De invloed van parasitisme op het microbioom van rupsen en implicaties voor parasitoïde-hyperparasitoïde-interacties

Although the microbiome of insects has been studied extensively over the past decade, little is still known about how microbial communities assemble in and on the bodies of insects. Furthermore, very little is known about the extent to which insect microbiomes are affected by the presence of parasitoids (i.e. insects that lay their eggs in or on other insects, and whose larvae will ultimately kill them), and whether parasitism-induced changes in the host microbiome cascade to affect higher trophic levels. In this PhD thesis, we assessed the hypothesis that parasitism on herbivore hosts by endoparasitoids alters the microbiome of the hosts and that these microbial changes influence parasitoid host-location by hyperparasitoids (i.e. parasitoids that develop at the expense of a primary parasitoid). Specifically, we aimed (i) to determine whether parasitism by an endoparasitoid affects the external and internal microbiomes of host populations; (ii) to assess how parasitism-induced microbial changes are influenced by the host species, the parasitoid species or their interaction; and (iii) to evaluate whether parasitism-induced microbial changes are correlated with alterations in the host's body odour, that in turn affect hyperparasitoid behaviour. To accomplish these research goals, a model system consisting of caterpillars of the large cabbage white butterfly (Pieris brassicae), its primary parasitoid (Cotesia glomerata), and the hyperparasitoid Baryscapus galactopus was used. Additionally, for the second objective the small cabbage white (Pieris rapae) and the primary parasitoid Cotesia rubecula were added to the study. Pieris brassicae and P. rapae are deleterious pest species whose caterpillars feed on various crucifers (Brassicaceae), including economically important crops like cabbage, broccoli, Brussels sprouts and kale. Cotesia parasitoids like C. glomerata and C. rubecula are well-known parasitoids of Lepidopteran pests, particularly those in the family Pieridae, such as P. brassicae and P. rapae. In Chapter 1, we provided a comprehensive literature overview of insect-associated microorganisms, their beneficial roles, the factors shaping insect microbial communities (microbiomes), how these microbiomes can be disrupted by parasitism, and the potential ecological consequences of such disruptions. In Chapter 2, we investigated whether parasitism by C. glomerata altered the microbial community within and on host caterpillars of P. brassicae, compared to non-parasitised caterpillars collected from various field sites or laboratory cultures. For this study, both bacterial and fungal communities were analysed. Our results indicated that fungal densities were low in both the external and internal host microbiomes, leading us to focus exclusively on bacteria for the remainder of the thesis. The findings clearly demonstrated that parasitism by C. glomerata significantly altered the host microbiome, reducing bacterial diversity in the internal microbiome of parasitised caterpillars, while the external microbiome remained unaffected. This pattern was consistent in laboratory-reared insects. The internal microbiome of all parasitised caterpillars and of the parasitoid larvae in the caterpillar hosts was dominated by a Wolbachia strain, which was completely absent in healthy caterpillars. Next, in Chapter 3, using lab-reared insects we assessed to which extent parasitised caterpillar host microbiomes are shaped by the caterpillar host species, the parasitoid species or their interaction. Specifically, we compared the internal and external microbiome of caterpillars of P. brassicae and P. rapae parasitised by C. glomerata or C. rubecula with non-parasitised caterpillars. Additionally, we investigated the internal and external microbiome of the developing parasitoid larvae inside their hosts. Both internal and external bacterial densities were significantly higher for P. brassicae than P. rapae, while no differences were found between parasitised and non-parasitised caterpillars. In contrast, parasitism significantly affected the composition of the internal and external microbiome of the caterpillars and the parasitoid larvae, but the effects were dependent on the host and parasitoid species. For example, we found a strong host-dependent variation in the occurrence of Enterococcus. While it remained at high relative abundance on and in parasitised P. brassicae caterpillars, its relative abundance was drastically lowered on and in parasitised P. rapae caterpillars compared to non-parasitised caterpillars, irrespective of the parasitoid species. Additionally, our results indicate that parasitism-induced changes in the host microbiome are also strongly determined by the parasitoid species, which was particularly clear for the internal microbiome. Irrespective of the host species, Wolbachia was exclusively found inside caterpillars parasitised by C. glomerata, as well as in the corresponding developing parasitoid larvae, while it was absent in the caterpillars parasitised by C. rubecula. Together with the presence of Wolbachia in adult females of C. glomerata, this suggests a possible transfer of the bacterium from the wasp to the host. Similarly, a Nosema species was abundantly present inside parasitised caterpillars and the parasitoid larvae, but this was independent of the host and the parasitoid species. In Chapter 4, we investigated the potential correlation between microbial changes and alterations in body odours in response to parasitism by C. glomerata in P. brassicae caterpillars, as well as with the olfactory behaviour of the hyperparasitoid B. galactopus. Furthermore, we assessed the effects of specific microbes linked with parasitism on the olfactory behaviour of the hyperparasitoid. We found that parasitism by C. glomerata led to the production of five characteristic volatile products (3-octanone, (Z)-3-hepten-1-ol, 3-methyl-3-buten-1-ol, 3-octanol and 3-pentone) and significantly affected the internal and external microbiome of the caterpillars, which were both found to have a significant correlation with caterpillar odours. The preference of the hyperparasitoid was correlated with the presence of the external microbiome. Likely, the changes in the external microbiome and body odour after parasitism were driven by the internal microbiome of the caterpillars, where Wolbachia was only present after parasitism. Micro-injection of Wolbachia in non-parasitised caterpillars increased hyperparasitoid attraction to the caterpillars compared to untreated caterpillars, while no differences were found compared to parasitised caterpillars, suggesting that Wolbachia is involved in host-location by the hyperparasitoid. Altogether, our results indicate that host-parasitoid interactions can affect multi-trophic interactions and hyperparasitoid olfaction through alterations of the microbiome. Further research should focus on the mechanisms underlying these effects, and how we can potentially translate these results to practice. This has been discussed in Chapter 5 and Chapter 6, along with the main conclusions drawn from this study and a perspective of the possible valorisation of these findings.