Spatial and morphological reorganization of endosymbiosis during metamorphosis accommodates adult metabolic requirements in a weevil Justin Mairea,1, Nicolas Parisota, Mariana Galvao Ferrarinia, Agnès Valliera, Benjamin Gilletb, Sandrine Hughesb, Séverine Balmanda, Carole Vincent-Monégata, Anna Zaidman-Rémya,2, and Abdelaziz Heddia,2 aUMR0203, Biologie Fonctionnelle, Insectes et Interactions (BF2i), Institut National des Sciences Appliquées de Lyon (INSA-Lyon), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Lyon (Univ Lyon), F-69621 Villeurbanne, France; and bUMR5242, Institut de Génomique Fonctionnelle de Lyon (IGFL), Ecole Normale Supérieure de Lyon, Centre National de la Recherche Scientifique (CNRS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon (Univ Lyon), F-69007 Lyon, France Edited by John R. Pringle, Stanford University Medical Center, Stanford, CA, and approved June 25, 2020 (received for review April 15, 2020) Bacterial intracellular symbiosis (endosymbiosis) is widespread in allows adaptive decoupling: for example, task specialization of nature and impacts many biological processes. In holometabolous growth at the larval stage versus reproduction at the adult stage symbiotic insects, metamorphosis entails a complete and abrupt (9, 10). However, metamorphosis is also associated with con- internal reorganization that creates a constraint for endosymbiont straints, including higher susceptibility to predators and patho- transmission from larvae to adults. To assess how endosymbiosis gens, as well as the conservation and adaptation of beneficial copes—and potentially evolves—throughout this major host-tissue symbionts (9, 11). In hemimetabolous insects, the relative mor- reorganization, we used the association between the cereal weevil phological stability associated with incomplete metamorphosis is Sitophilus oryzae and the bacterium Sodalis pierantonius as a model believed to facilitate symbiont maintenance and transmission system. S. pierantonius are contained inside specialized host cells, across developmental stages (11). On the other hand, complete the bacteriocytes, that group into an organ, the bacteriome. Cereal metamorphosis creates a double challenge for holometabolous weevils require metabolic inputs from their endosymbiont, particu- hosts: 1) Maintaining symbionts amid a drastic morphological EVOLUTION larly during adult cuticle synthesis, when endosymbiont load in- reorganization and 2) adapting the symbiotic relationship to a creases dramatically. By combining dual RNA-sequencing analyses new life stage that might have different metabolic needs. While and cell imaging, we show that the larval bacteriome dissociates at the evolution of gut symbionts through metamorphosis has been the onset of metamorphosis and releases bacteriocytes that un- addressed in several insects (12–14), very few insects harboring dergo endosymbiosis-dependent transcriptomic changes affecting cell motility, cell adhesion, and cytoskeleton organization. Remark- Significance ably, bacteriocytes turn into spindle cells and migrate along the midgut epithelium, thereby conveying endosymbionts to midgut Virtually all animals are associated with symbiotic bacteria. sites where future mesenteric caeca will develop. Concomitantly, How these associations are modulated across an animal’s life endosymbiont genes encoding a type III secretion system and a fla- cycle is a key question in understanding animal–bacteria in- gellum apparatus are transiently up-regulated while endosymbionts teractions, particularly in organisms that undergo metamor- infect putative stem cells and enter their nuclei. Infected cells then phosis during development. Here, we used the cereal weevil to turn into new differentiated bacteriocytes and form multiple new show how symbiosis is reorganized during metamorphosis, a bacteriomes in adults. These findings show that endosymbiosis re- developmental process entailing drastic tissue rearrangements. organization in a holometabolous insect relies on a synchronized In this insect, symbionts are housed within specialized host host–symbiont molecular and cellular “choreography” and illus- cells, the bacteriocytes, that form the bacteriome tissue. We trates an adaptive feature that promotes bacteriome multiplication show that the bacteriome is completely remodeled during to match increased metabolic requirements in emerging adults. metamorphosis through host–symbiont communication, in- volving adhesion and motility host proteins and a temporary endosymbiosis | bacteriome | metamorphosis | insect | dual RNA-seq symbiont infectious behavior. This interkingdom dialogue re- sults in the adaptation of the bacteriome to adulthood, high- ymbiotic associations between animals and bacteria are lighting the intertwining of symbiosis with host development. Swidespread in nature, impact organisms in different ways, and represent a driving force in evolution (1–4). Symbiosis is Author contributions: J.M., N.P., A.Z.