Fidelity in co-diversified symbiosis

Domain: neuroscience
Published: 2026-02-12
Editorial outcome: minor_revision

Abstract
Obligate co-dependence can arise in symbiosis, yielding heritable partnerships. These interactions are considered to be highly specific, but partner fidelity is difficult to quantify owing to the experimental constraints of symbiont exchange between host species. Here, we overcome this challenge by leveraging the unique transmission dynamics of Stammera capleta, the obligate digestive symbiont of tortoise beetles (Chrysomelidae: Cassidinae). Despite its extracellular localization, S. capleta possesses a drastically reduced genome (~0.25 Mb) and is vertically transmitted through egg-associated spheres. Manipulating these spheres allowed us to experimentally exchange S. capleta between beetle species to determine their impact on host development. We show that non-native S. capleta can successfully colonize the symbiotic organs of a novel host, but that the interaction outcome correlates with genetic relatedness to the native symbiont. Genetically distant species trigger a more pronounced transcriptional response and can only partially rescue host development. While more closely related symbionts proliferate similarly to the native one and induce a comparable host response, they fail to propagate to the next generation, underscoring how transmission fidelity, host-symbiont compatibility, and local adaptation can further specificity within a Paleocene-aged partnership.

Introduction
The metabolic intimacy of symbiosis demands the work of specialists, each evolving distinct yet complementary roles to sustain the partnership1,2. Obligate co-dependence can arise within these interactions3,4, necessitating adaptations that ensure the recognition, regulation, and persistence of mutualisms5–7. The resulting partnerships often feature heritable endosymbionts that are intertwined in the evolutionary history of their hosts8–10. As symbiont and host co-diversify, they exhibit parallel lineage splitting over evolutionary timescales11.

Among animals, obligately beneficial symbionts are typically acquired early in development2,4,12–14. For insects that host endosymbionts within specialized cells (i.e., bacteriocytes) and organs, microbial colonization can take place during embryogenesis or oogenesis13–15. For example, aphids acquire their nutritional endosymbiont, Buchnera aphidicola, during embryo development through calibrated cycles of exocytosis and endocytosis13. Endosymbiont cells released from maternal symbiotic organs colonize cells fated to become bacteriocytes in the embryo13. Ants similarly integrate their endosymbiont, Blochmannia floridanus, during embryogenesis through a shared regulatory network that is coopted by the microbe to facilitate infection14. Such models, and others6,15–18, continue to offer striking insights into the cellular mechanisms guiding symbiont colonization and proliferation12. But our inability to cultivate most obligate symbionts in pure culture18, compounded by the brief developmental window that their hosts spend aposymbiotically6,13,14, constrains the experimental exchange of symbionts and the ability to test fidelity within co-diversified partnerships.

In contrast to the experimental challenges posed by heritable endosymbionts, partnerships with beneficial microbes acquired from the environment have provided crucial insights into the mechanisms that govern specificity in animal-microbe symbioses19–25. For example, the Hawaiian bobtail squid (Euprymna scolopes) can differentiate its bioluminescent symbiont Vibrio fischeri from thousands of marine microbial species through an intricately honed process that relies on mechanical, chemical, immunological, and developmental exclusion26–29. Similarly, the bean bug (Riptortus pedestris) acquires its crypt-associated gut symbiont Caballeronia insecticola every generation from the soil19,30–32. Despite a horizontal acquisition mode30, several factors contribute to a highly selective recognition process, including a microbial-sorting structure within the gut20, the competitive exclusion of non-symbiotic strains33, and, finally, the morphological differentiation and closure of the symbiotic organ following passage by C. insecticola31. While horizontally transmitted symbionts lack evolutionary histories that mirror their hosts19,22, the partnerships between the squid-V. fischeri and bean bug-C. insecticola display striking adaptations for partner recognition at the genetic and molecular levels20,21,31,33–35. Whether these features extend to heritable endosymbionts, however, remains largely unexplored across study systems.

Here, we investigated the specificity of a co-diversified symbiosis by examining the partnership between tortoise leaf beetles (Chrysomelidae: Cassidinae) and their digestive bacterial symbiont, Candidatus Stammera capleta (Fig. 1A–H)36–38. The tortoise beetle-S. capleta partnership offers a tractable experimental framework to test fidelity in an obligate symbiosis given the microbe’s unique transmission route and colonization dynamics36,39,40.

Tortoise beetles maintain S. capleta in foregut symbiotic organs to facilitate folivory and in ovary-associated glands to ensure transmission (Fig. 1A–D)36–38,40–42. Despite possessing a drastically reduced genome (~0.2–0.3 Mb), S. capleta encodes and exports several plant cell wall-degrading enzymes, including polygalacturonase, a pectinase that underpins convergent nutritional symbioses across leaf beetles36–38,43–45. Tortoise beetles vertically transmit S. capleta by depositing an individual caplet at the anterior pole of each egg36,39 (Fig. 1E). This transmission mode is reflected in the strict co-cladogenesis between symbiont and host37,38,41. The caplet is populated with symbiont-bearing spheres where the microbe is embedded extracellularly (Fig. 1F–H)36,39. Embryos acquire S. capleta late in development by piercing the caplet membrane and consuming the spheres39, which initiates infection40.

Removing the egg caplet disrupts symbiont transmission, yielding S. capleta-free (aposymbiotic) larvae that exhibit a diminished digestive phenotype and drastically reduced survivorship36,39,40. However, reapplying the spheres to aposymbiotic eggs experimentally reconstitutes the symbiosis39,40, highlighting a potential experimental mechanism to exchange the microbe across different species of tortoise beetles by leveraging these structures.

In this study, we capitalized on the conserved symbiont transmission route shared by tortoise beetles to demonstrate that: (i) heritable endosymbionts can be exchanged between host species, (ii) non-native symbionts can colonize and differentially restore survivorship in a novel host, with outcomes ranging from full fitness recovery to reduced survival depending on genetic divergence among symbionts, (iii) but that, ultimately, a high level of fidelity governs this partnership during host development and the symbiont’s propagation to the next generation. Our findings highlight the key, complementary roles that partner recognition, transmission fidelity, and local adaptation all play to ensure specificity and stabilize an ancient, co-diversified symbiosis.

Results
Reciprocal symbiont exchange in tortoise beetles
Symbiont acquisition in tortoise beetles is governed by a strict developmental window39,40. Although the foregut symbiotic organs form 3 days before larval emergence from the egg, we previously identified the final 24 h of embryogenesis as the critical period for S. capleta colonization39,40. Reapplying symbiont-bearing spheres during this period can restore S. capleta infection in aposymbiotic embryos39,40. Given this, we explored whether the same approach could facilitate the exchange of S. capleta between different beetle species.

Using the tortoise beetle Chelymorpha alternans (Fig. 1A) as a model, we validated this method through four experimental treatments: (a) untreated control, (b) eggs with caplets removed (aposymbiotic), (c) eggs with caplets removed but re-supplied with their original symbiont-bearing spheres at the anterior pole (re-infected), and (d) eggs with caplets removed but instead supplied with spheres collected from Chelymorpha gressoria, a closely related species (cross-infected) (Fig. 2A).

Consistent with previous studies36,39,40, caplet removal disrupted S. capleta transmission, yielding aposymbiotic embryos, in contrast to the untreated and re-infected controls (Figs. 2A and S1i). Notably, cross-infected embryos (i.e., supplied with spheres from C. gressoria) were successfully colonized by non-native S. capleta (Figs. 2A and S1i). The symbiont occupied the foregut symbiotic organs of its new host, in line with the colonization dynamics observed for the native S. capleta of C. alternans (Figs. 2A and S1i).

To determine whether symbiont exchange is reciprocal across tortoise beetle species, we repeated these assays using C. gressoria as a host (Fig. 2B). The same four experimental treatments were applied: (a) untreated control, (b) aposymbiotic, (c) re-infected, and (d) cross-infected (i.e., aposymbiotic C. gressoria supplied with spheres collected from C. alternans). The colonization dynamics mirrored the findings observed in C. alternans (Figs. 2B and S1ii). Aposymbiotic C. gressoria lacked their native S. capleta, in contrast to the untreated and re-infected controls. On the other hand, cross-infected embryos were successfully colonized by the symbiont of C. alternans (Figs. 2B and S1ii), indicating that the cross-infection protocol was reciprocal.

To confirm whether these colonization dynamics persist beyond the embryo stage, we evaluated the localization of each symbiont in 5-day-old larvae of both C. alternans and C. gressoria (Fig. 2A, B). The same patterns were observed across both beetle species: aposymbiotic insects remained uncolonized, while untreated and re-infected controls maintained the native S. capleta of each species (Figs. 2A, B and S1iii, iv). Finally, non-native symbionts continued to occupy the foregut symbiotic organs of their new hosts (Figs. 2A, B and S1iii, iv).

These findings demonstrate that obligate, heritable symbionts can be experimentally exchanged between tortoise beetle species, and that reciprocal cross-infection persists beyond larval eclosion. With this established, we next asked: how do non-native symbionts influence the development of their novel hosts?

Non-native symbionts spur a gradient of mutualistic outcomes
Numerous insect traits are endowed through symbiosis46, with obligate dependence shaping interactions across diverse clades such as bugs47–49, ants14, termites50,51, bees52,53, and beetles5,6,54. Insects specializing on nutritionally imbalanced diets continue to offer important insights into these obligate interactions and the extent to which host development is stunted in their absence55,56. For example, aposymbiotic aphids deprived of Buchnera—and the essential amino acids it supplements—suffer significantly reduced survivorship and complete loss of fecundity57,58. Similarly, bed bugs, unable to balance the nutritional deficiencies of their blood meal, are developmentally constrained in the absence of their B vitamin-supplementing endosymbiont59. Such findings are consistent with consequences of aposymbiosis in tortoise beetles, where the experimental loss of S. capleta impairs the insects’ ability to process a leafy diet, resulting in low survivorship and failure to reach adulthood36,39. Given these pronounced effects, we asked whether non-native S. capleta could rescue aposymbiotic mortality. And if so, how do these beetles develop compared to larvae colonized by the native symbiont?

We pursued the above assays using six species of tortoise beetles: the aforementioned C. alternans and C. gressoria, and in addition, Chelymorpha bullata, Acromis sparsa, Aspidimorpha quinquefasciata, and Cassida rubiginosa (Fig. 3A, and Supplementary Data 1). Each of these beetles species harbors a single, genetically distinct species of S. capleta within its foregut symbiotic organs36–38 (Figs. 3B–D and S2–3), which is transmitted extracellularly via egg-associated spheres60.

Using C. alternans as a host, we assessed the colonization efficiency of different S. capleta species in a novel host and quantified their effects on larval survivorship. We structured our bioassays around eight experimental treatments: (a) untreated control, (b) aposymbiotic eggs, (c) eggs whose caplets were removed but re-infected with the native symbiont, or (d–h) eggs whose caplets were removed but cross-infected with S. capleta-bearing spheres collected from each of C. bullata, C. gressoria, A. sparsa, A. quinquefasciata, and C. rubiginosa.

Five-day-old larvae were dissected to assess their symbiotic status. Experimental manipulation significantly influenced S. capleta colonization (Fig. 4A; Fisher’s Exact Test, p=0.0005; Supplementary Data 2A). As demonstrated earlier (Fig. 2), removal of the egg caplet disrupted symbiont transmission relative to the untreated and re-infected controls (Fig. 4A; pairwise comparisons of proportions, p<0.001; Supplementary Data 2A). In contrast, cross-infected larvae were consistently symbiotic, irrespective of the donor host (Fig. 4A; pairwise comparisons of proportions, C. bullata symbiont: p=1, C. gressoria symbiont: p=1, A. sparsa symbiont: p=1, A. quinquefasciata symbiont: p=0.24, C. rubiginosa symbiont: p=0.67; Supplementary Data 2A).

Given the consistent cross-infection of S. capleta stemming from six donor beetle species, we further quantified symbiont titre to determine whether infection success was reflected in S. capeta abundance. Quantitative analysis revealed treatment-dependent variation in symbiont titer (Fig. 4B; GLM (quasi-Poisson), df=7, p<0.001, χ2=119.32; Supplementary Data 2B). S. capleta abundances were statistically comparable between larvae harboring their native symbiont and those cross-infected with C. bullata, C. gressoria, and A. sparsa symbionts (Fig. 4B; pairwise comparison, reinfection: p=1, C. bullata symbiont: p=1, C. gressoria symbiont: p=1, A. sparsa symbiont: p=0.084, Supplementary Data 2B). Conversely, larvae cross-infected with A. quinquefasciata and C. rubiginosa symbionts contained lower symbiont titers than the untreated larvae (Fig. 4B; pairwise comparison, A. quinquefasciata: p<0.001, C. rubiginosa: p<0.001, Supplementary Data 2B). Quantitative polymerase chain reaction (qPCR) amplification in aposymbiotic individuals was indistinguishable from background (NTC) levels, indicating that the reported copy numbers are likely overestimations and that aposymbiotic larvae are devoid of symbionts, as corroborated by the diagnostic PCR results.

We next quantified how these non-native symbionts shaped the development of C. alternans larvae and found that their effects on host survival varied, yielding a gradient of mutualistic outcomes (Fig. 4C; Mixed effects Cox regression model, df=7, p<0.001, χ2=229.044; Supplementary Data 2C). While all cross-infected treatments consistently outpaced aposymbiotic insects (Log-rank tests, p<0.001, Supplementary Data 2C), only two non-native S. capleta species completely rescued larval survivorship to levels mirroring to the untreated and re-infected controls (Fig. 4C). These symbionts stem from C. bullata (Log-rank tests, p=0.7 and 0.99; Supplementary Data 2C) and C. gressoria (Log-rank tests, p=0.38 and 0.76; Supplementary Data 2C), two beetle species that are congeneric (Chelymorpha) to the recipient host, C. alternans (Fig. 3B). In contrast, the three S. capleta species associated with A. sparsa, A. quinquefasciata, and C. rubiginosa could only partially rescue the survivorship of their novel host (Log-rank tests, A. sparsa, p=0.00029 and 0.0077; A. quinquefasciata, p=0.002 and 0.037, and C. rubiginosa, p=00013 and 0.00052, respectively; Supplementary Data 2C), underscoring the diverse developmental consequences of colonization by a non-native symbiont (Fig. 4C).

Collectively, this indicates that the observed mortality is only partly explained by a reduction in symbiont abundance, as seen in the cross-infection with the A. quinquefasciata symbiont (Fig. 4B). However, this pattern doesn’t hold for all high-mortality cross-infections, such as those involving the A. sparsa symbiont, where symbiont abundance is comparable to those of the native S. capleta (Fig. 4B). This variation suggests that host survival following our cross-infections may be influenced by factors beyond symbiont abundance, including reduced symbiont activity and/or the degree of compatibility between host and microbe.

To explore this possibility, we examined whether the mutualistic potential of S. capleta in a novel host correlates with its evolutionary relatedness to the native symbiont. S. capleta that are closely related to the original symbiont of C. alternans were more likely to rescue larval survivorship than more distantly related species (Fig. 4D, E, and Supplementary Data 2D). Using genome-wide pairwise comparisons of average nucleotide identity (ANI) and collinearity as measures of genetic distance, we observed a strong positive correlation between symbiont relatedness and host survivorship (Fig. 4D, E; Spearman’s rank correlations: ANI, r=0.89, p=0.0071; collinearity score, r=0.92, p=0.0034; Supplementary Data 2D).

How do different symbiont genotypes influence the survivorship of a novel host? Despite strong metabolic conservation across the S. capleta pangenome in categories related to informational processing (e.g., transcription, translation, and replication), different species encode and supplement two iterations of host-beneficial factors to tortoise beetles36–38. All genomes of S. capleta species sequenced to date encode polygalacturonase (PG), a pectinase that breaks down homogalacturonan, the most abundant pectic substrate36–38. However, different symbiont species also produce rhamnogalacturonan lyase (RL), a secondary pectinase that degrades the heteropolymer rhamnogalacturonan I, or α-glucuronidase (AG), a xylanase involved in hemicellulose digestion36–38.

