Dietary Implications of Interactions between Ants and Symbiotic Bacteria by Lina María Arcila Hernández A thesis submitted in conformity with the requirements for the degree of Masters of Science Graduate Department of Ecology and Evolutionary Biology University of Toronto © Copyright by Lina María Arcila Hernández, 2012 Dietary Implications of Interactions between Ants and Symbiotic Bacteria Lina María Arcila Hernández Masters of Science Graduate Department of Ecology and Evolutionary Biology University of Toronto 2012 ABSTRACT Studies of symbiotic bacteria have demonstrated that they provide multiple benefits to their hosts. These studies, however, have overlooked the importance of interactions with other bacteria and environmental factors that affect bacterial assemblages. To understand what shapes bacterial assemblages, I manipulated the diet of ants from the genus Cephalotes and disturbed their gut microbiome. I found that a deficit of nitrogen reduces bacterial densities. Furthermore, the data suggest that bacterial abundance may influence ant survival. I followed this experiment up by manipulating a putative protein source in the field. Our lab assigned Allomerus octoarticulatus ant colonies to treatments in which potential prey were present or absent. I collected data on foraging behaviour, colony performance, and composition of the bacterial community. The absence of prey increased ant recruitment to protein-rich baits; these ants were also less fit than ants that had insect prey but their bacterial assemblages were not affected. ii ACKNOWLEDGMENTS The last 16-18 months have been full of excitement and an immeasurable amount of learning, not only of nature and science but also of my own abilities. None of this would have been possible if my advisor Megan Frederickson had not been there for me. Megan a thank you may just not be enough this time but: un millón de gracias! To my advisory committee, Megan Frederickson, James Thomson, John Stinchcombe, and Stephen Wright, I owe this thesis. Although I was somehow overwhelmed by the presence of four smart and talented professors on my first meeting, I quickly understood that all of you were there willing to guide a new sheep on the great world of science. Thank you all for your valuable comments and guidance through the last year and a half. I want to acknowledge Benjamin Gilbert and Don Jackson who helped me with some of my data analysis. Furthermore, this project would not have been possible without the lab expertise, fieldwork assistance and home cooking of Jon Sanders. I am in debt to the Girguis lab at Harvard that allowed me to use their equipment for molecular work, especially Roxie Beinart and Kiana Franck. I also give special thanks to Greg Booth, Antonio Coral, René Escudero, Gabriel Miller, Alison Ravenscraft, and Lisseth Quispe for field assistance and fun times in Peru. I also want to recognize all the help and support I have had from the Frederickson current lab members, Kyle Turner and Adam Cembrowski; ex-lab technician, amazing Emma Hodgson; and undergrad students, Viviana Astudillo, Annabelle Ong, Margaret Thompson, Melissa Donnelly, and Ishita Aggarwal. Finally but not least, I am extremely grateful to my family, especially my parents and brother, and friends that always encourage me to keep moving forward. This project was funded by an NSERC Discovery grant to Megan Frederickson and a Sigma Xi Grant-in-aid of Research to Lina M. Arcila Hernández. iii TABLE OF CONTENTS ABSTRACT……………………………………………………………………………ii ACKNOWLEDGMENTS……………………………………………………………..iii LIST OF TABLES……………………………………………………………………..vi LIST OF FIGURES…………………………………………………………………...vii LIST OF APPENDICES……………………………………………………………….x CHAPTER ONE- General Introduction…………………………………………………….1 CHAPTER TWO- Effects of diet, antibiotics, and bacteria reintroduction on microbial assemblages in Cephalotes spinosus and repercussions for ant colony performance....…..5 ABSTRACT…………………………………………………………………………….5 INTRODUCTION……………………………………………………………………...5 METHODS……………………………………………………………………………..9 Study site and system…………………………………………………………...9 Collection of Cephalotes spinosus colonies and experimental design………...11 Abundance of gut microbes……………………………………………………13 Statistical analysis…………………………………………………………….14 RESULTS……………………………………………………………………………..14 Survival of adults and brood…………………………………………………..14 Abundance of gut microbes……………………………………………………15 DISCUSSION…………………………………………………………………………17 On interactions between ant survival and bacterial abundance……………...17 On mechanisms for change in bacterial abundance…………………………..20 CHAPTER THREE- The macronutrient requirements of a tropical arboreal ant…..31 ABSTRACT…………………………………………………………………………...31 iv INTRODUCTION…………………………………………………………………….32 METHODS……………………………………………………………………………35 Study site and system………………………………………………………….35 Experimental manipulation of insect herbivores/prey………………………...36 Ant diet………………………………………………………………………...37 Colony performance…………………………………………………………...38 Gaster microbiome…………………………………………………………….38 Statistical analysis…………………………………………………………….39 RESULTS……………………………………………………………………………..40 Diet and colony performance………………………………………………….40 Gaster microbiome…………………………………………………………….41 DISCUSSION…………………………………………………………………………42 Allomerus octoarticulatus diet………………………………………………...42 Colony performance…………………………………………………………...45 Gaster microbiome…………………………………………………………….46 Effects of multispecies interactions on the mutualism……...