Please cite this article in press as: Vigneron et al., Insects Recycle Endosymbionts when the Benefit Is Over, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.07.065 Current Biology 24, 1–7, October 6, 2014 ª2014 Elsevier Ltd All rights reserved
http://dx.doi.org/10.1016/j.cub.2014.07.065
Report Insects Recycle Endosymbionts when the Benefit Is Over Aure´lien Vigneron,1,2 Florent Masson,1 Agne`s Vallier,1 Se´verine Balmand,1 Marjolaine Rey,1 Carole Vincent-Mone´gat,1 Emre Aksoy,1 Etienne Aubailly-Giraud,1 Anna Zaidman-Re´my,1 and Abdelaziz Heddi1,* 1Biologie Fonctionnelle Insectes et Interactions, UMR203 BF2I, INRA, INSA-Lyon, Universite´ de Lyon, 69621 Villeurbanne, France
Summary Symbiotic associations are widespread in nature and represent a driving force in evolution. They are known to impact fitness, and thereby shape the host phenotype [1–4]. Insects subsisting on nutritionally poor substrates have evolved mutualistic relationships with intracellular symbiotic bacteria (endosymbionts) that supply them with metabolic components lacking in their diet [5–10]. In many species, endosymbionts are hosted within specialized host cells, called the bacteriocytes, and transmitted vertically across host generations [11]. How hosts balance the costs and benefits of having endosymbionts, and whether and how they adjust symbiont load to their physiological needs, remains largely unexplored. By investigating the cereal weevil Sitophilus association with the Sodalis pierantonius endosymbiont [8, 12], we discover that endosymbiont populations intensively multiply in young adults, before being rapidly eliminated within few days. We show that young adults strongly depend on endosymbionts and that endosymbiont proliferation after metamorphosis matches a drastic host physiological need for the tyrosine (Tyr) and phenylalanine (Phe) amino acids to rapidly build their protective exoskeleton. Tyr and Phe are precursors of the dihydroxyphenylalanine (DOPA) molecule that is an essential component for the cuticle synthesis. Once the cuticle is achieved, DOPA reaches high amounts in insects, which triggers endosymbiont elimination. This elimination relies on apoptosis and autophagy activation, allowing digestion and recycling of the endosymbiont material. Thus, the weevil-endosymbiont association reveals an adaptive interplay between metabolic and cellular functions that minimizes the cost of symbiosis and speeds up the exoskeleton formation during a critical phase when emerging adults are especially vulnerable. Results and Discussion Cereal weevils house endosymbionts permanently in the female germ cells from which they are transmitted to the progeny. Early during embryogenesis, endosymbionts induce the differentiation of bacteriocyte cells that form the bacteriome [7], a specific symbiotic organ that secludes endosymbionts and protect them against the host immune response [13]. Young adults house numerous bacteriomes at the apex of
2Present
address: Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06520-8034, USA *Correspondence: [email protected]
midgut caeca and in female ovaries (Figure 1A). Remarkably, an early observation has reported that gut bacteriomes, but not ovarian bacteriomes, are lost in adults a few days following adult metamorphosis and the ultimate insect molting (day 0 of adulthood) [14]. Using real-time quantitative PCR (qPCR), we unraveled an unexpected complex dynamic of endosymbiont populations in young adults (Figure 1B). Endosymbiont populations first drastically increase between the ultimate insect molting and day 6 of adulthood, suggesting a critical symbiotic requirement during this phase of development. Although weevils live up to 6 months under lab conditions, the gut endosymbiont population rapidly decreases, and is completely eliminated by day 14, whereas the ovarian symbiont population remains stable over time (Figure 1B). This suggests that from day 6, the benefit of gut endosymbiosis no longer compensates for the burden of symbiont maintenance and that weevils have evolved a mechanism to adjust symbiont load to their needs. The modulation of symbiont density during insect development has previously been reported [15–18], but the situation is extreme in weevils, as within a 2-week period only, the gut symbiotic population grows intensively and then completely disappears. Similar symbiotic dynamics also occur in two additional cereal weevil species, Sitophilus zeamais and Sitophilus granarius (Figures S1A and S1B available online), suggesting a conserved mechanism of symbiont load modulation. Importantly, endosymbiont dynamic is more rapid in the wild weevil species S. oryzae and S. zeamais, which thrive on cereal fields, than in the granary-associated weevil S. granarius species, which is encountered in cereal storerooms of temperate countries only, suggesting that endosymbiont load is also modulated according to animal life conditions. Thus, weevils have evolved an ability to finetune tissue-specific regulation of their symbiont populations, allowing them to rapidly eliminate the gut population while preserving the ovarian population that is essential for reproduction and for symbiont transmission to progeny. To gain insight into the cellular basis of symbiont modulation in the gut, we examined the morphology of this tissue over the first 2 weeks of adulthood. Fluorescent in situ hybridization (FISH) experiments and scanning electron microscopy (SEM) performed on S. oryzae, S. zeamais, and S. granarius intestines highlighted a structural plasticity of bacteriomes during symbiont growth. Bacteriomes drastically increase in size until day 6 (Figures 1C, 1D, and S1C), while their number remains unchanged (Figures 1C and S1B and Table S1). From day 7, and in parallel with the decrease in symbiont population, bacteriomes progressively degenerate from the posterior to the anterior midgut and from the base to the apex of the caeca (Figures 1C, 1D, and S1D). This oriented endosymbiont elimination and bacteriome atrophy is rapid, consistent, and occurs in the three cereal weevil species examined (Figures 1B, 1C, S1A, and S1B), indicating that a well-conserved genetic mechanism or convergent processes lead to the repeated elimination of the bacteriomes in several species. Apoptosis detection with YO-PRO staining, a nucleic acid stain that successfully passes the plasma membrane of apoptotic cells only, and immunohistochemistry against the activated form of the effector Caspase-3, a marker of apoptotic cascade activation, showed apoptosis activation in intestinal cells that correlates
Please cite this article in press as: Vigneron et al., Insects Recycle Endosymbionts when the Benefit Is Over, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.07.065 Current Biology Vol 24 No 19 2
Figure 1. Dynamics of Endosymbiont Density in S. oryzae (A) A diagram showing the location of the bacteriomes (in red) in the mesenteric caeca and ovaries of an adult weevil. (B) qPCR quantification of endosymbiont DNA from fourth-instar larvae, late nymphs, and adult males (whole insects) and from female ovaries. Mean (6SE) of five independent biological replicates is represented. In males (black line), endosymbiont density significantly increases until day 6 prior to symbiont elimination (generalized linear model [GLM], p < 0.0001). In ovaries (gray line), the symbiont population significantly increases until day 6 (GLM, p < 0.0001) and then remains stable. (C) FISH staining of endosymbionts in adult mesenteric caeca from day 3 to day 13. S. pierantonius (green) progressively disappears from the posterior to the anterior caeca (orientation indicated by arrows). In aposymbiotic insects, caeca remain morphologically similar over time. Blue, nuclei (DAPI). Anterior is to the left. (D) SEM of adult mesenteric caeca. Bacteriomes are present at the apex of caeca in symbiotic insects (indicated by a line on the day 3 picture). From day 6, bacteriomes regress from the caeca basis to its apex (see arrows). No bacteriome is observed in caeca of aposymbiotic insects. D, days after the ultimate insect molting (UIM). See also Figure S1 and Table S1.
in time and space with symbiont clearance (Figures 2A and 2B). Apoptosis is induced in bacteriocyte cells (Figure 2B), and apoptotic bodies were seen in the gut lumen or engulfed by adjacent epithelial cells (Figure 2C). Importantly, apoptosis was not detected in the gut of insects experimentally depleted
of endosymbionts (so-called ‘‘aposymbiotic weevils’’ [19]) (Figures 2A and 2B). This indicates that activation of cell death is most likely related to symbiont elimination. Transmission electronic microscopy (TEM) also revealed induction of autophagy in epithelial cells (Figure 2D). Autophagy is a process
Please cite this article in press as: Vigneron et al., Insects Recycle Endosymbionts when the Benefit Is Over, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.07.065 Bacterial Load Adjustment in Insect Symbiosis 3
Figure 2. Cell Apoptosis and Autophagy Activation during the Elimination of gut Endosymbiont Population (A) YO-PRO staining of whole guts in symbiotic adults (right) revealed apoptotic cells (green, arrowheads) in posterior caeca at day 7 (top, arrowheads) and in anterior caeca at day 11 (bottom, arrowheads). No apoptosis was detected in aposymbiotic insects (left). Blue, nuclei (Hoechst). (B) Cleaved-caspase-3 immunostaining (green) of gut sections indicates apoptosis at the basis of the bacteriome in symbiotic adults at day 7 (top right, arrowheads) and in fewer cells mainly located at the apex of caeca at day 11 (bottom). No signal was detected in aposymbiotic insects (left). Blue, nuclei (DAPI). (C and D) TEM of symbiotic midguts at day 6 and 8. (C) Apoptotic bodies are seen in epithelial cells (top) and in the midgut lumen (bottom). (D) Autophagy is observed in bacteriocytes and epithelial cells. Top: autophagosome digesting S. pierantonius in a bacteriocyte (*; left). An autophagosome fusing with vesicles containing symbiont and forming autophagic vesicles (middle), prompting symbiont digestion (*; right), is shown. Bottom: autophagy leads to the accumulation of lamellar bodies that cover the majority of the cell cytosol, here observed at different scales. (E) TEM of midguts from aposymbiotic adults at day 8, which are endosymbiont free. These cells present a dense vesicular network at their lumen pole and contain lipid droplets and peroxysomes, but no lamellar body was seen. EC, epithelial cell; Vc, vesicle; Symb, symbiont; Nu, nucleus; mu, muscle; mv, microvilli; Lumen, gut lumen; RER, rough endoplasmic reticulum; Rb, ribosomes.