The six S. capleta species used in our study encompass these two configurations of host-beneficial factors: PG and RL, or PG and AG (Supplementary Data 1). The symbiont of C. alternans, along with those of C. gressoria, C. bullata, and A. sparsa, all encode PG and AG36–38. In contrast, the symbionts of C. rubiginosa and A. quinquefasciata provide their beetle hosts with PG and RL36–38. Since the latter two species only partially restored survivorship in a novel beetle host that typically depends on PG and AG from its native symbiont (Fig. 4C, and Supplementary Data 2C), we hypothesized that differences in host-beneficial factors might explain the observed variation in mutualistic outcomes. However, this is unlikely to be the sole explanation, since the cross-infected symbiont from A. sparsa encodes the same set of plant cell wall-degrading enzymes (PG and AG) as the native S. capleta in C. alternans yet fail to restore larval survivorship relative to the untreated and re-infected controls (Fig. 4C, and Supplementary Data 2C). It is conceivable that differences in the expression or regulation of these host-beneficial factors could contribute to the observed variation. Alternatively, the mutualistic potential of S. capleta in a novel host may be constrained by other features of its genotype rather than the digestive enzymes it supplements. To explore this further, we investigated the role that partner recognition plays in shaping the symbiotic outcome between tortoise beetles and S. capleta. Specifically, we asked: are genetically distant species of S. capleta less likely to be recognized as symbionts by their host?

Recognition of an obligate symbiont
Animals that acquire their symbionts horizontally must winnow thousands of encountered microbes down to their selected partner29,30. These have shed light on the intricate molecular mechanisms behind symbiont acquisition and partner recognition22,26,32. In addition to enriching the environment with beneficial microbes30,61,62, the host deploys a honed immune system to facilitate symbiont colonization by removing environmental and potentially pathogenic microbes28,29,31,63–65. In bean bugs, hundreds of antimicrobial peptides are expressed in the gut, including crypt-specific cysteine-rich peptides65–67. These peptides damage the membranes of diverse bacteria but not the crypt-dwelling symbiont, C. insecticola, which is resistant65–67. Cnidarians, while indiscriminate in their uptake of microalgae from the environment, eject non-symbiotic dinoflagellates through vomocytosis (i.e., engulfment and expulsion by phagocytes)24. Similarly, the Hawaiian bobtail squid discriminates for bioluminescent V. fischeri through hemocyte binding of its outer membrane28,63. Non-symbionts induce an elevated immune response defined by the enrichment of antimicrobial peptides in the symbiotic light organ29, complementing mechanical and biochemical selective measures21,34.

Given the varied symbiotic outcomes of S. capleta colonization along a genetic gradient (Fig. 4C, D), we investigated whether non-native symbionts trigger a distinct host response. To address this question, we sequenced the transcriptome of the foregut symbiotic organs of C. alternans larvae (5-days-old) under three experimental conditions: (a) re-infection with the native symbiont, (b) cross-infection with the symbiont of C. gressoria and (c) cross-infection with the symbiont of A. quinquefasciata (Fig. 5A). The two non-native symbionts were chosen because of their contrasting effects on host development - while the C. gressoria symbiont fully restores larval development in its novel host, the A. quinquefasciata symbiont provides only partial rescue (Fig. 4C).

Paired-end, dual RNA sequencing (~30 million reads) was performed on an Illumina NextSeq 2000 and spanning three biological replicates for each treatment (Supplementary Data 3A). Strikingly, only two host genes were differentially expressed between larvae bearing the C. gressoria symbiont and insects colonised by their native S. capleta (Fig. 5A; Supplementary Data 3B, C, adjusted p<0.05). In contrast, 27 genes were differentially expressed in larvae cross-infected with the A. quinquefasciata symbiont compared to the re-infected control (Fig. 5A; Supplementary Data 3B, D, adjusted p<0.05). The A. quinquefasciata symbiont, which only partially rescues larval development, triggers a more pronounced host response compared to a non-native symbiont that fully restores it (Fig. 5A).

When the two cross-infected treatments were compared (A. quinquefasciata vs. C. gressoria), we found 26 genes to be differentially expressed (Fig. 5A; Supplementary Data 3B, E; adjusted p<0.05). Notably, 23 of these genes were also differentially expressed when larvae colonized by their native S. capleta were compared to the cross-infected treatment featuring the A. quinquefasciata symbiont (Fig. 5B; Supplementary Data 3F, PERMANOVA, p=0.025; Fig. S4 and Supplementary Data 4A). This substantial overlap indicates that the C. gressoria symbiont appears to recapitulate the metabolism of its novel host as would the native S. capleta.

Genes encoding Niemann-Pick type C2 (NPC2) proteins were highly expressed in cross-infected larvae colonized by the A. quinquefasciata symbiont compared to re-infected control and cross-infected larvae harboring the C. gressoria symbiont (Fig. 5C, D; Supplementary Data 3B–E; adjusted p<0.05; Npc2_1, NB-GLM, df=2, p<0.001, χ²=38.7; Npc2_2, NB-GLM, df=2, p<0.001, χ²=35.3; Supplementary Data 4B). NPC2 proteins are known to be involved in sterol homeostasis, steroid biosynthesis, and in innate immunity by modulating the IMD pathway in insects68–71. As demonstrated in Drosophila melanogaster and the Asian honeybee (Apis cerana), NPC2-encoding genes become highly expressed following pathogenic challenges69,71. These proteins actively combat microbial infections by directly binding to bacterial and fungal cell walls69,71, thereby stimulating the expression of antimicrobial peptides68–70. It is conceivable that S. capleta from A. quinquefasciata induces Npc2 expression because of structural differences in its cell wall compared to the symbionts of C. gressoria and C. alternans. Supporting this, while the S. capleta species from C. gressoria and C. alternans retained a subset of ligases involved in peptidoglycan biosynthesis (including murD, murE, D-alanine:D-alanine ligase) and outer membrane assembly proteins (including BamA and PhoE), the encoding genes are absent from the A. quinquefasciata symbiont genome37,38.

Colonization by the A. quinquefasciata symbiont also led to elevated expression of protease-encoding genes, including numerous cysteine cathepsins and a serine protease (Fig. 5C, D and S5, and Supplementary Data 3B–E; adjusted p<0.05; NB-GLM/LM, p<0.001; Supplementary Data 4B). These enzymes are known to have pleiotropic roles in digestion, development, and immune function. In response to bacterial pathogens, serine proteases are shown to activate the Spätzle-mediated Toll-like receptor and the prophenoloxidase pathway in D. melanogaster and the mealworm beetle Tenebrio molitor68–70. Cathepsins, on the other hand, are lysosomal proteases and are classified into over 10 subfamilies based on their primary sequence and enzymatic activity72. Many members of the cathepsin superfamily are biologically active and are deployed by insects to combat pathogenic infections and regulate microbial populations along epithelial cells in the gut64,73,74.

Our findings suggest that host immunity may contribute to symbiont recognition. Yet they also raise the possibility that a fine-tuned metabolic interplay between host and symbiont, mediated by transcriptional crosstalk, could be essential for proper symbiont growth. The results further reveal upregulation of genes involved in metabolic functions, such as lipase activity (Figs. 5C and S5, and Supplementary Data 3B–E; adjusted p<0.05; Supplementary Data 4B). Such changes could represent a compensatory host response to reduced nutrient access arising from colonization by a metabolically suboptimal symbiont. Consistent with this interpretation, the native (C. alternans) and A. quinquefasciata symbionts differ in their potential metabolic profiles, especially in lipid metabolism37. C. alternans and C. gressoria symbionts encode four and three genes, respectively, related to lipid transport and metabolism, whereas the A. quinquefasciata symbiont lacks genes associated with these functions.

We next quantified symbiont abundance using RNA-seq on cDNA and detected significant differences among treatments (Fig. 5E; LM, p<0.001, F(2,9)=42.12; Supplementary Data 4C), consistent with the previously observed proliferation levels of these non-native symbionts (Fig. 4B). S. capleta titers did not differ significantly between larvae colonized by their native symbiont or cross-infected with the C. gressoria symbiont (Fig. 5E; pairwise comparison, p=1; Supplementary Data 4C). In contrast, both groups contained higher symbiont abundance than larvae cross-infected with S. capleta from A. quinquefasciata (Fig. 5E; pairwise comparison, p<0.001 in both cases; Supplementary Data 4C). We also mapped S. capleta reads to measure the relative presence of symbiotic RNA. The proportion of symbiont transcripts differed significantly across the three experimental groups (Fig. 5F; Fisher’s Exact Test, p<0.001; Supplementary Data 4D). Larvae colonized by their native symbiont or cross-infected with the C. gressoria symbiont exhibited similar proportions of S. capleta transcripts (Fig. 5F; pairwise comparison of proportions, p=0.23; Supplementary Data 4D), and both groups showed significantly higher proportions than larvae cross-infected with S. capleta from A. quinquefasciata (Fig. 5F; pairwise comparison of proportions, p=0.00072 and p=0.023; Supplementary Data 4D). The pronounced reduction in A. quinquefasciata symbiont abundance suggests that an immune response may have inhibited its proliferation within the foregut symbiotic organ of a novel host. However, it remains unclear whether this decrease reflects bacterial killing or reduced growth in an unfavorable host environment.

Collectively, our findings indicate that (i) non-native S. capleta elicit distinct host responses, reflecting the varied symbiotic outcomes of their colonization in tortoise beetles, where (ii) genetically distant species induce a more pronounced host transcriptional response than those closely related to the native microbe. But given the strict pattern of co-cladogenesis between tortoise beetles and S. capleta37,38, even among closely related taxa such as C. alternans and C. gressoria, we next asked: is specificity reinforced at a finer resolution later in development?

Fidelity throughout host development and symbiont transmission
We quantified S. capleta colonization efficiency in adult beetles to determine whether non-native symbionts can persist throughout host development and propagate to future generations. We assessed the symbiotic status of C. alternans hosts that were either re-infected with their native symbiont or cross-infected with S. capleta from C. gressoria at two time points: 5 and 15 days after adult eclosion. In both cases, and despite the extensive transformation and internal reorganization that takes place during metamorphosis, the symbiont persisted throughout pupation and continued to occupy the foregut symbiotic organs of adult beetles (Fig. 6A; GLM (binomial), df=1, p=1, χ2=0; Supplementary Data 4E).

Among adult females, we observed that the ovary-associated glands were successfully colonized by non-native S. capleta, similar to the re-infected control (Figs. 6B–D and S6; GLM (binomial), df=1, p=1, χ2=0; Supplementary Data 4E). The mechanism by which symbionts translocate from the foregut symbiotic organs to the ovary-associated glands is still unknown, but it is conceivable that the non-native symbiont can coopt this pathway to reach the transmission organs of its novel host (Figs. 6B–D and S6). However, despite successfully colonizing the ovary-associated glands, the vertical transmission of S. capleta differed between native and non-native species. While the native symbiont was embedded into the spherical secretions within individual egg caplets, the non-native symbiont was not (Figs. 6E, F and S7).

Eggs laid by cross-infected females contained caplets, but these structures were populated with spheres that were strikingly devoid of S. capleta (Fig. 6F). Diagnostic PCR confirmed the absence of symbionts in the eight egg caplets tested, demonstrating that the non-native S. capleta failed to embed within the maternally secreted spheres. As a result, larvae emerging from these eggs were entirely aposymbiotic (Fig. 6G), in stark contrast to the re-infected treatment (GLM (binomial), df=1, p<0.001, χ2=88.98; Supplementary Data 4E). The ovary-associated glands cross-infected with the C. gressoria symbiont appear to undergo marked morphological remodeling compared to those infected by the native symbiont, from a complex labyrinthine architecture to a simpler conglomerate form between 5 and 15 days (Figs. 6D and S6). How, and whether, such a morphological change affects to the failure to transmit the non-native symbiont is unknown.

Extracellular symbiont transmission is widespread in insects22. These routes typically feature a maternal secretions that encapsulate the symbiont7,22,47,48,75–77, which are then consumed by offspring to initiate infection22. For instance, plataspid stink bugs ensure the vertical transmission of their nutritional symbiont, Ishikawaella capsulata, by embedding it within a proteinaceous capsule7,48,75. This capsule is primarily composed of an odorant-binding protein that stabilizes the symbiont’s metabolism during extracellular transfer7, compensating for its highly reduced genome78. Similarly, urostylidid stink bugs propagate their nutritional symbiont, Tachikawaea gelatinosa, through jelly-like secretions that are rich in galactan and essential amino acids47. It is conceivable that tortoise beetles rely on similar substrates to produce their S. capleta-bearing spheres (Fig. 1F–H). The embedding properties of these substrates may be specifically tailored to the unique cellular and physiological characteristics of each symbiont, which could explain why non-native S. capleta fail to populate the spherical secretions of their novel host (Fig. 6E, F). Similarly to tortoise beetles, transmission fidelity plays a crucial role for maintaining specificity between beewolves and their defensive ectosymbiont Streptomyces philanthi79. While non-native bacteria can colonize beewolf transmission glands, only the native symbiont can successfully embed within the maternal secretion, thereby ensuring the faithful propagation of this partnership79.

Since many tortoise beetle species are sympatric and often coexist on the same host plant60, we next examined the infection outcome of multiple S. capleta strains and explored whether local adaptation to the host environment offers a selective advantage to the native symbiont.

Competition-based selection reinforces specificity
Competition is a strong selective force shaping microbial community structure in a host environment33,80,81. The host can promote the dominant growth of locally adapted microbes in open systems like the gut, cuticle, or rhizosphere, by creating a specialized metabolic landscape33,80,81. Given recent examples of competition-based selection stabilizing specificity in symbioses33,82, we investigated whether this process offers an additional mechanism for tortoise beetles to promote the competitive advantage of their native S. capleta during mixed infections.

Using C. alternans as a host, we conducted dual-infection assays by introducing two S. capleta-bearing spheres to caplet-free eggs: one containing the native symbiont, and the other collected from the congeneric beetle, C. gressoria (Fig. 7A). Both symbionts successfully colonized the foregut symbiotic organs of 5-day-old larvae, demonstrating that tortoise beetles can support simultaneous infections by multiple S. capleta strains (Figs. 7Bi–iv and S8). We validated these findings by quantifying symbiont abundance throughout host development (Fig. 7C; LM, p<0.001, F(4,24)=83.61; Supplementary Data 4F).

In newly hatched larvae, native and non-native S. capleta initially co-colonized the host at similar titers (Fig. 7C; post-eclosion: day 1 (pairwise comparisons of contrasts, p=0.99), day 5 (pairwise comparisons of contrasts, p=0.41); Supplementary Data 4F). However, symbiont abundance diverged later in larval development (Fig. 7C; 12 days post hatching, pairwise comparisons of contrasts, p=0.0024; Supplementary Data 4F), despite encoding a near-identical (~97%) gene set38. Both symbionts share 226 genes but differ in only eight. The C. alternans symbiont possesses genes associated with translation and energy metabolism, whereas the C. gressoria symbiont encodes genes involved in amino acid and cell wall biosynthesis, reflecting differences in metabolic specialization (Table S1). The variations in symbiont population growth may be attributed to these specific genetic differences and/or to variations in the regulation of gene expression that led to distinct physiological capacities. To estimate the growth rate of the non-native S. capleta relative to the native one during larval development, we calculated a selection coefficient. This yielded a coefficient of −0.027 (Fig. 7D), indicating a selective disadvantage for the non-native microbe. A second, independent method leveraging Malthusian parameters corroborated this result, resulting in a similarly negative selection rate of −0.025 for the non-native species.

By adulthood, quantification of symbiont titers revealed the complete loss of the non-native species, while the native S. capleta continued to stably colonize both the foregut symbiotic organs and the ovary-associated glands, underscoring the role of microbe-microbe competition in reinforcing specificity between tortoise beetles and S. capleta (Fig. 7C; pairwise comparisons of contrasts: foregut symbiotic organs, p<0.001; ovary-associated glands, p<0.001; Supplementary Data 4F). This contrasts the infection dynamics observed when the C. gressoria symbiont colonized C. alternans alone—where it successfully persisted in both the adult foregut symbiotic organs and ovary-associated glands of its novel host (Fig. 6A–D). Reflecting this, offspring of dual-infected beetles were exclusively colonized by the native symbiont (Fig. 7E; GLM (binomial), df=1, p<0.001, χ2=133.08; Supplementary Data 4G).

