. Author manuscript; available in PMC: 2023 Apr 7.

Published in final edited form as:Curr Biol. 2022 Aug 12;32(18):3925–3938.e6. doi:10.1016/j.cub.2022.07.036

Rational engineering of a synthetic insect-bacterial mutualism

Yinghua Su^1,^*,Ho-Chen Lin¹,Li Szhen Teh¹,Fabienne Chevance¹,Ian James¹,Clara Mayfield¹,Kent G Golic¹,James A Gagnon¹,Ofer Rog¹,Colin Dale^1,^1,^*

¹School of Biological Sciences, University of Utah, Salt Lake City, UT, 84112, United States of America.

Lead contact

AUTHOR CONTRIBUTIONS

Y.S., H.L, K.G.G. and C.D. designed experiments. Y.S., H.L., L.S.T., I.J. F.C., J.G., O.R., C.M. and C.D performed experiments. Y.S. and C.D. analyzed data, wrote the manuscript and prepared figures. All authors read and provided edits for the manuscript and agree to its contents.

Correspondence:crystal.su@utah.edu (Y. Su),colin.dale@utah.edu (C. Dale)

Issue date 2022 Sep 26.

PMC Copyright notice

PMCID: PMC10080585 NIHMSID: NIHMS1884393 PMID:35963240

SUMMARY

Many insects maintain mutualistic associations with bacterial endosymbionts, but little is known about how they originate in nature. In this study, we describe the establishment and manipulation of a synthetic insect-bacterial symbiosis in a weevil host. Following egg injection, the nascent symbiont colonized many tissues, including prototypical somatic and germinal bacteriomes, yielding maternal transmission over many generations. We then engineered the nascent symbiont to overproduce the aromatic amino acids, tyrosine and phenylalanine, that facilitate weevil cuticle strengthening and accelerated larval development, replicating the function of mutualistic symbionts that are widely distributed among weevils and other beetles in nature. Our work provides empirical support for the notion that mutualistic symbioses can be initiated in insects by the acquisition of environmental bacteria. It also shows that certain bacterial genera, including theSodalis spp. used in our study, are predisposed to developing these associations due to an ability to maintain benign infections and undergo vertical transmission in diverse insect hosts, facilitating the partner fidelity feedback that is critical for the evolution of obligate mutualism. These experimental advances provide a new platform for laboratory studies focusing on the molecular mechanisms and evolutionary processes underlying insect-bacterial symbiosis.

Graphical Abstract

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INTRODUCTION

Insects are one of the most successful and diverse groups of animals with 10% of species estimated to harbor obligate mutualistic bacterial endosymbionts^1,2. Endosymbionts enhance insect fitness by providing essential nutritional supplements^3,4 or protection against enemies^5–8, stress⁹ or toxins^10,11. Acquisition of nutritional symbionts allows many insects (including the grain weevils,Sitophilus spp., highlighted in this study) to persist on diets that are nutritionally imbalanced or incomplete and has facilitated substantial niche expansion in insects, contributing greatly to the ecological success¹².

Insect endosymbionts are often derived from environmental progenitors with large gene inventories and capability to synthesize myriad nutrients (e.g. essential amino acids and vitamins) that many eukaryotes cannot synthesizede novo^13–15. Following establishment of mutualistic associations, endosymbionts undergo a degenerative mode of evolution, facilitating (i) loss of metabolic functions shared with the insect host and (ii) retention and potentiation of functions beneficial to the fitness of the association, including nutrient-provisioning pathways. These changes lead to establishment of associations in which partners are obligately co-dependent and metabolically integrated^15–17. Consequently, hosts often cannot be reared without their symbiotic partners, which often cannot be cultivated outside their hosts (e.g. in laboratory media), constraining experimentation. One essential and defining aspect that remains poorly understood is the transition to stable vertical symbiont transmission, requiring establishment of infection in reproductive tissues and developing oocytes. Interestingly, certain bacteria (e.g.Sodalis spp.,Arsenophonus spp.,Spiroplasma spp.) are predisposed to developing relationships with insects^15,18,19, suggesting maintenance of specialized properties that facilitate this outcome.