in which double-membrane autophagosomes sequester organelles or portions of cytosol and fuse with lysosomes, allowing digestion of the vacuole content [20]. In symbiotic weevils, all of these typical stages of autophagy were
observed surrounding endosymbionts, as well as prominent lamellar bodies that result from membrane accumulation and that are typical products of extensive autophagy [21–23] (Figure 2D). Such structures were not observed in aposymbiotic
Please cite this article in press as: Vigneron et al., Insects Recycle Endosymbionts when the Benefit Is Over, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.07.065 Current Biology Vol 24 No 19 4
Figure 3. Symbiosis Is Essential for Complete Cuticle Synthesis (A) Cuticle is darker in symbiotic (left) than in aposymbiotic (right) 26-day-old adults. (B) SEM of cuticle sections from symbiotic (left) and aposymbiotic (right) 15-day-old adults. Mean (6SE) of 11 measures of the epicuticle and exocuticle are presented. Exocuticle is thinner in aposymbiotic than in symbiotic individuals (double arrow). epi, epicuticle; exo, exocuticle; endo, endocuticle. (C) Progression of the cuticle color in adult weevils. The inverse of digital red color value was determined for both symbiotic (solid line, white square) and aposymbiotic insects (dotted line, black triangle). Symbiotic individuals are darker than aposymbiotic individuals (two-way ANOVA, symbiosis effect, p < 0.0001). Symbiotic and aposymbiotic individuals achieve their darker coloration by day 7 and day 17, respectively. Corresponding colors are indicated at the bottom. See also Figure S2.
insects (Figure 2E), supporting the idea that they result from symbiont digestion. Autophagy is a well-conserved mechanism used by cells to maintain homeostasis via the elimination and recycling of defective organelles. Lamellar bodies are thought to be involved in organelle recycling and serve as phospholipid storage [22, 23]. Autophagy is also a conserved defense mechanism against intracellular pathogens [24, 25], and it has recently been shown to control proliferation of the parasitic reproductive endosymbiont Wolbachia [24, 26]. Here, we report that autophagy controls the load of cooperative intracellular bacteria in weevils. Coordinated symbiont clearance by apoptosis and autophagy prevents the release of symbiont and cell material, thus avoiding tissue inflammation. By recycling endosymbiont biomaterials, these cellular processes may help insects recover the metabolic investments made during bacterial multiplication. S. pierantonius is known to provide the insect with many components that are poorly represented in wheat grains, such as pantothenic acid, biotin, riboflavin [27], and amino acids, particularly the aromatic amino acids phenylalanine (Phe) and tyrosine (Tyr) [8, 28]. Endosymbiont vitamin supply was shown to increase insect mitochondrial energetic metabolism and to improve the flight ability of adults [7, 29, 30]. Symbiosis also decreases larval developmental time and improves female fertility [31]. Here, the contrasting dynamic of gut endosymbiont population before and after day 6 (i.e., growth followed by elimination) strongly suggests that symbiosis is particularly important at the initial phase of adulthood but that it becomes advantageous to eradicate gut endosymbionts when the benefits of the association end. To experimentally address the benefits of the symbiosis at this early stage of adulthood, we reared weevils emerging from cereal grains on starch pellets that contain carbohydrates only. In contrast to symbiotic insects, aposymbiotic weevils rapidly died under
these conditions (Figure S2A). Strikingly, rearing of aposymbiotic weevils on cereal grains during the first 2 weeks of adulthood is sufficient to restore their ability to survive as long as their symbiotic counterparts if they are transferred to starch afterward (Figure S2A). This demonstrates that weevils require metabolic components other than carbohydrates after the ultimate insect molting and that symbiosis is essential to complement insect diets during this critical phase. As coleopterans, one remarkable adaptive trait of weevils is their hard, protective exoskeleton—the cuticle that preserves their body from desiccation, shocks, and pathogens [32, 33]. Symbiotic weevils exhibit a darker color than aposymbiotic individuals (Figure 3A), and, using SEM, we show a much thicker cuticle in symbiotic versus aposymbiotic weevils (Figure 3B). While symbiotic adults produce their cuticle rapidly after ultimate insect molting (Figures 3C and S2B), this process is slow and remains incomplete in aposymbiotic weevils (Figure 3C). In the three cereal weevil species, gut symbiont multiplication correlates with the progression of cuticle coloration, a process that ends after the beginning of symbiont elimination (Figure S2C). Taken together, these findings show that symbiosis is required for rapid cuticle synthesis, which may help insects to cope more effectively with environmental challenges. In line with this, we demonstrated that aposymbiotic insects die more rapidly than symbiotic individuals under high temperature and dry conditions (Figure S2D), which could be related to their defective cuticle. Cuticle biosynthesis and tanning are the result of sclerotization and melanization, two processes that require dihydroxyphenylalanine (DOPA) as a precursor [34]. This aromatic amino acid can be synthesized from Tyr, which itself can be derived from Phe (Figure 4A) [35]. Amino acid analysis with high-performance liquid chromatography (HPLC) indicated that levels of both free and protein-associated Tyr and Phe remain higher in symbiotic than in aposymbiotic insects, but decrease in the first 5 days after ultimate insect molting (Figures S3A and S3B), suggesting a redirection of these amino acids to other functions and their transformation into other products, including
Please cite this article in press as: Vigneron et al., Insects Recycle Endosymbionts when the Benefit Is Over, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.07.065 Bacterial Load Adjustment in Insect Symbiosis 5
Figure 4. DOPA Is Critical during the First Days of Adulthood (A) Scheme summarizing possible Phe and Tyr amino acid sources used by weevils, the DOPA synthesis pathway, and the origin of catalyzing enzymes. Phe is an essential amino acid that cannot be synthesized by insects, whereas insects are able to transform Phe into Tyr and Tyr into DOPA. (B) Kaplan-Meier representation of male aposymbiotic insect survival on wheat grain (black; control), starch pellets (red), starch pellets complemented with tryptophan (Trp, green), Phe (orange), Tyr (blue), all three aromatic amino acids (Trp+Tyr+Phe, purple), or DOPA (pink). Each curve represents the daily survival rate of 120 males until the death of the last individual. Data were analyzed using a log-rank test. Weevils reared on starch pellets complemented with all three amino acids exhibit higher rates of survival than weevils reared on plain starch pellets (c2 = 212, p < 0.0001) but lower survival than insects fed on wheat grains (c2 = 94, p < 0.0001). Phe complementation also partly restores weevil survival when compared with plain starch pellets (c2 = 190, p < 0.0001), in a range similar to the complementation with three amino acids (c2 = 0.6, p = 0.45). Tyr complementation partly restores weevil survival when compared with starch pellets (c2 = 83.3, p < 0.0001), but this restoration did not reach the level of Phe complementation (c2 = 24.4, p < 0.0001). Survival of insects reared on Trp-complemented pellets is not significantly different from survival of insects reared on starch pellets (c2 = 0.3, p = 0.589). DOPA complementation partly restores weevil survival, at a comparable level to Tyr complementation (c2 = 0.3, p = 0.582). (C) HPLC quantification of DOPA in adult gut (top) and remaining carcass (bottom). DOPA quantities are significantly higher in symbiotic (white squares) than in aposymbiotic (black triangles) insects, both in gut (two-way ANOVA, symbiosis effect, p < 0.0001) and carcass (two-way ANOVA, symbiosis effect, p < 0.0001). Symbiotic insects showed a significant increase of DOPA from day 5 to day 8 in both gut (two-way ANOVA, time effect, p < 0.0001) and carcass (two-way ANOVA, time effect, p < 0.0001). (D) qPCR quantification of symbiont DNA in adults emerging from grains (i.e., day 3 after ultimate insect molting) and reared on wheat flour pellets (white circle) or on wheat flour pellets complemented with 2% DOPA (black circle). Mean (6SE) of five independent biological replicates is represented. Elimination of endosymbionts in insects fed on DOPA-complemented pellets is accelerated (GLM, p < 0.0001). See also Figure S3.