Our findings show that once both symbionts colonize the foregut symbiotic organs, the native population rapidly supplants the non-native one, despite their near-functional equivalence and effects on host fitness (Fig. 4) and metabolic response (Fig. 5). This indicates that the native bacterium is better adapted to its host and reinforces the hypothesis that co-evolutionary history is a key driver of fidelity. We further observed spatial heterogeneity in symbiont distribution across the symbiotic organs. The native bacterium predominantly occupies regions adjacent to the epithelium, whereas the non-native bacterium remains restricted to the lumen (Fig. 7Bi-iv and S8). The observed distribution may partly explain the native symbiont’s competitive advantage, potentially through greater local adaptation and enhanced access to host-derived nutrients. We note that a reciprocal dual-infection experiment in C. gressoria would help rule out the alternative possibility that the C. gressoria symbiont is intrinsically a weaker competitor due to differences in its metabolic relative to S. capleta associated with C. alternans.

Overall, our findings align with recent studies on bean bugs33 and mice82, demonstrating that native symbionts consistently outcompete non-native microbes within host environments. This highlights the selective advantage of local adaptation, which can further strengthen specificity within symbiotic partnerships.

Collectively, we conducted experimental cross-infections in aposymbiotic eggs of the tortoise beetle C. alternans using S. capleta derived from five additional Cassidinae species. Our results demonstrate that larval survival is restored following cross-infections, but only when the non-native symbionts are closely genetically related to the native one. Consistent with this, the positive correlation between symbiont genetic distance and host survivorship highlight the strong influence of symbiont genotype on host development.

Our findings also demonstrate that while obligate, heritable endosymbionts can successfully establish and fully rescue development in a novel host, long-term persistence and successful vertical transmission are maintained by a robust framework of partner specificity. The introduction of a genetically distant, non-native symbiont triggered a strong transcriptional response in the host, including activation of immune genes. This was accompanied by a decrease in symbiont population, suggesting host discrimination between native and non-native partners. When both were introduced simultaneously, the native symbiont progressively outcompeted the non-native one and was exclusively transmitted to the next generation.

Together, these results highlight how the multilayered interplay of host recognition, transmission fidelity, and competitive exclusion acts to ensure that only the native symbiont persists, securing propagation to the next host generation. This selective advantage highlights the evolutionary fine-tuning of the S. capleta-tortoise beetle partnership, where co-diversification and local adaptation appear to reinforce a highly specific, heritable symbiosis. Testing whether these mechanisms act in concert to preserve specificity in other vertically transmitted, co-diversified systems would be valuable given the continued development of experimental approaches to clear and reconstitute endosymbionts, as demonstrated in aphids83, weevils18, and plataspid stink bugs84. Moreover, the application of microinjection into the host hemolymph to reproduce the mounted immune response is a worthy area for future study.

Our results align with and extend findings from horizontally acquired symbioses, such as the V. fischeri-Hawaiian bobtail squid and C. insecticola-bean bug systems, where host-driven selection-via immune filtering, niche restriction, and competitive exclusion-ensures the establishment of preferred microbial partners. In the squid, hemocyte-mediated recognition and antimicrobial peptide expression help winnow Vibrio from a diverse seawater microbiome21,26,28,29,34, while the bean bug’s gut crypts physically and chemically promote the retention of Caballeronia over environmental bacteria19,20,30,31,33. The S. capleta-tortoise beetle model demonstrates that even vertically transmitted endosymbionts are subject to similar competitive and immunological pressures, blurring the distinction between horizontal and vertical symbiont selection mechanisms. Future research comparing obligate, facultative, and environmentally acquired symbioses could uncover shared genetic and molecular pathways that mediate partner fidelity, with implications for the establishment and maintenance of complex microbial communities across ecological and evolutionary scales10,82.

Methods
This study complies with Nagoya Protocol regulations in the Republic of Panama and France per the following permits: ARG-027-2023 and TREL2302365S/693 for the collection and use of biological materials.

Insect rearing
Tortoise beetles were maintained in a greenhouse at the Max Planck Institute for Biology in Tübingen, Germany. The insects were reared in mesh containers along with their host plants: Ipomoea batatas for C. alternans, C. bullata, C. gressoria, and A. quinquefasciata beetles; Merremia umbellata for A. sparsa beetle; and Cirsium oleraceum for C. rubiginosa beetle (Supplementary Data 1). Experimental treatments were maintained in climate chambers at a constant temperature of 26°C, humidity of 60%, and long light regimes (14.30 h/9.30 h light/dark cycles).

Whole-mount fluorescence in situ hybridization
To visualize S. capleta in C. alternans eggs and adults, whole-mount fluorescence in situ hybridization (FISH) was performed, as previously described85,86. Eggs and a single dissection from an ovipositing female, containing the foregut, midgut, hindgut, and ovaries, were fixed in Carnoy’s solution (ethanol:chloroform:acetic acid = 6:3:1) at room temperature overnight. After thorough washing with 100% ethanol, the samples were bleached in an alcoholic 6% H2O2 solution to reduce autofluorescence and incubated at room temperature until they were decolorized. Samples were thoroughly washed with 100% and then 70% ethanol and rehydrated with PBSTx containing 0.3% Triton X-100. They were subsequently incubated with hybridization buffer (20 mM Tris-HCl [pH 8.0], 0.9 M NaCl, 0.01% sodium dodecyl sulfate, 30% formamide) containing 100 nM of each of the probes overnight. The female internal organs were hybridized with the oligonucleotide probes Atto550-SAL227 targeting the 16S rRNA sequence of S. capleta from C. alternans and Atto488-EUKCA164 targeting the 18S rRNA sequence of C. alternans (Supplementary Data 5A). Eggs were hybridized with the oligonucleotide probes Atto550-SAL227 targeting the 16S rRNA sequence of S. capleta from C. alternans and Atto488-EUK1195 targeting the 18S rRNA sequence of all eukaryotes (Supplementary Data 5A). All probes were dually labeled with fluorescent dyes at 5′ and 3′ ends. Samples were washed thoroughly with PBSTx, mounted using ProLong® Gold antifade mounting media (Thermo Fisher Scientific, USA), and observed using an LSM 980 NLO confocal microscope (Zeiss, Germany).

Fluorescence in situ hybridization on paraffin sections
To localize S. capleta in foregut symbiotic organs of the six tortoise beetle species and in C. alternans eggs, FISH was applied on paraffin sections. Adult female foregut symbiotic organs and eggs were dissected and fixed in Carnoy’s solution at room temperature overnight. Dehydration was performed by incubating the samples in 70 and 80% ethanol for 10 min each, followed by three 10-min incubations in 96 and 100% ethanol. Samples were gradually transferred into paraffin by passing through three incubations of Roti®-Histol (Carl Roth, Germany) at room temperature (two steps of 40 min and one step overnight), followed by incubations at 60°C in Roti®-Histol:paraffin (1:1 v/v) for 60 min and paraffin (Paraplast plus, Leica, Germany) (three steps of 60 min and one step overnight). The paraffin-embedded samples were cross-sectioned at 10 μm using a microtome and mounted on poly-L-lysine-coated glass slides (Epredia, Germany). Paraffin sections were dried at room temperature overnight and incubated at 60°C for 1 h to improve tissue adherence. They were dewaxed with Roti®-Histol in three consecutive steps for 10 min each, followed by ethanol 100% for 10 min. Slides were then dried at 37°C for 30 min. Foregut symbiotic organs were hybridized with the oligonucleotide probe Cya5-SCA600 targeting the 16S rRNA sequence of S. capleta from 52 cassidines (Supplementary Data 5A). Eggs were hybridized with the oligonucleotide probes Atto550-SAL227 targeting the 16S rRNA sequence of S. capleta from C. alternans and Atto488-EUKCA164 targeting the 18S rRNA sequence of C. alternans (Supplementary Data 5A). Probes, dually labeled with the fluorescent dye at their 5′ and 3′ ends, were dissolved at 900 nM in the hybridization buffer containing 35% formamide (v/v), 900 mM NaCl, 20 mM Tris-HCl pH 7.8, 1% blocking reagent for nucleic acids (v/v) (Roche, Switzerland), 0.02 SDS (v/v), and 10% dextran sulfate (w/v). Hybridization was performed at 46°C for 4 h. Sections were then transferred to fresh 48°C washing buffer for 15 min [70 mM NaCl, 20 mM Tris-HCl pH 7.8, 5 mM ethylenediaminetetraacetic acid pH 8.0, and 0.01% SDS (v/v)] followed by room-temperature washes in PBS (20 min) and Milli-Q® water (1 min). They were counterstained with DAPI (5 μg/ml) for 10 min at room temperature, dipped in Milli-Q® water, ethanol 100%, and dried at 30°C for 10 min. Slides were mounted using the ProLong® Gold antifade mounting media (Thermo Fisher Scientific, USA), cured overnight at room temperature, and visualized using an LSM 980 NLO confocal microscope (Zeiss, Germany).

Experimental symbiont exchange across beetle species
Egg masses containing 30 or more eggs with well-defined caplets were collected from C. alternans mating pairs to avoid pseudo-replication. Egg masses were then separated into different experimental treatments depending on the experiment: untreated control, aposymbiotic, re-infected with the native symbiont, and cross-infected with symbionts from a different beetle species. Aposymbiotic individuals were generated by removing caplets from eggs using sterile dissection scissors and without piercing the developing embryo36,39,40. Re-infection with the native bacterium was performed by eliminating caplets from eggs and reapplying all enclosed spheres to the anterior pole of the egg, as previously described39,40. Cross-infections with non-native symbionts were achieved by removing caplets from eggs and reapplying all enclosed spheres collected from eggs of different beetle species to the anterior pole of C. alternans eggs. As with the re-infection procedure, spheres were obtained from eggs of the same mass for each replicate.

To test whether S. capleta can be reciprocally exchanged across tortoise beetle species, whole-mount FISH was performed on entire embryos and larval foregut symbiotic organs of two beetle species C. alternans and C. gressoria, as described above. For each beetle species, two to four egg masses were collected from separate females and divided into four experimental treatments: untreated control, eggs with caplets removed (aposymbiotic), eggs with caplets removed but re-supplied with their original symbiont-bearing spheres, and eggs with caplets removed but supplied with spheres collected from different host species. A near-emergence embryo (~10.5 days following oviposition) and 5-day-old larvae were collected after experimental manipulation for each replicate. Samples were placed in 70% ethanol to remove the embryo legs and scoli and dissect the larval foregut symbiotic organs using sterilized scissors. The following oligonucleotide probes were used for in situ hybridization: Atto550-SAL227 targeting the 16S rRNA sequence of S. capleta from C. alternans, Cy5-SCG224 targeting the 16S rRNA sequence of S. capleta from C. gressoria, Atto488-EUKCA164 targeting the 18S rRNA sequence of C. alternans, and Atto488-EUKCG167 targeting the 18S rRNA sequence of C. gressoria (Supplementary Data 5A). Symbiont-targeting probes are specific and used simultaneously during hybridization. The formamide curve generator tool in mathFISH was used to analyze the formamide curves and check the specificity of each probe. All probes were dually labeled with fluorescent dyes at 5′ and 3′ ends. Controls consisted of the two beetle species infected with their native symbionts (Fig. S1).

Phylogenetic inference
Host mitochondrial genomes were extracted from metagenomic assemblies and aligned using MUSCLE (v3.8.1551)87, and as described by Garcia-Lozano and colleagues38 (Supplementary Data 5B). A concatenated alignment of 15 mitochondrial genes (13 protein-coding genes + two ribosomal rRNA genes) was partitioned to assign the most appropriate substitution model to each gene using PartitionFinder2 (branch lengths=unlinked, models = GTR+G and GTR+I+G, model_selection = bic; v2.1.1)88. Phylogenetic analyzes were performed using Maximum Likelihood (ML) in RAxML-NG (v1.2.0)89 (ngen=1000). Members of Spilopyrinae (Spilopyra sumptuosa) and Eumolpinae (Bromius obscurus, Colaspidea sp., and Parias sp.) subfamilies from the Chrysomelidae were used as outgroups for this analysis.

To infer the relationships between the 6 corresponding S. capleta symbionts, 61 single-copy core genes identified by anvi’o (v8.1-dev)90 and present in all symbionts were extracted from each S. capleta genome and aligned using MUSCLE (v3.8.1551)38,87. Concatenated alignments of these genes and four outgroups (Escherichia coli, B. aphidicola, Candidatus Ishikawaella capsulata, and V. fischeri) were included to construct a ML phylogeny using RAxML-NG (v1.2.0)89 (ngen=1000) (Supplementary Data 5B). The best-fit substitution model was selected using PartitionFinder2 (branch lengths=unlinked, models = GTR+G, GTR+I+G, model_selection = bic; v2.1.1)88. The tree reconciliation software eMPRess GUI (v1.0.)91 was applied to investigate the evolutionary relationship between the 6 beetle species and their symbionts. This software reconciles symbiont and host trees using the Duplication Transfer-Loss model. ML trees were used as input and the analysis was conducted using the following parameters: duplication cost=1, transfer cost=1, and loss cost=1. The significance of reconciliation between host and symbiont tree was calculated by randomizing the tips of the branches and then, re-calculating the cost to reconcile the phylogenies. Congruent phylogenies are obtained when the original cost of reconciliation is less than expected by chance (p<0.01).

S. capleta genetic distance
A dual approach was used to evaluate sequence similarity and structural gene organization across symbiont genomes38. The ANI between S. capleta genomes was calculated using PyANI (v2.4.0)92, and as implemented in anvi’o (v8.1-dev)90 to assess symbiont relatedness. Gene collinearity across different S. capleta lineages was analyzed with MCScanX (v1.0)93 and SynTracker (v1.1.3)94. Collinearity scores were then calculated using the collinearity scripts from Nowell et al.95. To adapt SynTracker for the comparison of different species we used modified parameters (minimal blast identity = 70%, minimal coverage = 70%). SynTracker comparison was carried out with the collection of six S. capleta genomes as potential references. The Average Pairwise Synteny score was calculated using all comparable regions, for each genome pair. Next, the score for each genome-pair was multiplied by the number of comparable regions, normalized by the length of the smaller genome in each genome-pair, and transformed to fit a scale of 1–100.

Experimental manipulation to quantify the impact of non-native S. capleta on beetle development
To estimate whether non-native S. capleta can successfully colonize the foregut symbiotic organs of C. alternans as a novel host and restore its survival, egg masses were collected from different female beetles and divided into eight experimental treatments: untreated control, aposymbiotic eggs, eggs whose caplets were removed but re-infected with the native bacterium, or eggs whose caplets were removed but cross-infected with S. capleta-bearing spheres collected from 5 different beetle species (C. bullata, C. gressoria, A. sparsa, A. quinquefasciata, and C. rubiginosa).

Symbiont infection frequencies were validated across all treatments using diagnostic PCR. Five days after hatching, foregut symbiotic organs of larvae were dissected, and DNA was extracted using the E.Z.N.A.® Insect DNA Kit from Omega Bio-tek. PCR primers targeting either specific S. capleta symbionts (groEL gene) or all symbionts collectively (16S rRNA gene) were used to verify the symbiotic status of the host beetles (Supplementary Data 5C). For cross-infected individuals, a groEL primer pair specific to the native symbiont was used to confirm its successful removal. The primer pairs were designed using Primer3 and Geneious software and synthesized by Sigma-Aldrich under standard conditions. PCR products were sequenced to confirm primer specificity in vitro. Additionally, a specific primer pair for the CO1 gene of C. alternans was used as a control for the DNA extraction (Supplementary Data 5C). Diagnostic PCR was conducted on an Analytik Jena Biometra T Advanced Thermal Cycler using a final volume of 20 µl containing 1 µl of DNA template, 0.5 µM of each primer, and 2× DreamTaq PCR Master Mix. The following cycle parameters were used: 5 min at 95°C, followed by 34 cycles of 95°C for 30 s, 54.7 to 62.5°C (depending on the primer pair, Supplementary Data 5C) for 30 s, 72°C for 1 min and a final extension time of 2 min at 72°C. Treatments were carried out in different experimental blocks. Four to five replicates were performed depending on treatment (i.e., n=13-to-25 larvae).

