Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Bacteriology
Non Technical Summary
We have recently discovered the production of a rare magnesium calcite biomineral on the fungus-growing ants and have shown that this biomineral layer serves as protective armor for the ants. Interestingly, despite human attempts to synthesize this biomineral requiring extreme amounts of energy, the formation of this biomineral by the ants appears to be a relatively low-kinetic process. Early evidence points to the ant's bacterial symbiosis, as well as the secreted protein layer of the ants, as being major factors in this efficiency. We propose investigating fundamental mechanisms that underlie this unique biomineralization process. First, we will test whether the calcium and magnesium ions, which are important building blocks of this biomineral, are coming from the ants themselves or from their bacterial symbionts. Next, we will begin examining the source of proteins that help facilitate the formation of the biomineral. Finally, we will characterize the occurrence of this biomineral throughout the fungus-growing ants in order to understand the widespread presence, as well as the historical origin, of this unique biomineral formation. Understanding biomineralization in the ants will provide diverse applications to national and state challenges put forth by the USDA and the state of Wisconsin, including energy-efficient bio-based material production, climate change mitigation, and bioremediation technologies.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Goals / Objectives
Biomineralization, the biogenic process of forming hard minerals, is widespread throughout plants, animals, and microbes. One of the most common biominerals in nature is calcium carbonate. Calcareous biogenic structures have been characterized in nearly all extant animal phyla, yet, until recently, had never been observed in the planet's most diverse group of animals, the insects. Work from our lab has identified the presence of a calcareous biomineral layer coating the exoskeleton of three genera of fungus-growing ants. Preliminary analysis has shown that this calcium carbonate biomineral layer in the ants is highly enriched with magnesium (Mg), where higher Mg content confers substantial increases in hardness of the ant's exoskeleton. Even though higher Mg content strengthens biogenic calcite structures, Mg is extremely rare as a major component in calcite biominerals due to the kinetic challenge of integrating Mg into calcite. This makes the biomineralization process of the fungus-growing ants both fascinating and of high scientific interest. In order to further investigate the breadth and underlying mechanisms of this Mg-calcite biomineralization process, we have proposed three main objectives: 1) determine the source of the metal ions in the formation of the ant's crystalline biomineral layer, 2) experimentally test the effects of the ant's protein layer and the host-symbiont partner fidelity on the low-kinetic biomineralization process, and 3) characterize the ultrastructural diversity of the cuticular biomineral layer across the fungus-growing ant phylogeny.In objective one, we will identify the source of the Ca and Mg ions that make up the crystalline biomineral layer. The potential roles of the three main mutualistic partners in the ant-fungus symbiosis (the fungus-farming ants, the Leucoagaricus fungal cultivar, and the bacterial symbiont Pseudonocardia) will be explored. We hypothesize that the ants themselves are the source of the ions.In objective two, we will focus on the effects of the specialized protein layer, as well as the host-symbiont partner fidelity, on the low-kinetic biomineral formation. The protein layer forms around nodules on the ant's cuticle, and co-localizes with the colonization sites of the bacterial symbiont Pseudonocardia.In objective three, we will characterize the taxonomic breadth of biomineralization throughout the fungus-growing ants. We have identified the biomineral layer in three genera of fungus-growing ants, including species in both of the major clades of the fungus-growing ants, the "neo-attines" and "paleo-attines", which diverged ~55 million years ago.
Project Methods
In objective one, we will identify the source of the Mg and Ca ions in the biomineralization process. Stable isotope tracking experiments will be conducted by following the passage of Mg and Ca ions through the fungus-growing symbiosis (i.e., from plant material fed to the fungal-cultivar, to the ants, and finally the biomineral). Previous studies have shown that certain elements are selectively enriched for one isotope type as they move up the food chain, including in this fungus-growing ant symbiosis. We will begin by incorporating the 26Mg and 40Ca stable isotope ions into an oak plant (Quercus) through water. Ants will be fed one organ type, leaves, as previous studies have shown that different plant organs may have varying levels of isotope enrichment. These isotope-labelled leaves will be fed to sub-colonies of ants. The sub-colonies will be raised according to standard lab procedures. The cotton balls used to control humidity will be wetted with deionized water in order to eliminate the introduction of outside ions into the system. The plant leaves will be analyzed for Mg and Ca content as well as 26Mg and 40Ca isotopic ratios. Additionally, the fungus-garden, ants, Pseudonocardia, and the biomineral layer will be separately analyzed for Mg and Ca isotopic ratios. To do this, specimens of each symbiont will be collected. Ants will be dissected in order to separate the following components: ant non-biomineral tissue, biomineral layer, and Pseudonocardia symbiont. Specimens will be euthanized and desiccated for 7-14 days to remove moisture. Samples will be milled into a powder and dissolved in HNO3. The concentration of Mg and Ca ions will be assessed using an inductively coupled plasma mass spectrometer (ICP-MS), housed