Ulrich Mueller - US grants

Workers in eusocial Hymenoptera colonies (relatedness asymmetry present) are predicted to favor a more female-biased sex-ratio as compared to the sex-ratio favored by workers in parasocial colonies (relatedness asymmetry absent). Experiments on the primitively eusocial sweat bee Augochlorella striata, with random assignment of colonies to a eusocial and parasocial condition confirmed the predicted female-bias in eusocial colonies, but were unable to test the hypothesis that worker reproduction of males is greater in parasocial than in eusocial colonies. The determination of maternity of males by means of a genetic analysis is essential to test this hypothesis. This study will involve genetically fingerprinting reproductives, workers, and male sexuals of the experimental nests in order to compare worker reproduction of males in eusocial and parasocial colonies. Because of parthenogenetic production of males, all bands in a fingerprint of a male must also be present in the fingerprint of his mother. Maternity of males can therefore be determined by matching fingerprints of males to fingerprints of putative mothers (e.g. foundresses and workers). If evidence from genetic fingerprinting refutes the hypothesis of worker reproduction, relatedness asymmetries deriving from haplodiploidy remain as the only explanation of the observed sex-ratio adjustments. This would imply that social behavior in A. striata evolved not only in response to ecological constraints limiting reproductive options, but also in relation to genetic variables (relatedness asymmetries) intrinsic to the haplodiploid system of sex-determination.

9707209 Mueller Among the more fascinating cases of symbiosis in the American tropics is the interaction between leafcutter ants of the tribe Attini (Family Formicidae) and fungi of the mushroom family Lepiotaceae (Basidiomycetes) in which the ants maintain "gardens" of freshly cut leaves on which the fungi are tended and harvested for food. There are some 200 or so described species of leafcutter Attini ants, and advanced members of the group are known to maintain extensive gardens in which propagation of the fungi is obligately linked to strict vertical transmission of inocula from parent to offspring nests. This obligate transmission of food inocula raises the intriguing question whether mutational changes and evolutionary diversification in the ants is mirrored by change and diversification in their cultivated fungi, and leads to conjoint patterns of speciation and coevolution. The interaction is known to be even more complicated because other groups of putatively primitive Attini ants are opportunistic in their acquisition of fungi for their gardens, and may regularly harvest fresh inocula from wild sources. Which ant groups do this regularly, which have switched over to the obligate mode of growth and transmission, and which act in an intermediate fashion are all unanswered questions. Correspondingly, which species and genera of Lepiotaceae fungi are associated with the various ant cohorts in "open" or "closed" systems of inocula uptake and transmission are also unknown. Preliminary molecular DNA analyses by Dr. Ulrich Mueller at the University of Maryland and his colleagues Ted Schultz at the Smithsonian and Stephen Rehner at the University of Puerto Rico have opened a major technological door to the study of this conceptually challenging case of symbiosis. Molecular methods are particularly necessary in study of the fungi because the cultivated forms rarely if ever sporulate, and in the absence of spore-bearing structures, taxonomic identification is difficult if not impossible. Diagn ostic DNA markers, from nuclear or mitochondrial genes, hold hope for solving this problem, while providing data that can be used to infer phylogenetic or genealogical associations among the fungi. This collaborative team of entomologists and mycologists continues field collecting in Central and South America, to augment the nearly 140 ant species thus far collected, and in parallel to collect fungal material from ant nests. Field work with Brazilian and Argentinian colleagues will facilitate collecting in rich areas in South America. Molecular DNA sequencing and morphological analyses of the ants and of those fungi that can be induced to sporulate in culture will provide additional taxonomic and phylogenetic information. Study of the phylogenetic patterns and ecological habitats of associated ants and fungi will create an accessible model of symbiont evolution in tropical terrestrial ecosystems, comparable in its richness of interactions with the coral reef symbioses involving corals and photosynthetic dinoflagellates.

9983879
Mueller

Fungus-growing ants comprise a group of about 200 species, all obligately dependent on the cultivation of fungus for food. Fungal cultivation by ants involves complex manuring regimes and use of antibiotic "herbicides" to control alien fungi and garden parasites. Recent genetic analyses indicate that fungus-growing ants domesticated fungal cultivars multiple times during their evolutionary history and frequently exchange these cultivars between ant species. The evolutionary ecology of cultivar exchange between ants will be investigated by testing a series of hypotheses on the ecological factors that govern exchanges. Field experiments will be conducted in Panama and the southwestern US, complemented by genetic analyses of the diversity of ants, their cultivars, and associated pathogens. Model ant-fungus systems will be developed to test specific hypotheses on the evolutionary and ecological relationships between ant farmers, their cultivars, associated garden parasites, and the ecological conditions modulating these relationships.

