Marshall-Colon and others will integrate molecular genetics with quantitative genetics and evolutionary ecology to address this current gap in knowledge. Combining additional analyses of sequenced genomes with exploration of transcriptional profiles and targeted genetic manipulations will allow us to address evolutionary responses of mutualisms at a mechanistic level, integrating molecular processes with ecological processes in this system for the first time.
Defining the functional diversity and evolution of phage-associated endosymbiont toxins
Bacterial endosymbionts hijack host cell biology by secreting effectors that mimic eukaryotic protein function. These effectors encode domains that are homologous to eukaryotic toxins and tend to cluster inside phage genomes that infect endosymbionts. Phage-associated toxins can have dramatic impacts on host cell biology. For example, expression of phage-associated toxins underlies cytoplasmic incompatibility (CI) between males and females infected with the maternally transmitted endosymbiont Wolbachia in Drosophila and mosquitoes. But, expression of non-phage-associated toxins does not appear to affect CI. It remains unclear, therefore, whether association with the phage genome itself impacts the function and evolution of endosymbiont toxins. Tandy Warnow and Irene Newton address this question by comparing the functional diversity and molecular evolution of phage- versus non-phage-associated toxins from Wolbachia. Over the last ten years, hundreds of diverse endosymbiont and associated phage genomes have been sequenced, but the impact of this diversity on the coevolution of host-symbiont interactions remains unclear. This project aims to capture much of the functional arms race between host, symbiont, and phage by characterizing the diversity and evolution of horizontally transferred endosymbiont toxins.
Modelling the impact of lateral gene transfer on strain diversity in host pathogen systems
Many endemic pathogens harbor highly diverse populations that lack the episodic fluctuations in density that are the hallmarks of infectious disease dynamics. Joy Bergelson, Mercedes Pascual, and Rachel Whitaker combine coevolutionary theory with empirical studies of plants, bacteria, and viruses to understand the eco-evolutionary forces maintaining genetic variation in symbiosis. The team aims to develop theory to explore how microbes’ non-core genomic elements, which are nevertheless critical to interactions with hosts, shape stable persistence of genomic diversity among pathogens via lateral gene transfer (LGT). Given LGT, these non-core elements can be viewed as “embedded” and symbiotic to the microbial pathogen genome. The team will model eco-evolutionary dynamics of host-virus systems mediated by CRISPR immunity of microbes, and influence on the structure of strain diversity, whose predictions will be assessed for surveying P. syringae on clover in the field and leveraging ongoing surveys of P. syringae on A. thaliana. The theory will be extended from lytic to chronic viruses to incorporate long-term symbiotic and beneficial interactions in addition to pathogenic ones.
Fungal contributions to GEMS systems
Fungi are underappreciated components of microbial communities and can contribute to community assembly dynamics through microbe-microbe and host-microbe interactions. Specifically, Aspergillus species are ubiquitous environmental fungi that can play significant roles in plant, bee, and soil health. These species also unify the GEMS systems and can be potentially trafficked between them. However, little is known about the phenotypic and genetic diversity of Aspergillus strains in any of the GEMS systems. For example, previously, the Berenbaum and Dolezal labs have shown that a specific Aspergillus flavus strain, isolated from bee bread, can detoxify pesticides, providing a benefit to the bee. However, a different isolate of Aspergillus flavus, cultivated from a dead honey bee by the Newton lab, is a potent pathogen of bee brood and is inhibited by the B. apis antifungal metabolite. These disparate results lead to questions such as: What is the genetic diversity of Aspergillus strains across these environments? How does this diversity relate to the ability of these organisms to interact with resident microbes and the bee colony? Is there environmental filtering that occurs as the bees traffic in these environmental microbes from gathered pollen? What is the spillover effect on bee health? May Berenbaum, Irene Newton, Adam Dolezal, and Tony Yannarell focus on the bee system as preliminary data from the group suggests Aspergillus may play significant roles in the health of the bee colony. However, because the colony is an open system, the project and framework easily extend to other GEMS systems, with the potential for this project to expand to the soil, and plant interactions in subsequent years.
Nested evolution of Streptomyces populations and their phages
Streptomyces produce an immense array of useful secondary metabolic products (i.e., antibiotics), as a response to the complex communities harbored in the soil where they are typically found. These secondary metabolites are highly variable within Streptomyces genomes and are likely associated in populations with mobile DNA, phage, and other mobile elements. Streptomyces species contain CRISPR-Cas loci within their genomes that likely control mobile DNA mobility and thereby impact the balance between selection and gene flow of these important interaction loci. Rachel Whitaker and Bill Metcalf propose that examining CRISPR-Cas diversity in Streptomyces will reveal the assortment of phages commonly infecting and interacting with Streptomyces and may elucidate the evolutionary relationships between the environment, the phages present, and the development of CRISPR defense systems.
From sequence to structure workflow for analysis of CRISPR-Cas immunity dynamics in microbial populations
Predictive models for the epidemiology of infection by genetic elements of microbes are necessary to address both their ecological and evolutionary impacts. Metrics have been developed that describe the diversity of the CRISPR spacers and their outcome in simple microbial communities; however, their comparative application to empirical data sets is limited. Liudmila Mainzer, Rachel Whitaker, and Irene Newton will develop an accessible genomics tool for analysis of CRISPR population diversity in populations of bacterial genomes.
Closed genomes of Rhizobia populations for evolutionary analysis
Molecular understanding of microbial genomes shows that complete genomes will be required for eco-evolutionary analyses that include variable elements of microbial genomes, including viruses, plasmids, defense islands, CRISPRs, and large indels. The polished genomes will integrate evolutionary dynamics with genome architecture and mutation and recombination mechanisms and HGT balanced with selection. Rachel Whitaker and the GEMS core team propose long-read sequencing of 65 strains of Rhizobia isolated from 2008 that form the basis of GEMS mutualism breakdown with N addition. An additional 60 strains isolated in 2018 will also be added for full analysis as well as a growing set of strains (up to 50) from leaves and soil in N addition and control plots isolated by the GEMS culture club.