DOPA. The level of protein-associated Tyr and Phe increases from day 5 to 11, while cuticle synthesis is being completed (Figures S3A and S3B), indicating that insects redirect free amino acids to general protein synthesis and replenish the molecules hydrolyzed during the first days of adulthood. S. pierantonius has recently established intracellular symbiosis with Sitophilus weevils after a replacement of Nardonella, the ancestral endosymbiont of the Dryophthoridae family, to which cereal weevils belong [12, 36–38]. Relaxed selection on S. pierantonius genome at this initial phase of symbiosis integration has resulted in a large number of gene pseudogenization and rearrangements, associated with an expansion of transposable elements [8]. However, although the S. pierantonius genome has lost several metabolic functions, Phe and Tyr pathways have been conserved [8]. This suggests that endosymbionts are essential for providing their hosts with these amino acids to boost the cuticle synthesis after molting. To address this hypothesis, we monitored aposymbiotic insect survival on starch pellets complemented with different amino acids (Figure 4B). While aposymbiotic weevils cannot survive on starch or on starch complemented with tryptophan (another aromatic amino acid), their survival is partially restored when starch is complemented with Tyr and even more so with Phe. This result demonstrates that Tyr and Phe
are two important amino acids supplemented by endosymbionts. They are required for the host to survive on restricted diets, and their supplementation partly compensates the absence of endosymbionts. However, rearing weevils on highly concentrated Tyr and Phe does not prematurely trigger endosymbiont clearance (Figure S3C). Concordant with the idea that Phe and Tyr are required for DOPA production at this critical stage of cuticle biosynthesis, HPLC quantification of DOPA revealed a strong increase in the level of this amino acid in symbiotic weevils from day 5, whereas its level remains low in aposymbiotic insects (Figure 4C). This means that symbiosis allows for high levels of DOPA production. Moreover, using a complementation test, we showed that feeding aposymbiotic weevils directly on DOPA-complemented starch is sufficient to restore their survival to the same extent as feeding them on Tyr-complemented starch diet (Figure 4B). This finding strongly suggests that Tyr is mainly required for DOPA production at this stage of development, while Phe is also involved in other processes impacting the insect survival. Remarkably, symbiotic weevils transferred to DOPA-complemented wheat flour pellets after grain emergence complete symbiont elimination 2 days earlier than weevils maintained on control wheat flour pellets (Figure 4D). Thus, increased access of weevils to DOPA is
Please cite this article in press as: Vigneron et al., Insects Recycle Endosymbionts when the Benefit Is Over, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.07.065 Current Biology Vol 24 No 19 6
sufficient to accelerate gut endosymbiont elimination. DOPA level directly impacts endosymbiont density, demonstrating the existence of a molecular mechanism that allows weevils to adjust their symbiotic load to their specific physiological needs in DOPA at the critical phase of cuticle synthesis. Interestingly, the only Sitophilus species known to be naturally free of endosymbionts so far is Sitophilus linearis [38], which feeds on tamarind seeds that contain a high level of DOPA [39]. Therefore, in addition to the previously described S. pierantonius effects on weevil physiology [7, 10, 27, 29– 31, 40], this work shows that S. pierantonius drastically interferes with DOPA production and cuticle synthesis. Further, weevils appear to have evolved a mechanism to tightly synchronize endosymbiont elimination and recycling with the completion of cuticle deposition. In this evolutionary context, it would be of interest to figure out whether bacterial Tyr and Phe pathways are adaptive for other symbiotic coleopterans, by analyzing Tyr and Phe pathway conservation in the longlasting endosymbiont Nardonella. Future investigations will also tell us about the precise function of DOPA in the interplay between metabolism and host cellular responses to endosymbionts and whether the growth and subsequent recycling of endosymbionts is relevant for other processes occurring during this stage of development, including reproduction maturity. Conclusions Examining the association between cereal weevils, Sitophilus spp. and their intracellular symbiotic bacterium, S. pierantonius, we have described an extreme modulation of endosymbiont load in these coleopteran insects, ending with a complete elimination of symbionts from the host ‘‘somatic’’ tissues. We report that weevil endosymbionts intensively multiply in young adults to support the massive need for Tyr and Phe that are the precursors of the DOPA molecule required for cuticle tanning and hardening. Comparison between weevil species living in different ecological niches showed that endosymbiont dynamics and cuticle completion are more rapid in field- than in the granary-living species, suggesting that endosymbiont load is also modulated according to animal environmental conditions. Once the cuticle is achieved, endosymbionts are rapidly recycled through apoptosis and autophagy in a tissue-dependent manner that preserves female germline-associated endosymbionts and thus their transmission to next generations. We show that complementation of insect diets with DOPA decreased symbiont abundance and hastened their clearance. In addition to bacteriocyte immune regulations [13, 41, 42] and symbiont control by antimicrobial peptides recently described in weevils [41], we report here that cellular apoptosis and autophagy are involved in bacterial load regulation according to insect development. These data present a striking example of how host-endosymbiont coevolution has shaped mechanisms that adjust symbiont load to the host physiological needs, while allowing a return of investment through natural symbiont recycling. While mutualism significantly impacts organism life history traits, enhancing adaptive features such as the cuticle thickness, cooperative associations remain under the control of a fine-tuned integration of costs and benefits according to host developmental requirements. The synchronization of a ‘‘somatic’’ breakdown of symbiosis with the achievement of a specific developmental trait minimizes insect investment in symbiosis while preserving transmission to the progeny.
Supplemental Information Supplemental Information includes Supplemental Experimental Procedures, three figures, one table, and one movie and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2014.07.065. Author Contributions A. Vigneron designed and performed experiments, analyzed data, and wrote the manuscript; F.M. conducted biological experiments and analyzed data; A. Vallier carried out dissections and molecular biology experiments; S.B. carried out histological experiments; M.R. performed HPLC experiments; C.V.-M. participated in the design and the analysis of experiments; E.A. measured the cuticle color; E.A.-G. performed FISH experiments; A.Z.-R. performed biological experiments, analyzed the data, and wrote the manuscript; and A.H. conceived the study, coordinated the work, and drafted and wrote the manuscript. Acknowledgments This work was supported by INRA, INSA-Lyon, and the French ANR-10 BSV7-170101-03 (ImmunSymbArt) and ANR-13-BSV7-0016-01 (IMetSym). We thank G. Febvay for advices on amino acids, S. Schaack, J. Regan, and B. Weiss for their critical comments on the manuscript, and A. Gilles, J. Schinko, and D. Compagnon for weevil image capture. The authors would also like to thank the anonymous reviewers for their constructive criticisms. Electron microscopy was carried out in Centre Technologique des Microstructures (CTm, Universite´ Lyon I), and qPCR in the DTAMB (De´veloppement de Techniques et Analyse Mole´culaire de la Biodiversite´). Received: June 19, 2014 Revised: July 23, 2014 Accepted: July 24, 2014 Published: September 18, 2014 References 1. Margulis, L. (1993). Origins of species: acquired genomes and individuality. Biosystems 31, 121–125. 2. Moran, N.A. (2006). Symbiosis. Curr. Biol. 16, R866–R871. 3. Tsuchida, T., Koga, R., Horikawa, M., Tsunoda, T., Maoka, T., Matsumoto, S., Simon, J.C., and Fukatsu, T. (2010). Symbiotic bacterium modifies aphid body color. Science 330, 1102–1104. 4. Zilber-Rosenberg, I., and Rosenberg, E. (2008). Role of microorganisms in the evolution of animals and plants: the hologenome theory of evolution. FEMS Microbiol. Rev. 32, 723–735. 5. Akman Gu¨ndu¨z, E., and Douglas, A.E. (2009). Symbiotic bacteria enable insect to use a nutritionally inadequate diet. Proc. Biol. Sci. 276, 987–991. 6. Douglas, A.E. (1998). Nutritional interactions in insect-microbial symbioses: aphids and their symbiotic bacteria Buchnera. Annu. Rev. Entomol. 43, 17–37. 7. Heddi, A., Grenier, A.M., Khatchadourian, C., Charles, H., and Nardon, P. (1999). Four intracellular genomes direct weevil biology: nuclear, mitochondrial, principal endosymbiont, and Wolbachia. Proc. Natl. Acad. Sci. USA 96, 6814–6819. 8. Oakeson, K.F., Gil, R., Clayton, A.L., Dunn, D.M., von Niederhausern, A.C., Hamil, C., Aoyagi, A., Duval, B., Baca, A., Silva, F.J., et al. (2014). Genome degeneration and adaptation in a nascent stage of symbiosis. Genome Biol. Evol. 6, 76–93. 9. Snyder, A.K., Deberry, J.W., Runyen-Janecky, L., and Rio, R.V. (2010). Nutrient provisioning facilitates homeostasis between tsetse fly (Diptera: Glossinidae) symbionts. Proc. Biol. Sci. 277, 2389–2397. 10. Wicker, C., Guillaud, J., and Bonnot, G. (1985). Comparative composition of free, peptide and protein amino acids in symbiotic and aposymbiotic Sitophilus oryzae (Coleoptera, Curculionidae). Insect Biochem. 15, 537–541. 11. Koga, R., Meng, X.Y., Tsuchida, T., and Fukatsu, T. (2012). Cellular mechanism for selective vertical transmission of an obligate insect symbiont at the bacteriocyte-embryo interface. Proc. Natl. Acad. Sci. USA 109, E1230–E1237. 12. Charles, H., Heddi, A., and Rahbe, Y. (2001). A putative insect intracellular endosymbiont stem clade, within the Enterobacteriaceae, infered
Please cite this article in press as: Vigneron et al., Insects Recycle Endosymbionts when the Benefit Is Over, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.07.065 Bacterial Load Adjustment in Insect Symbiosis 7
13.