The attine ant-fungus symbiosis represents a unique case of evolutionary and ecological complexity. This complexity derives from the interaction of pathogenic and mutualistic microbes that evolved to form integrated parts of the ecological fabric of a social animal, the ant farmers. The research will yield a synthetic understanding of this 50-million-year antfarmer-cultivar association.

Ant agriculture, exemplified by the symbiosis between fungus-growing (attine) ants and their fungal cultivars, is intensively studied as a model system for understanding the general ecological and coevolutionary mechanisms that shape highly integrated symbioses. The attine ant agricultural symbiosis encompasses both mutualistic and parasitic interactions between (1) ant farmers, (2) fungal cultivars, (3) parasitic fungal "weeds" that infest ant gardens, and (4) antibiotic-producing bacteria that control the garden parasites and are cultured on the bodies of ants. This project specifically explores the ecological complexity of the attine ant-fungus-bacterium symbiosis and elucidates its 50-million-year coevolutionary history by testing hypotheses posed at multiple ecological and evolutionary levels, including: (1) the local ecological level within ant colonies; (2) the population and community levels; (3) the biogeographic level; and the phylogenetic level, including (4) coevolutionary processes and historical ecology; (5) symbiont-mediated speciation; and (6) the effects of symbiotic life histories on rates of evolutionary change. Because symbiosis is a major recurring theme in the history of life, this research is expected to generate general insights into the broad mechanisms that drive biological complexity and diversity. Such insights could in some cases impact issues directly relevant to human welfare, including the evolution of antibiotic resistance and the practice of sustainable agriculture.

Speciation is the basic process that generates biodiversity. In the tropics, leaf-cutter ants and their sister species the lower attine ants comprise a significant proportion of tropical insects, but little is known about their diversification. Patterns of speciation by switching fungal cultivar types between ant species will be examined in three Central American species complexes of lower attine ants (Cyphomyrmex longiscapus, Mycocepurus smithi, and Apterostigma pilosum). The goals of this study are to: split each species complex into valid species using molecular genetic techniques; assess genetic diversity of each species; examine biogeographic patterns of each species complex in Central America; and differentiate between allopatric and sympatric modes of speciation.

This research examines the fundamental process of forming new species. By investigating patterns of diversification in cryptic attine ant species, the attine-fungus mutualism may emerge as a new model system for speciation studies, thus contributing to our understanding of biodiversity. This research will provide training for a graduate student in molecular genetic, entomological, and field techniques, and the graduate student will train and supervise several undergraduate assistants. These results will add new information to the already astonishing level of complexity that has evolved within the attine system over the fifty million years that these ancient farming ants have been cultivating fungi.

DESCRIPTION (provided by applicant): The long-term goal of my laboratory is to elucidate the mechanisms that control mechanotransduction in hair cells, and the defects in this process that cause deafness. We propose here to identify and study proteins that interact with PCDH15 and TMHS/ LHFPL5 (referred to in the following as LHFPL5), two components of the hair cell's mechanotransduction machinery. Based on preliminary data, we hypothesize that PCDH15 and LHFPL5 are components of a larger protein complex that regulates the activity of mechanically gated ion channels in hair cells. We predict that mutations in complex components lead to auditory impairment. To test our hypothesis, we will: (i) Determine the function of LHFPL5 and some of its close homologues for mechano-transduction and auditory perception; (ii) continue our identification of hair cell proteins that interact with PCDH15 and/or LHFPL5; (iii) functionall characterize proteins that interact with PCDH15 and/or LHFPL5; (iv) determine their relevance for auditory impairment in humans. Our preliminary data show the feasibility of our approach. We have already identified hair cell proteins that interact with PCDH15 and LHFPL5, at least one of which is linked to auditory impairment in humans.

This study uses fungus-growing ant species to address host specialization by a virulent pathogen of the ants' fungus gardens. Molecular analyses of host-parasite phylogenetic relationships will be combined with analyses of population differences to determine if parasites specialize on particular host fungal species. Subsequent experiments will be initiated to determine the consequences of host specialization, and analyses of the chemical mechanisms underlying resistance will explain how specialization is maintained. The project will show how mechanisms of resistance and infectivity shape parasite-host specialization and the dynamics of these interacting populations. The research includes collaboration with scientists at domestic and foreign institutions and deposition of collections at museums in Latin America. Undergraduate students will be trained in molecular and experimental techniques.