Grain weevils provide an excellent model to study establishment of symbiosis because it is possible to remove their native bacterial symbiont (Sodalis pierantonius) through antibiotic treatment and maintain resulting aposymbiotic (symbiont-free) weevils in the laboratory²⁰. Previous studies have shown thatS. pierantonius supplements its host with vitamins and amino acids²¹. Notably, it secretes tyrosine and phenylalanine during larval and early adult stages to facilitate cuticle strengthening²⁰. In addition, it triggers development of bacteriomes, housing symbionts and protecting them from insect innate immunity^22,23. Further, these bacteriomes are absent or markedly reduced in size in aposymbiotic insects, indicating that symbiotic interactions influence host developmental processes²⁰.

Interestingly, the symbiosis involving weevils andS. pierantonius is recent in origin^15,24,25. In addition, a diverse range of insects harborSodalis-allied symbionts that perform distinct nutritional functions (e.g. mealybugs²⁶, tsetse flies²⁷, seal lice²⁸, louse flies²⁹, stinkbugs³⁰, lygaeoid bug³¹, psyllids³²). This suggests that free-livingSodalis spp. have repeatedly and independently colonized insects inhabiting a wide range of niches³³, catalyzing novel mutualistic relationships with diverse functions. Several studies have exploited the use of a close free-living relative of theSodalis-allied symbionts, namedS. praecaptivus²⁴. This bacterium has a relatively large genome with a high coding density and few pseudogenes, consistent with the notion that it evolves under strong stabilizing selection in a free-living/opportunistic lifestyle. Comparative studies indicate that related insect symbionts have gene inventories that are subsets ofS. praecaptivus. They are substantially reduced in coding content, indicating that they have evolved degeneratively, under a relaxed selection pressure facilitating loss of gene functions that lack adaptive value in symbiosis. BecauseS. praecaptivus is amenable to culture³⁴ and genetic manipulation³⁵ and yields stable and benign infections in insect hosts that naturally harborSodalis-allied symbionts^36,37, it has proved useful in studying the mechanistic interactions underpinning symbiosis. These studies are performed by microinjecting adult insects with mutant strains ofS. praecaptivus and examining their effects. However, in the case of grain weevils, which are oviparous and therefore require bacterial infection of oocytes in female ovaries to facilitate vertical transmission,S. praecaptivus is not observed to be maternally transmitted following adult microinjection³⁶. Tsetse flies, which are viviparous and nourish developing larvae via milk gland secretions during pregnancy³⁸, undergo a low frequency of vertical transmission ofS. praecaptivus following adult microinjection³⁷, but it is insufficient to facilitate experimentation.

One explanation for the inability ofS. praecaptivus to achieve vertical transmission in grain weevils following adult microinjection is that bacteria may need to establish infection in germline stem cells. This makes sense considering our understanding of the natural association between grain weevils andS. pierantonius, in which adult weevils maintain two populations of symbiotic bacteria²⁰: “germinal” (facilitating maternal transmission) and “somatic” (facilitating nutrient production).

In this study, we describe a protocol for microinjection ofS. praecaptivus into eggs of the grain weevilSitophilus zeamais, resulting in sustained vertical transmission over multiple insect generations, providing a means for partner-fidelity feedback to facilitate evolution of mutualistic functions. We use this new experimental platform to introduce mutant strains ofS. praecaptivus with modified tyrosine and phenylalanine biosynthetic capabilities. Notably, these strains significantly impact weevil cuticle sclerotization and larval development time, providing a clear genetic validation of the role of aromatic amino acid production in this symbiosis. This work demonstrates that aS. praecaptivus mutant with a singletyrR gene knockout can overproduce tyrosine and phenylalanine to impact host cuticle sclerotization and reduce larval development time, signifying that the relationship is mutualistic.

RESULTS

Egg injection establishes a synthetic, insect-bacterial symbiosis

We developed a procedure for microinjection ofS. praecaptivus into grain weevil eggs to test the hypothesis that egg infection leads to vertical transmission of bacteria. This procedure uses a modifiedDrosophila egg microinjection protocol, followed by transplantation of larvae into grain, facilitating development to adulthood. The procedural efficiency was monitored for a batch of injections performed on 96 aposymbiotic weevil eggs. Herein, 40% (38/96) of eggs incurred lethal damage during isolation and preparation for injection. Out of the remaining 58, 50% (29/58) survived and yielded larvae. Following transplantation into maize, 21% (6/29) of larvae completed development and emerged as adults. All six demonstrated mCherry fluorescence, indicative ofS. praecaptivus MC1 infection. First instar larvae maintained 3.74 × 10⁴ bacterial CFU / larva (Mean; SD = 2.88 × 10⁴), increasing to 7.95 × 10⁶ CFU / weevil (Mean; SD = 2.19 × 10⁶ ) in newly emerged adults.