14.
15.
16.
17.
18.
19.
20. 21. 22.
23.
24.
25.
26.
27.
28.
29. 30.
31.
32.
33.
from phylogenetic analysis based on a heterogeneous model of DNA evolution. C. R. Acad. Sci. III 324, 489–494. Anselme, C., Pe´rez-Brocal, V., Vallier, A., Vincent-Monegat, C., Charif, D., Latorre, A., Moya, A., and Heddi, A. (2008). Identification of the weevil immune genes and their expression in the bacteriome tissue. BMC Biol. 6, 43. Mansour, K. (1930). Preliminary studies on the bacterial cell mass (accessory cell-mass) of Calandra oryzae: the rice weevil. Q. J. Microsc. Sci. 73, 421–436. Kim, J.K., Han, S.H., Kim, C.H., Jo, Y.H., Futahashi, R., Kikuchi, Y., Fukatsu, T., and Lee, B.L. (2014). Molting-associated suppression of symbiont population and up-regulation of antimicrobial activity in the midgut symbiotic organ of the Riptortus-Burkholderia symbiosis. Dev. Comp. Immunol. 43, 10–14. Laughton, A.M., Fan, M.H., and Gerardo, N.M. (2014). The combined effects of bacterial symbionts and aging on life history traits in the pea aphid, Acyrthosiphon pisum. Appl. Environ. Microbiol. 80, 470–477. Nishikori, K., Morioka, K., Kubo, T., and Morioka, M. (2009). Age- and morph-dependent activation of the lysosomal system and Buchnera degradation in aphid endosymbiosis. J. Insect Physiol. 55, 351–357. Stoll, S., Feldhaar, H., Fraunholz, M.J., and Gross, R. (2010). Bacteriocyte dynamics during development of a holometabolous insect, the carpenter ant Camponotus floridanus. BMC Microbiol. 10, 308. Nardon, P. (1973). Obtention d’une souche asymbiotique chez le charanc¸on Sitophilus sasakii Tak: diffe´rentes me´thodes d’obtention et comparaison avec la souche symbiotique d’origine. C. R. Acad. Sci. Paris 277, 981–984. He, C., and Klionsky, D.J. (2009). Regulation mechanisms and signaling pathways of autophagy. Annu. Rev. Genet. 43, 67–93. Clarke, M., Bennett, M., and Littlewood, T. (2007). Cell death in the cardiovascular system. Heart 93, 659–664. Schmitz, G., and Mu¨ller, G. (1991). Structure and function of lamellar bodies, lipid-protein complexes involved in storage and secretion of cellular lipids. J. Lipid Res. 32, 1539–1570. Weaver, T.E., Na, C.L., and Stahlman, M. (2002). Biogenesis of lamellar bodies, lysosome-related organelles involved in storage and secretion of pulmonary surfactant. Semin. Cell Dev. Biol. 13, 263–270. Le Clec’h, W., Braquart-Varnier, C., Raimond, M., Ferdy, J.B., Bouchon, D., and Sicard, M. (2012). High virulence of Wolbachia after host switching: when autophagy hurts. PLoS Pathog. 8, e1002844. Yano, T., Mita, S., Ohmori, H., Oshima, Y., Fujimoto, Y., Ueda, R., Takada, H., Goldman, W.E., Fukase, K., Silverman, N., et al. (2008). Autophagic control of listeria through intracellular innate immune recognition in Drosophila. Nat. Immunol. 9, 908–916. Voronin, D., Cook, D.A., Steven, A., and Taylor, M.J. (2012). Autophagy regulates Wolbachia populations across diverse symbiotic associations. Proc. Natl. Acad. Sci. USA 109, E1638–E1646. Wicker, C. (1983). Differential vitamin and choline requirements of symbiotic and aposymbiotic Sitophilus oryzae (Coleoptera: Curculionidae). Comp. Biochem. Physiol. A 76A, 177–182. Wicker, C., and Nardon, P. (1982). Development responses of symbiotic and aposymbiotic weevils Sitophilus oryzae L. (Coleoptera, Curculionidae) to a diet supplemented with aromatic amino acids. J. Insect Physiol. 28, 1021–1024. Grenier, A.M., Nardon, C., and Nardon, P. (1994). The role of symbionts in flight activity of Sitophilus weevils. Entomol. Exp. Appl. 70, 201–208. Heddi, A., Lefebvre, F., and Nardon, P. (1993). Effect of endocytobiotic bacteria on mitochondrial enzymatic activities in the weevil Sitophilus oryzae (Coleoptera: Curculionidae). Insect Biochem. Mol. Biol. 23, 403–411. Heddi, A. (2003). Endosymbiosis in the weevil of genus Sitophilus: genetic, physiological and molecular interactions among associated genomes. In Insect Symbiosis, K. Bourtzis and A. Miller, eds. (New York: CRC Press), pp. 67–82. Brey, P.T., Lee, W.J., Yamakawa, M., Koizumi, Y., Perrot, S., Franc¸ois, M., and Ashida, M. (1993). Role of the integument in insect immunity: epicuticular abrasion and induction of cecropin synthesis in cuticular epithelial cells. Proc. Natl. Acad. Sci. USA 90, 6275–6279. Marmaras, V.J., Bournazos, S.N., Katsoris, P.G., and Lambropoulou, M. (1993). Defense mechanisms in insects: certain integumental proteins and tyrosinase are responsible for nonself-recognition and immobilization of Escherichia coli in the cuticle of developing Ceratitis capitata. Arch. Insect Biochem. Physiol. 23, 169–180.
34. Andersen, S.O. (2010). Insect cuticular sclerotization: a review. Insect Biochem. Mol. Biol. 40, 166–178. 35. Andersen, S.O. (2007). Involvement of tyrosine residues, N-terminal amino acids, and beta-alanine in insect cuticular sclerotization. Insect Biochem. Mol. Biol. 37, 969–974. 36. Conord, C., Despres, L., Vallier, A., Balmand, S., Miquel, C., Zundel, S., Lemperiere, G., and Heddi, A. (2008). Long-term evolutionary stability of bacterial endosymbiosis in curculionoidea: additional evidence of symbiont replacement in the dryophthoridae family. Mol. Biol. Evol. 25, 859–868. 37. Heddi, A., Charles, H., Khatchadourian, C., Bonnot, G., and Nardon, P. (1998). Molecular characterization of the principal symbiotic bacteria of the weevil Sitophilus oryzae: a peculiar G + C content of an endocytobiotic DNA. J. Mol. Evol. 47, 52–61. 38. Lefe`vre, C., Charles, H., Vallier, A., Delobel, B., Farrell, B., and Heddi, A. (2004). Endosymbiont phylogenesis in the dryophthoridae weevils: evidence for bacterial replacement. Mol. Biol. Evol. 21, 965–973. 39. Vadivel, V., and Pugalenthi, M. (2010). Evaluation of nutritional value and protein quality of an under-utilized tribal food legume. Indian J. Tradit. Know. 9, 791–797. 40. Charles, H., Heddi, A., Guillaud, J., Nardon, C., and Nardon, P. (1997). A molecular aspect of symbiotic interactions between the weevil Sitophilus oryzae and its endosymbiotic bacteria: over-expression of a chaperonin. Biochem. Biophys. Res. Commun. 239, 769–774. 41. Login, F.H., Balmand, S., Vallier, A., Vincent-Mone´gat, C., Vigneron, A., Weiss-Gayet, M., Rochat, D., and Heddi, A. (2011). Antimicrobial peptides keep insect endosymbionts under control. Science 334, 362–365. 42. Vigneron, A., Charif, D., Vincent-Mone´gat, C., Vallier, A., Gavory, F., Wincker, P., and Heddi, A. (2012). Host gene response to endosymbiont and pathogen in the cereal weevil Sitophilus oryzae. BMC Microbiol. 12 (Suppl 1 ), S14.
