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Role of the oxidative environment in the stability of symbiotic associations

Final Report Summary - SYMBIOX (Role of the oxidative environment in the stability of symbiotic associations)

Symbiotic associations between eukaryotes and microorganisms are omnipresent in nature, with outcomes ranging from parasitism to mutualism. The presence of a symbiont constitutes an important selective force, as demonstrated by the specific adaptations that have evolved to accommodate the presence of microorganisms (e.g. the evolution of the immune system, the morphogenesis of organs dedicated to symbiosis). As little research has, to date, integrated the molecular mechanisms of host-symbiont interactions within a broader evolutionary context, SymbiOx approaches this topic through the development of a comparative analysis between monospecific symbiotic interactions that present very different characteristics in terms of symbiont localization, transmission mode, and effect on the host. Increasing evidence shows that the regulation of the oxidative environment plays an important role in the establishment, the maintenance and the breakdown of symbiotic associations, through its pleiotropic role in immunity, development and metabolism. SymbiOx thus provides an in-depth focus on the role of the oxidative environment as a force in the evolution of symbiotic interactions.

Biological systems:
The two biological systems used as models for SymbiOx have proved to be powerful models to study the regulation and the evolution of symbiotic interactions.
• The squid-vibrio system is a facultative mutualistic association in which the bioluminescent bacteria protect the squid from predation. The acquisition of Vibrio fischeri from the seawater induces important morphological changes in the squid light organ, dedicated in harboring the symbionts. Another unique quality is the daily expulsion / re-colonization of the bacterial population, a rhythm that is mostly correlated with the pattern of symbiont luminescence. Luminescence results from the bacterial luciferase activity, which requires oxygen.
• The insect-Wolbachia systems are a variety of symbioses ranging from parasitism to mutualism, with various degrees of dependence between partners. Wolbachia are intracellular bacteria that are transmitted from mother to offspring. Two main systems are studied: the Asobara tabida-Wolbachia association, in which bacteria are necessary for the wasp oogenesis, and the Drosophila-Wolbachia association, in which the bacteria are reproductive parasites of the fruitfly.

Project objectives:
The first objective was to determine the molecular mechanisms involved in the control of the oxidative environment over symbiosis, through the study of gene regulation. Two main approaches were developed in both model systems: a general comparative transcriptomic analysis using next-generation sequencing techniques, and the study of specific pathways known to interfere with the regulation of the oxidative environment (oxygen availability and detoxification processes).
The second objective was to study the influence of the presence of a symbiont on the oxidative environment by analyzing the cell phenotype in symbiotic and aposymbiotic animals, in the different symbiotic associations. These comparisons helped to determine if the presence of a symbiont generally increases or limits the host oxidative environment, and what could be the consequences of these perturbations.
The third objective was to determine if the regulation of the oxidative environment plays a role in the establishment and stability of the symbiotic association. Because both the host and the symbiont can modulate the oxidative environment, we can wonder if the oxidative environment is due to a bacterial manipulation, a host resistance or a by-product of the symbiotic interaction.

Main results:
Squid/Vibrio system:
• The most complete transcriptomes of the squid Euprymna scolopes light organ have been built at two major time points: 3 h after hatching, when the symbionts first enter in contact with host tissues; and at 4 weeks, when the establishment of the symbiosis is over and the maintenance process is underway. Numerous genes involved in oxidative stress regulation have been identified.
• Squid response to an initial contact with V. fischeri (3 h) has been investigated. A set of genes was differentially regulated, which suggests that the animal can sense very few specific partners during their initial engagement. We characterized more deeply one of these genes (although not directly linked to oxidative stress), which encodes a protein that promotes the establishment of a chemo-attractant gradient that favors the migration of V. fischeri in host tissues.
• The effect of the initiation of the symbiosis (< 2 days) on the expression of genes involved in the regulation of the oxidative environment has been investigated using antibiotics and bacterial mutants involved in light production and bacterial density. These experiments showed that the presence of symbionts highly influences the oxidative regulation. The oxidative environment of the light organ has also been characterized by confocal microscopy, and the oxidative pattern appears to be highly affected by symbiosis and these patterns evolve over time.
• The effect of the maintenance of the symbiosis (4 weeks) on gene expression has been investigated, and showed that numerous genes are differentially expressed between symbiotic and aposymbiotic squid. Some genes are related to the regulation of the oxidative environment and these have been studied more deeply in relation to the change in bacterial density over the day/night cycle.
• The respiratory protein hemocyanin has been deeply characterized. Hemocyanin is present in the highly vascularized light organ and efficiently transports oxygen, suggesting that it modulates oxygen access to symbionts. In addition, hemocyanin is expressed in the appendages of the light organ that are in direct contact with the symbionts, and exhibits a phenol-oxidase activity, which suggests that hemocyanin plays a role during the initiation of the symbiosis as an antimicrobial agent, and participates in the selection of V. fischeri among the bacteria from the plankton.

Insect/Wolbachia system:
• A general signature of the presence of Wolbachia has been characterized in several insect-Wolbachia associations, and includes immunity, apoptosis, autophagy, oxidative stress, transport, lipid metabolism and transport and hormonal regulation.
• A modulation of the oxidative homeostasis differentially affects the life-history traits of flies, depending on their Wolbachia-infection status. These results suggest that the oxidative homeostasis may be a target of selection following symbiosis establishment.
• The regulation of the oxidative environment could play a major role in the evolution of symbioses, as it is involved in the establishment, the maintenance and the breakdown of several symbiotic associations.

Potential impact and use of the results:
Studying jointly the influence of a symbiont on the evolution of the immune system and the developmental program is essential to build an understanding of the consequences of symbiosis on the host fitness. Interactions between symbionts and pathogens are well known, and it is necessary to first characterize the mechanisms involved in the maintenance of a symbiotic association to assess later how they can affect the host response to a secondary pathogenic infection.
The biomedical community is becoming increasingly aware that long-term associations with beneficial bacteria are a central requirement for human health and development. For this reason, less complex models, like the squid/Vibrio mutualistic association, have been developed to discover universal themes and mechanisms that characterize host-symbiont associations.
Finally, many arthropod pests and vector of animal/human diseases live in multipartite association with viruses and bacteria, and their control strategy poses significant challenges for the medical and agriculture communities. Public concern over pesticide use and more stringent environmental regulations creates the need for more sustainable technologies, which will undoubtedly be built upon a better understanding of insect/symbiont interactions. Wolbachia-host interactions, through their broad range along the parasitism-mutualism continuum, constitute the model of choice to study mechanisms involved in insects/symbiont interactions and are important candidates for development of new control methods.
The permanent position I got at the Centre National de la Recherche Scientifique (CNRS) will thus give me the opportunity to work on this latter aspect. I will indeed work on Wolbachia-mediated protection in insects, and address in particular the possible evolution of protection in a context of control strategies related to vector-borne disease transmission. The training I got and the network I acquired thanks to this fellowship have played a major role in the success of my job applications. I will now continue to work on symbiosis and combine functional approaches in the study of evolutionary processes.