Following injection, weevils were monitored using fluorescence microscopy to track bacteria. Uninjected weevils demonstrated no mCherry fluorescence in egg, larval or adult stages (Figure S1A–D). Following injection into the egg posterior pole (Figure 1A), bacteria proliferated at the injection site (Figure 1B) and then migrated through the embryo, achieving dense infection in the developing gut (Figure 1C) and resulting first instar larvae (Figure 1E). Following metamorphosis, adults demonstrated widespread mCherry fluorescence (Figure 1F), consistent with the presence ofS. praecaptivus MC1 in hemolymph (Figure S2A), and other tissues. Adult ovaries harboredS. praecaptivus MC1 in several regions (Figure 1I) including the tropharium apex where the native symbionts of grain weevils are localized²⁰.

Figure 1: — (A) Schematic and micrograph showing microinjection into the egg posterior pole (PP). Subsequent images (B-I) are shown under normal light and under mCherry fluorescence. (B) Egg one day post injection (PI) showing infection at PP. (C) Egg four days post injection, with infection progressing. (D) Egg four days post injection with a Δ*ypeI* mutant, showing extensive pathogenesis. (E) First instar larva, immediately following emergence from microinjected egg. (F) Adult weevil, injected at egg stage following emergence from grain. (G) Egg derived from microinjected parents that acquiredS. praecaptivus via maternal transmission at five days post deposition (PD). (H) First instar larva derived from egg-microinjected parents. (I) Ovaries from mated aposymbiotic female derived from microinjected egg, showing extensive colonization. See alsoFigure S1 andVideo S1.

To further explore the utility of this technique, we injected a mCherry-expressingS. praecaptivus strain lackingypeI, encoding an N-acyl homoserine lactone synthase involved in quorum sensing. This strain kills weevils following microinjection into adults because quorum sensing represses expression of virulence factors, including insecticidal toxins³⁶. Following egg injection, this strain proliferated rapidly in eggs, revealing dense infection after four days (Figure 1D). Out of 55 eggs injected with this strain, only one (uninfected) larva emerged, indicating that the ΔypeI strain efficiently kills eggs. This illustrates the utility of the egg microinjection procedure in exploring molecular mechanisms of symbiosis throughout the entire developmental cycle of the host.

Egg injection yields sustained vertical transmission ofS. praecaptivus

To determine ifS. praecaptivus MC1 undergoes vertical transmission following egg injection, we tracked ten generations of weevils derived from a single isofemale and isomale aposymbiotic weevil pairing that were successfully infected by egg microinjection. From this line, ten randomly selected F₁ offspring were found to be infected withS. praecaptivus MC1, having an average of 2.5 × 10⁶ CFU / weevil (SD = 2.09 × 10⁶). Fluorescence microscopy revealedS. praecaptivus MC1 in those F₁ eggs and larvae, confirming that bacteria had been acquired vertically (Figure 1G–H). In the F₂ generation, the adult infection frequency declined to 50% (n = 20), with an average number of 1.39 × 10⁶ CFU / weevil (SD = 1.04 × 10⁶), excluding two samples that were considered outliers having very low densities of bacteria. Based on the decline in the F₂ generation, we elected to maintain only weevils showing mCherry fluorescence to serve as parents for the F₃ generation. This selection was repeated at generational intervals throughout the experiment to ensure that sufficient number of weevils maintainedS. praecaptivus MC1. The rate of vertical transmission and infection density remained relatively constant in subsequent generations (Figure 2A). The high level of transmission from P₀-F₁ stage is likely explained by high numbers of bacterial cells in eggs following injection, yielding a high level of infection in female ovaries, consistent with the observation that bacterial infection densities in adult weevils were higher in P₀ individuals and settled to a lower consistent level in subsequent generations. Nine additionalS. praecaptivus MC1-injected aposymbiotic isofemale lines were established to assess repeatability of the procedure. While all yieldedS. praecaptivus MC1-infected offspring, the transmission rate varied from 20% to 100%, with a median of 95% and mean of 78% (Figure 2B) and an aggregate average of 1.21 × 10⁶ CFU / weevil (SD = 1.83 × 10⁶). Differences may arise due to variation in the age of the eggs (0–24 h), a factor known to affectDrosophila egg microinjection as well³⁹. Alternatively, variation in the bacterial inoculum or the precise site of the injection may affect the success of the procedure.