http://dx.doi.org/10.1016/j.cub.2014.07.065
Report Insects Recycle Endosymbionts when the Benefit Is Over Aure´lien Vigneron,1,2 Florent Masson,1 Agne`s Vallier,1 Se´verine Balmand,1 Marjolaine Rey,1 Carole Vincent-Mone´gat,1 Emre Aksoy,1 Etienne Aubailly-Giraud,1 Anna Zaidman-Re´my,1 and Abdelaziz Heddi1,* 1Biologie Fonctionnelle Insectes et Interactions, UMR203 BF2I, INRA, INSA-Lyon, Universite´ de Lyon, 69621 Villeurbanne, France
Summary Symbiotic associations are widespread in nature and represent a driving force in evolution. They are known to impact fitness, and thereby shape the host phenotype [1–4]. Insects subsisting on nutritionally poor substrates have evolved mutualistic relationships with intracellular symbiotic bacteria (endosymbionts) that supply them with metabolic components lacking in their diet [5–10]. In many species, endosymbionts are hosted within specialized host cells, called the bacteriocytes, and transmitted vertically across host generations [11]. How hosts balance the costs and benefits of having endosymbionts, and whether and how they adjust symbiont load to their physiological needs, remains largely unexplored. By investigating the cereal weevil Sitophilus association with the Sodalis pierantonius endosymbiont [8, 12], we discover that endosymbiont populations intensively multiply in young adults, before being rapidly eliminated within few days. We show that young adults strongly depend on endosymbionts and that endosymbiont proliferation after metamorphosis matches a drastic host physiological need for the tyrosine (Tyr) and phenylalanine (Phe) amino acids to rapidly build their protective exoskeleton. Tyr and Phe are precursors of the dihydroxyphenylalanine (DOPA) molecule that is an essential component for the cuticle synthesis. Once the cuticle is achieved, DOPA reaches high amounts in insects, which triggers endosymbiont elimination. This elimination relies on apoptosis and autophagy activation, allowing digestion and recycling of the endosymbiont material. Thus, the weevil-endosymbiont association reveals an adaptive interplay between metabolic and cellular functions that minimizes the cost of symbiosis and speeds up the exoskeleton formation during a critical phase when emerging adults are especially vulnerable. Results and Discussion Cereal weevils house endosymbionts permanently in the female germ cells from which they are transmitted to the progeny. Early during embryogenesis, endosymbionts induce the differentiation of bacteriocyte cells that form the bacteriome [7], a specific symbiotic organ that secludes endosymbionts and protect them against the host immune response [13]. Young adults house numerous bacteriomes at the apex of
2Present
address: Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06520-8034, USA *Correspondence: [email protected]
midgut caeca and in female ovaries (Figure 1A). Remarkably, an early observation has reported that gut bacteriomes, but not ovarian bacteriomes, are lost in adults a few days following adult metamorphosis and the ultimate insect molting (day 0 of adulthood) [14]. Using real-time quantitative PCR (qPCR), we unraveled an unexpected complex dynamic of endosymbiont populations in young adults (Figure 1B). Endosymbiont populations first drastically increase between the ultimate insect molting and day 6 of adulthood, suggesting a critical symbiotic requirement during this phase of development. Although weevils live up to 6 months under lab conditions, the gut endosymbiont population rapidly decreases, and is completely eliminated by day 14, whereas the ovarian symbiont population remains stable over time (Figure 1B). This suggests that from day 6, the benefit of gut endosymbiosis no longer compensates for the burden of symbiont maintenance and that weevils have evolved a mechanism to adjust symbiont load to their needs. The modulation of symbiont density during insect development has previously been reported [15–18], but the situation is extreme in weevils, as within a 2-week period only, the gut symbiotic population grows intensively and then completely disappears. Similar symbiotic dynamics also occur in two additional cereal weevil species, Sitophilus zeamais and Sitophilus granarius (Figures S1A and S1B available online), suggesting a conserved mechanism of symbiont load modulation. Importantly, endosymbiont dynamic is more rapid in the wild weevil species S. oryzae and S. zeamais, which thrive on cereal fields, than in the granary-associated weevil S. granarius species, which is encountered in cereal storerooms of temperate countries only, suggesting that endosymbiont load is also modulated according to animal life conditions. Thus, weevils have evolved an ability to finetune tissue-specific regulation of their symbiont populations, allowing them to rapidly eliminate the gut population while preserving the ovarian population that is essential for reproduction and for symbiont transmission to progeny. To gain insight into the cellular basis of symbiont modulation in the gut, we examined the morphology of this tissue over the first 2 weeks of adulthood. Fluorescent in situ hybridization (FISH) experiments and scanning electron microscopy (SEM) performed on S. oryzae, S. zeamais, and S. granarius intestines highlighted a structural plasticity of bacteriomes during symbiont growth. Bacteriomes drastically increase in size until day 6 (Figures 1C, 1D, and S1C), while their number remains unchanged (Figures 1C and S1B and Table S1). From day 7, and in parallel with the decrease in symbiont population, bacteriomes progressively degenerate from the posterior to the anterior midgut and from the base to the apex of the caeca (Figures 1C, 1D, and S1D). This oriented endosymbiont elimination and bacteriome atrophy is rapid, consistent, and occurs in the three cereal weevil species examined (Figures 1B, 1C, S1A, and S1B), indicating that a well-conserved genetic mechanism or convergent processes lead to the repeated elimination of the bacteriomes in several species. Apoptosis detection with YO-PRO staining, a nucleic acid stain that successfully passes the plasma membrane of apoptotic cells only, and immunohistochemistry against the activated form of the effector Caspase-3, a marker of apoptotic cascade activation, showed apoptosis activation in intestinal cells that correlates
Please cite this article in press as: Vigneron et al., Insects Recycle Endosymbionts when the Benefit Is Over, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.07.065 Current Biology Vol 24 No 19 2
Figure 1. Dynamics of Endosymbiont Density in S. oryzae (A) A diagram showing the location of the bacteriomes (in red) in the mesenteric caeca and ovaries of an adult weevil. (B) qPCR quantification of endosymbiont DNA from fourth-instar larvae, late nymphs, and adult males (whole insects) and from female ovaries. Mean (6SE) of five independent biological replicates is represented. In males (black line), endosymbiont density significantly increases until day 6 prior to symbiont elimination (generalized linear model [GLM], p < 0.0001). In ovaries (gray line), the symbiont population significantly increases until day 6 (GLM, p < 0.0001) and then remains stable. (C) FISH staining of endosymbionts in adult mesenteric caeca from day 3 to day 13. S. pierantonius (green) progressively disappears from the posterior to the anterior caeca (orientation indicated by arrows). In aposymbiotic insects, caeca remain morphologically similar over time. Blue, nuclei (DAPI). Anterior is to the left. (D) SEM of adult mesenteric caeca. Bacteriomes are present at the apex of caeca in symbiotic insects (indicated by a line on the day 3 picture). From day 6, bacteriomes regress from the caeca basis to its apex (see arrows). No bacteriome is observed in caeca of aposymbiotic insects. D, days after the ultimate insect molting (UIM). See also Figure S1 and Table S1.
in time and space with symbiont clearance (Figures 2A and 2B). Apoptosis is induced in bacteriocyte cells (Figure 2B), and apoptotic bodies were seen in the gut lumen or engulfed by adjacent epithelial cells (Figure 2C). Importantly, apoptosis was not detected in the gut of insects experimentally depleted
of endosymbionts (so-called ‘‘aposymbiotic weevils’’ [19]) (Figures 2A and 2B). This indicates that activation of cell death is most likely related to symbiont elimination. Transmission electronic microscopy (TEM) also revealed induction of autophagy in epithelial cells (Figure 2D). Autophagy is a process
Please cite this article in press as: Vigneron et al., Insects Recycle Endosymbionts when the Benefit Is Over, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.07.065 Bacterial Load Adjustment in Insect Symbiosis 3
Figure 2. Cell Apoptosis and Autophagy Activation during the Elimination of gut Endosymbiont Population (A) YO-PRO staining of whole guts in symbiotic adults (right) revealed apoptotic cells (green, arrowheads) in posterior caeca at day 7 (top, arrowheads) and in anterior caeca at day 11 (bottom, arrowheads). No apoptosis was detected in aposymbiotic insects (left). Blue, nuclei (Hoechst). (B) Cleaved-caspase-3 immunostaining (green) of gut sections indicates apoptosis at the basis of the bacteriome in symbiotic adults at day 7 (top right, arrowheads) and in fewer cells mainly located at the apex of caeca at day 11 (bottom). No signal was detected in aposymbiotic insects (left). Blue, nuclei (DAPI). (C and D) TEM of symbiotic midguts at day 6 and 8. (C) Apoptotic bodies are seen in epithelial cells (top) and in the midgut lumen (bottom). (D) Autophagy is observed in bacteriocytes and epithelial cells. Top: autophagosome digesting S. pierantonius in a bacteriocyte (*; left). An autophagosome fusing with vesicles containing symbiont and forming autophagic vesicles (middle), prompting symbiont digestion (*; right), is shown. Bottom: autophagy leads to the accumulation of lamellar bodies that cover the majority of the cell cytosol, here observed at different scales. (E) TEM of midguts from aposymbiotic adults at day 8, which are endosymbiont free. These cells present a dense vesicular network at their lumen pole and contain lipid droplets and peroxysomes, but no lamellar body was seen. EC, epithelial cell; Vc, vesicle; Symb, symbiont; Nu, nucleus; mu, muscle; mv, microvilli; Lumen, gut lumen; RER, rough endoplasmic reticulum; Rb, ribosomes.