The impact of the cross-infection procedure on symbiont abundances was assessed by qPCR using an Analytik Jena® qTOWER³ G cycler. DNA samples used previously served as templates. Symbiont abundance was estimated by quantifying the copy number of the S. capleta 16S rRNA gene, targeting all Cassidinae species studied, using the following primer pair: TTGACCGCCTGGGGAGTA and TTCGCGTTGCATCGAATTAAAC (Supplementary Data 5D). The primer pair was designed using Geneious software and synthesized by Sigma-Aldrich under standard conditions. Verification of primer specificity was conducted in silico by comparison with reference sequences of all symbionts. qPCR reactions were performed in a final volume of 25 µl, containing 1 µl of DNA template, 2.5 µl of each primer (10 µM), 6.5 µl of autoclaved distilled H2O, and 12.5 μl of the Platinum SYBR Green qPCR SuperMix-UDG (Thermo Fisher Scientific). Cycling conditions consisted of 3 min activation at 95°C, followed by 45 cycles of 95°C for 15 s, 62°C for 45 s, 72°C for 20 s. A melting curve analysis was conducted from 60 to 95°C, increasing the temperature by 1° every 30 s. Gene copy numbers were estimated from standard curves (tenfold dilution series from 10−1 to 10−8 ng/µl) generated from purified PCR products, whose DNA concentrations were measured with a NanoDropTM 1000 spectrophotometer. Since a slight amplification signal was detected in the negative controls (NTCs), the gene copy numbers were corrected by subtracting the average copy number obtained for the NTCs. Treatments were carried out in separate experimental blocks, with four to five replicates per treatment (n=13-to-25 larvae).

Emerged larvae were observed daily, and survival until adulthood was recorded to assess the impact of non-native symbionts on larval survivorship. Treatments were carried out in different experimental blocks. Five-to-ten replicates were performed depending on treatment (i.e., n=27 to 109 larvae). The relationship between the percentage of survivors after adult emergence and symbiont genetic distance (ANI or collinearity score) was also tested.

RNA sequencing
The transcriptome of foregut symbiotic organs of 5-day-old C. alternans larvae was sequenced across three experimental treatments: (a) re-infection with the native symbiont, (b) cross-infection with the symbiont of C. gressoria and (c) cross-infection with the symbiont of A. quinquefasciata. Three egg masses were collected from separate females and divided into these experimental treatments. Two larvae from each replicate were sampled 5 days after hatching, and foregut symbiotic organs were dissected using sterile scissors and snap-frozen in liquid nitrogen. RNA extraction was performed for each sample immediately after collection using the QIAGEN RNeasy Mini Kit according to the protocol 4: Enzymatic Lysis and Proteinase K Digestion of Bacteria starting from step 7. This protocol is included in the RNAprotect® Bacteria Reagent Handbook from Qiagen and as referenced in Ref. 38. Total RNA was quantified using the QubitTM RNA High Sensitivity (HS) kit. The two samples were pooled to obtain 40 ng of total RNA, which was used as input to prepare nine RNA sequencing libraries (3 biological replicates). Ribosomal RNA was depleted from total RNA using the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat). Libraries were constructed using the NEBNext® UltraTM Directional II RNA Library Prep kit, and their size was confirmed in a 2100 Bioanalyzer system using the Agilent Technologies High Sensitivity DNA Kit. Sequencing of the final libraries was performed on an Illumina NextSeq 2000 system at the Max Planck Institute for Biology (Tübingen, Germany) using paired-end chemistry (2×100 bp) with a depth of ~30 million reads.

Adapter removal and quality filtering of raw reads were performed in Trimmomatic (v0.36)96 and FastQC (v0.12.0)97. Filtered reads were mapped to the C. alternans genome (Garcia-Lozano et al. personal communication) using HISAT2 (v2.2.0)98. Gene counts were obtained using htseq-count (v0.11.5)99 (stranded=reverse-rpos). After applying a filter, read counts were normalized by the DESeq2’s median of ratios established in the DESeq2 package100. The likelihood ratio test (LRT), implemented in DESeq2, was used to test for differences in host gene expression across treatments. Host genes were considered significantly differentially expressed at adjusted p-value (padj)<0.05 and a fold-change > 2. Volcano plots were generated using the ggplot2101 and dplyr102 packages for visualizing transcriptome data. We compared the differentially expressed gene (DEG) profile between treatments by testing for significant clusters using a permuted multivariate analysis of variance (PERMANOVA) and applying the function vegan::adonis(). The batch effect was removed using the LIMMA package103. DEGs were further annotated for immune functions using the C. alternans immune gene reference set (Garcia-Lozano et al. personal communication), which was derived from the 4IN database (Innate Immunity Genes in Insects: http://bf2i300.insa-lyon.fr:443/home). A heatmap of the log2(x+1) normalized counts was then constructed to illustrate host DEG expression patterns in response to colonization by S. capleta symbionts. Analyzes were performed in R v. 3.5.3104.

Symbiont abundances across treatments were quantified by qPCR on an Analytik Jena® qTOWER³ G cycler. cDNA generated from the corresponding RNA samples were used as templates. For cDNA synthesis, 10 ng of total RNA per sample was reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) with oligo(dT) primers. qPCR was performed with the same primer pair (16S rRNA) and conditions as described earlier, except that the standard curve was constructed from a tenfold serial dilution of cDNA (10−1 to 10−8 ng/μl). Three replicates were performed per treatment (n=6 larvae). In addition, the proportion of S. capleta symbionts within each treatment was estimated as the ratio of RNA-seq reads mapped to the genome of the target symbiont relative the total number of mapped reads (i.e., reads aligned to both the symbiont and host genomes).

Persistence of non-native S. capleta across host development and during transmission
To determine whether non-native S. capleta can be maintained during host development, and transmitted to the next generation, egg masses were collected from different C. alternans females and divided into two experimental treatments: re-infected with the native symbiont and cross-infected with the symbiont of C. gressoria.

Emerged larvae were maintained through pupation and until adulthood. S. capleta infection frequency was investigated in the female foregut symbiotic organs and ovary-associated glands 5- and 15-days after adult eclosion, using diagnostic PCR specific to each S. capleta-species and as described above. Three to six replicates were performed depending on treatment and sampling time.

Additionally, whole-mount FISH was performed to visualize each S. capleta species in the ovary-associated glands, as described above. For each treatment, one female was collected 5 and 15 days after emergence and dissected using sterile scissors. The following oligonucleotide probes were used for in situ hybridization: Atto550-SAL227 targeting the 16S rRNA sequence of S. capleta from C. alternans, Cy5-SCG224 targeting the 16S rRNA sequence of S. capleta from C. gressoria, and Atto488-EUKCA164 targeting the 18S rRNA sequence of C. alternans (Supplementary Data 5A). Symbiont-targeting probes are specific to each species and were used simultaneously during hybridization.

Three re-infected and two cross-infected females were maintained until sexual maturation. Egg masses laid were recorded daily and subsampled for 1 month to assess their symbiotic status (7 days post oviposition). FISH was applied to paraffin sections to localize symbionts in the egg caplets, as described above. Eggs were dissected and fixed in Carnoy’s solution at room temperature overnight. The following oligonucleotide probes were used for in situ hybridization: Atto550-SAL227 targeting the 16S rRNA sequence of S. capleta from C. alternans, Cy5-SCG224 targeting the 16S rRNA sequence of S. capleta from C. gressoria, and Atto488-EUKCA164 targeting the 18S rRNA sequence of C. alternans (Supplementary Data 5A). Controls consisted of untreated eggs from C. alternans and C. gressoria beetles (Fig. S7). Two replicates with one-to-four eggs per replicate were carried out for each FISH treatment.

To confirm the FISH results, four egg masses from cross-infected females were tested by diagnostic PCR targeting the non-native S. capleta, pooling two egg caplets per egg mass. Egg masses laid by re-infected and cross-infected females were then maintained until larval emergence. The larvae were observed daily and subsampled (3-day-old larvae) to assess their symbiotic status using S. capleta-specific diagnostic PCR, as described above. Two and three replicates were performed for cross-infected (n=30 larvae) and re-infected (n=48 larvae) treatments, respectively.

Dual-infection assays
Dual-infection assays were performed to investigate whether non-native symbionts can colonize C. alternans foregut symbiotic organs and propagate in the presence of the native symbiont. Two symbiont-bearing spheres, one bearing the native symbiont and the other collected from a C. gressoria egg caplet, were reintroduced into caplet-free eggs. Both spheres were deposited on the anterior pole of each egg and were observed hourly to ensuring that they were both consumed by the embryo before hatching.

To visualize the symbionts in foregut symbiotic organs of dual-infected C. alternans larvae, FISH was applied on paraffin sections, as described above. Foregut symbiotic organs from 5-day-old larvae were dissected and fixed in Carnoy’s solution at room temperature overnight. The following oligonucleotide probes were used for in situ hybridization: Atto550-SAL227 targeting the 16S rRNA sequence of S. capleta from C. alternans, Cy5-SCG224 targeting the 16S rRNA sequence of S. capleta from C. gressoria, and Atto488-EUKCA164 targeting the 18S rRNA sequence of C. alternans (Supplementary Data 5A). Symbiont-targeting probes are specific and were used simultaneously during hybridization.

Population dynamics of native and non-native symbionts in foregut symbiotic organs of dual-infected C. alternans beetles during development was estimated through qPCR on an Analytik Jena® qTOWER³ G cycler. Dual-infected larvae were sampled 1-, 5- and 12 days after hatching, and adults (females and males) were sampled 50 days after pupation. Foregut symbiotic organs were dissected, and DNA was extracted using the E.Z.N.A.® Insect DNA Kit from Omega Biotek. To estimate symbiont abundance, the copy number of the S. capleta groEL gene from C. alternans was quantified with the following primer pair (AGAAGGGATGCAATTTGATAGAGGT and GCAACAGATTCTAATATAGGTAAT, Supplementary Data 5D), and the copy number of the S. capleta groEL gene from C. gressoria was quantified with the following primer pair (TGAAGGAATGCAATTTGATAGAGGA and AGCAACAGCTTCTAATATAGGTAAC, Supplementary Data 5D). The primer pairs were designed using Geneious software, and synthesized by Sigma-Aldrich under standard conditions. Primers were designed to be specific to each symbiont but sharing the same amplicon and efficiency. qPCRs were performed using a final reaction volume of 25 µl containing 1 µl of DNA template, 2.5 µl of each primer (10 µM), 6.5 µl of autoclaved distilled H2O, and 12.5 μl of the Platinum SYBR Green qPCR SuperMix-UDG (Thermo Fisher Scientific). The cycling conditions were 3 min of activation at 95°C, followed by 45 cycles at 95°C for 15 s, 60°C for 45 s, 72°C for 20 s, and a melting curve analysis was conducted by increasing the temperature by 1° every 30 s, from 60 to 95°C. The gene copy number was estimated from the standard curves (tenfold dilution series from 10−1 to 10−8 ng/µl), generated using purified PCR products and measuring their DNA concentration using a NanoDropTM 1000 spectrophotometer. Treatments were carried out in different experimental blocks. Three to five replicates were performed per treatment.

The relative growth rate of the non-native S. capleta compared to the native one in foregut symbiotic organs of C. alternans larvae was measured assuming that the mixed infections started at 1:1. The plot of ln(non-native symbiont abundance/native symbiont abundance) over time was generated with a slope equal to ln ω105:

ln(non-native symbiont abundance(t)/native symbiont abundance(t)) = ln(non-native symbiont abundance(0)/native symbiont abundance(0)) + t*ln(ω)

The selection coefficient (ω) for non-native bacterium versus native one was calculated using the e raised to the slope of the line fit to the plot of relative S. capleta abundance over time105. The linear regression to the non-native symbiont growth curve was fitted using R v. 3.5.3104 and ggplot2101. Another method was performed, using the selection rate (r) defined as the difference in Malthusian parameters as follows106:

r = [ln(density non-native symbiont at day t+1) - ln(density native symbiont at day t+1)] / day t

Egg masses laid by dual-infected adults were maintained until larval emergence. Larvae were observed daily and subsampled (3-day-old larvae) to assess their symbiotic status using S. capleta-specific diagnostic PCR, as described above. Three replicates were performed per treatment (n=48 larvae).

Statistical analyses
To assess the colonization efficiency of different S. capleta species in C. alternans as a novel host, Fisher’s Exact test was used to assess the effect of treatment (untreated, aposymbiotic, re-infected, or cross-infected) on symbiont infection frequency in larval foregut symbiotic organs. Pairwise comparisons between treatments were performed using the pairwise.prop.test() function with Holm correction to adjust for multiple testing. Abundances of different S. capleta symbionts in the foregut symbiotic organs of 5-day-old C. alternans larvae were analyzed using a generalized linear model with a quasi-Poisson error structure and a log-link function. The treatment, replicate, and experimental block were included in the model as fixed factors. Tukey’s HSD pairwise comparisons were performed using the glht() function with Bonferroni corrections. The survival of C. alternans beetles into adulthood was analyzed with a Cox mixed-effect model testing for the effect of experimental treatments (untreated, aposymbiotic, re-infected or cross-infected) and replicates with experimental blocks as a random effect, and using the coxme package107. A pairwise comparison between treatments was performed using the pairwise_survdiff() function with Bonferroni corrections in the survminer package. The survival data were visualized by computing the Kaplan–Meier survival functions108. Spearman’s rank correlations were used to test the relationship between larval survivorship and symbiont genetic distance (ANI or collinearity score) after testing the data distribution.

To compare the expression level of DEGs after colonization of foregut symbiotic organs by S. capleta symbionts, normalized transcripts were analyzed using either a negative binomial generalized linear model implemented by the glm.nb() function, or a general linear model framework depending on the data distribution and after verification that model assumptions were respected. In these statistical models, treatment and replicate were considered as fixed factors. Tukey’s HSD pairwise comparisons were performed using the glht() function with Bonferroni corrections. Abundances of different S. capleta symbionts in the foregut symbiotic organs of 5-day-old C. alternans larvae (RNA-seq experiment) were analyzed using a general linear model after appropriate data transformation and verification of model assumptions. Treatment, replicate, and their interaction were included as fixed factors, and pairwise comparisons were conducted using Tukey’s HSD with Bonferroni corrections via the glht() function. Fisher’s Exact test was used to assess the effect of treatment (re-infected or cross-infected) on the proportion of S. capleta reads mapped to the symbiont genome relative to total reads mapped. Pairwise comparisons between treatments were performed using the pairwise.prop.test() function with Holm correction to adjust for multiple testing. Akaike information criterion was used for model selection.

The effect of experimental treatments (re-infected or cross-infected) on symbiont infection frequencies in foregut symbiotic organs or ovary-associated glands of C. alternans females 5- and 15-days post emergence was analyzed using generalized linear models with a binomial error structure and a logit-link function. In these statistical models, treatment, time, and their interaction were considered as fixed factors. The effect of experimental treatments (re-infected or cross-infected adults) on symbiont infection frequency in their offspring was analyzed using a generalized linear model with a binomial error structure and a logit-link function, and the treatment was considered a fixed factor. Akaike information criterion was used to select all previous models.

To compare the population dynamics of native and non-native bacteria in the foregut symbiotic organs of dual-infected beetles during development, their abundance was analyzed using a general linear model framework after verification that model assumptions were respected. The time, symbiont, replicate, experimental block, and the interaction between the time and symbiont were used as fixed factors. A pairwise comparison of the interaction was performed using least-squares means with Tukey corrections. Given that the data associated with adult foregut symbiotic organs include both females and males, the effect of sex on S. capleta population dynamics was determined by fitting a negative binomial generalized linear model implemented by the glm.nb() function. Beetle sex and symbiont were considered as fixed factors. The effect of beetle development time on the abundance of native and non-native symbionts was analyzed using general linear models after checking that model assumptions were respected and using time as a fixed factor. Tukey’s HSD pairwise comparisons were performed using the glht() function with Bonferroni corrections. The impact of dual-infected beetles on S. capleta infection frequencies in their offspring was analyzed using a generalized linear model with a binomial error structure and a logit-link function, and the treatment was considered a fixed factor. Akaike information criterion was used to select all previous models.

