Insects rely on their innate immune system to successfully mediate complex interactions with their internal microbiota, as well as the microbes present in the environment. Given the variation in microbes across habitats, the challenges to respond to them are likely to result in local adaptations in the immune system. Here we focus upon phagocytosis, a mechanism by which pathogens and foreign particles are engulfed in order to be contained, killed, and processed. We investigated the phenotypic and genetic variation related to phagocytosis in two allopatric populations of the butterfly Pieris napi. Populations were found to differ in their hemocyte composition and overall phagocytic capability, driven by the increased phagocytic propensity of each cell type. Yet, genes annotated to phagocytosis showed no large genomic signal of divergence. However, a gene set enrichment analysis on significantly divergent genes identified loci involved in glutamine metabolism, which recently have been linked to immune cell differentiation in mammals. Together these results suggest that heritable variation in phagocytic capacity arises via a quantitative trait architecture with variation in genes affecting the activation and/or differentiation of phagocytic cells, suggesting them as potential candidate genes underlying these phenotypic differences.

Endoparasitoid wasps are important natural enemies of many insect species and are major selective forces on the host immune system. Despite increased interest in insect antiparasitoid immunity, there is sparse information on the evolutionary dynamics of biological pathways and gene regulation involved in host immune defense outside Drosophila species. We de novo assembled transcriptomes from two beetle species and used time-course differential expression analysis to investigate gene expression differences in closely related species Galerucella pusilla and G. calmariensis that are, respectively, resistant and susceptible against parasitoid infection by Asecodes paividava parasitoids. Approximately 271 million and 224 million paired-ended reads were assembled and filtered to form 52,563 and 59,781 transcripts for G. pusilla and G. calmariensis, respectively. In the whole-transcriptome level, an enrichment of functional categories related to energy production, biosynthetic process, and metabolic process was exhibited in both species. The main difference between species appears to be immune response and wound healing process mounted by G. pusilla larvae. Using reciprocal BLAST against the Drosophila melanogaster proteome, 120 and 121 immune-related genes were identified in G. pusilla and G. calmariensis, respectively. More immune genes were differentially expressed in G. pusilla than in G. calmariensis, in particular genes involved in signaling, hematopoiesis, and melanization. In contrast, only one gene was differentially expressed in G. calmariensis. Our study characterizes important genes and pathways involved in different immune functions after parasitoid infection and supports the role of signaling and hematopoiesis genes as key players in host immunity in Galerucella against parasitoid wasps.

Parasitoid fitness is influenced by the ability to overcome host defense strategies and by the ability of parasitoid females to select high-quality host individuals. When females are unable to differentiate among hosts, their fitness will decrease with an increasing abundance of resistant hosts. To understand the effect of mixed host populations on female fitness, it is therefore necessary to investigate the ability of female parasitoids to select among hosts. Here, we used behavioral assays, headspace volatile collection, and electrophysiology to study the ability of Asecodes parviclava to use olfactory cues to select between a susceptible host (Galerucella calmariensis) and a resistant host (Galerucella pusilla) from a distance. Our studies show that parasitoid females have the capacity to distinguish the two hosts and that the selection behavior is acquired through experiences during earlier life stages. Further, we identified two volatiles (-terpinolene and [E]--ocimene) which amounts differ between the two plant-herbivore systems and that caused behavioral and electrophysiological responses. The consequence of this selection behavior is that females have the capacity to avoid laying eggs in G.pusilla, where the egg mortality is higher due to much stronger immune responses toward A.parviclava than in larvae of G.calmariensis.

1. Host-parasitoid systems are characterized by a continuous development of new defence strategies in hosts and counter-defence mechanisms in parasitoids. This co-evolutionary arms race makes host-parasitoid systems excellent for understanding trade-offs in host use caused by evolutionary changes in host immune responses and parasitoid virulence. However, knowledge obtained from natural host-parasitoid systems on such trade-offs is still limited.

2. In this study, the aim was to examine trade-offs in parasitoid virulence in Asecodes parviclava (Hymenoptera: Eulophidae) when attacking three closely related beetles: Galerucella pusilla, Galerucella calmariensis and Galerucella tenella (Coleoptera: Chrysomelidae). A second aim was to examine whether geographic variation in parasitoid infectivity or host immune response could explain differences in parasitism rate between northern and southern sites.