-R., and A.H. designed research; J.M., A.V., B.G., S.H., found in all ecological niches and occurs at different levels of S.B., and C.V.-M. performed research; J.M., N.P., A.V., B.G., and S.H. contributed new host integration, culminating in intracellular symbiosis (endo- reagents/analytic tools; J.M., N.P., M.G.F., C.V.-M., A.Z.-R., and A.H. analyzed data; and J.M., N.P., M.G.F., A.Z.-R., and A.H. wrote the paper. symbiosis) in which the genomes of both partners are present within specific host cells, the bacteriocytes. Endosymbiosis is The authors declare no competing interest. prevalent in insects thriving on nutritionally unbalanced diets, This article is a PNAS Direct Submission. where endosymbionts provide nutritional complementation to Published under the PNAS license. their hosts and thereby increase their fitness and their adaptive Data deposition: Raw sequencing data from this study have been deposited at the Na- – tional Center for Biotechnology Information Sequence Read Archive, https://www.ncbi. capacities (5 7). Endosymbiosis occurs both in insects going nlm.nih.gov/sra (accession no. PRJNA484327). through incomplete metamorphosis (Hemimetabola) and com- 1Present address: School of BioSciences, The University of Melbourne, Parkville, VIC 3010, plete metamorphosis (Holometabola). Metamorphosis is a major Australia. postembryonic developmental process that represents a drastic 2To whom correspondence may be addressed. Email: [email protected] or anatomical remodeling of internal and external morphological [email protected]. organization (8). Such extreme changes allow the discrete life This article contains supporting information online at https://www.pnas.org/lookup/suppl/ stages of a given species to adapt to distinct environmental doi:10.1073/pnas.2007151117/-/DCSupplemental. niches, hence limiting intraspecies competition (8, 9). It also www.pnas.org/cgi/doi/10.1073/pnas.2007151117 PNAS Latest Articles | 1of12 Downloaded by guest on September 27, 2021 intracellular symbionts have been studied in this regard (15–17). individuals, no bacteriome-like structure is observed at either One of the most extreme cases reported to date is the olive fly larval or pupal stages (SI Appendix, Fig. S2), consistent with Bactrocera oleae, whose endosymbiont transitions from being previous observations in aposymbiotic adults (22). Mesenteric intracellular in larvae to extracellular in adults (17), although the caeca still form during metamorphosis of aposymbiotic individ- underlying mechanisms and associated advantages are yet to be uals but contain only gut epithelial cells and no bacteriocyte (SI established. Appendix, Fig. S2). This suggests that in both larvae and pupae, To address how endosymbiosis is shaped by a holometabolous bacteriocyte differentiation and bacteriome formation are lifestyle, we studied the cereal weevil Sitophilus oryzae. This co- bacteria-dependent, unlike what has been reported in the pea leopteran species houses its obligatory bacterial endosymbiont aphid Acyrthosiphon pisum (30). Interestingly, endosymbiont Sodalis pierantonius within specialized cells, the bacteriocytes, quantification by flow cytometry showed that endosymbiont that group together into an organ, the bacteriome (18–21). S. density remains stable during metamorphosis and increases only pierantonius provides S. oryzae with vitamins and amino acids once metamorphosis is achieved (SI Appendix, Fig. S1B), sup- that are at very low abundance in cereals (22–25). While endo- porting the idea that the formation of multiple gut bacteriomes symbiont load remains relatively stable during the larval stages, it in pupae may be a prerequisite to the symbiotic burst required to drastically and rapidly increases during 1 wk following the end of fulfill the increased metabolic demands in young adults. metamorphosis (22). It was shown that endosymbionts supply the In the fourth and last nymphal instar of the hemimetabolous host with large amounts of the aromatic amino acids, phenylal- whitefly Bemisia tabaci, a few bacteriocytes were described to anine and tyrosine, which are necessary components for cuticle migrate from the bacteriome to the ovaries, where they are in- synthesis following final ecdysis (22). Once the cuticle is com- corporated into developing oocytes (32). In contrast, we ob- plete, endosymbionts are rapidly eliminated and recycled from served in the cereal weevil that ovarian bacteriomes, from which the gut bacteriomes (22, 26). Hence,