Statistical analyzes were performed in R v. 3.5.3104 using the prop.test() function to obtain the 95% confidence intervals for a binomial distribution, the survival package for survival analyzes109, multcomp and lsmeans packages for pairwise comparisons110,111, MASS package for negative binomial generalized linear models112, and ggplot2 for producing figures101.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "By employing an experimental symbiont exchange, this study aims to elucidate the evolution of partner recognition and\nfidelity in symbiotic microorganisms that are strictly vertically transmitted and have reduced genomes. Symbiotic systems\nwith vertical transmission are generally difficult to manipulate experimentally because separating the host and its symbionts\nis very challenging, making partner-exchange experiments rare. The authors overcame this technical barrier by employing a\nunique model system, tortoise beetles, wherein symbionts are transmitted via caplets. The primary focus is on a single\nspecies of tortoise beetle (Chelymorpha alternans), from which the native symbiotic caplet was removed and replaced with\ncaplets from five different tortoise beetle species. This allowed the authors to investigate in detail how co-diversified and\ngenome-reduced symbionts infect, colonize, establish symbiotic associations with different hosts, and are transmitted to the\nnext generation.\nSurprisingly (very surprisingly for me), the authors found a high degree of host–symbiont recognition in this vertically\ntransmitted and coevolving symbiotic system. While the native symbiont was well established in the symbiotic organ,\nconferred substantial fitness benefits to the host, and was reliably transmitted to offspring, non-native symbionts from\ndifferent species of tortoise beetles were not normally maintained. Although the non-native bacteria initially colonized the\nsymbiotic organ, the host’s survivability was reduced, and vertical transmission was unsuccessful. Notably, when non-native\nsymbionts were introduced, immune-related genes were upregulated in the symbiotic organ, suggesting that the host insect\ndiscriminates between native and non-native symbionts, even though all have co-diversified and are vertically transmitted. In\naddition, when native and non-native symbionts were provided simultaneously, the native symbiont gradually became\ndominant in the symbiotic organ, and only the native symbiont was transmitted to the next generation, despite their initially\nequal establishment. Through this series of experiments, the authors demonstrate that even in symbiotic systems with strict\nvertical transmission and co-diversifying, genome-reduced symbionts, host selection and microbial competition are pivotal\nfor maintaining partner fidelity.\nAs the authors note, similar studies have been conducted in aphids, weevils, and plataspid stinkbugs, but never before has\nsuch a precise and extensive experimental framework been applied. In particular, the evidence implicating both microbe–\nmicrobe competition and host immune responses as mechanisms underpinning partner fidelity is novel. Furthermore, the\nresults showing that host fitness correlates with the evolutionary distance among symbiotic bacteria (Figure 4C,D), as well\nas the insights into how hosts respond to coevolving symbionts (Figure 5), are especially noteworthy. These points could be\nemphasized more explicitly in the abstract and conclusion.\nOverall, the manuscript is well written and clearly organized, although there are a few points of concern:\nIn Figure 4, the authors present experiments in which symbiotic bacteria from different beetle species were introduced into\nChelymorpha alternans, and all symbionts appeared to colonize the symbiotic organ. In this symbiont exchange\nexperiments, for symbionts from four host species (Chelymorpha gressoria, Acromis sparsa, Aspidimorpha quinquefasciata,\nand Cassida rubiginosa), there was a gradual decline in host survival, and most insects eventually died, similar to the\naposymbiotic controls. This result could be due to poor colonization by non-native symbionts, but no quantitative data on\nsymbiont titer in the symbiotic organ are provided. Is the observed mortality a consequence of reduced symbiont density, or\nis it due to adequate colonization but low activity (e.g., reduced gene expression, as shown in figure 5E)?\nThe competitive interactions among microorganisms shown in Figure 7 are particularly interesting. One possible mechanism\ninvolves the immune response observed in Figure 5. Does simultaneous colonization by native and non-native symbionts\nalso enhance immune activity in the symbiotic organ, thereby eliminating non-native symbionts? (But it seems likely that\nsuch immune activation would also impact the native symbiont, too.) Alternatively, does the native symbiont simply\noutcompete non-native symbionts through superior nutrient acquisition or other fitness advantages within the symbiotic\norgan?\nOther points:\nL69: Spell out Stammera in full at first mention.\nL92–94: This is a clear summary, but given that multiple symbionts from different lineages were tested, a more detailed\ndiscussion of evolutionary distance, fitness effects, and transmission rates (as shown in Figure 4C,D) could be emphasized\nin this part. This could also be reiterated in the conclusion.\nL229: I remain curious about the density of symbiotic bacteria inside the symbiotic organ before this discussion.\nFigure 4A: The methods section does not clearly describe how symbiont presence was investigated. It would be helpful to\ninclude this information in the text and/or figure legend.\nFigure 4: Clearly state the number of biological replicates (n=).\nL315–331: Regarding the differential immune responses, it might be informative to inject symbiotic bacteria into the\nhemolymph and examine immune gene expression dynamics.\nL403: It is intriguing that the symbiotic bacteria do not enter the caplet despite colonizing the transmitting organ. Do non-\nnative symbionts attempt to transmit but lose viability within the caplet? Since the authors investigated the caplets only by\nFISH, the distinction between the hypothesis that symbionts fail to enter the caplet and the hypothesis that they enter but do\nnot survive (less active) is not fully discussed.", "reviewer_2": "In this particularly elegant study, Pons et al. use a fascinating example of symbiosis as an experimental system to address a\nfundamental question in the field: what makes partners in a symbiotic relationship so specific to each other?\nFirst, they demonstrate how tortoise beetle symbioses offer an experimental platform that allows transmission of their\nrespective symbionts to be manipulated and various hypotheses to be tested following reciprocal exchanges. These\nreciprocal exchanges successfully lead to the infection of their hosts.\nThey then demonstrate that some of these symbiont exchanges maintain the host's fitness at a level similar to that with its\nown symbiont, while others lead to a clear decrease in survival rate. The outcomes of the infections therefore differ greatly\nfrom one symbiont to another, which correlates with genetic differences between the symbionts.\nThey then demonstrate that infection with another symbiont can lead to different outcomes in terms of gene expression, even\nwhen partially rescuing fitness. They also highlight the potential role of immunity in interactions with non-native symbionts.\nAnother striking result of the study is that, despite successful infection of larvae and adults, non-native symbionts do not get\ntransmitted to the next generation.\nFinally, they demonstrate that, in the context of co-infection with a native symbiont, the non-native symbiont is rapidly\noutcompeted and lost later on during adulthood.\nThis is a beautiful and highly original work, offering a striking demonstration of how studying an adequate model system can\nprovide a powerful experimental foundation for addressing a fundamental question in the field, shedding light on the\nmechanisms behind fidelity in symbiotic associations. I believe this article will contribute significantly to the field of\nsymbiosis and will appeal to a wider audience beyond this (already substantial) area of research.\nOverall, I believe the study and its methodology are sound, and the methods are well described. However, I have a few\nremarks that I would like to be taken into account before publication.\nMain remarks\nMy main concern is the section on how the host recognises the symbiont.\nWhile the results clearly show that the host responds differently to one of the cross-infected symbionts than to its native\nsymbiont, I think the conclusions stretch the immunity hypothesis too far at the expense of other alternative hypotheses.\nWhile the data presented here support the immunity hypothesis, they do not fully demonstrate it or rule out other hypotheses,\nsuch as fine metabolic interplay between the symbiont and host. While I agree that differential recognition likely occurs, I\ndon’t agree that the proposed data demonstrate that this recognition is immunity-based, nor that the resulting effect is\nimmunity-mediated. For example, the assertion in line 344 of a 'heightened immune response' is, in my opinion, overly\nassertive without providing evidence of increased AMP expression or ROS production, for example.\nI think the authors could either conduct more experiments to strengthen the evidence behind this conclusion, or they could\nbe more open in their conclusions, including other hypotheses and developing the potential immunity mechanisms in a\nfollow-up story.\nMore specifically, I would like to see a more thorough discussion about what NPC2 is. It’s also a sterol-binding protein that is\ninvolved in many pathways, not just immune-related ones. For instance, it appears to play a part in maintaining intestinal\nstem cells in Drosophila. I think it is too reductive to conclude that the immune pathway is involved based on the change in\nexpression of this gene. To make this claim, I would like to see a measure of the immune response, starting with AMP\nexpression, in the gut of the host in different infection experiments. It would also be interesting to demonstrate whether the\ndecrease in bacterial load is due to the killing of bacteria or the inhibition of their growth. If confirmed, the immune response\nitself could also damage the host.\nAnother possibility is that the fine pairing of the host and symbiont relies on transcriptional crosstalk, which could be\nnecessary for the symbiont to grow properly. This crosstalk could involve NPC2, or it could be independent. Only a loss-of-\nfunction experiment (for example, RNA interference against NPC2) could resolve this issue.\nThe host responds differently to its symbiont and cross-symbiont. However, the link with fitness outcomes could be due to\nthe host not expressing as many digestive enzymes because it is not 'prompted' by the presence of its native symbiont. The\ntranscriptomic data also seem to point towards genes that are more strictly linked to metabolic function than immune\nfunction, such as lipase activity. Could the metabolic interplay be disturbed when the symbiont is not the native one?\nWith regard to cathepsins and serine proteases, while some of them can be involved in immune responses, not all of them are,\nand they also have pleiotropic functions, including digestion. Their increased expression may also be the host's reaction to\nreduced nutrient accessibility due to a change in symbionts. Apart from the plant digestive enzymes described by the\nauthors, I would be interested to see whether the comparison between the native symbiont and the symbiont from A.\nquinquefasciata differs in terms of the metabolic advantages they could provide to their hosts, perhaps in relation to lipid\nmetabolism.\nIn conclusion, I would like to see a more nuanced interpretation of these transcriptomic data.\nFinally, while the number of reads is an indication of a decrease in the population of S. capleta, a qPCR measurement is\nrequired to confirm these results and rule out an effect on the transcriptional activity of the bacteria (which would also be an\ninteresting phenotype).\nMinor points\nThis manuscript is written particularly elegantly, and I enjoyed reading it very much. That said, I have a few comments below\nthat I hope will contribute to improving the manuscript.\nThe first three sentences of the abstract are somewhat difficult to follow as they encompass many important and complex\nconcepts. I know the abstract is limited in length, but perhaps try rephrasing or simplifying which ones need to be displayed\nin this short abstract.\nLine 16 : why « despite » ?\nLines 22–24: I find it a bit oversimplified to include symbiont recognition and partial rescue in the same sentence, as the link\nbetween the two is not fully demonstrated.\nLine 30: The first sentence is abrupt; the term 'specialists' could be explained in more detail.\nLine 34: Please define or explain what co-diversification is. Also, I’m curious: what is the author's take on the distinction\nbetween co-diversification and co-evolution? The term 'co-diversification' is used throughout the manuscript, so it is\nimportant to define it clearly.\nLine 68 : explain what is meant by « streamlined »\nLine 87 : Add 'experimental' before 'mechanism', or reformulate to make it clear that the authors are referring to a way of\nmanipulating their experimental model here.\nLine 134-135 : the sentence is not complete\nLine 208 : « their effects » : please precise which one(s)\nLine 24§-248 : The symbionts share the same genes related to plant digestion. Is there any possibility that these genes are\nnot expressed as efficiently in one species as in the other? Apart from the different host response, this could be one\nhypothesis that the author cannot exclude (at least with the current dataset).\nFigure 4B. I wondered why the survival lines stopped even for the insect population that was still alive. I think I now\nunderstand that this is because we are only following survival until metamorphosis; these individuals are still alive, but are\nno longer recorded in the assay. It would be easier for readers if this explanation were included in the figure legend.\nLine 469: “Both symbionts share 226 genes but differ in only eight.” This is fascinating. I’d love to see a short discussion of\nwhat are these 8 genes. Also, is it possible that the genomic difference is very small, but the symbionts regulate differentially\ntheir expression? (it is maybe not known yet, but I’m curious to know what the authors think)\nLine 481-484: I was a bit lost in this argument that competition increases the specificity of transmission, as the non-native\nsymbiont was shown to not be able to be transmitted to the progeny even when alone. Hence the problem with transmission\ndoes not seem to be linked to competition but to an incompatibility between the symbiont and host.\nFigure 7A: the native symbiont seems to be closer to the epithelial cells in these FISH examples, compared to the other\nbacteria. Is it a consistent pattern? If yes can the authors mention it in the text? It could be in link with the symbiont\nrecognition, and/or the out-competitiveness (for example through gaining access to nutrients the out-competed symbiont\ncannot access because of its regional exclusion).\nConclusion and outlook: I was missing a bit of conclusion on the effect of symbiont replacement on the host fitness, which is\nalso a very important result of this article.", "reviewer_3": "In this work, the authors experimentally test the role of long-term evolutionary codiversification in determining the functional\ncompatibility of mutualistic partners. Using the ancient and specialized symbiosis between Stammera bacterial symbionts\nand their cassidine beetle hosts, they test the hypothesis that symbiotic functon(host fitness, microbial fitness, host gene\nregulation) is associated with the phylogenetic relatedness of the symbiont to the host’s native strain. They accomplish this\nby artificially inoculating eggs with symbiont strains from conspecific and heterospecific beetles, measuring shifts in gene\nexpression and fitness consequences for both host and microbe.\nThis manuscript was a pleasure to review. The authors designed and undertook experiments that exhibit conceptual\nelegance and technical virtuosity, which merit the publication of this work in Nature Communications. While other research\nin the field of host-microbe symbiosis have studied coevolution by undertaking similar approaches of subjecting a panel of\nmicrobes to a single host, this work is groundbreaking in accomplishing this in a system with a long history of vertical\ninheritance. In many ways, this is what makes this paper an important and novel contribution to the field. Often\ncoevolutionary interactions and specificity are assumed in these highly co-diversified interactions, but it is often nearly\nimpossible to test these assumptions because host and symbiont are so tightly metabolically integrated. Remarkably, the\nauthors test this important underlying assumption of co-diversified symbiosis by leveraging a naturally occurring interaction.\nThey demonstrate a capacity for creative experimental design, exquisite attention to detail, and commitment to implementing\ndifficult and delicate manipulations in this non-model system. Beyond this, the demonstrate a novel and remarkable feature\nof this system – the capacity for coinfection. We believe this is a novel contribution for this system, but it is also rare to\ndemonstrate such dynamics broadly in vertically transmitted interactions, especially with the same level of visual spatial\nresolution as demonstrated here. This paper contributes to a small number of studies showing such coinfection potential.\nThe manuscript was extremely well-written, and the authors do a fantastic job placing this study within the broader context of\nthe field.\nThe results overall, while not particularly surprising, establish an essential empirical verification of underlying evolutionary\nassumptions. They not only set the stage for intriguing future experiments but address long-standing questions about the\nrole of coevolution in symbiosis. Perhaps the most striking result is the fact that the co-infected C. alternans host still favors\nthe native symbiont over cross-infecting symbionts despite their functional near-equivalency in terms of host fitness and host\ngene expression, which lends the most credence to their argument that coevolutionary history drives fidelity. The authors\ncould actually emphasize the impact of these results more in their discussion of the results – a symbiont can provide the\nsame level of functional benefit to the host and yet have its transmission inhibited. We would like some discussion of what\nthis means for evolutionary stability and potential. Overall, this is a pretty remarkable result, and this could be brought out\nmore in the discussion.