in which double-membrane autophagosomes sequester organelles or portions of cytosol and fuse with lysosomes, allowing digestion of the vacuole content [20]. In symbiotic weevils, all of these typical stages of autophagy were
observed surrounding endosymbionts, as well as prominent lamellar bodies that result from membrane accumulation and that are typical products of extensive autophagy [21–23] (Figure 2D). Such structures were not observed in aposymbiotic
Please cite this article in press as: Vigneron et al., Insects Recycle Endosymbionts when the Benefit Is Over, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.07.065 Current Biology Vol 24 No 19 4
Figure 3. Symbiosis Is Essential for Complete Cuticle Synthesis (A) Cuticle is darker in symbiotic (left) than in aposymbiotic (right) 26-day-old adults. (B) SEM of cuticle sections from symbiotic (left) and aposymbiotic (right) 15-day-old adults. Mean (6SE) of 11 measures of the epicuticle and exocuticle are presented. Exocuticle is thinner in aposymbiotic than in symbiotic individuals (double arrow). epi, epicuticle; exo, exocuticle; endo, endocuticle. (C) Progression of the cuticle color in adult weevils. The inverse of digital red color value was determined for both symbiotic (solid line, white square) and aposymbiotic insects (dotted line, black triangle). Symbiotic individuals are darker than aposymbiotic individuals (two-way ANOVA, symbiosis effect, p < 0.0001). Symbiotic and aposymbiotic individuals achieve their darker coloration by day 7 and day 17, respectively. Corresponding colors are indicated at the bottom. See also Figure S2.
insects (Figure 2E), supporting the idea that they result from symbiont digestion. Autophagy is a well-conserved mechanism used by cells to maintain homeostasis via the elimination and recycling of defective organelles. Lamellar bodies are thought to be involved in organelle recycling and serve as phospholipid storage [22, 23]. Autophagy is also a conserved defense mechanism against intracellular pathogens [24, 25], and it has recently been shown to control proliferation of the parasitic reproductive endosymbiont Wolbachia [24, 26]. Here, we report that autophagy controls the load of cooperative intracellular bacteria in weevils. Coordinated symbiont clearance by apoptosis and autophagy prevents the release of symbiont and cell material, thus avoiding tissue inflammation. By recycling endosymbiont biomaterials, these cellular processes may help insects recover the metabolic investments made during bacterial multiplication. S. pierantonius is known to provide the insect with many components that are poorly represented in wheat grains, such as pantothenic acid, biotin, riboflavin [27], and amino acids, particularly the aromatic amino acids phenylalanine (Phe) and tyrosine (Tyr) [8, 28]. Endosymbiont vitamin supply was shown to increase insect mitochondrial energetic metabolism and to improve the flight ability of adults [7, 29, 30]. Symbiosis also decreases larval developmental time and improves female fertility [31]. Here, the contrasting dynamic of gut endosymbiont population before and after day 6 (i.e., growth followed by elimination) strongly suggests that symbiosis is particularly important at the initial phase of adulthood but that it becomes advantageous to eradicate gut endosymbionts when the benefits of the association end. To experimentally address the benefits of the symbiosis at this early stage of adulthood, we reared weevils emerging from cereal grains on starch pellets that contain carbohydrates only. In contrast to symbiotic insects, aposymbiotic weevils rapidly died under
these conditions (Figure S2A). Strikingly, rearing of aposymbiotic weevils on cereal grains during the first 2 weeks of adulthood is sufficient to restore their ability to survive as long as their symbiotic counterparts if they are transferred to starch afterward (Figure S2A). This demonstrates that weevils require metabolic components other than carbohydrates after the ultimate insect molting and that symbiosis is essential to complement insect diets during this critical phase. As coleopterans, one remarkable adaptive trait of weevils is their hard, protective exoskeleton—the cuticle that preserves their body from desiccation, shocks, and pathogens [32, 33]. Symbiotic weevils exhibit a darker color than aposymbiotic individuals (Figure 3A), and, using SEM, we show a much thicker cuticle in symbiotic versus aposymbiotic weevils (Figure 3B). While symbiotic adults produce their cuticle rapidly after ultimate insect molting (Figures 3C and S2B), this process is slow and remains incomplete in aposymbiotic weevils (Figure 3C). In the three cereal weevil species, gut symbiont multiplication correlates with the progression of cuticle coloration, a process that ends after the beginning of symbiont elimination (Figure S2C). Taken together, these findings show that symbiosis is required for rapid cuticle synthesis, which may help insects to cope more effectively with environmental challenges. In line with this, we demonstrated that aposymbiotic insects die more rapidly than symbiotic individuals under high temperature and dry conditions (Figure S2D), which could be related to their defective cuticle. Cuticle biosynthesis and tanning are the result of sclerotization and melanization, two processes that require dihydroxyphenylalanine (DOPA) as a precursor [34]. This aromatic amino acid can be synthesized from Tyr, which itself can be derived from Phe (Figure 4A) [35]. Amino acid analysis with high-performance liquid chromatography (HPLC) indicated that levels of both free and protein-associated Tyr and Phe remain higher in symbiotic than in aposymbiotic insects, but decrease in the first 5 days after ultimate insect molting (Figures S3A and S3B), suggesting a redirection of these amino acids to other functions and their transformation into other products, including
Please cite this article in press as: Vigneron et al., Insects Recycle Endosymbionts when the Benefit Is Over, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.07.065 Bacterial Load Adjustment in Insect Symbiosis 5
Figure 4. DOPA Is Critical during the First Days of Adulthood (A) Scheme summarizing possible Phe and Tyr amino acid sources used by weevils, the DOPA synthesis pathway, and the origin of catalyzing enzymes. Phe is an essential amino acid that cannot be synthesized by insects, whereas insects are able to transform Phe into Tyr and Tyr into DOPA. (B) Kaplan-Meier representation of male aposymbiotic insect survival on wheat grain (black; control), starch pellets (red), starch pellets complemented with tryptophan (Trp, green), Phe (orange), Tyr (blue), all three aromatic amino acids (Trp+Tyr+Phe, purple), or DOPA (pink). Each curve represents the daily survival rate of 120 males until the death of the last individual. Data were analyzed using a log-rank test. Weevils reared on starch pellets complemented with all three amino acids exhibit higher rates of survival than weevils reared on plain starch pellets (c2 = 212, p < 0.0001) but lower survival than insects fed on wheat grains (c2 = 94, p < 0.0001). Phe complementation also partly restores weevil survival when compared with plain starch pellets (c2 = 190, p < 0.0001), in a range similar to the complementation with three amino acids (c2 = 0.6, p = 0.45). Tyr complementation partly restores weevil survival when compared with starch pellets (c2 = 83.3, p < 0.0001), but this restoration did not reach the level of Phe complementation (c2 = 24.4, p < 0.0001). Survival of insects reared on Trp-complemented pellets is not significantly different from survival of insects reared on starch pellets (c2 = 0.3, p = 0.589). DOPA complementation partly restores weevil survival, at a comparable level to Tyr complementation (c2 = 0.3, p = 0.582). (C) HPLC quantification of DOPA in adult gut (top) and remaining carcass (bottom). DOPA quantities are significantly higher in symbiotic (white squares) than in aposymbiotic (black triangles) insects, both in gut (two-way ANOVA, symbiosis effect, p < 0.0001) and carcass (two-way ANOVA, symbiosis effect, p < 0.0001). Symbiotic insects showed a significant increase of DOPA from day 5 to day 8 in both gut (two-way ANOVA, time effect, p < 0.0001) and carcass (two-way ANOVA, time effect, p < 0.0001). (D) qPCR quantification of symbiont DNA in adults emerging from grains (i.e., day 3 after ultimate insect molting) and reared on wheat flour pellets (white circle) or on wheat flour pellets complemented with 2% DOPA (black circle). Mean (6SE) of five independent biological replicates is represented. Elimination of endosymbionts in insects fed on DOPA-complemented pellets is accelerated (GLM, p < 0.0001). See also Figure S3.
