Nancy A. Moran, Ph.D. - US grants

Host and symbiont determinants of colonization by a co-evolved gut community Project Summary Host-specific gut bacteria are central to the biology and health of animals, but elucidating the processes that govern normal and atypical assembly of gut communities is challenging. Most gut communities, and specifically those of humans, are dauntingly complex; whereas others, such as that of Drosophila, have variable compositions dominated by opportunistic bacterial species from other environments. In the honey bee (Apis mellifera), the ileum region of the hindgut contains a dense but simple community dominated by only three specialized bacterial species that comprise >95% of bacteria despite the continual entry of diverse environmental microbes present in food. These three species are found only in bee guts, and distinct but related strains are found in related bee species. Our pilot work established axenic culture conditions, official nomenclature, and reference strains for all bee gut community members, transposon-based mutagenesis methods, protocols for controlling colonization with specific isolates or mutants, and methods for quantitative description of community composition. Furthermore, the host is amenable to genetic studies: sequenced genome and associated resources are available for the honey bee, and RNAi methods have been established. Building on these foundations, we will use the honey bee ileum as a model gut community to determine the specific bacterial and host factors that underlie the establishment of a persistent gut community characteristic of a host species. We will use expression analyses and genome-wide mutagenesis and mutant screening based on high-throughput sequencing to identify symbiont genes affecting colonization of the bee ileum and to elucidate the effects of host genes and pathways on colonization. We will examine how a focal pioneer symbiont influences the assembly and subsequent composition of the gut community. This will be achieved by monitoring succession in experimental gut communities and by identifying symbiont genes that affect colonization by other bacterial species and strains, including both the symbionts normally present in the community and the opportunistic or pathogenic bacteria that are typically excluded. These results will reveal host and symbiont-based processes that govern development of a normal, host- specific gut microbiota and will illuminate how and why gut communities sometimes develop abnormally, to the detriment of host health. !

Intimate associations between bacteria and animals have evolved frequently and form the basis for many specialized animal lifestyles. Insects that feed only on sap of vascular plants depend on bacterial partners that live within specialized cells of their insect hosts and that provide nutrients to the insects. These relationships have persisted for hundreds of millions of years, yet little is known about what mechanisms allow both insects and bacteria to rely on each other. This project addresses the question of how these insects tolerate and foster their resident bacterial populations. The project will incorporate experiments on laboratory populations of leafhoppers, which have some of the most specialized symbiotic associations. Typically leafhoppers harbor two different symbiont types that provide complementary sets of nutrients required by the hosts. Investigators will use new genomic and molecular technologies to determine the mechanisms that leafhopper species use to maintain their symbiotic bacteria. To understand the specific host mechanisms that differentiate among bacterial types, investigators will make use of comparisons between related leafhopper species that have different bacteria as symbionts.
This project will give in-depth research experiences to numerous undergraduates at the University of Texas. It will also be the basis for the long-term research program of a postdoctoral researcher who plans future work on insect-bacterial relationships. In addition, leafhoppers are primary vectors of many viral and bacterial diseases of plants, including many major agricultural crops in the United States. For this reason, leafhoppers are some of the most severe insect pests of agricultural systems. Thus, investigators will perform all experiments on two notorious North American pest species. These results will provide new insight into how these pest species interact with microorganisms, including both symbionts and disease organisms.

Many insects, including pest species that harm crops and transmit disease, depend on symbiotic bacteria for survival. This community of symbiotic bacteria, known as the microbiome, lives inside their host's cells and tissues and is passed directly from mother to offspring. These bacteria often benefit their hosts, for example, by producing required nutrients missing from the diet. But symbionts also can undergo genetic changes detrimental to hosts. This project addresses whether and how detrimental evolution of symbionts affects their host insects. The focus here is on one such group, aphids, which attack most of our agricultural crops and require symbiotic bacteria in order to grow and reproduce. The research will be used as a basis for teaching basic biology and genetics to University of Texas undergraduate students, including participants in a large freshman research program. Also, the results will further understanding of a significant group of agricultural pests, potentially contributing to the development of new methods of crop protection.

The experiments address how genetic variation of pea aphids affects their ability to regulate symbiotic bacteria and how genetic variation of the symbiotic bacteria affects their own multiplication within aphid hosts. The researchers measure how variation in symbiont regulation affects aphid growth and reproduction. Experimental genetic crosses of aphids that vary in levels of symbiotic bacteria will be combined with modern DNA sequencing methods to identify the parts of the aphid genome that affect symbiont numbers. In addition, symbiont strains will be transferred between aphid hosts using microinjection, to determine how different symbiont strains affect hosts.