\nWhile we believe this manuscript will make an important contribution, we have some suggestions for improving the overall\nimpact of the findings. First, we believe the discussion of coinfection dynamics could be made stronger if the authors\ndemonstrate the same phenomenon using C. gressoria co-infected with the C. alternans and native symbionts, ruling out the\npossibility that the C. gressoria symbiont is intrinsically a weaker competitor, perhaps due to differences in metabolic\nefficiency or localization that cannot be predicted from a comparison of their genomic contents. As is, the coinfection study\nstill offers interesting and important insights, but the reciprocal cross-infection experiment would strengthen these insights.\nAlternatively, the authors can address this limitation in the text.\nBeyond this suggested experiment we also recommend two points of discussion, and one small assay, which could elevate\nthis manuscript, based on our interpretation of the beautiful microscopy that accompanies this work. First, based on Figure 6\nand Figure S6 in the supplement, it seems that C. alternans OAGs cross-infected with C. gressoria Stammera exhibit striking\nmorphological remodeling, from a complex labyrinthine architecture to a simpler conglomerate form between 5 and 15 days-\nit does not seem to occur in C. alternans OAGs reinfected with the native Stammera. This morphological change is only\nsomewhat clear from the FISH images but is evinced by the Stammera signal, which does not shift markedly for the\nnative/magenta Stammera but does shift to colocalization with the nuclei(?) for the gressorial/green Stammera. Is this an\naccurate interpretation of these images? Or is this an experimental artifact? The images do appear markedly distinct, so if\nthese morphological differences between treatments at day 15 are not a consequence of a biological effect of symbiont\nstrain, the authors should keep their manuscript as is but could briefly explain the methodological cause of these apparent\ndifferences in their response here. If these differences are real, we would appreciate some brief discussion on the potential\nrelevance of this consistent morphological change (as evidenced by Figure S6) for symbiont transmission, and if possible, a\nconventional bright-field microscopy image that displays at least the surface morphology of CIC vs RI OAGs at 15 days.\nIdeally, some higher-magnification FISH images of OAGs demonstrating more clearly the spatial shift in the Stammera probe\nsignal would be appreciated as well.\nSecond, based on Figure 7 and Figure S8 in the supplement, it seems that the native symbiont exhibits a distinctive pattern\nof localization from the gressoria symbiont in the co-colonized foregut organ. The native symbiont seems to monopolize an\nassociation with the DAPI-heavy host nuclei as well as being found lining the lumen of the organ; the gressoria symbiont is\nfound exclusively in the lumen. If the authors agree that this is also the case, we suggest discussing the potential\nimplications of differential localization for these symbionts in competition- specifically, whether one might be more prone to\ndisplacement over host development than the other. If the authors feel a discussion of strain-specific symbiont localization is\ninformative, I also think it is worth devoting some space in Figure 7 to a panel depicting just an overlay of panels A4 and A5\n(or B4 and B5) from Figure S8. The space is there- 7D could be compressed lengthwise, 7E has a lot of white space for just\n3 points, and 7F recapitulates the results of a prior experiment.\nMinor comments:\nLine 63. The authors cite examples of host responses to specialized symbionts that facilitate their selective acquisition.\nWhile in these interactions, host do demonstrate striking specificity for recognition of certain symbionts at a broad taxonomic\nlevel (i.e. Vibrio fischeri or Caballeronia spp.), they do not demonstrate fidelity or specificity at a lineage scale. Given\nthese interactions are horizontally transmitted and there is no co-diversification, there is inherently a lack of partner fidelity in\nthe canonical sense (i.e. at a lineage level). As such, we find the phrasing “fidelity… at the genetic and molecular levels”\nsomewhat awkward. We agree that these interactions are maintained by unique molecular and genetics interactions, and\nthat there is fidelity at the species and genus levels respectively, but there are not recorded patterns of fidelity/specificity that\ntypically associated with patterns of local adaptation. Perhaps consider “display striking adaptations for partner recognition\nat the genetic and molecular levels.”\nLine 63 – Reference 37 is a poor fit for the argument made here. While this manuscript does demonstrates the native\nCaballeronia symbiont gains a competitive advantage over non-native symbionts when colonizing Riptortus hosts, they do\nnot demonstrate molecular and genetic mechanisms underpinning the competitive success of native Caballeronia\nsymbionts. They also do not demonstrate broad patterns of partner specificity by comparing the competitive success\nbetween multiple Caballeronia strains.\nLine 69. This is the first time the name of the symbiont is mentioned outside the abstract, so provide the entire name:\nCandidatus Stammera capleta.\nLines 397-400: If the mechanism for symbiont translocation from the foregut to the OAGs is unknown, the authors could\nconsider weakening this statement to make it clearer that they are speculating the route by which symbiont translocation\noccurred. The non-native symbiont may use the same unidentified pathway, but it very well may have used an alternative\npathway.\nLine 811: “considering”- perhaps “assuming”?\nLines 813-815: In its current format this math is cumbersome to interpret. Consider reformatting as an equation.\nAcross all figures, I strongly urge the inclusion of individual points for the data from each trial on top of all bar plots and box\nplots using ggplot, to provide the reader with information on experimental consistency within replicate trials for each\ntreatment.\nFigure 4: Legend of this figure using inconsistent terminology with the rest of the manuscript and the figure caption. It refers\nto the untreated control as “caplet intact.” This seems to be the only place in the text where this term is used.\nFigure S1ivA, B: These images do not appear to be at the same scale as all the other larval foregut FISH images- provide a\nrevised scale bar.\nFigure S5: I am curious about the differential expression of insect OBP A10 in the foregut organs. Do you think this is ectopic\nexpression of an otherwise tissue-specific protein elicited by the symbiont? Or is this just a misannotated transcript?\nFigure S6: Is the caption correct? It seems that the top left image corresponds to the merged channels, the top right is host\nprobe, bottom left is gressoria green probe, and bottom right is alternans magenta probe. In addition, the layout of the panels\nis somewhat confusing- I would be consistent with Day 5 to Day 15 from left to right.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ">>>>> We thank the reviewer for the positive assessment and helpful suggestions of our\nstudy. We agree that these points could have been emphasized more explicitly in the Abstract\nand Conclusion sections. We have done so here:\nAbstract: ‘We show that non-native S. capleta can successfully colonize the symbiotic organs\nof a novel host, but that the interaction outcome correlates with genetic relatedness to the\nnative symbiont (Lines 19-21).’\nConclusion:\n‘We conducted experimental cross-infections in aposymbiotic eggs of the tortoise beetle C.\nalternans using S. capleta derived from five additional Cassidinae species. Our results\ndemonstrate that larval survival is restored following cross-infections, but only when the non-\nnative symbionts are closely genetically related to the native one. Consistent with this, the\npositive correlation between symbiont genetic distance and host survivorship highlights the\nstrong influence of symbiont genotype on host development (Lines 595-600).’\n‘The introduction of a genetically distant, non-native symbiont triggered a strong transcriptional\nresponse in the host, including activation of immune genes. This was accompanied by a\ndecrease in symbiont population, suggesting host discrimination between native and non-\nnative partners. When both were introduced simultaneously, the native symbiont progressively\noutcompeted the non-native one and was exclusively transmitted to the next generation (Lines\n603-609).’\nOverall, the manuscript is well written and clearly organized, although there are a few points\nof concern:\nIn Figure 4, the authors present experiments in which symbiotic bacteria from different beetle\nspecies were introduced into Chelymorpha alternans, and all symbionts appeared to colonize\nthe symbiotic organ. In this symbiont exchange experiments, for symbionts from four host\nspecies (Chelymorpha gressoria, Acromis sparsa, Aspidimorpha quinquefasciata, and\nCassida rubiginosa), there was a gradual decline in host survival, and most insects eventually\ndied, similar to the aposymbiotic controls. This result could be due to poor colonization by non-\nnative symbionts, but no quantitative data on symbiont titer in the symbiotic organ are\nprovided. Is the observed mortality a consequence of reduced symbiont density, or is it due to\nadequate colonization but low activity (e.g., reduced gene expression, as shown in figure 5E)?\n>>>>> We thank the reviewer for this valuable comment. We fully agree that quantitative data\non symbiont titer would greatly improve the study and therefore additionally investigated\nStammera abundance using quantitative PCR. Our results showed that the observed mortality\nis partly explained by a reduction in symbiont titers, as observed in the cross-infection with\nthe A. quinquefasciata symbiont. However, this pattern does not hold for all high-mortality\ncross-infections, such as those involving the A. sparsa symbiont. This suggests that factors\nbeyond symbiont density, such as low metabolic activity, may affect host survival. The\nmanuscript has been revised accordingly:\n(Lines 207-220): ‘Given the consistent cross-infection of S. capleta stemming from six donor\nbeetle species, we further quantified symbiont loads to determine whether infection success\nwas reflected in S. capeta abundance. Quantitative analyses revealed treatment-dependent\nvariation in symbiont titer (Figure 4B; GLM (quasi-Poisson), χ2 = 119.32, d.f. = 7, p < 0.001;\nTable S2B). S. capleta abundances were significantly comparable between larvae harboring\ntheir native symbiont and those cross-infected with C. bullata, C. gressoria, and A. sparsa\nsymbionts (Figure 4B; pairwise comparison, reinfection: p =1, C. bullata symbiont: p = 1, C.\ngressoria symbiont: p = 1, A. sparsa symbiont: p = 0.084, Table S2B). Conversely, larvae\ncross-infected with A. quinquefasciata and C. rubiginosa symbionts contained lower symbiont\ntiters than the untreated larvae (Figure 4B; pairwise comparison, A. quinquefasciata: p <\n0.001, C. rubiginosa: p < 0.001, Table S2B). qPCR amplification in aposymbiotic individuals\nwas indistinguishable from background (NTC) levels, indicating that the reported copy\nnumbers are likely overestimations and that aposymbiotic beetles may be devoid of symbionts,\nas corroborated by the PCR results.’\n(Lines 234-241): ‘Collectively, this indicates that the observed mortality is only partly explained\nby a reduction in symbiont abundance, as seen in the cross-infection with the A.\nquinquefasciata symbiont (Figure 4B). However, this pattern does not hold for all high-\nmortality cross-infections, such as those involving the A. sparsa symbiont, where symbiont\nabundance is comparable to those of the native S. capleta (Figure 4B). This variation suggests\nthat host survival following our cross-infections may be influenced by factors beyond symbiont\nabundance, including reduced symbiont activity and/or the degree of compatibility between\nhost and microbe. To explore this possibility, we examined whether the mutualistic potential\nof S. capleta in a novel host correlates with its evolutionary relatedness to the native symbiont.’\nWe have revised the manuscript accordingly to reflect these new results.\nWe have also added the corresponding figure (Figure 4B) and updated the Methods section\n(Lines 787-805). The revised raw data, statistical tables, and script are included in the\nsupplementary data (Table S2B).\nThe competitive interactions among microorganisms shown in Figure 7 are particularly\ninteresting. One possible mechanism involves the immune response observed in Figure 5.\nDoes simultaneous colonization by native and non-native symbionts also enhance immune\nactivity in the symbiotic organ, thereby eliminating non-native symbionts? (But it seems likely that\nsuch immune activation would also impact the native symbiont, too.) Alternatively, does the\nnative symbiont simply outcompete non-native symbionts through superior nutrient acquisition or\nother fitness advantages within the symbiotic organ?\n>>>>> We agree that a potential mechanism underlying the native symbiont’s competitive\nadvantage over the non-native symbiont may involve enhanced nutrient acquisition or other\nfitness-related advantages within the symbiotic organ. To address this point, we have revised\nthe manuscript in line with suggestions from the other reviewers (Lines 558-560): ‘The\nobserved distribution may partly explain the native symbiont’s competitive advantage,\npotentially through greater local adaptation and enhanced access to host-derived nutrients.’\nOther points:\nL69: Spell out Stammera in full at first mention.\n>>>>> Done. We included the entire name, Candidatus Stammera capleta (Line 70).\nL92–94: This is a clear summary, but given that multiple symbionts from different lineages\nwere tested, a more detailed discussion of evolutionary distance, fitness effects, and\ntransmission rates (as shown in Figure 4C,D) could be emphasized in this part. This could\nalso be reiterated in the conclusion.\n>>>>> We agree and have now expanded the discussion about the effects of symbiont\nreplacement on host fitness at the end of the introduction (Lines 93-95): ‘non-native symbionts\ncan colonize and differentially restore survivorship in a novel host, with outcomes ranging from\nfull fitness recovery to reduced survival depending on genetic divergence among symbionts.’\nWe also expanded on our findings in the Conclusions section (Lines 595-600): ‘We conducted\nexperimental cross-infections in aposymbiotic eggs of the tortoise beetle C. alternans using\nS. capleta derived from five additional Cassidinae species. Our results demonstrate that larval\nsurvival is restored following cross-infections, but only when the non-native symbionts are\nclosely genetically related to the native one. Consistent with this, the positive correlation\nbetween symbiont genetic distance and host survivorship highlights the strong influence of\nsymbiont genotype on host development.’\nL229: I remain curious about the density of symbiotic bacteria inside the symbiotic organ\nbefore this discussion.\n>>>>> We were curious as well! Per an earlier response, we have quantified Stammera titers\nacross all treatments (Lines 207-220 and 234-241).\nFigure 4A: The methods section does not clearly describe how symbiont presence was\ninvestigated. It would be helpful to include this information in the text and/or figure legend.\n>>>>> Thank you for your comment. The sentence has been changed to clearly describe how\nsymbiont presence was investigated (Lines 774-778): ‘PCR primers targeting either specific S.\ncapleta symbionts (groEL gene) or all symbionts collectively (16S rRNA gene) were used to\nverify the symbiotic status of the host beetles (Table S5C). For cross-infected individuals, a\ngroEL primer pair specific to the native symbiont was used to confirm its successful removal.’\nFigure 4: Clearly state the number of biological replicates (n=).\n>>>>> Done. We added the number of biological replicates to the Figure 4 legend and\nelsewhere throughout the manuscript.\nL315–331: Regarding the differential immune responses, it might be informative to inject\nsymbiotic bacteria into the hemolymph and examine immune gene expression dynamics.\n>>>>> We thank the reviewer for this suggestion and agree that such microinjection assays\ncan be extremely informative. We hope they are the basis of a future study, and have revised\nthe manuscript as following (Lines 617-618): ‘Moreover, the application of microinjection into\nthe host hemolymph to reproduce the mounted immune response is a worthy area for future\nstudy.’\nL403: It is intriguing that the symbiotic bacteria do not enter the caplet despite colonizing the\ntransmitting organ. Do non-native symbionts attempt to transmit but lose viability within the\ncaplet? Since the authors investigated the caplets only by FISH, the distinction between the\nhypothesis that symbionts fail to enter the caplet and the hypothesis that they enter but do not\nsurvive (less active) is not fully discussed.\n>>>>> Thank you for this interesting point. We agree that the distinction is highly informative.\nAlthough not elaborated on in our previous submission, we had collected this data as part of\nour study. Our objective was to assess whether the non-native symbiont can be vertically\ntransmitted to subsequent generations. However, we tested four egg masses from cross-\ninfected females by diagnostic PCR targeting the non-native S. capleta, pooling two egg\ncaplets per egg mass. All samples were found to be devoid of symbionts, consistent with the\nFISH observations and indicating that the non-native symbiont fails to embed within the caplet-\nassociated spheres.\nWe have added this information (Lines 460-462): ‘Diagnostic PCR confirmed the absence of\nsymbionts in eight egg masses tested, demonstrating that the non-native S. capleta failed to\nembed within the maternally secreted spheres.’\nAnd in the Methods section (Lines 889-890): ‘To confirm the FISH results, four egg masses\nfrom cross-infected females were tested by diagnostic PCR targeting the non-native S.\ncapleta, pooling two egg caplets per egg mass.’\nReviewer #1 (Remarks on code availability):\nRaw data and scripts were uploaded appropriately. I don't think there is any problem.\n>>>>> Thank you for your thorough and positive evaluation of our study.\nReviewer #2 (Remarks to the Author):\nIn this particularly elegant study, Pons et al. use a fascinating example of symbiosis as an\nexperimental system to address a fundamental question in the field: what makes partners in\na symbiotic relationship so specific to each other?\nFirst, they demonstrate how tortoise beetle symbioses offer an experimental platform that\nallows transmission of their respective symbionts to be manipulated and various hypotheses\nto be tested following reciprocal exchanges. These reciprocal exchanges successfully lead to\nthe infection of their hosts. They then demonstrate that some of these symbiont exchanges\nmaintain the host's fitness at a level similar to that with its own symbiont, while others lead to\na clear decrease in survival rate. The outcomes of the infections therefore differ greatly from\none symbiont to another, which correlates with genetic differences between the symbionts.