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Microorganisms living in and on macroorganisms may produce microbial volatile compounds (mVOCs) that characterise organismal odours. The mVOCs might thereby provide a reliable cue to carnivorous enemies in locating their host or prey. Parasitism by parasitoid wasps might alter the microbiome of their caterpillar host, affecting organismal odours and interactions with insects of higher trophic levels such as hyperparasitoids. Hyperparasitoids parasitise larvae or pupae of parasitoids, which are often concealed or inconspicuous. Odours of parasitised caterpillars aid them to locate their host, but the origin of these odours and its relationship to the caterpillar microbiome are unknown. Here, we analysed the odours and microbiome of the large cabbage white caterpillar Pieris brassicae in relation to parasitism by its endoparasitoid Cotesia glomerata. We identified how bacterial presence in and on the caterpillars is correlated with caterpillar odours and tested the attractiveness of parasitised and unparasitised caterpillars to the hyperparasitoid Baryscapus galactopus. We manipulated the presence of the external microbiome and the transient internal microbiome of caterpillars to identify the microbial origin of odours. We found that parasitism by C. glomerata led to the production of five characteristic volatile products and significantly affected the internal and external microbiome of the caterpillar, 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 external microbiome and body odour after parasitism were driven by the resident internal microbiome of caterpillars, where the bacterium Wolbachia sp. was only present after parasitism. Micro-injection of Wolbachia in unparasitised caterpillars increased hyperparasitoid attraction to the caterpillars compared to untreated caterpillars, while no differences were found compared to parasitised caterpillars. In conclusion, our results indicate that host-parasite interactions can affect multi-trophic interactions and hyperparasitoid olfaction through alterations of the microbiome.

Abstract Although microbial communities of insects from larval to adult stage have been increasingly investigated in recent years, little is still known about the diversity and composition of egg‐associated microbiomes. In this study, we used high‐throughput amplicon sequencing and quantitative PCR to get a better understanding of the microbiome of insect eggs and how they are established using the Southern green stinkbug Nezara viridula (L.) (Hemiptera: Pentatomidae) as a study object. First, to determine the bacterial community composition, egg masses from two natural populations in Belgium and Italy were examined. Subsequently, microbial community establishment was assessed by studying stinkbug eggs of different ages obtained from laboratory strains (unlaid eggs collected from the ovaries, eggs less than 24 h old, and eggs collected 4 days after oviposition). Both the external and internal egg‐associated microbiomes were analyzed by investigating egg washes and surface‐sterilized washed eggs, respectively. Eggs from the ovaries were completely devoid of bacteria, indicating that egg‐associated bacteria were deposited on the eggs during or after oviposition. The bacterial diversity of deposited eggs was very low, with on average 6.1 zero‐radius operational taxonomic units (zOTUs) in the external microbiome and 1.2 zOTUs in internal samples of egg masses collected from the field. Bacterial community composition and density did not change significantly over time, suggesting limited bacterial growth. A Pantoea‐like symbiont previously found in the midgut of N. viridula was found in every sample and generally occurred at high relative and absolute densities, especially in the internal egg samples. Additionally, some eggs harbored a Sodalis symbiont, which has previously been found in the abdomen of several insects, but so far not in N. viridula populations. We conclude that the egg‐associated bacterial microbiome of N. viridula is species‐poor and dominated by a few symbionts, particularly the species‐specific obligate Pantoea‐like symbiont.

Abstract Background The microbiome of many insects consists of a diverse community of microorganisms that can play critical roles in the functioning and overall health of their hosts. Although the microbial communities of insects have been studied thoroughly over the past decade, little is still known about how biotic interactions affect the microbial community structure in and on the bodies of insects. In insects that are attacked by parasites or parasitoids, it can be expected that the microbiome of the host insect is affected by the presence of these parasitic organisms that develop in close association with their host. In this study, we used high-throughput amplicon sequencing targeting both bacteria and fungi to test the hypothesis that parasitism by the endoparasitoid Cotesia glomerata affected the microbiome of its host Pieris brassicae. Healthy and parasitized caterpillars were collected from both natural populations and a laboratory culture. Results Significant differences in bacterial community structure were found between field-collected caterpillars and laboratory-reared caterpillars, and between the external and the internal microbiome of the caterpillars. Parasitism significantly altered the internal microbiome of caterpillars, but not the external microbiome. The internal microbiome of all parasitized caterpillars and of the parasitoid larvae in the caterpillar hosts was dominated by a Wolbachia strain, which was completely absent in healthy caterpillars, suggesting that the strain was transferred to the caterpillars during oviposition by the parasitoids. Conclusion We conclude that biotic interactions such as parasitism have pronounced effects on the microbiome of an insect host and possibly affect interactions with higher-order insects.

Abstract The evolution of host range drives diversification in phytophagous insects, and understanding the female oviposition choices is pivotal for understanding host specialization. One controversial mechanism for female host choice is Hopkins’ host selection principle, where females are predicted to increase their preference for the host species they were feeding upon as larvae. A recent hypothesis posits that such larval imprinting is especially adaptive in combination with anticipatory transgenerational acclimation, so that females both allocate and adapt their offspring to their future host. We study the butterfly Pieris rapae, for which previous evidence suggests that females prefer to oviposit on host individuals of similar nitrogen content as the plant they were feeding upon as larvae, and where the offspring show higher performance on the mother's host type. We test the hypothesis that larval experience and anticipatory transgenerational effects influence female host plant acceptance (no‐choice) and preference (choice) of two host plant species (Barbarea vulgaris and Berteroa incana) of varying nitrogen content. We then test the offspring performance on these hosts. We found no evidence of larval imprinting affecting female decision‐making during oviposition, but that an adult female experience of egg laying in no‐choice trials on the less‐preferred host Be. incana slightly increased the P. rapae propensity to oviposit on Be. incana in subsequent choice trials. We found no transgenerational effects on female host acceptance or preference, but negative transgenerational effects on larval performance, because the offspring of P. rapae females that had developed on Be. incana as larvae grew slower on both hosts, and especially on Be. incana. Our results suggest that among host species, preferences are guided by hard‐wired preference hierarchies linked to species‐specific host traits and less affected by larval experience or transgenerational effects, which may be more important for females evaluating different host individuals of the same species.