Exploring the transcriptomics of polyphagy in plant-eating insects by Nicholas Christodoulides A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Master of Science Auburn, Alabama August 5, 2017 Keywords: gene expression, plasticity, host use, generalist Copyright 2017 by Nicholas Christodoulides Approved by Nate B. Hardy, Chair, Assistant Professor, Department of Entomology and Plant Pathology Tonia S. Schwartz, Assistant Professor, Department of Biological Sciences Alana L. Jacobson, Assistant Professor, Department of Entomology and Plant Pathology Abstract The aim of my thesis research was to gain a better understanding of the genetics underlying insect polyphagy. My thesis consisted of three objectives, each exploring a different aspect of plant-feeding insect genetics. For my first objective, I assembled and analyzed transcriptome data to study how gene expression varies across hosts of an extremely generalist insect, the lobate lac scale (Paratachardina pseudolobata). For my second objective, I examined published transcriptomes of 42 species of plant-feeding insects to test the simple idea that more generalist insects have more biochemical tools for herbivory, and express more herbivory genes. As a final part of my thesis research, I lay the foundation for an experiment to examine the role of phenotypic plasticity in the evolution of host use in aphid species. ii Table of Contents Abstract ...........................................................................................................................................ii List of Tables ..................................................................................................................................v List of Figures ................................................................................................................................vi Chapter 1: Introduction ...................................................................................................................1 Overview …………………………………………………………………………….........1 Theoretical Background……………………………….......................................................2 Host Plant Defenses against Herbivores…………………………………………..2 Detoxification Genes………………………………………………………….......3 Genomics of Generalism…………………………………………………………..4 Thesis Research Design…………………………………………………………………...7 References………………………………………………………………………………..10 Chapter 2: Gene expression plasticity across hosts of an invasive scale insect species…………14 Abstract..............................................................................................................................15 Introduction .......................................................................................................................16 Materials and Methods.......................................................................................................19 Results……………………………………………………………………………………22 Discussion………………………………………………………………………………..26 Conclusion……………………………………………………………………………….30 References………………………………………………………………………………..32 Chapter 3: Genetic architecture of evolvability: Generalist insect herbivores have more detoxification genes………….......................................................................................................39 Abstract………………………..........................................................................................39 Background........................................................................................................................40 Methods .............................................................................................................................41 iii Results……………………………………………………………………………………43 Discussion……………………………………………………………………………......44 Conclusion……………………………………………………………………………….45 References………………………………………………………………………………..46 Chapter 4: Experimental Evolution of Host-Use Plasticity in Two Aphid Species……………..54 Abstract…………………………………………………………………………………..54 Background………………………………………………………………………………55 Proposed Methods………………………………………………………………………..60 Conclusion…………………………………………………………………………….....63 References………………………………………………………………………………..64 iv List of Tables Table 2.1 ………………………………………………………………………………………...19 Table 2.2 ……………………………………………………………………………………...…23 Table 2.3 ………………………………………………………………………………………...25 Table 3.1 ………………………………………………………………………………………...49 Table 3.2 ………………………………………………………………………………………...50 v List of Figures Figure 2.1 ………………………………………………………………………………………..24 Figure 3.1 ………………………………………………………………………………………..52 Figure 3.2 ………………………………………………………………………………………..53 Figure 4.1 ………………………………………………………………………………………..62 vi Chapter 1: Introduction Overview Plant-feeding insects comprise about a quarter of the described terrestrial species on earth [1]. The extreme biodiversity of herbivorous insects is commonly attributed to coevolution with their host plants, which they depend on for food, shelter, and mating sites [2]. About 90% of plant- feeding insect species are specialists and only use one type of host plant, or a few similar types. Some species however, are generalists with diets ranging up to hundreds of different plant species [3]. Although generalists play important roles in both human and natural environments, little is known about the genetics underlying generalism and its evolution in insect species. What genetic mechanisms might enable polyphagy in generalist plant-feeding insects? Why are some generalist insects able to consume significantly more hosts than others? Are there heritable gene expression patterns that are associated with generalism? With the advent of next-generation sequencing technologies and the number of available insect gene sequences steadily increasing, we are now in a position to potentially answer these questions and test different hypothesis about the genetic architecture of generalism. My MS thesis was motivated by the need for general knowledge of the genetics of niche generalism. I was interested in 1) obtaining and analyzing plant-eating insect transcriptome data to find specific biological processes and molecular functions that are affected by the host environment, 2) testing the idea that broad diets depend on the proliferation of herbivory genes, and 3) designing an experimental evolution study to assess the role of phenotypic plasticity in host-use adaptation. 1 Through my work on this research, I have gained insights into the genetics of generalist insect herbivory, and have become adept at differential gene expression analysis. I have also learned how to use comparative statistical methods to test hypotheses about host-use adaptation, as well as design experimental evolution studies of generalist gene expression plasticity. Theoretical Background About a third of metazoan species are plant-feeding insects. Most are specialists. Previous studies on specialist insect diversity have linked their high diversification rates with the adoption of phytophagy [4]. Despite being the minority of herbivorous insect species, generalists consume a disproportionate amount of plant material relative to what their species richness would suggest. Generalist insects are also important because they are enriched for pest and invasive species that can damage and transmit plant diseases to hosts that are important to natural ecosystems and human agricultural activities [5]. In addition, they serve as a good model for understanding how broad ecological niches can develop in a group of organisms with typically very narrow ones [6]. We are just beginning to understand the molecular basis of insect-host interactions. Host Plant Defenses against Herbivores In order to understand the broad diets of some plant-eating insect species, we need to know what limits diet breadth. Over their history of antagonism with herbivorous insects, plants have evolved numerous defensive mechanisms to protect themselves from being eaten [7]. Some plant defenses are constitutive, that is, they are always “on” and continuously deter feeding by insects, regardless of whether or not feeding is actually happening. Constitutive defenses may be gross structural barriers, such as bark or waxy cuticles. They may also take the form of secondary plant 2 metabolites such as terpenes, cardenolides, and alkaloids [8]. Because such chemicals may be costly to constantly produce, plant defenses may also be inducible, that is activated only in response to stresses, such as insect feeding. Inducible defense mechanisms are expressed as a part of plant innate immunity, and are triggered in response to foreign proteins and pathogens. In response, some insects are able to secrete effector molecules into their host plants to disable systemic resistance responses such as the synthesis of antimicrobial secondary metabolites. These inducible defenses tend to be more efficient for plants that only occasionally encounter stress [9]. Detoxification Genes In order for plants to be a part of a generalist insects’ host range, the insect must be able to circumvent their defenses, both constitutive and inducible. Early research suggested that plant- feeding insects use detoxification genes as their main tools in circumventing plant defense chemicals [10]. If this is true, then the evolution of detoxification genes could play an important role in the evolution of plant-eating insect niches. Moreover, the same enzymes are thought to be critical for the evolution of resistance to man-made insecticides [11]. Thus, an understanding of the evolutionary genetics of