DOPA. The level of protein-associated Tyr and Phe increases from day 5 to 11, while cuticle synthesis is being completed (Figures S3A and S3B), indicating that insects redirect free amino acids to general protein synthesis and replenish the molecules hydrolyzed during the first days of adulthood. S. pierantonius has recently established intracellular symbiosis with Sitophilus weevils after a replacement of Nardonella, the ancestral endosymbiont of the Dryophthoridae family, to which cereal weevils belong [12, 36–38]. Relaxed selection on S. pierantonius genome at this initial phase of symbiosis integration has resulted in a large number of gene pseudogenization and rearrangements, associated with an expansion of transposable elements [8]. However, although the S. pierantonius genome has lost several metabolic functions, Phe and Tyr pathways have been conserved [8]. This suggests that endosymbionts are essential for providing their hosts with these amino acids to boost the cuticle synthesis after molting. To address this hypothesis, we monitored aposymbiotic insect survival on starch pellets complemented with different amino acids (Figure 4B). While aposymbiotic weevils cannot survive on starch or on starch complemented with tryptophan (another aromatic amino acid), their survival is partially restored when starch is complemented with Tyr and even more so with Phe. This result demonstrates that Tyr and Phe
are two important amino acids supplemented by endosymbionts. They are required for the host to survive on restricted diets, and their supplementation partly compensates the absence of endosymbionts. However, rearing weevils on highly concentrated Tyr and Phe does not prematurely trigger endosymbiont clearance (Figure S3C). Concordant with the idea that Phe and Tyr are required for DOPA production at this critical stage of cuticle biosynthesis, HPLC quantification of DOPA revealed a strong increase in the level of this amino acid in symbiotic weevils from day 5, whereas its level remains low in aposymbiotic insects (Figure 4C). This means that symbiosis allows for high levels of DOPA production. Moreover, using a complementation test, we showed that feeding aposymbiotic weevils directly on DOPA-complemented starch is sufficient to restore their survival to the same extent as feeding them on Tyr-complemented starch diet (Figure 4B). This finding strongly suggests that Tyr is mainly required for DOPA production at this stage of development, while Phe is also involved in other processes impacting the insect survival. Remarkably, symbiotic weevils transferred to DOPA-complemented wheat flour pellets after grain emergence complete symbiont elimination 2 days earlier than weevils maintained on control wheat flour pellets (Figure 4D). Thus, increased access of weevils to DOPA is
Please cite this article in press as: Vigneron et al., Insects Recycle Endosymbionts when the Benefit Is Over, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.07.065 Current Biology Vol 24 No 19 6
sufficient to accelerate gut endosymbiont elimination. DOPA level directly impacts endosymbiont density, demonstrating the existence of a molecular mechanism that allows weevils to adjust their symbiotic load to their specific physiological needs in DOPA at the critical phase of cuticle synthesis. Interestingly, the only Sitophilus species known to be naturally free of endosymbionts so far is Sitophilus linearis [38], which feeds on tamarind seeds that contain a high level of DOPA [39]. Therefore, in addition to the previously described S. pierantonius effects on weevil physiology [7, 10, 27, 29– 31, 40], this work shows that S. pierantonius drastically interferes with DOPA production and cuticle synthesis. Further, weevils appear to have evolved a mechanism to tightly synchronize endosymbiont elimination and recycling with the completion of cuticle deposition. In this evolutionary context, it would be of interest to figure out whether bacterial Tyr and Phe pathways are adaptive for other symbiotic coleopterans, by analyzing Tyr and Phe pathway conservation in the longlasting endosymbiont Nardonella. Future investigations will also tell us about the precise function of DOPA in the interplay between metabolism and host cellular responses to endosymbionts and whether the growth and subsequent recycling of endosymbionts is relevant for other processes occurring during this stage of development, including reproduction maturity. Conclusions Examining the association between cereal weevils, Sitophilus spp. and their intracellular symbiotic bacterium, S. pierantonius, we have described an extreme modulation of endosymbiont load in these coleopteran insects, ending with a complete elimination of symbionts from the host ‘‘somatic’’ tissues. We report that weevil endosymbionts intensively multiply in young adults to support the massive need for Tyr and Phe that are the precursors of the DOPA molecule required for cuticle tanning and hardening. Comparison between weevil species living in different ecological niches showed that endosymbiont dynamics and cuticle completion are more rapid in field- than in the granary-living species, suggesting that endosymbiont load is also modulated according to animal environmental conditions. Once the cuticle is achieved, endosymbionts are rapidly recycled through apoptosis and autophagy in a tissue-dependent manner that preserves female germline-associated endosymbionts and thus their transmission to next generations. We show that complementation of insect diets with DOPA decreased symbiont abundance and hastened their clearance. In addition to bacteriocyte immune regulations [13, 41, 42] and symbiont control by antimicrobial peptides recently described in weevils [41], we report here that cellular apoptosis and autophagy are involved in bacterial load regulation according to insect development. These data present a striking example of how host-endosymbiont coevolution has shaped mechanisms that adjust symbiont load to the host physiological needs, while allowing a return of investment through natural symbiont recycling. While mutualism significantly impacts organism life history traits, enhancing adaptive features such as the cuticle thickness, cooperative associations remain under the control of a fine-tuned integration of costs and benefits according to host developmental requirements. The synchronization of a ‘‘somatic’’ breakdown of symbiosis with the achievement of a specific developmental trait minimizes insect investment in symbiosis while preserving transmission to the progeny.
Supplemental Information Supplemental Information includes Supplemental Experimental Procedures, three figures, one table, and one movie and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2014.07.065. Author Contributions A. Vigneron designed and performed experiments, analyzed data, and wrote the manuscript; F.M. conducted biological experiments and analyzed data; A. Vallier carried out dissections and molecular biology experiments; S.B. carried out histological experiments; M.R. performed HPLC experiments; C.V.-M. participated in the design and the analysis of experiments; E.A. measured the cuticle color; E.A.-G. performed FISH experiments; A.Z.-R. performed biological experiments, analyzed the data, and wrote the manuscript; and A.H. conceived the study, coordinated the work, and drafted and wrote the manuscript. Acknowledgments This work was supported by INRA, INSA-Lyon, and the French ANR-10 BSV7-170101-03 (ImmunSymbArt) and ANR-13-BSV7-0016-01 (IMetSym). We thank G. Febvay for advices on amino acids, S. Schaack, J. Regan, and B. Weiss for their critical comments on the manuscript, and A. Gilles, J. Schinko, and D. Compagnon for weevil image capture. The authors would also like to thank the anonymous reviewers for their constructive criticisms. Electron microscopy was carried out in Centre Technologique des Microstructures (CTm, Universite´ Lyon I), and qPCR in the DTAMB (De´veloppement de Techniques et Analyse Mole´culaire de la Biodiversite´). Received: June 19, 2014 Revised: July 23, 2014 Accepted: July 24, 2014 Published: September 18, 2014 References 1. Margulis, L. (1993). Origins of species: acquired genomes and individuality. Biosystems 31, 121–125. 2. Moran, N.A. (2006). Symbiosis. Curr. Biol. 16, R866–R871. 3. Tsuchida, T., Koga, R., Horikawa, M., Tsunoda, T., Maoka, T., Matsumoto, S., Simon, J.C., and Fukatsu, T. (2010). Symbiotic bacterium modifies aphid body color. Science 330, 1102–1104. 4. Zilber-Rosenberg, I., and Rosenberg, E. (2008). Role of microorganisms in the evolution of animals and plants: the hologenome theory of evolution. FEMS Microbiol. Rev. 32, 723–735. 5. Akman Gu¨ndu¨z, E., and Douglas, A.E. (2009). Symbiotic bacteria enable insect to use a nutritionally inadequate diet. Proc. Biol. Sci. 276, 987–991. 6. Douglas, A.E. (1998). Nutritional interactions in insect-microbial symbioses: aphids and their symbiotic bacteria Buchnera. Annu. Rev. Entomol. 43, 17–37. 7. Heddi, A., Grenier, A.M., Khatchadourian, C., Charles, H., and Nardon, P. (1999). Four intracellular genomes direct weevil biology: nuclear, mitochondrial, principal endosymbiont, and Wolbachia. Proc. Natl. Acad. Sci. USA 96, 6814–6819. 8. Oakeson, K.F., Gil, R., Clayton, A.L., Dunn, D.M., von Niederhausern, A.C., Hamil, C., Aoyagi, A., Duval, B., Baca, A., Silva, F.J., et al. (2014). Genome degeneration and adaptation in a nascent stage of symbiosis. Genome Biol. Evol. 6, 76–93. 9. Snyder, A.K., Deberry, J.W., Runyen-Janecky, L., and Rio, R.V. (2010). Nutrient provisioning facilitates homeostasis between tsetse fly (Diptera: Glossinidae) symbionts. Proc. Biol. Sci. 277, 2389–2397. 10. Wicker, C., Guillaud, J., and Bonnot, G. (1985). Comparative composition of free, peptide and protein amino acids in symbiotic and aposymbiotic Sitophilus oryzae (Coleoptera, Curculionidae). Insect Biochem. 15, 537–541. 11. Koga, R., Meng, X.Y., Tsuchida, T., and Fukatsu, T. (2012). Cellular mechanism for selective vertical transmission of an obligate insect symbiont at the bacteriocyte-embryo interface. Proc. Natl. Acad. Sci. USA 109, E1230–E1237. 12. Charles, H., Heddi, A., and Rahbe, Y. (2001). A putative insect intracellular endosymbiont stem clade, within the Enterobacteriaceae, infered
Please cite this article in press as: Vigneron et al., Insects Recycle Endosymbionts when the Benefit Is Over, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.07.065 Bacterial Load Adjustment in Insect Symbiosis 7
13.