…………………49 CHAPTER FOUR- Concluding Remarks………………………………………………….59 LITERATURE CITED……………………………………………………………………...61 v LIST OF TABLES Table 2.1. Parametric survival analysis of (A) adults and (B) brood…………………………29 Table 2.2. Mixed model ANOVA results showing the factors affecting the number of bacterial 16S copies found in the guts of C. spinosus workers on day 13 (A) and day 28 (B)..….30 Table 3.1. MANOVA results for the different data sets of stable isotope ratios. H&T indicates samples with only heads and thoraces and Whole indicates samples with whole ants. Statistically significant values in bold…………………………………………………..57 Table 3.2. Adonis test for A) herbivore treatments and B) block effects…………………….57 Table 3.3. Model selection of biological and environmental factors using AIC……………..58 vi LIST OF FIGURES Figure 2.1. A) A Cephalotes spinosus worker. Photo © J. Sanders. B) Ten workers and five larvae lived in small plastic containers for four weeks. Disposable pipettes were cut in half; one half was used to store water and the other half simulated a nest chamber. The red tent provided darkness to the workers………………………………………………24 Figure 2.2. Survival plot for Cephalotes spinosus A) adult workers and B) brood…………..25 Figure 2.3. Interaction effects of diet (‘Com’ refers to a complete diet and ‘Noaa’ to a no amino acids diet), gut bacteria reintroduction (‘N’ for no reintroduction, and ‘Y’ for introduction) and presence of antibiotics on the proportion of adults (A and B) and brood (C and D) surviving to the last day of the experiment………………………………….26 Figure 2.4. Effects of diet, antibiotics, reintroduction of guts and their interactions on number of bacterial cells on the 13th day of the experiment (A and B; note that reintroduction of bacteria had not taken place at this point, hence the division of treatments Y and N is artificial) and on the last day of the experiment (C and D). ‘Com’ refers to a complete diet and ‘Noaa’ to a no amino acids diet. ‘Y’ codes for treatments that received guts on day 14 and ‘N’ codes for treatments that did not……………………………………….27 Figure 2.5. Relationship between the number of 16S copies (square-root transformed) and the proportion of surviving adults (logit transformed) on day 28 of the experiment (r2= 0.22, P = 0.02)………………………………………………………………………………...28 Figure 3.1. A) A C. nodosa domatium. B) A food body on the underside of a C. nodosa leaf. C) A. octoarticulatus workers foraging on C. nodosa leaves. Photos © G. Miller……..51 vii Figure 3.2. Morphological measurements taken on A. octoarticulatus workers. HL: head length, ML: mesosoma length, P: petiole and postpetiole length, G: gaster length, MW: mesosoma width, HW: head width, SL: scape length, and MaL: mandible length. Photos © A. Nobile……………………………………………………………………………..51 Figure 3.3. Mean (± SE) number of ants that recruited to protein-rich (P) baits after five hours on herbivore-excluded (H-) and control (H+) plants……………………………………52 Figure 3.4. Carbon and nitrogen stable isotope ratios for ants in the H+ (closed circles) and H- (open circles) treatments. H&T indicates samples with only heads and thoraces; Whole indicates samples with entire ants. Isotope ratios for naturally occurring C. nodosa, A. octoarticulatus workers, insect herbivores (coleopterans, hemipterans, etc.), and predatory spiders found on C. nodosa are given for reference………………………….52 Figure 3.5. A) Mean (± SE) worker mass and B) worker length of ants that developed with access to insect herbivores (H+) and in the absence of insect herbivores (H-)…………53 Figure 3.6. Mean (± SE) number of workers produced in colonies with access to insect herbivores (H+) and in the absence of insect herbivores (H-) after 11 months………...53 Figure 3.7. Relationship between worker weight (log-transformed) and the residual values of the number of reproductives (alates; square-root transformed) per colony (r2= 0.56)….54 Figure 3.8. Phylogenetic diversity of microbes in the gaster of ants with access to insect herbivores (closed circles) and in the absence of insect herbivores (open circles) (P>> 0.05). Rarefaction plot obtained