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Few bacteria are able to colonize eukaryotic host cells, and even fewer can invade organelles. Among the latter, those which colonize the nucleus (intranuclear bacteria) are a majority. The nucleus is considered as a replication niche that offers bacteria shelter, the opportunity to manipulate nuclear processes and nutrients in form of chromatin. Previous studies have suggested that intranuclear bacteria consume host chromatin, but this would inevitably lead to a collapse of their replication niche. Nuclear colonization has intrinsic risks for bacteria, as eukaryotic cells are able to sense nuclear deformation and respond to it activating apoptosis. Therefore, intranuclear bacteria must count on molecular mechanisms to prevent their host cells to shut down. Although many intranuclear bacteria have been visually characterized, little is known about how they colonize the nucleus, how they thrive within, and which effects they have on their host cells. [...] My findings challenge previous hypotheses about nutritional and survival strategies of intranuclear bacteria, and point out the genomic innovations that might define intranuclear lifestyle in bacteria.

Endosymbionts harbored inside insects play critical roles in the biology of their insect host and can influence the transmission of pathogens by insect vectors. Bactericera trigonica infests umbelliferous plants and transmits the bacterial plant pathogen Candidatus Liberibacter solanacearum (CLso), causing carrot yellows disease. To characterize the bacterial diversity of B. trigonica, as a first step, we used PCR-restriction fragment length polymorphism (PCR-RFLP) and denaturing gradient gel electrophoresis (DGGE) analyses of 16S rDNA to identify Sodalis and Spiroplasma endosymbionts. The prevalence of both symbionts in field-collected psyllid populations was determined: Sodalis was detected in 100% of field populations, while Spiroplasma was present in 82.5% of individuals. Phylogenetic analysis using 16S rDNA revealed that Sodalis infecting B. trigonica was more closely related to symbionts infecting weevils, stink bugs and tsetse flies than to those from psyllid species. Using fluorescent in situ hybridization and immunostaining, Sodalis was found to be localized inside the nuclei of the midgut cells and bacteriocytes. Spiroplasma was restricted to the cytoplasm of the midgut cells. We further show that a recently reported Bactericera trigonica densovirus (BtDNV), a densovirus infecting B. trigonica was detected in 100% of psyllids and has reduced titers inside CLso-infected psyllids by more than two-fold compared to CLso uninfected psyllids. The findings of this study will help to increase our understanding of psyllid–endosymbiont interactions.

Endosymbionts harbored inside insects play critical roles in the biology of their insect host and can influence the transmission of pathogens by insect vectors. Bactericera trigonica infests umbelliferous plants and transmits the bacterial plant pathogen Candidatus Liberibacter solanacearum (CLso), causing carrot yellows disease. To characterize the bacterial diversity of B. trigonica, as a first step, we used PCR-restriction fragment length polymorphism (PCR-RFLP) and denaturing gradient gel electrophoresis (DGGE) analyses of 16S rDNA to identify Sodalis and Spiroplasma endosymbionts. The prevalence of both symbionts in field-collected psyllid populations was determined: Sodalis was detected in 100% of field populations, while Spiroplasma was present in 82.5% of individuals. Phylogenetic analysis using 16S rDNA revealed that Sodalis infecting B. trigonica was more closely related to symbionts infecting weevils, stink bugs and tsetse flies than to those from psyllid species. Using fluorescent in situ hybridization and immunostaining, Sodalis was found to be localized inside the nuclei of the midgut cells and bacteriocytes. Spiroplasma was restricted to the cytoplasm of the midgut cells. We further show that a recently reported Bactericera trigonica densovirus (BtDNV), a densovirus infecting B. trigonica was detected in 100% of psyllids and has reduced titers inside CLso-infected psyllids by more than two-fold compared to CLso uninfected psyllids. The findings of this study will help to increase our understanding of psyllid&ndash, endosymbiont interactions.

Symbiotic associations are complex partnerships that can lead to new metabolic capabilities and the establishment of novel organisms. The diversity of these associations is very broad and there are still many mysteries about the origin and the exact relationship between the organisms that are involved in a symbiosis (host and symbiont). Some of these associations are essential to the hosts, such as the chemosynthetic symbioses occurring in invertebrates of the deep-sea. In others the host probably would rather not be the host, as in the case of parasitic microbes. My PhD research focuses on symbiotic and parasitic associations in chemosynthetic and non-chemosynthetic invertebrates. This thesis describes and discusses three different aspects of associations between bacteria and marine invertebrates. The first aspect focuses on chemosynthetic associations from a unique asphalt seep called Chapopote in the Gulf of Mexico (GoM). Phylogenetic analyses of host genes (cytochrome-c-oxidase subunit I) and bacterial genes (16S rRNA) in two Bathymodiolus mussel species and an Escarpia tubeworm showed that both the hosts and their chemosynthetic symbionts are very similar to their congeners from the northern GoM. Unexpectedly, a novel symbiont most closely related to hydrocarbon degrading bacteria of the genus Cycloclasticus was discovered in B. heckerae. Stable carbon isotope values in B. heckerae tissues of lipids typical for Cycloclasticus spp. were consistently heavier by 2.5permil than other lipids indicating that the novel symbiont might use isotopically heavy hydrocarbons from the asphalt seep as an energy and carbon source. The discovery of a novel symbiont that may be able to metabolize hydrocarbons is particularly intriguing because until now only methane and reduced sulfur compounds have been identified as energy sources in chemosynthetic symbioses. The large amounts of hydrocarbons available at Chapopote would provide these mussel symbioses with a rich source of nutrition. The second aspect of this thesis deals with bacteria that infect the nuclei of marine invertebrates and were recently found to be widespread in deep-sea Bathymodiolus mussels. Because of their potentially lethal effect on bivalve populations, I looked for the presence of intranuclear bacteria in economically important and commercially available bivalve species, i.e. oysters (Crassostrea gigas), razor clams (Siliqua patula and Ensis directus), blue mussels (Mytilus edulis), Manila clams (Venerupis philippinarum), and common cockles (Cerastoderma edule). Fluorescence in situ hybridization (FISH) revealed the presence of intranuclear bacteria in all investigated bivalves except oysters and blue mussels. Preliminary tests with real-time PCR showed massive amounts of intranuclear bacteria in some of the bivalve species, raising the question if these might affect not only the health of the bivalves but possibly also of the humans that eat them. In the third and final aspect of my thesis, I examined the general diversity of bacteria in the gill tissues of deep-sea and shallow-water mussels and clams. Comparative 16S rRNA sequence analysis and cultivation experiments revealed a much higher diversity than previously recognized. This thesis shows that bivalves are ideal models for studying the microbiota of marine invertebrates because of the high diversity of both highly specific and more generalized symbiotic and parasitic bacteria in their gill tissues.

International audience; Pentastiridius leporinus is an important vector of sugar beet pathogens in eastern France. An electron microscope survey on the insectassociated microflora revealed the occurrence of intranuclear prokaryotic cells in every internal organ analysed. These bacteria, which could also be found in the cytoplasm surrounding the nucleus, had a homogeneous coccoid (ca. 0.45 lm) or rod (0.45–1 lm) shape. The presence of three membrane layers was observed, the outermost forming a kind of vacuole containing generally a single microorganism. No cytopathological abnormalities were detected in the infected cells. To our knowledge, this is the first report of a hemipteran species infected by intranuclear bacteria. The possible identity of this microorganism is discussed.