This study will test three hypotheses concerning the processes that drive diversification of tropical organisms. These hypotheses have been widely discussed and have specific predictions that should be broadly applicable to tropical organisms. Dr. Ulrich Mueller and Scott Solomon will use three species of leafcutter ants in the genus Atta for this project. Specimens will be collected throughout the geographic range of each species, and genetic analyses, conducted in the United States and in Brazil, will test each prediction in turn.

This project will be the first to test these hypotheses with invertebrates, a group that comprises the majority of diversity in many tropical ecosystems. Furthermore, leafcutter ants are a fundamental component of many tropical habitats, and some species are major agricultural pests. By documenting genetic diversity within three such species, this analysis that could reveal the presence of new, cryptic species. Finally, the elucidation of the patterns by which new species are formed is critical for the establishment of effective conservation strategies, since the goal of conservation is to preserve not only existing diversity but also the processes by which diversity is generated.

DESCRIPTION (provided by applicant): Developmental defects and degenerative changes that affect the central nervous system (CNS) lead to neurological disorders including dementia, epilepsy, schizophrenia, Parkinson's and Alzheimer's disease. An understanding of the mechanisms that regulate CNS development and function is expected to provide insights into the molecular pathogenesis of neurological disorders. The development and physiology of neurons is regulated by their interactions with neighboring cells and with extracellular matrix (ECM) molecules. The mechanisms by which receptors that mediate these interactions regulate neuronal development and health are poorly defined. Our long-term goal is to understand the mechanisms by which cell-cell and celI-ECM interactions regulate CNS development, maintenance, and function. We propose here to study ECM receptors of the beta1-integrin family in the CNS. We hypothesis that beta1-integrins are part of a regulatory network that controls cell cycle progression of cortical precursor cells in the CNS. To test our hypothesis, we will use genetically modified mice carrying floxed alleles, Cre, and GFP to analyze by BrdU labeling and immunohistochemistry defects in cell proliferation and differentiation of beta1 integrin-deficient CNS precursors in vivo, and after FACS sorting in vitro. Our preliminary data validate our hypothesis. They show that beta1-integrins regulate proliferation in neurogenic zones of the CNS. We expect that beta1 -integrins are part of the regulatory circuit that coordinates cell-cycle exit with differentiation and migration. The identification of the mechanisms that regulate these events is important to understand CNS development and disease progression, as well as to control the behavior of neural stem cells in order to use them as therapeutic agents.

To a large extent, the history of life is a history of symbiotic interactions, where organisms exist together, either competing or collaborating. The extent to which selfish or cooperative behavior dominates a symbiosis depends on whether the interacting partners' genes are co-transmitted from generation to generation. If strict co-inheritance does not occur, cheating and other forms of virulent behavior can evolve. Using the well-studied intimately co-evolved attine ant-fungal cultivar symbiosis, this study will examine conflict and cooperation between the ants and the cultivar fungi from the perspective of mutualistic and antagonistic evolution theory. In particular, this study will focus on the outcome of the ancient conflict over cultivar escape through fruiting.

Results will be broadly applicable to other symbioses, such as evolution of disease virulence. Additionally, as fungus-gardening ants (the leaf-cutters) are the most important plant pests of the Neotropics, the economic consequences of the research permit collaboration with a Brazilian research group. This collaboration will foster international academic exchange, building a framework for field research and lab visits. Students from both the US and Brazil will participate in the project, receiving training and opportunities for conducting independent research. These international activities are supported by funds from the Office of International Science and Engineering.

Social parasitism, the exploitation of a society by other social organisms, is a
widespread phenomenon that has evolved independently numerous times within
many social systems. The monophyletic Megalomyrmex genus is an excellent
system to investigate how parasitism evolves in a social society. Using an integrative
approach, phylogenic analysis will reveal the origins of social parasitism, while
behavioral, ecological, and venom alkaloid information will help distinguish the
characteristics necessary for social parasitism to evolve and be maintained. The
following questions will be investigated: 1) Did social parasitism evolve once in
the Megalomyrmex genus? 2) Did social parasitism arise from behaviors
related to a primarily predatory lifestyle?

Merit:
By gathering these data the Megalomyrmex system will be comparable with other
parasite systems, and will thus broaden our understanding of the evolutionary
processes associated with the onset and maintenance of host-parasite associations.