DESCRIPTION (provided by applicant): Host and symbiont determinants of colonization by a co-evolved gut community Project Summary Host-specific gut bacteria are central to the biology and health of animals, but elucidating the processes that govern normal and atypical assembly of gut communities is challenging. Most gut communities, and specifically those of humans, are dauntingly complex; whereas others, such as that of Drosophila, have variable compositions dominated by opportunistic bacterial species from other environments. In the honey bee (Apis mellifera), the ileum region of the hindgut contains a dense but simple community dominated by only three specialized bacterial species that comprise >95% of bacteria despite the continual entry of diverse environmental microbes present in food. These three species are found only in bee guts, and distinct but related strains are found in related bee species. Our pilot work established axenic culture conditions, official nomenclature, and reference strains for all bee gut community members, transposon-based mutagenesis methods, protocols for controlling colonization with specific isolates or mutants, and methods for quantitative description of community composition. Furthermore, the host is amenable to genetic studies: sequenced genome and associated resources are available for the honey bee, and RNAi methods have been established. Building on these foundations, we will use the honey bee ileum as a model gut community to determine the specific bacterial and host factors that underlie the establishment of a persistent gut community characteristic of a host species. We will use expression analyses and genome-wide mutagenesis and mutant screening based on high-throughput sequencing to identify symbiont genes affecting colonization of the bee ileum and to elucidate the effects of host genes and pathways on colonization. We will examine how a focal pioneer symbiont influences the assembly and subsequent composition of the gut community. This will be achieved by monitoring succession in experimental gut communities and by identifying symbiont genes that affect colonization by other bacterial species and strains, including both the symbionts normally present in the community and the opportunistic or pathogenic bacteria that are typically excluded. These results will reveal host and symbiont-based processes that govern development of a normal, host- specific gut microbiota and will illuminate how and why gut communities sometimes develop abnormally, to the detriment of host health.

Pollinator health is important for both agricultural and natural ecosystems, and one of the most important pollinators, especially in agriculture, is the honey bee. In this project, researchers will focus on bacteria in honey bee guts, because gut bacteria affect animals' health. They will study how these bacteria interact with one another, because this is important for how stable helpful gut bacterial communities are, and how they interact with the host animal. The researchers will continue working with two critical bacteria in the bee gut. They have already shown how these bacteria have toxins with which they can attack one another. However, it is likely that these bacteria also help one another in some situations. The researchers will use recently developed methods to look at whether and how these bacteria pass nutrients to one another and to the host bee. This will contribute to understanding how bee-gut bacterial communities stay stable through time. It will help to develop new routes to improving bee health. In addition, during the project graduate and undergraduate researchers will be trained in state-of-the-art methods in microbiology.

As insects can take up and utilize byproducts of carbohydrate fermentation, honeybees may benefit from the metabolic activities of members of their gut microbial communities through both the degradation of toxic sugars and the production of useful byproducts. To better understand the role of cooperative metabolic interactions in gut communities, this project will trace nutrient exchange in the honeybee gut. Genomic evidence suggests that the bee gut microbes Snodgrassella alvi and Gilliamella apicola depend upon one another for production of nutrients. G. apicola metabolizes carbohydrates that S. alvi cannot, and metabolizes sugars found in nectar that are toxic to honey bees. This project will use isotopically-labeled monosaccharides that can be utilized by G. apicola, but not by S. alvi or the host, to trace metabolic interactions in the bee gut. DNA-stable isotope probing (DNA-SIP) will be used to track incorporation of labeled carbon atoms into bacterial DNA, while incorporation of these atoms into host tissues will be measured through elemental analysis and isotope ratio mass spectrometry. This research will contribute to a greater fundamental understanding of the processes that govern cooperative metabolic interactions among members of the gut microbiota and between the microbiota and the host, and will demonstrate the important potential role of SIP methods in hypothesis testing in microbial community ecology, a framework in which it is uncommonly used.