Figure 2: — (A) Infection frequency and average bacterial density (with error bars showing standard deviation) in adult weevils over ten generations. (B) Dynamics of F₁ infection in multiple replicated egg injection experiments involving aposymbiotic (apo) and symbiotic (sym) grain weevils (n=10 for each line). (C) Kaplan-Meier analysis of association between infection and developmental status. (D) Infection status of the first seven and last seven offspring obtained from six individual aposymbiotic F₆ females infected withS. praecaptivus MC1, demonstrating no significant difference.

While the majority of our experiments were performed on aposymbiotic weevils, we were also interested to determine how symbiotic weevils, harboring their native symbiont (Sodalis pierantonius), responded to introduction ofS. praecaptivus MC1 into their eggs. Notably, the procedure was also successful with symbiotic weevils, yielding P₀ adults with average infection density of 9.95 × 10⁶ CFU / weevil (SD = 1.25 × 10⁷). However, a substantially lower level of transmission was observed relative to aposymbiotic weevils (Mean = 19%) with only four of nine isofemale lines producingS. praecaptivus MC1-infected offspring (with aggregated average of 3.47 × 10⁶ CFU / weevil; SD = 3.91 × 10⁶;Figure 2B). This indicates thatS. praecaptivus andS. pierantonius can coexist and be transmitted simultaneously but thatS. praecaptivus transmission is constrained by the presence of the native symbiont, which is transmitted with ~100% efficiency in our laboratory population.

Throughout our experiments, in order to identify weevils infected withS. praecaptivus MC1, we employed a simple screening method in which live insects were inspected for mCherry fluorescence. However, this detection method could fail to identify weevils that maintain low-density infections. Yet, a low-density infection could be sufficient to lead to transmission of bacteria to offspring, leading to an underestimate of transmission frequency. To evaluate this, 30 offspring from 30 non-fluorescent parents (F₆ derivatives) were checked forS. praecaptivus MC1 by homogenization and plating. Notably, none of those weevil homogenates yieldedS. praecaptivus MC1 colonies, indicating that absence of fluorescence in parents is strongly correlated with the absence of bacteria in offspring.

Dynamics ofS. praecaptivus transmission

To determine if transmission/maintenance ofS. praecaptivus MC1 is biased towards offspring sex, we selected 100 random offspring from generation F₆ of the infected aposymbiotic weevils, checked them for mCherry fluorescence and dissected them to determine sex. No significant difference existed between sexes with 29/40 males and 37/60 females harboring infections,X² (1,N = 100) = 1.255,p > 0.05. To determine ifS. praecaptivus MC1 is associated with increased development time, 30 matedS. praecaptivus MC1-infected F₆ weevils oviposited for three days and their offspring emergence time and infection status were tracked. Kaplan-Meier analysis revealed no significant difference in development time between infected and uninfected weevils (Figure 2C;p = 0.34). To investigate stability of theS. praecaptivus infection throughout development, 20 1^st instar larvae and 20 adult offspring were collected from 30 infected F₇ parents, for bacterial enumeration. Their infection frequencies demonstrated no significant difference between larvae (40%; 8/20) and adults (45%; 9/20;X² (1,N = 40) = 0.102,p > 0.05), indicating robustness over the course of development. To check if transmission ofS. praecaptivus MC1 is influenced by female reproductive age, sixS. praecaptivus MC1-infected F₆ females in the first 14 days of adulthood were allowed to oviposit for three weeks on fresh maize and adult offspring were collected until no more emerged. Offspring were homogenized and plated to determine infection. We then compared the first seven and last seven offspring from each female, revealing no significant difference in infection frequency (Figure 2D;X² (1,N = 43) = 8.712,p > 0.05.). Finally, we performed a crossbreeding experiment using unmated F₆ weevils to determine sexual dynamics ofS. praecaptivus transmission. Six pairs were assembled for mating, three of which comprised an uninfected male and infected female and three of which comprised the reciprocal combination. Offspring from each pairing (n = 30) were homogenized and plated to check for infection. All offspring maintainingS. praecaptivus MC1 were derived from infected females, indicating exclusively maternal transmission.