Impacts:
This project will continue to incorporate undergraduate mentorship and training.
Ongoing collaborations will strengthen ties between Brazilian and American
institutions while expediting and developing this research. In addition, voucher
specimens have and will be deposited at one domestic and five foreign museums.
These collaborations and associations will not only make the proposed research
feasible but will effectively disseminate scientific knowledge.

DESCRIPTION (provided by applicant): Deafness is a major health problem. ~1 in 1000 children is born deaf and a large part of the aging population is afflicted by age-related hearing loss. Many forms of hearing loss are of genetic origin, but the majority of genes that are linked to the disease still need to be identified. There is also a pressing need for animal models to study gene function in the auditory system and to develop therapeutic approaches for treating hearing loss. The long-term goal of my laboratory is to elucidate the molecular mechanisms that control sound perception and the defects in this process that cause hearing loss. As a step towards attaining this goal, we propose here to extend our forward genetic screen in mice that was initiated in the previous funding period with the aim to generate mouse lines afflicted with congenital deafness. We have in the meantime optimized our screen and hypothesize that it will provide valuable animal models for studying the molecular pathogenesis of congenital, progressive, and late-onset forms of hearing loss in humans. This hypothesis is based on our published and preliminary data, which show that we have already generated in our screen mouse lines afflicted with various forms of hearing loss caused by mutations in genes linked to the human disease. To achieve our overall goal, we will generate by ENU mutagenesis additional mouse lines with hearing impairment, positionally clone the affected genes, characterize the mice phenotypically and search for mutation in human genes orthologous to the mouse genes that we identify in our screen. As more than 60% of the genes that are linked to hearing loss still need to be identified, we anticipate that we will identify additional genes that are linked to hearing loss in humans and generate mouse models for the human disease. Our mouse lines will be valuable for testing therapeutic approaches towards treating hearing loss.

DESCRIPTION (Provided by Applicant): A major goal in neurobiology is identifying functional neuronal pathways. Classical anatomical tracing techniques allow connectivity between brain regions to be determined. Lesion studies and anatomically restricted infusion of pharmacological agents have helped to identify the functional role of these brain regions at a gross level. The development of techniques for the genetic manipulation of the mouse during the past 15 years has greatly expanded our ability to probe the molecular mechanisms of biological function in a mammalian model system. However, one of the difficulties in the application of the genetic approach to the nervous system is the relative lack of ability to map molecular genetic changes onto the complex neuroanatomy of the brain. The goal of the current application is to develop a series of 48 driver lines for the generation of anatomically restricted and inducible gene knock-outs. For greatest genetic utility to the neuroscience community the strains will be developed on a pure C57BL/6 background. The most widely used tools for anatomically restricted and time dependent manipulation of gene function in the mouse are the CRE-recombinase and the tTA-transactivator, respectively, but very few driver lines are available to the neuroscience community. Furthermore, most CRE-driver lines do not allow strict temporal control, such as the ability to knock-out genes in adult tissues. We plan to use transgenic approaches to express CRE and tTA driven by the regulatory elements of 24 chosen genes. The default approach is targeting via homologous recombination in ES cells;vectors for pronuclear injections (including BACs) will also be utilized. The driver lines will allow us to achieve temporal and spatial control of recombinase activity in disparate regions of the nervous system. Driver lines will be validated by monitoring cell type and time dependent CRE activity. Behavioral studies will ensure that the transgenes do not affect neuronal function. The choice of the 24 driver loci represents the expertise of the four investigators involved in this application and are of relevance for the neuroscience community, covering sensory biology and pain, CMS and stem cell development, the limbic system, and learning and memory. From a clinical perspective the lines generated should be particularly relevant to the study of Neuropsychiatric disorders and addiction (eg. targeting the dopamine system) and neurodegenerative disorders (e.g. targeting the limbic system and adult stem cells).

