On the composition and function of the gut microbiome of two insect species, the generalist Spodoptera littoralis and the specialist Melolontha hippocastani Dissertation to Fulfill the Requirements for the Degree of “doctor rerum naturalium” (Dr.rer.nat) Submitted to the Council of the Faculty of Biology and Pharmacy of the Friedrich Schiller University By M.Sc. Pol Alonso Pernas, born on 12.12.1986 in Barcelona Dissertation, Friedrich-Schiller-Universität Jena, [2018] Reviewers: 1. Prof. Wilhelm Boland (Max Planck Institute for Chemical Ecology, Jena) 2. Prof. Erika Kothe (Institute of Microbiology, Friedrich Schiller University, Jena) 3. Prof. Dieter Spiteller (University of Konstanz, Konstanz) Date of the public defense: July 4th 2018 Table of contents Abbreviations and symbols 1. General introduction ............................................................................................................... 7 1.1. General structure and physiochemical conditions of insect guts ............................... 7 1.2. Types of symbionts and mechanisms of transmission .............................................. 10 1.3. Correlation between location and role of insect symbiotic bacteria ......................... 12 1.4. Melolontha hippocastani: life cycle, gut physiochemical conditions and known bacterial symbionts .......................................................................................................... 18 1.5. Spodoptera littoralis: life cycle, gut physiochemical conditions and known bacterial symbionts ......................................................................................................................... 20 1.6. Goals of the thesis ..................................................................................................... 21 2. Thesis outline: list of articles with author’s contribution .................................................. 23 3. Articles with author’s contribution ...................................................................................... 27 3.1. Article I ..................................................................................................................... 27 3.2. Article II ................................................................................................................... 48 3.3.Article III ................................................................................................................... 54 4. Unpublished results ............................................................................................................... 73 5. General discussion ............................................................................................................... 103 5.1. Summary of the findings ........................................................................................ 103 5.2. Function of the gut bacterial symbionts of a coleopteran (M. hippocastani).......... 104 5.3. Function of Enterococcus mundtii within the gut of a lepidopteran (S. littoralis) . 106 5.4. Dynamics of the bacterial community inhabiting the hindgut wall of larval Melolontha hippocastani ............................................................................................... 108 5.5. Bacterial community and possible biological role of the pockets .......................... 111 5.6. Comparison of the symbiotic communities of Spodoptera littoralis and Melolontha hippocastani .................................................................................................................. 113 6. Future perspectives .............................................................................................................. 116 7. Summary ............................................................................................................................... 118 7.1. English .................................................................................................................... 118 7.2. German .................................................................................................................... 120 8. References ............................................................................................................................. 124 9. Eigenständigkeiterklärung .................................................................................................. 136 10. Acknowledgments .............................................................................................................. 137 Abbreviations and symbols AMP antimicrobial peptide APSE Acyrthosiphon pisum secondary endosymbiont BLAST Basic Local Alignment Search Tool cDNA complementary DNA CoA coenzyme A CPM counts per million dd-H2O double-distilled water DE differentially expressed DEPC diethyl pyrocarbonate DUOX dual oxidase FACS fluorescence-activated cell sorting FDR false discovery rate FISH fluorescence in situ hybridization GC-MS gas chromatography – mass spectrometry GFP green fluorescent protein HDE highly and differentially expressed IRMS isotope ratio mass spectrometry KEGG Kyoto Encyclopedia of Genes and Genomes NCBI National Center for Biotechnology Information NSTI nearest sequenced taxon index OTU operational taxonomic unit PBS phosphate-buffered saline PHB polyhydroxybutyrate PICRUSt Phylogenetic Investigation of Communities by Reconstruction of Unobserved States PP pyrophosphate qPCR quantitative polymerase chain reaction rDNA ribosomal DNA RNAseq RNA sequencing ROS reactive oxygen species RPKM reads per kilobase per million mapped reads rRNA ribosomal RNA SIP stable isotope probing TCA tricarboxylic acid TEM transmission electron microscopy TMM trimmed mean of M-values 1. General introduction_________________________________________________________7 1. General introduction Insects are the largest class of animals on Earth. Of all the plants and animal species described till date, insects occupy approximately 60% of it. [1]. They can be found in water, on land, in almost any latitude, even southern of the Antarctic circle [2]. Such an extreme widespread distribution implies that insects must embrace a variety of habitats and life styles, which would not be possible without a corresponding physiological adaptation. Concretely, the gut is of particular interest, as this organ is responsible for the processing of the food as well as for excretion of digestive waste. The vast range of diets consumed by the class Insecta is reflected in the diversification of gut structures among different insect species [3]. Also, the digestive tract harbors symbiotic microorganisms, without which insects possibly would not have achieved their ecological success. These partners (microbiome) belong to a wide range of taxonomic affiliations spanning through several domains, encompassing fungi, bacteria, viruses and protozoa [4], [5], [6], [7]. Diet and host taxon are the two most significant factors that influence the composition of the symbiotic community [8], [9]. 1.1 General structure and physiochemical conditions of insect guts The insect gut is a continuous tube divided into three main sections: the foregut, the midgut, and the hindgut (Fig. 1A). The most proximal one, the foregut, is of ectodermal origin. Thus, its cells secrete a chitinous cuticle, known as intima, which is continuous with that covering the outside of the body [10]. The foregut is not involved either in secretion of digestive enzymes or absorption of nutrients. Its main functions are pushing the ingested material towards the midgut and its storage, principally in a subsection called crop [3]. The following section, the midgut, is of endodermal origin and its cells do not secrete cuticle but a more fragile membrane named the peritrophic matrix. Since it is in this region of the digestive tract where most digestive processes take place, the midgut cells actively secrete digestive enzymes into the lumen. Also, the epithelium is covered with microvilli which optimize absorption by increasing the area of contact of the cells with the ingested material up to two orders of magnitude [10]. Usually, the microvilli are covered by a layer of filamentous glycoproteins called glycocalyx, lining the so called ectoperitrophic space, delimited by the other side by the above mentioned peritrophic matrix. In some cases, this membrane packages the food bolus as it moves thought the digestive tract. Either way, the peritrophic matrix is composed of a number of laminae made of a network of 8_________________________________________________________1. General introduction chitin, proteins and glycoproteins. Its main purpose is the separation of the food material from the midgut epithelium, thus protecting the microvilli from abrasion and rendering possible the compartmentalization of enzymatic activity in the endo- and ectoperitrophic spaces [10]. To some extent, the peritrophic matrix also provides protection against harmful chemicals and pathogenic microorganisms, as the pores formed by the proteoglycans (less than 100 nm in diameter) are too small to permit the passage of bacteria [3], [10]. Thus, insects harboring symbiotic bacteria in the midgut confine them to the endoperitrophic space. After the midgut lies the pylorus, sometimes forming a valve between the midgut and the hindgut. Arising from it, the Malpighian tubules collect wastes of different nature from the hemolymph, such as uric acid or alkaloids, and release them to the anterior hindgut [10]. Figure 1. The insect gut. (A) Representation of the general design of the insect gut. (B) Representation of