3. More specifically, we wanted to examine whether the capacity to infect host larvae differed depending on the previous host species of the parasitoids and if such differences were connected to differences in the induction of host immune systems. This was achieved by combining controlled parasitism experiments with cytological studies of infected larvae.

4. Our results reveal that parasitism success in A. parviclava differs both depending on previous and current host species, with a higher virulence when attacking larvae of the same species as the previous host. Virulence was in general high for parasitoids from G. pusilla and low for parasitoids from G. calmariensis. At the same time, G. pusilla larvae had the strongest immune response and G. calmariensis the weakest. These observations were linked to changes in the larval hemocyte composition, showing changes in cell types important for the encapsulation process in individuals infected by more or less virulent parasitoids.

5. These findings suggest ongoing evolution in parasitoid virulence and host immune response, making the system a strong candidate for further studies on host race formation and speciation.

Herbivore insects use a variety of search cues during host finding and mate recognition, including visual, gustatory, and olfactory stimuli, leaving multiple traits for evolution to act upon. However, information about differences or similarities in search pattern amongst closely related insect herbivore species is still scarce. Here, we study the production of and the response to pheromone in Galerucella (Coleoptera: Chrysomelidae) to investigate the beetles' search behaviour. Males of G. pusilla and G. calmariensis, two closely related species, are known to produce the aggregation pheromone dimethylfuran-lactone when feeding on their host plant, whereas no pheromones have been identified in other Galerucella species. We show that dimethylfuran-lactone is produced also by males of G. tenella, a species phylogenetically close to G. pusilla and G. calmariensis, whereas the more distantly related species G. lineola and G. sagittariae were not found to produce the same compound. To investigate the beetles' behavioural response to dimethylfuran-lactone, the pheromone was synthesized using a partly novel method and tested in olfactometers, showing that G. pusilla, G. calmariensis, and G. tenella were all attracted to the pheromone, whereas G. lineola and G. sagittariae did not respond. This suggests that the production of and the response to pheromone could be linked to the phylogenetic relatedness between the species.

In host-parasitoid systems, there is a continuous coevolutionary arms race where each species imposes a strong selection pressure on the other. The host needs to develop defence strategies in order to escape parasitism and the parasitoid must evolve counter-defence strategies in order to overcome the host’s immune defence and successfully reproduce. This makes host-parasitoid systems excellent model systems for understanding evolutionary processes underlying host race formation and speciation. In order to gain a better understanding of the complexity of host-parasitoid interactions several aspects must be considered, such as search behaviour and host selection in the parasitoid, the development of immune responses in the host and counter-defence strategies in the parasitoid. In this thesis, I investigate interactions and coevolution in a natural host-parasitoid system, consisting of five species of Galerucella leaf beetles and three species of Asecodes parasitoids, by combining behavioural ecology with chemical ecology and immunology. In the studies performed, I found that pheromone production and responses in the beetles are connected to the phylogenetic relatedness between the Galerucella species (Paper I). I found no evidence that Asecodes exploits the adult pheromone to locate host larvae, but observed an ability in the parasitoids to distinguish a better host from a less suitable one based on larval odors (Paper II). The studies also revealed large differences in immune competence between the Galerucella species, which were linked to differences in hemocyte composition in the beetle larvae (Paper III, IV). Further, the results suggest that parasitism success in polyphagous Asecodes is strongly affected by former host species of the parasitoid (Paper IV). In conclusion, the results of this thesis suggest an on-going evolution in both parasitoid virulence and host immune responses in the Asecodes-Galerucella system.