14.
15.
16.
17.
18.
19.
20. 21. 22.
23.
24.
25.
26.
27.
28.
29. 30.
31.
32.
33.
from phylogenetic analysis based on a heterogeneous model of DNA evolution. C. R. Acad. Sci. III 324, 489–494. Anselme, C., Pe´rez-Brocal, V., Vallier, A., Vincent-Monegat, C., Charif, D., Latorre, A., Moya, A., and Heddi, A. (2008). Identification of the weevil immune genes and their expression in the bacteriome tissue. BMC Biol. 6, 43. Mansour, K. (1930). Preliminary studies on the bacterial cell mass (accessory cell-mass) of Calandra oryzae: the rice weevil. Q. J. Microsc. Sci. 73, 421–436. Kim, J.K., Han, S.H., Kim, C.H., Jo, Y.H., Futahashi, R., Kikuchi, Y., Fukatsu, T., and Lee, B.L. (2014). Molting-associated suppression of symbiont population and up-regulation of antimicrobial activity in the midgut symbiotic organ of the Riptortus-Burkholderia symbiosis. Dev. Comp. Immunol. 43, 10–14. Laughton, A.M., Fan, M.H., and Gerardo, N.M. (2014). The combined effects of bacterial symbionts and aging on life history traits in the pea aphid, Acyrthosiphon pisum. Appl. Environ. Microbiol. 80, 470–477. Nishikori, K., Morioka, K., Kubo, T., and Morioka, M. (2009). Age- and morph-dependent activation of the lysosomal system and Buchnera degradation in aphid endosymbiosis. J. Insect Physiol. 55, 351–357. Stoll, S., Feldhaar, H., Fraunholz, M.J., and Gross, R. (2010). Bacteriocyte dynamics during development of a holometabolous insect, the carpenter ant Camponotus floridanus. BMC Microbiol. 10, 308. Nardon, P. (1973). Obtention d’une souche asymbiotique chez le charanc¸on Sitophilus sasakii Tak: diffe´rentes me´thodes d’obtention et comparaison avec la souche symbiotique d’origine. C. R. Acad. Sci. Paris 277, 981–984. He, C., and Klionsky, D.J. (2009). Regulation mechanisms and signaling pathways of autophagy. Annu. Rev. Genet. 43, 67–93. Clarke, M., Bennett, M., and Littlewood, T. (2007). Cell death in the cardiovascular system. Heart 93, 659–664. Schmitz, G., and Mu¨ller, G. (1991). Structure and function of lamellar bodies, lipid-protein complexes involved in storage and secretion of cellular lipids. J. Lipid Res. 32, 1539–1570. Weaver, T.E., Na, C.L., and Stahlman, M. (2002). Biogenesis of lamellar bodies, lysosome-related organelles involved in storage and secretion of pulmonary surfactant. Semin. Cell Dev. Biol. 13, 263–270. Le Clec’h, W., Braquart-Varnier, C., Raimond, M., Ferdy, J.B., Bouchon, D., and Sicard, M. (2012). High virulence of Wolbachia after host switching: when autophagy hurts. PLoS Pathog. 8, e1002844. Yano, T., Mita, S., Ohmori, H., Oshima, Y., Fujimoto, Y., Ueda, R., Takada, H., Goldman, W.E., Fukase, K., Silverman, N., et al. (2008). Autophagic control of listeria through intracellular innate immune recognition in Drosophila. Nat. Immunol. 9, 908–916. Voronin, D., Cook, D.A., Steven, A., and Taylor, M.J. (2012). Autophagy regulates Wolbachia populations across diverse symbiotic associations. Proc. Natl. Acad. Sci. USA 109, E1638–E1646. Wicker, C. (1983). Differential vitamin and choline requirements of symbiotic and aposymbiotic Sitophilus oryzae (Coleoptera: Curculionidae). Comp. Biochem. Physiol. A 76A, 177–182. Wicker, C., and Nardon, P. (1982). Development responses of symbiotic and aposymbiotic weevils Sitophilus oryzae L. (Coleoptera, Curculionidae) to a diet supplemented with aromatic amino acids. J. Insect Physiol. 28, 1021–1024. Grenier, A.M., Nardon, C., and Nardon, P. (1994). The role of symbionts in flight activity of Sitophilus weevils. Entomol. Exp. Appl. 70, 201–208. Heddi, A., Lefebvre, F., and Nardon, P. (1993). Effect of endocytobiotic bacteria on mitochondrial enzymatic activities in the weevil Sitophilus oryzae (Coleoptera: Curculionidae). Insect Biochem. Mol. Biol. 23, 403–411. Heddi, A. (2003). Endosymbiosis in the weevil of genus Sitophilus: genetic, physiological and molecular interactions among associated genomes. In Insect Symbiosis, K. Bourtzis and A. Miller, eds. (New York: CRC Press), pp. 67–82. Brey, P.T., Lee, W.J., Yamakawa, M., Koizumi, Y., Perrot, S., Franc¸ois, M., and Ashida, M. (1993). Role of the integument in insect immunity: epicuticular abrasion and induction of cecropin synthesis in cuticular epithelial cells. Proc. Natl. Acad. Sci. USA 90, 6275–6279. Marmaras, V.J., Bournazos, S.N., Katsoris, P.G., and Lambropoulou, M. (1993). Defense mechanisms in insects: certain integumental proteins and tyrosinase are responsible for nonself-recognition and immobilization of Escherichia coli in the cuticle of developing Ceratitis capitata. Arch. Insect Biochem. Physiol. 23, 169–180.
34. Andersen, S.O. (2010). Insect cuticular sclerotization: a review. Insect Biochem. Mol. Biol. 40, 166–178. 35. Andersen, S.O. (2007). Involvement of tyrosine residues, N-terminal amino acids, and beta-alanine in insect cuticular sclerotization. Insect Biochem. Mol. Biol. 37, 969–974. 36. Conord, C., Despres, L., Vallier, A., Balmand, S., Miquel, C., Zundel, S., Lemperiere, G., and Heddi, A. (2008). Long-term evolutionary stability of bacterial endosymbiosis in curculionoidea: additional evidence of symbiont replacement in the dryophthoridae family. Mol. Biol. Evol. 25, 859–868. 37. Heddi, A., Charles, H., Khatchadourian, C., Bonnot, G., and Nardon, P. (1998). Molecular characterization of the principal symbiotic bacteria of the weevil Sitophilus oryzae: a peculiar G + C content of an endocytobiotic DNA. J. Mol. Evol. 47, 52–61. 38. Lefe`vre, C., Charles, H., Vallier, A., Delobel, B., Farrell, B., and Heddi, A. (2004). Endosymbiont phylogenesis in the dryophthoridae weevils: evidence for bacterial replacement. Mol. Biol. Evol. 21, 965–973. 39. Vadivel, V., and Pugalenthi, M. (2010). Evaluation of nutritional value and protein quality of an under-utilized tribal food legume. Indian J. Tradit. Know. 9, 791–797. 40. Charles, H., Heddi, A., Guillaud, J., Nardon, C., and Nardon, P. (1997). A molecular aspect of symbiotic interactions between the weevil Sitophilus oryzae and its endosymbiotic bacteria: over-expression of a chaperonin. Biochem. Biophys. Res. Commun. 239, 769–774. 41. Login, F.H., Balmand, S., Vallier, A., Vincent-Mone´gat, C., Vigneron, A., Weiss-Gayet, M., Rochat, D., and Heddi, A. (2011). Antimicrobial peptides keep insect endosymbionts under control. Science 334, 362–365. 42. Vigneron, A., Charif, D., Vincent-Mone´gat, C., Vallier, A., Gavory, F., Wincker, P., and Heddi, A. (2012). Host gene response to endosymbiont and pathogen in the cereal weevil Sitophilus oryzae. BMC Microbiol. 12 (Suppl 1 ), S14.
