Introduction: The midgut epithelium functions as tissue for nutrient uptake as well as physical barrier against pathogens. Additionally, it responds to pathogen contact by production and release of various factors including antimicrobial peptides, similar to the systemic innate immune response. However, if such a response is restricted to a local stimulus or if it appears in response to a systemic infection, too is a rather underexplored topic in insect immunity. We addressed the role of the midgut and the role of systemic immune tissues in the defense against gut-borne and systemic infections, respectively. Methods: Manduca sexta larvae were challenged with DAP-type peptidoglycan bacteria – Bacillus thuringiensis for local gut infection and Escherichia coli for systemic stimulation. We compared the immune response to both infection models by measuring mRNA levels of four selected immunity-related genes in midgut, fat body, hematopoietic organs (HOs), and hemocytes, and determined hemolymph antimicrobial activity. Hemocytes and HOs were tested for presence and distribution of lysozyme mRNA and protein. Results: The midgut and circulating hemocytes exhibited a significantly increased level of lysozyme mRNA in response to gut infection but did not significantly alter expression in response to a systemic infection. Conversely, fat body and HOs responded to both infection models by altered mRNA levels of at least one gene monitored. Most, but not all hemocytes and HO cells contain lysozyme mRNA and protein. Discussion: These data suggest that the gut recruits immune-related tissues in response to gut infection whereas systemic infections do not induce a response in the midgut. The experimental approach implies a skewed cross-talk: An intestinal infection triggers immune activity in systemic immune organs, while a systemic infection does not elicit any or only a restricted immune response in the midgut. The HOs, which form and release hemocytes in larval M. sexta, i) synthesize lysozyme, and ii) respond to immune challenges by increased immune gene expression. These findings strongly suggest that they not only provide phagocytes for the cellular immune response but also synthesize humoral immune components.

Wolbachien sind weitverbreitete bakterielle Symbionten von Arthropoden. Sie werden überwiegend durch maternale Vererbung übertragen, können aber auch horizontal von Art zu Art übertragen werden. Wolbachien sind berüchtigt dafür, die Wirtsreproduktion zu manipulieren, können aber auch Mutualismen mit ihren Wirten evolvieren. In dieser Arbeit untersuche ich, welche Rolle horizontale Transmission und mutualistische Effekte bei der Wolbachien-Pandemie unter Arthropoden spielen. Zunächst schätze ich, dass Millionen Arthropodenarten mit Wolbachien infiziert sind. Um diese erstaunliche Verbreitung zu verstehen, entwickele ich ein Modell zur horizontalen zwischenartlichen Transmission von Wolbachien, das auf epidemiologischer und Netzwerk-Theorie aufbaut. Die Ergebnisse weisen auf die Bedeutung von horizontaler Transmission über große phylogenetische Distanzen hin. Da eine erfolgreiche Transmission wahrscheinlich durch symbionteninduzierte Wirtsvorteile begünstigt wird, betrachte ich dann umfassend und kritisch Wolbachien-Arthropoden-Mutualismen und finde diese in vielfältigen Kontexten, aber nur begrenzt Hinweise auf Wolbachien-induzierten Wirtsschutz. Mithilfe eines populationsgenetischen Modells untersuche ich dann den Einfluss von Wirtsvorteilen auf die Infektionsdynamik von Wolbachien. Erstmalig leite ich Invasionsbedingungen und Gleichgewichtsfrequenzen für Wolbachien-Doppelinfektionen her. Die Ergebnisse bestätigen, dass Wirtsvorteile die Invasion von Wolbachien in neue Wirte erheblich erleichtern. Schließlich untersuche ich die Wechselwirkungen zwischen einer Wolbachien-Infektion und dem Immunsystem des Wirtes, wobei ein Schwerpunkt auf reaktiven Sauerstoffspezies liegt. Ich schlage eine Hypothese vor, die unterschiedliche Immunantworten in neuen und ko-evolvierten Assoziationen erklärt. Insgesamt sprechen die Ergebnisse dieser Arbeit für einen wesentlichen Anteil von horizontaler Transmission und mutualistischen Effekten an der Wolbachien-Pandemie in Arthropoden. / Wolbachia are widespread bacterial symbionts of arthropods. They are transmitted predominantly via maternal inheritance, but are