at the ICP-TIMS Isotope Laboratory at UW-Madison. Using a cation exchange resin and HNO3 as an elution agent, Mg and Ca will be separated from the remaining solution. These purified fractions will be diluted with HNO3 to a known concentration and analyzed on the multi-collector ICP-MS. A blank HNO3 will be measured, and Mg and Ca isotope ratios will then be calculated.In objective two, we will experimentally test the effects of the ant's protein layer, and the host-symbiont partner fidelity, on the low-kinetic biomineralization process. Symbiont-switching experiments will be performed on Acromyrmex echinatior ants, allowing workers to acquire either: 1) their native symbiont, as a positive control, 2) non-native Pseudonocardia symbiont from another genus of fungus-growing ants (inter-generic symbiont transfer), or 3) no symbiont. We have extensive experience conducting symbiont switching experiments and will follow our previously established methods. Briefly, pupa of A. echinatior will be reared to adult stage in isolation with controlled exposure conditions. Shortly after eclosion, within the 2-3-hour window necessary for symbiont acquisition, we will inoculate with either their native Pseudonocardia or Pseudonocardia isolated from Trachymyrmex sp, a closely related genus of fungus-growing ants. For our aposymbiotic workers, we will prevent exposure to bacteria for 24 hours, after which workers are no longer able to acquire the symbiont. Rearing the pupae with no symbiont will allow us to determine which proteins are ant specific. To identify proteins produced by Pseudonocardia, we will culture it under a range of in vitro conditions (e.g., low and high Ca and Mg medium, medium supplemented with ant exoskeleton, etc.). This will allow us to examine if any of the proteins that are secreted in the presence of high Mg and Ca are also present in the symbiont-switching experiment. Successful symbiont acquisition will be confirmed by qPCR using Pseudonocardia-specific EF-tu primers. The new workers will then be analyzed for the presence of biomineral and protein layers starting from four to 14 days post-eclosion.Following successful cross-fostering of the pupae, and in vitro culturing of the isolated Pseudonocardia, we will extract the protein layers following previously established lab protocols. Samples will be submersed in 1x phosphate-buffered saline and immediately frozen in a dry ice and alcohol bath. Proteomics analyses on each sample will be performed at the UW Biotechnology Center or in collaboration with the Pacific Northwest National Laboratory (PNNL), with whom we have an active collaboration on fungus-growing ants. For each condition, we will run eight replicates. Spectra from our proteomic analysis will be compared to a protein database generated from publicly available genomic data of the ants and Pseudonocardia. Additional replicates of the reared pupae will be used for observational analysis. Scanning electron microscopy (SEM) will be used to observe the presence/absence of the biomineral layer, and X-ray diffraction analysis (XRD) will be used to characterize the composition of the biomineral layer. Energy-dispersive X-ray spectroscopy (EDS) will be used to determine if the glandular secretions are still enriched with Mg and Ca ions when Pseudonocardia is switched and/or absent.In objective three, we will use a combination of ant specimens already available in our lab, as well as newly collected specimens. Our lab currently has a NSF Dimension of Biodiversity grant which can be used to fund field collections. In addition, we will use specimens from other labs studying fungus-growing ants. In previous research, we were able to successfully obtain a variety of ant specimens using similar methods. These chosen taxa will then be subjected to SEM and XRD analyses. SEM will be carried out at the UW Geology Specimen Prep Facility, and will allow us to visibly characterize the presence, coverage, and crystallization of the biomineral layer. Additionally, SEM will provide visualizations of the biomineral-cuticle interface, allowing us to construct the morphology of the biomineral layer, and view how they vary between taxa. To determine the composition of the biomineral layers, XRD will be performed using the Rigaku Small-angle X-ray Scattering System, located at the UW Nanoscale Imaging and Analysis Center. XRD allows for identification of crystalline structures based on previously determined 2theta peaks. An intense peak at d104(Å) indicates the presence of a Mg-calcite crystalline structure. Based on historic data collection of Mg-calcite, it is well established that the intensity of the d104(Å) peak is negatively correlated with the Mol% Mg present in the sample, with geologic and biogenic dolomite having slightly different negative trend lines. Work from our lab has recorded XRD and Mol% Mg measurements from several biomineral layers, and found that they do fit to the previously determined trend of biogenic Mg-calcite. Furthermore, we have shown an increase in Mol% Mg in the Mg-calcite corresponds with an increase in hardness of the biomineral layer.Therefore, our XRD experiments will give us a rough indication of the diversity of composition and structural hardness of the biomineral layer across the fungus-growing ant phylogeny. Since the formation of dolomite biomineral is extremely unusual, we will conduct further analyses to determine the presence of partial Ca-Mg ordering for species we determine to have more than 40 mol% Mg. This will include Raman spectroscopy, which we have used previously to identify spectra congruent with those of geologic dolomite, with peaks of lattice mode (L), in-plane bending mode (v4), and symmetric stretching mode (v1) shifting toward higher wavenumbers, as compared to both calcite and high-Mg calcite. Also, selected area electron diffraction analyses of individual cuticular crystals will be used to confirm that the calcite lattice includes Ca-Mg ordering.