Identifying how the Ecological and Evolutionary Interactions between Host and Symbiont shape Holobiont Biodiversity Brianna M. Busscher and John T. Wertz, PhD Department of Biology, Calvin College, Grand Rapids, MI 49546 Ants are one of the most abundant, diverse, and ecologically important insects on the planet and the center of an extensive web of interactions. Of particular interest to us are herbivorous ants, specifically within the genus Cephalotes (turtle ants). Interestingly, the evolution of herbivory in ants led to extensive diversification. This is a somewhat surprising occurrence, as an herbivorous diet tends to be limiting. Most plants do not provide enough of certain necessary nutrients, like amino acids and nitrogen, for an organism to survive solely on a plant-­‐based diet. In the case of the Cephalotes ants, bacterial provisioning of nutrients is likely connected to their survival and speciation. Carnivorous and herbivorous ants host bacterial symbionts within their gut, but the herbivorous ants have a much tighter association with their gut bacteria than do the carnivorous ants. Burkholderia, Pseudomonads, Rhizobia, Verrucomicrobia, and Xanthomonads are five orders of bacteria conserved in the guts of all herbivorous ants, with Rhizobia being the group most closely linked to herbivory. The ant host with its bacterial symbionts is called a holobiont, and they are together considered one organism. From the holobiont perspective, it is improbable that ant evolution was independent of the evolution of their symbiotic bacteria. In order to better understand the interaction between the two organisms, we are studying the metabolic characteristics of the gut bacteria, and we hypothesize that the bacteria supplement the ant’s diet with their metabolic products. To test this hypothesis, bacteria were isolated from ant gasters and characterized using a variety of methods. Biolog plates and API strips were used to test for utilization of a range of substrates, urease and pectinase assays tested for the ability to degrade urea to ammonia and pectin to simpler carbohydrates, respectively, and Balch tube experiments were used to produce generation times and growth curves. Thus far, all Rhizobia inoculated on Biolog plates have been found to utilize α-­‐D-­‐glucose, α-­‐hydroxybutyric acid, and D,L-­‐
lactic acid, and many are able to utilize additional simple sugars. PCR amplification and gel electrophoresis revealed that certain Enterobacteria from Cephalotes varians contain the nifH gene, which encodes a nitrogenase enzyme. It is unknown whether or not the gene is expressed, but if so, the bacteria would have the ability to fix atmospheric nitrogen to ammonia, a form of nitrogen that could then be used by the ant or other gut bacteria. Preliminary data from the pectinase assay suggests some bacteria may be able to degrade pectin. A major component of some herbivorous ants’ diet is pollen, which consists mainly of pectin, so the ability of gut bacteria to degrade pectin would support the hypothesis that the bacteria provide nutritional supplements to the ants’ diet. This summer research experience has allowed me to delve into the realm of microbiology and evaluate whether it is a topic I would like to pursue in the future. I have gained valuable knowledge of basic microbiological techniques – such as Gram staining and a plethora of characterization methods – and a greater appreciation for the importance of symbiotic bacteria in all organisms. Developing my ability to work independently while effectively communicating and discussing results with others has been another benefit. Additionally, I have the privilege of continuing to work on this project with Dr. Wertz during the academic year, and I look forward to that experience.