also able to move between different species (horizontal transmission). Wolbachia are notorious for selfishly interfering with host reproduction, but they can also evolve mutualistic associations with their hosts. In this thesis, we analyze the role of horizontal transmission and mutualistic effects in the Wolbachia pandemic among arthropods. First, we derive an estimate of the number of Wolbachia-infected arthropod species and find that millions of species are infected. In order to explain this striking distribution, we develop a model of Wolbachia horizontal transmission between species, building on epidemiological theory and network theory. Our findings point to the importance of transmission over large phylogenetic distances. Given that successful horizontal transmission is likely to be facilitated by symbiont-induced host benefits, we then perform a comprehensive review of Wolbachia-arthropod mutualisms and find that these occur in diverse contexts, although the evidence of Wolbachia-induced host protection in nature is limited so far. By means of a population genetic model, we then analyze the influence of host benefits on the infection dynamics of Wolbachia. For the first time, we derive invasion conditions and equilibrium frequencies for Wolbachia double infections. Our results corroborate that host benefits substantially facilitate invasion of Wolbachia into novel hosts. Finally, we examine the interactions between Wolbachia infection and the host immune system, with a focus on reactive oxygen species. We propose a hypothesis that explains differential immune responses in novel and coevolved associations. Taken together, the findings presented in this thesis argue for a significant involvement of horizontal transmission and mutualistic effects in the Wolbachia pandemic among arthropods.

Arthropod hemocyanin and phenoloxidase are members of a group of proteins called the Type-3 copper oxygen-binding proteins, both possessing a highly conserved oxygen-binding site containing two copper atoms each coordinated by three histidine residues (Decker and Tuczek, 2000). Despite similarities in their active site, these proteins have very different physiological functions. Phenoloxidase possesses both tyrosinase and o-diphenoloxidase activity, and is predominantly involved in reactions which protect insects from infection (Kopàcek et al., 1995). Hemocyanin is a large multi-subunit protein with a primary function as a respiratory protein, reversibly binding and transporting molecular O2 (Decker and Rimke, 1998; Decker and Tuczek, 2000). Recently, it has been demonstrated in vitro that arthropod hemocyanin possesses an inducible phenoloxidase activity when incubated with denaturants, detergents, phospholipids or proteolytic enzymes. This activity appears to be restricted to only a few subunit types, and it has been hypothesised that it may be accompanied by conformational change which opens the active site increasing access for larger phenolic substrates (Decker and Jaenicke, 2004; Decker et al., 2001; Decker and Tuczek, 2000). This possibly suggests a dual role of hemocyanin in arthropods. The presented thesis deals with two distinct aims. The first was to isolate and sequence a phenoloxidase gene from the insect Spodoptera littoralis (Egyptian Cottonleaf Worm). Despite efforts, progress was hindered by a number of experimental problems which are outlined within the relevant chapters. The second aim was to characterise the mode of SDS induced phenoloxidase activity in arthropod hemocyanin from the ancient chelicerates Limulus polyphemus (horseshoe crab) and Eurypelma californicum (tarantula) and the more modern chelicerate Pandinus imperator (scorpion), using a number of biophysical techniques. The results indicated that the SDS induced phenoloxidase activity is associated with localised tertiary and secondary conformational changes in hemocyanin, most likely in the vicinity of the dicopper centre, thus enhancing access for larger phenolic substrates. Experiments indicate that copper remains associated with the protein during these structural changes; however the nature of the association is unclear. SDS concentrations approximating the CMC appeared critical in causing the necessary structural changes required for a significant increase in the detectable phenoloxidase activity to be exhibited.