Project Summary/Abstract Microbial communities exert major impacts on animal biology and human health, and disruption of these communities is associated with multiple disease states. To date, the field of microbiome research has been dominated by surveys of microbial community compositions and by analyses correlating composition with host phenotypes. But there have been few attempts to directly link the specific, causal processes that determine colonization dynamics and success of host-associated bacteria, and how these interactions ultimately affect hosts. The proposed research plan is motivated by the need for experimental systems to identify the mechanisms that control the composition and consequences of host-associated bacterial communities. This work focuses on two model systems that provide complementary approaches to examining host-associated communities, and that offer new opportunities to identify the mechanisms underlying host colonization. The honey bee and its specialized gut microbiota provides an exceptional model for multispecies gut communities as it shares many features with the human gut microbiota. In both human and bee guts, a stable, healthy community bestows ?colonization resistance?, the exclusion of foreign microorganisms; in both systems, disruption can result in dysbiosis and expansion of atypical communities, including enteric pathogens. The human system is highly complex and not amenable to experiments, but, for the bee gut, we are able to culture isolates of all component bacterial species and to introduce these to microbiota-free hosts to establish defined communities. We have already developed genetic tools for experimental manipulation of the dominant species. One set of experiments will identify the direct host-bacterial interactions that determine colonization success or failure of particular strains that vary in ability to colonize honey bees. Existing results from a mutagenesis screen indicate that features of the outer cell envelope play essential roles during host colonization, and we will use new genetic tools to determine which of these factors are key to acceptance by hosts. In addition to elucidating the mechanisms that enable specific bacterial strains to mono-colonize specific hosts, a second set of experiments will investigate how the interactions between microbial strains, which range from metabolic co-dependency to direct toxin-mediated antagonism, determine community membership. The pea aphid and its endosymbionts provide an effective model for how intracellular bacterial associates stably colonize host cells. Our newly devised techniques allow isolation, manipulation and inter-host transfer of endosymbionts. To address how hosts control endosymbiont replication and persistence, we will perform genomic comparisons, biochemical experiments to test effects of host-produced gene products on endosymbiont cells, and physical and structural characterization of endosymbiont outer membrane proteins. Through focus on experimental models that provide tractable examples of host-associated bacteria, this work will illuminate the mechanisms that underlie ability of bacterial symbionts to colonize hosts.

Insects are among the most widespread and diverse animals on our planet. They have critical roles in natural ecosystems and agriculture and have evolved unique biomaterials and lifestyles. Scientific tools for studying the functions of the genes responsible for these traits are well-established for only a few types of insects, such as fruit flies. Genetic tools do not exist for millions of other species. Honey bees and bumblebees are economically important as widespread pollinators of food crops and are scientifically interesting due to the complex social behaviors observed in bee colonies. Currently, there are few effective tools for studying the functions of bee genes. This project will develop and disseminate a toolkit that allows researchers to alter the expression of bee genes by engineering their native symbiotic gut bacteria. This technology will enable studies of how specific genes contribute to bee physiology, development, and behavior. This work will contribute to understanding bee ecology and health in ways that are expected to benefit biodiversity and US food security in the long term. The technology for engineering symbiotic bacteria is expected to be widely applicable to studying other insect species. These research and outreach activities will be integrated with education by supporting two experiential learning courses that are part of the Freshman Research Initiative program at The University of Texas at Austin. Supporting this program will foster the development of a diverse science and technology workforce by involving underrepresented and first-generation college students in genuine research experiences.

Silencing the expression of a gene by inducing an RNA interference (RNAi) response is a common approach for performing studies of gene function in invertebrates. However, delivering enough double-stranded RNA to achieve sufficient gene knockdown through injection or feeding is expensive and ineffective in many insects, including bees. In this project, a FUnctional Genomics Using Engineered Symbionts (FUGUES) methodology will be developed and applied to honey bees (Apis mellifera) and bumblebees (Bombus spp.). In FUGUES, microbial symbionts are engineered to continuously produce and deliver double-stranded RNA to induce a targeted RNAi response in their host. Newly emerged bees colonized with an engineered bacterial symbiont exhibit reduced expression of a target gene throughout the bee body, enabling one to ascertain the function of a bee gene and its role in determining specific phenotypes. There are key advantages of using FUGUES to study gene function over current techniques that generate transgenic animals: it can be accomplished more quickly, it can be conducted in high-throughput when coupled with insect colonization via feeding, and it can be applied to species, such as bees, with mating systems and collective behaviors that complicate using genome editing techniques. Improving and disseminating the FUGUES tools created in this work will broadly enable studies of genes underlying bee behavior, development, and physiology. These tools will likely also be useful for studying many other insect species and other organisms that harbor symbiotic bacteria.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.