Deciphering the Functional Organization of Cross-kingdom 'metabolic factories’ in insect endoSymbiosis – FOCuS

Firstly, we focused on studying the subcellular organisation that enables the intensive metabolic exchanges necessary for effective nutritional endosymbiosis. We analysed the three-dimensional ultrastructure of bacteriocytes and endosymbionts, paying particular attention to cellular membrane networks and the localisation of metabolites and potential transporters. Key methods used were: high-pressure cryo-fixation, electron microscopy, volume electron microscopy, STXM, immunostaining/immunogold and fluorescence microscopy.

We then studied the various cell types that constitute the bacteriome organ, as well as the endosymbionts present within it. We analysed the host and endosymbiont transcriptomes using a dual RNA-seq approach on microdissected cells. The transcriptomic-based identification of key genes in this interactome allowed the further analysis of the functions of some of them using RNA interference, combined with molecular biology classical approaches such as RT-qPCR and imaging and biochemical methods.

Finally, we analysed the impact of host nutrition on the transcriptomes of bacteriocytes and their bacterial inhabitants.

We demonstrated that intracellular symbionts form extensive tubular membrane networks. These structures act as conduits for nutrient exchange, allowing the bacteria to efficiently acquire host-derived carbohydrates. This analysis relied heavily on a combination of volume electron microscopy, which allowed us to understand the organisation of these structures in three dimensions, and spectromicroscopy (STXM), which demonstrated their carbohydrate content (Balmand et al., Cell 2025).

To address what defines differentiated bacteriocytes we performed microdissection of different cell subtypes in the bacteriome of symbiotic insects and their closest aposymbiotic equivalents, and combined this approach with low-input dual RNA-seq. We showed that bacteriocytes have a distinct programme from nearby epithelial cells and aposymbiotic counterparts. Furthermore, we identified two subcategories of bacteriocytes with different functions. We also demonstrated that the bacteria infecting epithelial cells express a different set of genes to those present in bacteriocytes. Analysing the impact of nutritional stress on the bacteriocyte and bacterial transcriptomes, we showed that nutritional stress modifies the metabolic strategies of both partners (Galambos et al., Microbiome, 2025).

The discovery of novel membrane features produced by intracellular symbiotic bacteria within host cells opens up a new, ultrastructural level of understanding of metabolic exchange mechanisms. Many questions remain regarding the molecular basis of structure formation and regulation, which will form the focus of future projects.

Pesticides are intensively used to protect crops from pests. However, pesticides are known to increase pest resistance and affect the environment and non-target organisms, including humans, which points to the need for sustainable alternatives. Cereal weevils Sitophilus spp. are one of the main cereal pests. Their capacity to thrive on a cereal-exclusive diet relies on their mutualistic association with the intracellular symbiotic bacterium (endosymbiont) Sodalis pierantonius, which provides them with amino acids, vitamins and cofactors that are scarce in the grains. Instead of targeting the insect itself, and because weevils rely on this symbiosis to survive, a new, specific and sustainable control strategy could be to target the nutritional functioning that has evolved between associated partners in these long-term successful relationships.
Metabolic exchanges between the cereal weevils and S. pierantonius are at the core of this symbiosis. Both partners have evolved towards a metabolic complementarity and dependency on each other: the bacteria benefit from carbohydrates that insects process from the grain; in return, bacteria produce and provide to the host metabolites that complement its diet. While the importance and nature of the metabolic exchanges between symbiotic partners is well documented, how the nutrients are exchanged between partners remains an open question. The metabolic integration between Sitophilus weevils and Sodalis takes place into dedicated host cells, the bacteriocytes, which house the endosymbionts. To date, the mechanisms by which bacteriocytes are turned into highly specialized cross-kingdom ‘metabolic factories’ remain unclear, despite the fact that this adaptation is a key element in bacteria-insect partnership.
The FOCuS project aims at deciphering the functional and ultrastructural organization of these ‘cross-kingdom metabolic factories’, thanks to recent advances in forefront imaging and molecular biology techniques. Work package 1 will address how bacteriocyte subcellular organization allows for the intensive metabolic exchanges required in an efficient nutritional endosymbiosis. We will analyze the three-dimensional ultrastructure of bacteriocyte and endosymbiont, with a focus on the cell membranous networks and the localization of transporters and exchanged metabolites. Work package 2 will investigate how bacteriocytes differentiate into fully functional specialized cells, and how the bacteria participate in this process. The impact of host nutrition on these structures will also be analyzed. Cellular and developmental biology approaches will be used to better characterize the differentiation process. Microdissection and Dual RNAseq will be conducted to uncover the host and endosymbiont interactome (paired transcriptomes) during bacteriocyte differentiation. The key genetic elements of this interactome will be functionally studied by using complementary tools, including RNA interference. Work package 3 will analyze whether and how the bacteriocyte structural organization is impacted by changes in the host diet.
We expect with FOCuS to identify novel target mechanisms to disturb either bacteriocyte differentiation or function and, consequently, impact host or endosymbiont fitness as a novel strategy to control crop pests.