\nThey then demonstrate that infection with another symbiont can lead to different outcomes in\nterms of gene expression, even when partially rescuing fitness. They also highlight the\npotential role of immunity in interactions with non-native symbionts. Another striking result of\nthe study is that, despite successful infection of larvae and adults, non-native symbionts do\nnot get transmitted to the next generation. Finally, they demonstrate that, in the context of co-\ninfection with a native symbiont, the non-native symbiont is rapidly outcompeted and lost later\non during adulthood.\nThis is a beautiful and highly original work, offering a striking demonstration of how studying\nan adequate model system can provide a powerful experimental foundation for addressing a\nfundamental question in the field, shedding light on the mechanisms behind fidelity in\nsymbiotic associations. I believe this article will contribute significantly to the field of symbiosis\nand will appeal to a wider audience beyond this (already substantial) area of research. Overall,\nI believe the study and its methodology are sound, and the methods are well described.\nHowever, I have a few remarks that I would like to be taken into account before publication.\n>>>>> Thank you very much for the positive assessment of our study and constructive input\nwhich improved the manuscript.\nMain remarks\nMy main concern is the section on how the host recognises the symbiont.\nWhile the results clearly show that the host responds differently to one of the cross-infected\nsymbionts than to its native symbiont, I think the conclusions stretch the immunity hypothesis\ntoo far at the expense of other alternative hypotheses. While the data presented here support\nthe immunity hypothesis, they do not fully demonstrate it or rule out other hypotheses, such\nas fine metabolic interplay between the symbiont and host. While I agree that differential\nrecognition likely occurs, I don’t agree that the proposed data demonstrate that this recognition\nis immunity-based, nor that the resulting effect is immunity-mediated. For example, the\nassertion in line 344 of a 'heightened immune response' is, in my opinion, overly assertive\nwithout providing evidence of increased AMP expression or ROS production, for example.\nI think the authors could either conduct more experiments to strengthen the evidence behind\nthis conclusion, or they could be more open in their conclusions, including other hypotheses\nand developing the potential immunity mechanisms in a follow-up story.\n>>>>> We agree with the suggestion to adopt a more nuanced approach in our discussion,\nproviding a more balanced interpretation of our transcriptomic data. We elaborated on our\nchanges more specifically below, so please see our response to your queries.\nMore specifically, I would like to see a more thorough discussion about what NPC2 is. It’s also\na sterol-binding protein that is involved in many pathways, not just immune-related ones. For\ninstance, it appears to play a part in maintaining intestinal stem cells in Drosophila. I think it is\ntoo reductive to conclude that the immune pathway is involved based on the change in\nexpression of this gene. To make this claim, I would like to see a measure of the immune\nresponse, starting with AMP expression, in the gut of the host in different infection\nexperiments. It would also be interesting to demonstrate whether the decrease in bacterial\nload is due to the killing of bacteria or the inhibition of their growth. If confirmed, the immune\nresponse itself could also damage the host.\nAnother possibility is that the fine pairing of the host and symbiont relies on transcriptional\ncrosstalk, which could be necessary for the symbiont to grow properly. This crosstalk could\ninvolve NPC2, or it could be independent. Only a loss-of-function experiment (for example,\nRNA interference against NPC2) could resolve this issue.\nThe host responds differently to its symbiont and cross-symbiont. However, the link with\nfitness outcomes could be due to the host not expressing as many digestive enzymes because\nit is not 'prompted' by the presence of its native symbiont. The transcriptomic data also seem\nto point towards genes that are more strictly linked to metabolic function than immune function,\nsuch as lipase activity. Could the metabolic interplay be disturbed when the symbiont is not\nthe native one?\nWith regard to cathepsins and serine proteases, while some of them can be involved in\nimmune responses, not all of them are, and they also have pleiotropic functions, including\ndigestion. Their increased expression may also be the host's reaction to reduced nutrient\naccessibility due to a change in symbionts. Apart from the plant digestive enzymes described\nby the authors, I would be interested to see whether the comparison between the native\nsymbiont and the symbiont from A. quinquefasciata differs in terms of the metabolic\nadvantages they could provide to their hosts, perhaps in relation to lipid metabolism.\nIn conclusion, I would like to see a more nuanced interpretation of these transcriptomic data.\n>>>>> We thank the reviewer again for the helpful feedback. Per our comment above, we\nagree and have refined our discussion and developed alternative hypotheses to offer a more\nbalanced approach that extends beyond the immunity angle:\n(Lines 346-352): ‘Genes encoding Niemann-Pick type C2 (NPC2) proteins were highly\nexpressed in cross-infected larvae colonized by the A. quinquefasciata symbiont compared to\nre-infected control and cross-infected larvae harboring the C. gressoria symbiont (Figures 5,\nC and D; Tables S3, B-E; adjusted p < 0.05; Npc2_1, NB-GLM, χ² = 38.7, d.f. = 2, p <\n0.001; Npc2_2, NB-GLM, χ² = 35.3, d.f. = 2, p < 0.001; Table S4B). NPC2 proteins are known\nto be involved in sterol homeostasis, steroid biosynthesis, and in innate immunity by\nmodulating the IMD pathway in insects 64–67.’\n(Lines 362-366): ‘Colonization by the A. quinquefasciata symbiont also led to elevated\nexpression of protease-encoding genes, including numerous cysteine cathepsins and a serine\nprotease (Figures 5, C and D; Figure S5; Tables S3, B-E; adjusted p < 0.05; NB-GLM/GLM-\nquasipoisson, p < 0.001; Table S4B). These enzymes are known to have pleiotropic roles in\ndigestion, development, and immune function.’\n(Lines 373-383): ‘Our findings suggest that host immunity may contribute to symbiont\nrecognition. Yet they also raise the possibility that a fine-tuned metabolic interplay between\nhost and symbiont, mediated by transcriptional crosstalk, could be essential for proper\nsymbiont growth. The results further reveal upregulation of genes involved in metabolic\nfunctions, such as lipase activity (Figure 5C, Figure S5, Tables S3, B-E; adjusted p < 0.05;\nTable S4B). Such changes could represent a compensatory host response to reduced nutrient\naccess arising from colonization by a metabolically suboptimal symbiont. Consistent with this\ninterpretation, the native (C. alternans) and A. quinquefasciata symbionts differ in their\npotential metabolic profiles, especially in lipid metabolism 37. C. alternans and C. gressoria\nsymbionts encode four and three genes, respectively, related to lipid transport and\nmetabolism, whereas the A. quinquefasciata symbiont lacks genes associated with these\nfunctions.’\n(Line 388): The beginning of the sentence ‘Given a heightened immune response …’ has been\nremoved.\n(Lines 398-402): ‘The pronounced reduction in A. quinquefasciata symbiont abundance\nsuggests that an immune response may have inhibited its proliferation within the foregut\nsymbiotic organ of a novel host. However, it remains unclear whether this decrease reflects\nbacterial killing or reduced growth in an unfavorable host environment.’\n(Line 403): ‘elevated’ has been removed.\n(Lines 411-412): ‘Surprisingly, non-native S. capleta that are closely related to the original\nsymbiont seem to evade immunorecognition by their host.’ has been removed.\nMoreover, we have revised the manuscript accordingly to reflect these new interpretations\n(see the Abstract and the Conclusions).\nFinally, while the number of reads is an indication of a decrease in the population of S. capleta,\na qPCR measurement is required to confirm these results and rule out an effect on the\ntranscriptional activity of the bacteria (which would also be an interesting phenotype).\n>>>>> We thank the reviewer for this valuable comment. We agree and have investigated\nsymbiont abundance using quantitative PCR, and our results confirmed a reduction in the A.\nquinquefasciata symbiont population relative to the native symbiont or the C. gressoria\nsymbiont populations. The manuscript has been revised accordingly:\n(Lines 384-492): ‘We next quantified symbiont abundance using RNA-seq on cDNA and\ndetected significant differences among treatments (Figure 5E; LM, F = 42.12, p < 0.001;\n(2,9)\nTable S4C), consistent with the previously observed proliferation levels of these non-native\nsymbionts (Figure 4B). S. capleta titers did not differ significantly between larvae colonized by\ntheir native symbiont or cross-infected with the C. gressoria symbiont (Figure 5E; pairwise\ncomparison, p = 1; Table S4C). In contrast, both groups contained higher symbiont\nabundance than larvae cross-infected with S. capleta from A. quinquefasciata (Figure 5E;\npairwise comparison, p < 0.001 in both cases; Table S4C). We also mapped S. capleta reads\nto measure the relative presence of symbiotic RNA.’\n(Lines 398-402): ‘The pronounced reduction in A. quinquefasciata symbiont abundance\nsuggests that an immune response may have inhibited its proliferation within the foregut\nsymbiotic organ of a novel host. However, it remains unclear whether this decrease reflects\nbacterial killing or reduced growth in an unfavorable host environment.’\nWe have also added the corresponding figure (Figure 5E) and updated the methods section\n(Lines 858-865). The revised raw data, statistical tables (Table S4C), and script are included\nin the supplementary data.\nMinor points\nThis manuscript is written particularly elegantly, and I enjoyed reading it very much. That said,\nI have a few comments below that I hope will contribute to improving the manuscript.\nThe first three sentences of the abstract are somewhat difficult to follow as they encompass\nmany important and complex concepts. I know the abstract is limited in length, but perhaps\ntry rephrasing or simplifying which ones need to be displayed in this short abstract.\n>>>>> We thank the reviewer and have sought to simplify these three sentences as following\n(Lines 11-13): ‘Obligate co-dependence can arise in symbiosis, yielding heritable\npartnerships. These interactions are considered to be highly specific, but partner fidelity is\ndifficult to quantify owing to the experimental constraints of symbiont exchange between host\nspecies.’\nLine 16 : why « despite » ?\n>>>>> Thank you for the correction. The sentence has been restructured to explain why it is\nunusual for Stammera to possess such a limited metabolism despite existing extracellularly\n(Lines 16-17): ‘Despite its extracellular localization, S. capleta possesses a drastically reduced\ngenome (~0.25 Mb) and is vertically transmitted through egg-associated spheres.’\nLines 22–24: I find it a bit oversimplified to include symbiont recognition and partial rescue in\nthe same sentence, as the link between the two is not fully demonstrated.\n>>>>> We agree and have sought to discuss both findings separately. We have revised both\nthe sentence and the abstract to improve clarity, as follows (Lines 19-26): ‘We show that non-\nnative S. capleta can successfully colonize the symbiotic organs of a novel host, but that the\ninteraction outcome correlates with genetic relatedness to the native symbiont. Genetically\ndistant species trigger a more pronounced transcriptional response and can only partially\nrescue host development. While more closely related symbionts proliferate similarly to native\none and induce a comparable host response, they fail to propagate to the next generation,\nunderscoring how transmission fidelity, host-symbiont compatibility, and local adaptation can\nfurther specificity within a Paleocene-aged partnership.’\nLine 30: The first sentence is abrupt; the term 'specialists' could be explained in more detail.\n>>>>> Done. The sentence has been changed and explained in more detail (Lines 30-31):\n‘The metabolic intimacy of symbiosis demands the work of specialists, each evolving distinct\nyet complementary roles to sustain the partnership.’\nLine 34: Please define or explain what co-diversification is. Also, I’m curious: what is the\nauthor's take on the distinction between co-diversification and co-evolution? The term 'co-\ndiversification' is used throughout the manuscript, so it is important to define it clearly.\n>>>>> We completely agree that defining ‘co-diversification’ is key to many aspects of our\nmanuscript and have done so in lines 34–35: ‘As symbiont and host co-diversify, they exhibit\nparallel lineage splitting over evolutionary timescales.’\nLine 68 : explain what is meant by « streamlined »\n>>>>> We meant to convey that the symbiosis is predicated on few host-beneficial factors.\nHowever, we see how the word may not be immediately helpful and have removed from the\ntext (Line 68).\nLine 87 : Add 'experimental' before 'mechanism', or reformulate to make it clear that the\nauthors are referring to a way of manipulating their experimental model here.\n>>>>> We agree and have added ‘experimental’ before ‘mechanism’ (Line 88).\nLine 134-135 : the sentence is not complete\n>>>>> Thank you, the sentence has been changed (Lines 136-138): ‘On the other hand,\ncross-infected embryos were successfully colonized by the symbiont of C. alternans (Figure\n2B; Figure S1ii), indicating that the cross-infection protocol was reciprocal.’\nLine 208 : « their effects » : please precise which one(s)\n>>>>> We agree and have changed the sentence as following (Lines 221-223): ‘We next\nquantified how these non-native symbionts shaped the development of C. alternans larvae\nand found that their effects on host survival varied, yielding a gradient of mutualistic outcomes\nin larvae.’\nLine 24§-248 : The symbionts share the same genes related to plant digestion. Is there any\npossibility that these genes are not expressed as efficiently in one species as in the other?\nApart from the different host response, this could be one hypothesis that the author cannot\nexclude (at least with the current dataset).\n>>>>> We thank the reviewer for this comment. We agree, we have added this hypothesis to\nthe revised manuscript (Lines 269–271): ‘It is conceivable that differences in the expression\nor regulation of these host-beneficial factors could contribute to the observed variation.’\nFigure 4B. I wondered why the survival lines stopped even for the insect population that was\nstill alive. I think I now understand that this is because we are only following survival until\nmetamorphosis; these individuals are still alive, but are no longer recorded in the assay. It\nwould be easier for readers if this explanation were included in the figure legend.\n>>>>> Absolutely. Thank you for this helpful comment. We agree that additional clarification\nwas required, and we have now added the following explanation to the Figure 4 legend: ‘Beetle\nsurvival was monitored until metamorphosis; truncated lines denote surviving individuals\ncensored from the assay’.\nLine 469: “Both symbionts share 226 genes but differ in only eight.” This is fascinating. I’d love\nto see a short discussion of what are these 8 genes. Also, is it possible that the genomic\ndifference is very small, but the symbionts regulate differentially their expression? (it is maybe\nnot known yet, but I’m curious to know what the authors think)\n>>>>> Thank you very much for this valuable comment. We now included this information in\nour revised manuscript. As shown in Table S9, the C. alternans symbiont uniquely possesses\ngenes associated with translation and energy metabolism (serS, gapA, fldA), suggesting roles\nin protein synthesis and energy production. In contrast, the C. gressoria symbiont encodes\ngenes involved in amino acid and cell wall biosynthesis (glyA, murA, rlmE), indicating\ncontributions to structural maintenance and metabolic support. Although only eight genes\ndiffer between the two symbionts, the competitive advantage of the C. alternans symbiont may\nbe attributed to these specific genetic differences and/or to variations in gene expression\nregulation that lead to distinct physiological capacities. However, since we did not generate a\nmulti-RNA-seq dataset for this study, this remains a hypothesis.\nWe have added this discussion to the revised manuscript (Lines 530-535): ‘The C. alternans\nsymbiont additionally possesses genes associated with translation and energy metabolism,\nwhereas the C. gressoria symbiont encodes genes involved in amino acid and cell wall\nbiosynthesis, reflecting differences in metabolic specialization (Figure S9). The variations in\nsymbiont population growth may be attributed to these specific genetic differences and/or to\nvariations in the regulation of gene expression that led to distinct physiological capacities.’\nLine 481-484: I was a bit lost in this argument that competition increases the specificity of\ntransmission, as the non-native symbiont was shown to not be able to be transmitted to the\nprogeny even when alone. Hence the problem with transmission does not seem to be linked\nto competition but to an incompatibility between the symbiont and host.\n>>>>> Thank you. We have removed the sentence and revised the discussion accordingly,\nconsistent with this and other reviewers’ comments (Lines 551-557): ‘Our findings show that\nonce both symbionts colonize the foregut symbiotic organs, the native population rapidly\nsupplants the non-native one, despite their near-functional equivalence and effects on host\nfitness (Figure 4) and metabolic response (Figure 5). This indicates that the native bacterium\nis better adapted to its host and reinforces the hypothesis that co-evolutionary history is a key\ndriver of fidelity.’\nFigure 7A: the native symbiont seems to be closer to the epithelial cells in these FISH\nexamples, compared to the other bacteria. Is it a consistent pattern? If yes can the authors\nmention it in the text? It could be in link with the symbiont recognition, and/or the out-\ncompetitiveness (for example through gaining access to nutrients the out-competed symbiont\ncannot access because of its regional exclusion).\n>>>>> We thank the reviewer for this important observation. Spatial heterogeneity between\nboth symbiont populations indeed seems to occur, with the native symbiont occupying the\narea adjacent to the epithelium, whereas the C. gressoria symbiont is localized exclusively in\nthe lumen.