Mueller, Ulrich
Klein, Barrett Anthony
NSF DDIG proposal number IOS-0710142


SLEEP IN A SOCIETY: the behavioral ecology of sleep within colonies of insects

Sleep is a phenomenon that greatly impacts the lives of organisms, yet aspects of it are little understood, or greatly ignored by biologists. The very functions of sleep are still in question and few studies have explored how social organization and individual need for sleep may interact. This research will address questions relating to uniquely social aspects of sleep within colonies of honey bees (Apis mellifera). Objectives of this research include mapping sleep, spatially and through the day and night, and testing possible functions of sleep related to communication and acquisition of food. Researchers will individually mark and examine bees in glass observation hives and train bees to food sources, recording behaviors resulting from different experimental regimes within the colony. The expectation is that maps of sleep will reveal differences in sleep, depending on bees' age or function. Further, the researchers will test the hypothesis that sleep-deprived bees will show reduced communication and food collecting performance, suggestive of sleep's specific importance within a social context. The proposed research will contribute to the demystification of sleep and to promote integration across behavioral, societal, ecological, physiological, and medical disciplines. Many undergraduate students will be trained in various aspects of this research and scientific progress will be presented to a wide range of audiences. Sleep is a widespread phenomenon and the proposed research direction will strive to address its relevance across all audiences.

A major unresolved problem in biology is explaining the maintenance of cooperation and mutualism. One unstudied mechanism that stabilizes mutualisms between species is partner choice, where one of the mutualists discriminates between superior versus inferior partners, and rewards the superior types through preferential association. This proposal develops the attine ant-fungus mutualism as a model for the study of symbiont choice. Attine ants cultivate monocultures of fungal clones as their major food source, whereas the fungus receives from the ants both nourishment and protection from pathogens. The proposal develops several ant-fungus systems, including leafcutter ants from the southwestern USA, to study the genetic consequences of symbiont choice exerted by the ants on their fungi. In essence, the proposed experiments ask whether cultivar diversity in ant gardens, arising through genetic mutation, may evolve under an analog of ant-driven "artificial selection" (symbiont choice).

Because symbiosis is a major recurring theme in the history of life, this research is expected to generate insights into the mechanisms that drive biological complexity and diversity. Such insights could impact issues directly relevant to human welfare, including disease evolution and the practice of sustainable agriculture. The case study of the attine ant-fungus mutualism will also be used to promote education of students and the public on the importance of ecological, symbiotic, and ultimately all biological processes.

DESCRIPTION (provided by applicant): The long-term goal of my laboratory is to elucidate the mechanisms that control mechanoelectrical transduction (MET) in hair cells, and the defects in this process that cause deafness. We propose here to study the function of the cell adhesion molecules cadherin 23 (CDH23) and protocadherin 15 (PCDH15) in hair cell function and disease. Previous studies have shown that CDH23 and PCDH15 are required for hair bundle development. Recent findings show that CDH23 and PCDH15 are components of extracellular filaments that connect stereocilia not only in developing but also in functionally mature hair cells. Based on these findings and new preliminary data, we hypothesis that CDH23 and PCDH15 are components of larger transmembrane signaling complexes in hair cells that control not only hair bundle morphogenesis but also MET. We predict that alternative splicing regulates the assembly and function of these protein complexes. To test our hypothesis, we will: (i) Determine the function of CDH23 for MET using genetically modified mice that were designed to circumvent developmental hair cell defects that are associated with CDH23 null alleles; (ii) define the function of PCDH15 splice variants for hair cell development and MET; (iii) isolate by yeast-two-hybrid assays novel components of CDH23 and PCDH15 dependent adhesion complexes in hair cells; (iv) analyze mouse lines with mutations in CDH23 that mimic mutations in patients suffering from Usher Syndrome 1D and autosomal recessive deafness DFNB12. We anticipate that mutations that are associated with different disease phenotypes affects distinct aspects of CDH23 function in hair cells. Deafness is a major health problem. 1 in 1000 children is born with hearing impairment and large parts of the aging population are afflicted by age-related hearing loss. In recent years, dramatic progress has been made in identifying gene mutations that cause deafness, but we know comparatively little about the mechanisms by which mutations in the affected genes cause deafness. We propose here to study the function of two genes that have been linked to deafness, Cdh23 and Pcdh15, in hair cells. Based on published and preliminary data, we anticipate that the two genes are required both for the development and function of mechanosensory hair cells, and that different mutations in the two genes that cause syndromic and non-syndromic forms of deafness affect distinct aspects of gene function in hair cells.

SYSTEMATICS, PHYLOGENETICS AND THE EVOLUTION OF ASEXUALITY IN
THE FUNGUS-GARDENING ANT GENUS MYCOCEPURUS

The fungus-gardening ant Mycocepurus smithii reproduces exclusively via unfertilized eggs. Strict asexual reproduction makes M. smithii almost unique among insects. Studying M. smithii contributes to our understanding of why sexuality is so common among animals, and why asexuality is rare. The proposed research will test theoretical predictions of obligate and long-term asexuality in an evolutionary context. First, a modern taxonomic revision of the genus will identify existing species and describe new species. Second, a molecular phylogenetic analysis will infer the evolutionary transition from sexuality to asexuality and identify the sexual sister species of M. smithii. Third, M. smithii will be tested for the presence (or absence) of genetic signatures indicative of obligate, long-term asexuality.