The immune defence of an organism is evolving continuously, causing counteradaptations in interacting species, which in turn affect other ecological and evolutionary processes. Until recently comparative studies of species interactions and immunity, combining information from both ecological and immunological fields, have been rare. The cellular immune defense in insects, mainly mediated by circulating hemocytes, has been studied primarily in Lepidoptera and Diptera, whereas corresponding information about coleopteran species is still scarce. In the study presented here, we used two closely related chrysomelids, Galerucella pusilla and G. calmariensis (Coleoptera), both attacked by the same parasitoid, Asecodes parviclava (Hymenoptera). In order to investigate the structure of the immune system in Galerucella and to detect possible differences between the two species, we combined ecological studies with controlled parasitism experiments, followed by an investigation of the cell composition in the larval hemolymph. We found a striking difference in parasitism rate between the species, as well as in the level of successful immune response (i.e. encapsulation and melanisation of parasitoid eggs), with G. pusilla showing a much more potent immune defense than G. calmariensis. These differences were linked to differences in the larval cell composition, where hemocyte subsets in both naive and parasitised individuals differed significantly between the species. In particular, the hemocytes shown to be active in the encapsulation process; phagocytes, lamellocytes and granulocytes, differ between the species, indicating that the cell composition reflects the ability to defend against the parasitoid.

Background: To understand the ecological and evolutionary consequences of species interactions in food webs necessitates that interactions are properly identified. Genetic analyses suggest that many supposedly generalist parasitoid species should rather be defined as multiple species with a more narrow diet, reducing the probability that such species may mediate indirect interactions such as apparent competition among hosts. Recent studies showed that the parasitoid Asecodes lucens mediate apparent competition between two hosts, Galerucella tenella and G. calmariensis, affecting both interaction strengths and evolutionary feedbacks. The same parasitoid was also recorded from other species in the genus Galerucella, suggesting that similar indirect effects may also occur for other species pairs. Methods: To explore the possibility of such interactions, we sequenced mitochondrial and nuclear genetic markers to resolve the phylogeny of both host and parasitoid and to test the number of parasitoid species involved. We thus collected 139 Galerucella larvae from 8 host plant species and sequenced 31 adult beetle and 108 parasitoid individuals. Results: The analysis of the Galerucella data, that also included sequences from previous studies, verified the five species previously documented as reciprocally monophyletic, but the Bayesian species delimitation for A. lucens suggested 3-4 cryptic taxa with a more specialised host use than previously suggested. The gene data analyzed under the multispecies coalescent model allowed us to reconstruct the species tree phylogeny for both host and parasitoid and we found a fully congruent coevolutionary pattern suggesting that parasitoid speciation followed upon host speciation. Conclusion: Using multilocus sequence data in a Bayesian species delimitation analysis we propose that hymenopteran parasitoids of the genus Asecodes that infest Galerucella larvae constitute at least three species with narrow diet breath. The evolution of parasitoid Asecodes and host Galerucella show a fully congruent coevolutionary pattern. This finding strengthens the hypothesis that the parasitoid in host search uses cues of the host rather than more general cues of both host and plant.

Ecological systems are usually complex, with a number of interacting species. These species interactions are commonly divided into two major groups: mutualistic and antagonistic. If the interactions are mutualistic, they are beneficial for all species involved, as in specialized relationships between certain plants and their pollinators. Antagonistic interactions, on the other hand, can be either competitive or trophic. Trophically interacting species are for example plants and their associated herbivores, predators and their prey or parasites and their hosts. In many of these interactions, some species are depending on others in order to survive. If one species changes, other species associated to it may have to adapt to the changes. This may lead to a process of reciprocal evolution between the interacting species, called coevolution. In this paper I start with a brief background on coevolution and local adaptation, and then describe some interactions in tritrophic systems. The tritrophic systems I focus on consist of plants, herbivore insects and parasitoids. I discuss some processes and mechanisms in these systems, such as host search, plant defense and the immune response in insects. In the end of the paper, a short description of my PhD-project is included.

Insects rely on their innate immune system to successfully mediate complex interactions with their internal microbiota, as well as the microbes present in the environment. Given the variation in microbes across habitats, the challenges to respond to them is likely to result in local adaptation in the immune system. Here we focus upon phagocytosis, a mechanism by which pathogens and foreign particles are engulfed in order to be contained, killed and processed for antigen presentation. We investigated the phenotypic and genetic variation related to phagocytosis, in two allopatric populations of the butterfly Pieris napi. We found that the populations differ in their hemocyte composition, and overall phagocytic capability, driven by the increased phagocytic propensity of each cell type. However, no evidence for divergence in phagocytosis-related genes was observed, though an enrichment of genes involved in glutamine metabolism was found, which have recently been linked to immune cell differentiation in mammals.