\nWe have clarified this point in the revised manuscript and discussed its potential implications\nfor symbiont competition (Lines 555-560): ‘We further observed spatial heterogeneity in\nsymbiont distribution across the symbiotic organs. The native bacterium predominantly\noccupies regions adjacent to the epithelium, whereas the non-native bacterium remains\nrestricted to the lumen (Figures 7, Bi-iv; Figure S8). The observed distribution may partly\nexplain the native symbiont’s competitive advantage, potentially through greater local\nadaptation and enhanced access to host-derived nutrients.’\nConclusion and outlook: I was missing a bit of conclusion on the effect of symbiont\nreplacement on the host fitness, which is also a very important result of this article.\n>>>>> Thank you for this comment. We have expanded the discussion about the effects of\nsymbiont replacement on the host fitness (Lines 595-600): ‘We conducted experimental cross-\ninfections in aposymbiotic eggs of the tortoise beetle C. alternans using S. capleta derived\nfrom five additional Cassidinae species. Our results demonstrate that larval survival is restored\nfollowing cross-infections, but only when the non-native symbionts are closely genetically\nrelated to the native one. Consistent with this, the positive correlation between symbiont\ngenetic distance and host survivorship highlights the strong influence of symbiont genotype\non host development.’\nReviewer #3 (Remarks to the Author):\nIn this work, the authors experimentally test the role of long-term evolutionary codiversification\nin determining the functional compatibility of mutualistic partners. Using the ancient and\nspecialized symbiosis between Stammera bacterial symbionts and their cassidine beetle\nhosts, they test the hypothesis that symbiotic functon(host fitness, microbial fitness, host gene\nregulation) is associated with the phylogenetic relatedness of the symbiont to the host’s native\nstrain. They accomplish this by artificially inoculating eggs with symbiont strains from\nconspecific and heterospecific beetles, measuring shifts in gene expression and fitness\nconsequences for both host and microbe.\nThis manuscript was a pleasure to review. The authors designed and undertook experiments\nthat exhibit conceptual elegance and technical virtuosity, which merit the publication of this\nwork in Nature Communications. While other research in the field of host-microbe symbiosis\nhave studied coevolution by undertaking similar approaches of subjecting a panel of microbes\nto a single host, this work is groundbreaking in accomplishing this in a system with a long history of vertical\ninheritance. In many ways, this is what makes this paper an important and novel contribution to the field. Often\ncoevolutionary interactions and specificity are assumed in these highly co-diversified interactions, but it is often nearly\nimpossible to test these assumptions because host and symbiont are so tightly metabolically integrated. Remarkably,\nthe authors test this important underlying assumption of co-diversified symbiosis by leveraging\na naturally occurring interaction. They demonstrate a capacity for creative experimental\ndesign, exquisite attention to detail, and commitment to implementing difficult and delicate\nmanipulations in this non-model system. Beyond this, the demonstrate a novel and remarkable feature\nof this system – the capacity for coinfection. We believe this is a novel contribution for this system, but it is also rare to\ndemonstrate such dynamics broadly in vertically transmitted interactions, especially with the same level of visual spatial\nresolution as demonstrated here. This paper contributes to a small number of studies showing such coinfection potential.\nThe manuscript was extremely well-written, and the authors do a fantastic job placing this study within the broader context of\nthe field.\n>>>>> Thank you very much for the positive assessment and constructive input throughout.\nThe results overall, while not particularly surprising, establish an essential empirical\nverification of underlying evolutionary assumptions. They not only set the stage for intriguing\nfuture experiments but address long-standing questions about the role of coevolution in\nsymbiosis. Perhaps the most striking result is the fact that the co-infected C. alternans host\nstill favors the native symbiont over cross-infecting symbionts despite their functional near-\nequivalency in terms of host fitness and host gene expression, which lends the most credence\nto their argument that coevolutionary history drives fidelity. The authors could actually\nemphasize the impact of these results more in their discussion of the results – a symbiont can\nprovide the same level of functional benefit to the host and yet have its transmission inhibited.\nWe would like some discussion of what this means for evolutionary stability and potential.\nOverall, this is a pretty remarkable result, and this could be brought out more in the discussion.\n>>>>> We thank the reviewers for the constructive feedback. We have emphasized the impact\nof the co-infection experiment from an evolutionary stability perspective in our discussion\n(Lines 555-559): ‘Our findings show that once both symbionts colonize the foregut symbiotic\norgans, the native population rapidly supplants the non-native one, despite their near-\nfunctional equivalence and effects on host fitness (Figure 4) and metabolic response (Figure\n5). This indicates that the native bacterium is better adapted to its host and reinforces the\nhypothesis that co-evolutionary history is a key driver of fidelity.’\nWhile we believe this manuscript will make an important contribution, we have some\nsuggestions for improving the overall impact of the findings. First, we believe the discussion\nof coinfection dynamics could be made stronger if the authors demonstrate the same\nphenomenon using C. gressoria co-infected with the C. alternans and native symbionts, ruling\nout the possibility that the C. gressoria symbiont is intrinsically a weaker competitor, perhaps\ndue to differences in metabolic efficiency or localization that cannot be predicted from a\ncomparison of their genomic contents. As is, the coinfection study still offers interesting and\nimportant insights, but the reciprocal cross-infection experiment would strengthen these\ninsights. Alternatively, the authors can address this limitation in the text.\n>>>>> We thank the reviewers for this valuable comment. We agree that a reciprocal cross-\ninfection experiment would further strengthen our conclusions and have thus addressed this\nlimitation in the revised manuscript (Lines 560-562): ‘We note that a reciprocal dual-infection\nexperiment in C. gressoria would help rule out the alternative possibility that the C. gressoria\nsymbiont is intrinsically a weaker competitor due to differences in its metabolic relative to S.\ncapleta associated with C. alternans.’\nBeyond this suggested experiment we also recommend two points of discussion, and one\nsmall assay, which could elevate this manuscript, based on our interpretation of the beautiful\nmicroscopy that accompanies this work. First, based on Figure 6 and Figure S6 in the\nsupplement, it seems that C. alternans OAGs cross-infected with C. gressoria Stammera\nexhibit striking morphological remodeling, from a complex labyrinthine architecture to a\nsimpler conglomerate form between 5 and 15 days- it does not seem to occur in C. alternans\nOAGs reinfected with the native Stammera. This morphological change is only somewhat clear\nfrom the FISH images but is evinced by the Stammera signal, which does not shift markedly\nfor the native/magenta Stammera but does shift to colocalization with the nuclei(?) for the\ngressorial/green Stammera. Is this an accurate interpretation of these images? Or is this an\nexperimental artifact? The images do appear markedly distinct, so if these morphological\ndifferences between treatments at day 15 are not a consequence of a biological effect of\nsymbiont strain, the authors should keep their manuscript as is but could briefly explain the\nmethodological cause of these apparent differences in their response here. If these differences\nare real, we would appreciate some brief discussion on the potential relevance of this\nconsistent morphological change (as evidenced by Figure S6) for symbiont transmission, and\nif possible, a conventional bright-field microscopy image that displays at least the surface\nmorphology of CIC vs RI OAGs at 15 days. Ideally, some higher-magnification FISH images\nof OAGs demonstrating more clearly the spatial shift in the Stammera probe signal would be\nappreciated as well.\n>>>>> We thank the reviewers for raising this point and agree that the ovary-associated\nglands cross-infected with the C. gressoria symbiont seem to undergo marked morphological\nremodeling, shifting from a complex labyrinthine architecture to a simpler conglomerate form\nbetween 5 and 15 days.\nWe have clarified this point in the revised manuscript and discussed its potential implications\nfor symbiont transmission (Lines 464-4469): ‘The ovary-associated glands cross-infected with\nthe C. gressoria symbiont appear to undergo marked morphological remodeling compared to\nthose infected by the native symbiont, from a complex labyrinthine architecture to a simpler\nconglomerate form between 5 and 15 days (Figures 6D; Figure S6). How, and whether, such\na morphological change affects to the failure to transmit the non-native symbiont is unknown.’\nSecond, based on Figure 7 and Figure S8 in the supplement, it seems that the native symbiont\nexhibits a distinctive pattern of localization from the gressoria symbiont in the co-colonized\nforegut organ. The native symbiont seems to monopolize an association with the DAPI-heavy\nhost nuclei as well as being found lining the lumen of the organ; the gressoria symbiont is\nfound exclusively in the lumen. If the authors agree that this is also the case, we suggest\ndiscussing the potential implications of differential localization for these symbionts in\ncompetition- specifically, whether one might be more prone to displacement over host\ndevelopment than the other. If the authors feel a discussion of strain-specific symbiont\nlocalization is informative, I also think it is worth devoting some space in Figure 7 to a panel\ndepicting just an overlay of panels A4 and A5 (or B4 and B5) from Figure S8. The space is\nthere- 7D could be compressed lengthwise, 7E has a lot of white space for just 3 points, and\n7F recapitulates the results of a prior experiment.\n>>>>> We thank the reviewers for this insightful observation. We completely agree. Spatial\nheterogeneity indeed seems to occur, with the native symbiont occupying the area adjacent\nto the epithelium, whereas the C. gressoria symbiont is localized exclusively in the lumen.\nReviewer 2 similarly commented on this pattern.\nWe have clarified this point in the revised manuscript and discussed its potential implications\nfor symbiont competition (Lines 555-560): ‘We further observed spatial heterogeneity in\nsymbiont distribution across the symbiotic organs. The native bacterium predominantly\noccupies regions adjacent to the epithelium, whereas the non-native bacterium remains\nrestricted to the lumen (Figures 7, Bi-iv; Figure S8). The observed distribution may partly\nexplain the native symbiont’s competitive advantage, potentially through greater local\nadaptation and enhanced access to host-derived nutrients.’\nIn addition, we changed Figure 7 by adding panels B4 and B5 from Figure S8.\nMinor comments:\nLine 63. The authors cite examples of host responses to specialized symbionts that facilitate\ntheir selective acquisition. While in these interactions, host do demonstrate striking specificity\nfor recognition of certain symbionts at a broad taxonomic level (i.e. Vibrio fischeri or\nCaballeronia spp.), they do not demonstrate fidelity or specificity at a lineage scale. Given\nthese interactions are horizontally transmitted and there is no co-diversification, there is\ninherently a lack of partner fidelity in the canonical sense (i.e. at a lineage level). As such, we\nfind the phrasing “fidelity… at the genetic and molecular levels” somewhat awkward. We agree\nthat these interactions are maintained by unique molecular and genetics interactions, and that\nthere is fidelity at the species and genus levels respectively, but there are not recorded\npatterns of fidelity/specificity that typically associated with patterns of local adaptation.\nPerhaps consider “display striking adaptations for partner recognition at the genetic and\nmolecular levels.”\n>>>>> We agree and have changed the sentence as requested (Line 64).\nLine 63 – Reference 37 is a poor fit for the argument made here. While this manuscript does\ndemonstrates the native Caballeronia symbiont gains a competitive advantage over non-\nnative symbionts when colonizing Riptortus hosts, they do not demonstrate molecular and\ngenetic mechanisms underpinning the competitive success of native Caballeronia symbionts.\nThey also do not demonstrate broad patterns of partner specificity by comparing the\ncompetitive success between multiple Caballeronia strains.\n>>>>> Thank you for the comment. We have removed the reference from that sentence.\nLine 69. This is the first time the name of the symbiont is mentioned outside the abstract, so\nprovide the entire name: Candidatus Stammera capleta.\n>>>>> Thank you. We wrote the entire name, Candidatus Stammera capleta (Line 70).\nLines 397-400: If the mechanism for symbiont translocation from the foregut to the OAGs is\nunknown, the authors could consider weakening this statement to make it clearer that they\nare speculating the route by which symbiont translocation occurred. The non-native symbiont\nmay use the same unidentified pathway, but it very well may have used an alternative\npathway.\n>>>>> We agree with your comment. The statement has been modified to clarify that this is\nspeculation (Lines 451-454): ‘The mechanism by which symbionts translocate from the foregut\nsymbiotic organs to the ovary-associated glands is still unknown, but it is conceivable that the\nnon-native symbiont can coopt this pathway to reach the transmission organs of its novel host\n(Figures 6, B-D; Figure S6).’\nLine 811: “considering”- perhaps “assuming”?\n>>>>> Done. We replaced ‘considering’ with ‘assuming’ (Line 933).\nLines 813-815: In its current format this math is cumbersome to interpret. Consider\nreformatting as an equation.\n>>>>> We agree. The math has been reformatted as equations (Line 937 and Line 945).\nAcross all figures, I strongly urge the inclusion of individual points for the data from each trial\non top of all bar plots and box plots using ggplot, to provide the reader with information on\nexperimental consistency within replicate trials for each treatment.\n>>>>> We agree. Dots indicating replicates or the average of each replicate have been added\nto each bar plot and box plot. The figure legends and scripts have been modified accordingly.\nFigure 4: Legend of this figure using inconsistent terminology with the rest of the manuscript\nand the figure caption. It refers to the untreated control as “caplet intact.” This seems to be the\nonly place in the text where this term is used.\n>>>>> We agree. The terminology in Figure 4 and its legend has been changed to be\nconsistent with the rest of the manuscript.\nFigure S1ivA, B: These images do not appear to be at the same scale as all the other larval\nforegut FISH images- provide a revised scale bar.\n>>>>> Thank you. These images have been modified to be on the same scale as all other\nlarval foregut FISH images.\nFigure S5: I am curious about the differential expression of insect OBP A10 in the foregut\norgans. Do you think this is ectopic expression of an otherwise tissue-specific protein elicited\nby the symbiont? Or is this just a misannotated transcript?\n>>>>> Excellent point. We are confident that this is not a misannotated transcript. In addition\nto our manual annotation of this gene, OBP has been previously associated with symbiont\nsurvival and vertical transmission in plataspid stinkbugs (Koga et al. 2021, PNAS) and with\nhemocyte regulation by the obligate mutualist Wigglesworthia in tsetse flies (Benoit et al. 2017,\neLife). These findings suggest that this gene may play a role in mediating the specificity of the\nS. capleta – Cassidinae symbiosis.\nFigure S6: Is the caption correct? It seems that the top left image corresponds to the merged\nchannels, the top right is host probe, bottom left is gressoria green probe, and bottom right is\nalternans magenta probe. In addition, the layout of the panels is somewhat confusing- I would\nbe consistent with Day 5 to Day 15 from left to right.\n>>>>> Thank you very much. The legend has been corrected and the layout of the panels\nredesigned.\nReviewer #3 (Remarks on code availability):\nWe just briefly examined the data files and scripts to ensure they were present and the authors\ndemonstrated transparency. We did not run the scripts ourselves. The R scripts were well\nannotated and the .csv files appear complete and readable. They did not include a README\nof the files included themselves, but overall this dataset appears straightforward. These are\nsimply datafiles and scripts for plots and stats. There are no models to review. The data/scripts\nuploaded is fine as is.\n>>>>> Thank you for your thorough and positive evaluation of our study.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\n>>>>> Thank you for helping us improve our study."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have provided precise responses to all my comments, and I am satisfied. The qPCR results are very clear and sufficiently reinforce the results of this study. I have no further comments to add. I really look forward to more in-depth analysis of the mechanisms underlying these symbiotic specificities in the future.", "reviewer_2": "The revised version has been largely improved. All the concerns that I had raised have been addressed satisfactorily.\nAll in one, I think this is a very interesting contribution.", "reviewer_3": "We are pleased with the revisions made to the manuscript and your thoughtful responses to each of our suggestions.\nOverall, we believe this is interesting work and is ready for publication. We do have one very minor edit. In line 327, you refer to Figure 4B when we think you should refer to Figure 4C. In the text, you refer to the effect of A. quinquefasciata on larval rescue, but Figure 4B shows symbiont titer within host. Figure 4C shows the effect or symbiont on survival.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ">>>>> We thank you for your positive feedback on our study.\n>>>>> Thank you very much for your favorable evaluation of our work.\n>>>>> Thank you very much for this positive assessment and for pointing out this error. The\nreference to Figure 4B has now been updated to Figure 4C.\n>>>>> We thank you for your favorable evaluation of our study."}]

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