The presence of sexual reproduction among most animals is a longstanding puzzle in evolutionary biology. If asexual organisms are able to persist over evolutionary time, they may have evolved a different solution insuring the same benefits of sexuality with two sexes. The proposed research will foster the exchange of information and technology through collaboration with scientists in Latin America. Several undergraduate research assistants will be trained in molecular genetic techniques. Scientific progress will be presented in public outreach programs and scientific meetings. In addition, a taxonomy & phylogeny workshop will be held at the partner Institute in Rio Claro, Brazil.

Certain ants cultivate fungi as their major food source. The ants collect and transport vegetable substrate to a "garden," usually a sheltered chamber excavated in the ground, then plant fungus on this new substrate. Gardens of fungus-growing ants are foci of interactions within a community of ants, the cultivated fungus, and a great diversity of additional microbes. Specifically, pathogens attacking the ants or the fungal gardens can cause the death of the nest community, but a variety of auxiliary microbes contribute disease-suppressing properties that control such pathogens. Disease resistance to pathogens is shaped by the genetic identity of both the ant and the fungus and the interaction of these two dominant community members with the auxiliary microbes. This research will elucidate how these nest community members interact and co-evolve in response to pathogen presence. To understand disease dynamics, the research will (a) characterize the inheritance of assemblages of auxiliary microbes from maternal to offspring ant nest; (b) determine the importance of co-inheritance of ant-fungus-microbe combinations to disease resistance of the community; and (c) evaluate the relative contribution to disease-resistance by ant, fungus, and auxiliary microbes. The research integrates a variety of experimental approaches within an ecological-genetics and quantitative-genetics framework, contributing towards a unification of ecology and evolutionary biology.

Inheritance of auxiliary microbes between generations occurs in diverse hosts, including humans, but the importance of communities of auxiliary microbes in their contribution to the health of a host is incompletely understood. This research develops novel experimental approaches to elucidate the role of auxiliary microbe communities in disease suppression. Because microbial communities also confer disease resistance for humans (e.g., the microbiome of human skin or gut) and for crops (e.g., the microbiome of roots), this research on fungus-growing ants will contribute to the understanding of general microbial principles with applications to human and agricultural disease management. Fungus-growing ants and their microbes will also be used to promote education of students and the public on the importance of ecological, evolutionary, and all biological processes. A postdoctoral researcher and multiple undergraduates will be trained and mentored in this research project.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).


Mutualistic alliances between different organisms generate common goods for the interacting organisms that often exceed the sum of the parts. For example, attine fungus-growing ants provide their fungal cultivars with growth material, shelter, and protection from pathogens, while the fungus in turn serves as the ants' main food source. The fungus also provides the ants with digestive enzymes. Fungal cultivars are transferred to offspring ant nests from parental nests, but on rare occasions cultivars are also passed from one ant species to another ant species, creating new ant-fungus combinations with novel properties and novel common goods. This proposal aims to elucidate the novel common goods that are generated in natural and experimentally induced ant-fungus associations, focusing on enzyme efficiency, growth rate, and health (pathogen resistance) of different ant-fungus combinations. The project therefore elucidates general principles governing mutualism and cooperation in a social, symbiotic organism. Because the project focuses specifically on several ant-fungus mutualisms occurring in the USA, the research also presents rich opportunities in teaching and outreach (e.g., workshops at public schools and nature centers) to promote education of students and the public on the importance of symbiosis in local biodiversity and local environments.

The first ant was a hunter-gatherer. But around 50 million years ago, one ant species discovered agriculture and began cultivating fungus gardens for food. Today over 230 species of 'fungus-farming ants', all descended from that single ant pioneer, participate in intricate symbioses with their cultivated fungi. These symbioses also include a fungal 'crop disease' and bacteria that produce antibiotics useful to the ants. Using DNA sequence data and computer algorithms, this research will focus on reconstructing the shared evolutionary histories of the farming ants and the fungi that they cultivate, shedding light on the origin and evolution of this spectacular biological system.

The project will benefit the education and career of one postdoctoral researcher, two graduate students, and a number of undergraduate interns. Ant workshops will foster enthusiasm for science in secondary and high-school students and museum exhibits and family festivals will educate the general public. Because ant agriculture has become a model system for the study of symbiosis and coevolution, the results of this project will benefit a wide range of biologists. Finally, the results could benefit society in general because fungus-growing ants provide a rare non-human model system for the emerging field of 'Darwinian agriculture', aimed at improving human agricultural methods through the study of natural systems.

This dissertation research seeks to understand how brain anatomy correlates with task-specialization within ant societies. The research develops a model experimental system of acacia ants, which nest symbiotically in hollow spines of acacia plants, feed on food produced by the plant, and in return protect the plant. Acacia ants are ideal for this study because workers are similar in shape but exhibit distinct behavioral groups that are specialized on different tasks critical for the coordinated functioning of the society. The research specifically tests (1) whether task-specialization correlates with different brain structures; and (2) how society size affects the behavioral and neuro-anatomical specialization among workers. The research contributes to the understanding of animal sociality. The research will be the first to use multiple behavioral groups of an ant species to explore the links between brain and behavior, while controlling for worker morphology. The study will also provide insights into the poorly understood brain anatomy of ants that rely heavily on visual stimuli and visual communication. The investigations involve collaborating researchers in Panama, Costa Rica, and the USA, as well as field sites in Costa Rica and Panama. As part of the research, the doctoral trainee is supervising the Master's thesis of a female student from the Universidad de Costa Rica. Undergraduate students from the University of Texas and a high school student are digitally reconstructing brain anatomy. The trainee also disseminates research results through regular presentations to students, schoolteachers, park rangers, and naturalist guides.

This research will elucidate how the ecological and evolutionary processes of interacting species will respond to climate change at their range limits, with implications for conservation of species in marginal habitat. Climate change alters species distributions, with major consequences for species embedded in complex ecological interactions, such as mutualistic symbioses (e.g., plant-pollinator or host-microbe mutualisms). This project focuses on an insect-fungus mutualism, leafcutter ants cultivating fungi for food, which are agricultural pests in the southwestern USA and throughout the New World. Leafcutter mutualisms are ideal to study how symbioses respond to climate change because leafcutter ants are dominant components in ecosystems, and because experimental ant-fungus combinations can be manipulated under laboratory conditions simulating the altered temperature stresses expected under climate change. This research adapts techniques developed for the study of gene-by-gene interactions within an organism to test whether ant-by-fungus synergy enhances temperature-stress adaptations and thus determines range-limits at the northern (USA) and southern (Uruguay/Argentina) distributional limits of leafcutter ants.

Improved understanding of how interacting species respond to climate change has scientific and societal benefits, contributing to development of models for conservation of species in marginal habitat, and to models predicting whether mutualistic pest species may become more problematic under climate change. Collaborations with researchers in Uruguay and Argentina will provide training of US researchers in an international setting. This project is a collaboration among a major research institution, a regional institution, and an institution serving underrepresented students where some of the field research will be conducted, and will strengthen long term interactions and help foster enhanced STEM education initiatives. Furthermore, collaborations with researchers in Uruguay and Argentina will provide training of US researchers in an international setting. Workshops will be conducted at public schools, museums, and nature centers to foster understanding of local biodiversity, conservation, and challenges under environmental change.

? DESCRIPTION (provided by applicant): The long-term goal of my laboratory is to elucidate the mechanisms that regulate the development and function of the auditory sense organ and how inner ear defects cause hearing loss. We propose here to study the function of the Ig-superfamily member neuroplastin (Nptn) in the inner ear. Based on our preliminary data we hypothesize that Nptn regulates hair cell function and their interactions with sensory afferent neurons by mechanisms that are regulated by alternative splicing of the primary Nptn transcript. To test our hypothesis, we will: (i) determine the expression pattern and subcellular localization of Nptn isoforms in the inner ear; (ii) Study the consequences of inactivation of Nptn isoforms in hair cells and afferent neurons of the inner ear for hearing function; (iii) Determine the mechanisms by which Nptn isoforms carry out their function in hair cells and afferent neurons; (iv) determine the extent to which mutations in the gene encoding NPTN and its close homologues cause hearing impairment in humans. We anticipate that our findings will reveal novel insights into the mechanisms that regulate the function of hair cells and their innervating sensory afferent neurons, and how mutations in NPTN cause disease.

Project Summary The long-term goal of our laboratories is to elucidate the mechanisms that control hair cell development and function, and ascertain the defects in this process that cause deafness. We propose here to identify and study proteins that physically and functionally interact with MYO7A and therefore mediate its function in hair cells. Based on published and preliminary data, we hypothesize that MYO7A has a three-fold function in hair cells, regulating the transport of proteins critical for hair bundle adhesion and actin polymerization and directly controlling mechanotransduction. To test our hypothesis, we will: (i) define the protein complexes that mediate stereocilia adhesion, focusing on those consisting of PCDH15 and GRP98; (ii) determine how MYO7A complexes regulate stereocilia length, focusing initially on the complex with CAPZ that we have defined; (iii) specify how MYO7A and its interacting proteins control mechanotransduction. Our preliminary data show the feasibility of our approach. We have already identified hair bundle proteins that interact with MYO7A and mediate its function. We anticipate that some of the novel interaction partners of MYO7A will be affected in genetic diseases that cause hearing impairment.

Deafness is a major health problem. A major cause of deafness is defects in hair cells, the mechanosensory cells of the cochlea that convert sound induced vibrations into electrical signals to provide our sense of hearing. Mutations in the genes encoding protocadherin (PCDH15) and cadherin 23 (CDH23) cause hearing loss. Both genes are expressed in the hair bundles of the mechanosensory hair cells of the inner ear where they form heterophilic adhesion complexes that are important for hair bundle morphogenesis and mechanotransduction. Significantly, different mutation in both PCDH15 and CDH23 lead to different disease outcomes. While some mutations cause profound congenital deafness with retinal impairment (Usher Syndrome) others lead to recessive and progressive hearing loss without visual involvement. Gene-association studies also suggest a link of CDH23 polymorphisms with age- and noise-induce hearing loss. The mechanisms by which different mutations lead to distinct disease outcomes are poorly defined. We propose here to combine high-resolution structural studies with functional studies in hair cells to gain insights into the mechanisms by which PCDH15 and CDH23 regulate hair cell function and to define disease mechanisms. To achieve this goal, a laboratory with expertise in studying the biophysical and structural properties of cadherins and a laboratory dedicated to the study of auditory neuroscience have combined their efforts to achieve what either could not accomplish alone. Unlike previous studies that have focused on structural analysis of small monomeric fragments of CDH23 and PCDH15 expressed in bacteria, the team proposed to define the high- resolution structure of natively assembled PCDH15-CDH23 complexes using crystallography and cryo-EM. Structural data will be validated biochemically and by functional interrogation of mutant cadherins in the physiologically relevant mechanosensory hair cells paying attention to mutations associated with disease. We anticipate that our studies will provide the first high-resolution native structure of any protein complex important for mechanotransduction and provide mechanistic insights into its functional properties and pathophysiological mechanisms that are associated with different forms of hearing impairment.

What are the rules to engineer microbiomes to improve health of animals and plants? What organisms use microbiome engineering to improve their own health? Do these organisms use specific mechanisms to assemble beneficial microbes into their microbiomes? Addressing these questions will elucidate general principles of microbiome engineering, with implications for medicine and agriculture. The proposed work will synthesize research on one group of organisms that have practiced microbiome engineering for millions of years, the fungus-growing insects, specifically fungus-growing ants. Fungus-growing insects are farmers. They cultivate gardens of fungal food embedded in complex communities of beneficial microbes that serve nutritive and health functions, but can also include microbial pathogens. Research during the last three decades vastly increased genomic, microbial, physiological, and biochemical understanding of such insect-microbe interactions. There exists therefore now an interest in a comparative synthesis of these diverse host-microbiome associations and the underlying rules of microbiome engineering.

The proposed work will generate two syntheses to elucidate general principles of microbiome assembly and microbiome interactions with the host. The first synthesis will summarize recent microbial, genomic, and biochemical insights on fungus-growing insects to (i) generate an integrative understanding of ecological and evolutionary processes shaping such complex mutualisms; (ii) elucidate unresolved controversies; (iii) evaluate alternate hypotheses underlying these controversies; (iv) outline experimental work to test these hypotheses; and thus (v) identify promising novel research directions. The second synthesis will summarize the biology of fungus-growing ants occurring in the US to enable researchers to address the promising research directions that emerge from the first synthesis. The second synthesis will also integrate 25 years of unpublished observations and photography by the collaborating investigators on the fungus-growing ants of the USA. The second synthesis will therefore enable US researchers to take advantage of their local biodiversity and develop local study systems. The project will also generate visual media on all species of fungus-growing ants in the US and disseminate commons-use images to the public.

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.