Throughout this study, no morphological or behavioral abnormalities were observed in either aposymbiotic or symbiotic weevils at any life cycle stage following establishment ofS. praecaptivus MC1. Further, following microinjection, adult weevils emerged at a median 45 days for aposymbiotic weevils and 41 days for symbiotic weevils following transfer into grain. This is comparable to the uninfected weevils subjected to identical husbandry lacking only the microinjection (aposymbiotic median 44.5 days; symbiotic median 39 days).

S. praecaptivus colonizes prototypical bacteriomes in grain weevils

The grain weevils native symbiont,S. pierantonius, resides in specialized bacteriomes at the anterior of the midgut in larvae, the midgut mesenteric caeca in young adults and ovaries of adult females²⁰. To determine ifS. praecaptivus MC1 infects the same tissues in aposymbiotic weevils, we visualized tissues of F₁ larvae and adults. Both larval and adult bacteriomes that are potentiated in symbiotic weevils are colonized intracellularly byS. praecaptivus MC1 in both aposymbiotic and symbiotic weevils, albeit at higher density in the latter (Figure 3;Figure S2B). Thus, onlyS. pierantonius, induces larval and gut bacteriome cell proliferation^40,41. Symbiotic weevils featured fully formed larval and adult bacteriomes, densely infected with bothS. praecaptivus MC1 andS. pierantonius (Figure 3A and3D), mimicking experimental outcomes observed in aphids^42,43. BecauseS. pierantonius, has a distinct morphology (Figure 3B), microscopy clearly revealed both bacterial species inside the same larval and adult bacteriome cells (Figure 3C and3E). Bacteriomes from uninjected weevils demonstrated no mCherry fluorescence (Figure S1E andF).

Figure 3: — (A) Larval gut with white circle highlighting the bacteriome that develop only in sym weevils, shown under normal (left) and fluorescent (right) light. (B) Scanning electron micrograph (SEM) of the weevil symbiont,*S. pierantonius,* isolated from uninjected symS. zeamais bacteriome, showing distinctive spiral morphology. (C) Confocal image of larval gut bacteriome from sym weevil, stained with Hoechst 33342 (blue; targeting nucleic acid), showing co-habitation ofS. praecaptivus MC1 (red) andS. pierantonius (blue spirals). (D) Adult gut from newly emerged weevils with white circles highlighting cecal bacteriomes that form only in sym weevils. (E) Confocal image of cecal bacteriome from sym weevil, stained with Hoechst 33342 (blue; targeting nucleic acid) and CellMask Green (yellow: targeting cell membranes), showing co-habitation ofS. praecaptivus MC1 (red) andS. pierantonius (blue spirals). Inset images in panels C&E are zoomed and enhanced in contrast. See alsoFigure S1 andS2.

S. praecaptivus infects weevil eggs at early stage of oogenesis

Figure 1I shows widespread infection ofS. praecaptivus MC1 in the ovary. For more in depth characterization, we performed confocal microscopy on ovaries fromS. praecaptivus MC1-infected F₁ females (Figure 4). In the telotrophic weevil reproductive system, germ cells are localized in a transition zone between the tropharium and vitellarium. The developing oocytes receive nutrients from nurse cells in the tropharium via nutritive cords⁴⁴.S. praecaptivus MC1 was present in tropharium cells in both aposymbiotic (Figure 4A) and symbiotic (Figure 4B) weevils, suggesting bacteria could be transmitted to developing oocytes from nurse cells. However,S. praecaptivus MC1 also infected the zone between the tropharium and vitellarium (Figure 4C), containing pro-oocytes, along with central and lateral prefollicular cells⁴⁵. To facilitate oocyte development, pro-oocytes are encapsulated by prefollicular cells in the vitellarium to form egg chambers. Even the most proximal oocytes in the weevil vitellarium maintainedS. praecaptivus in the oocyte and surrounding follicular cells, indicating that oocytes are infected at a very early stage of development.

Figure 4: — Specimens were stained with Hoechst 33342 (blue: targeting nucleic acid) and CellMask Green (yellow: targeting cell membranes). (A) Tropharium from adult apo weevil, showingS. praecaptivus MC1 inside tropharium cells. (B) Tropharium from adult sym weevil, showing co-existence ofS. praecaptivus MC1 andS. pierantonius. (C) Vitellarium from adult apo weevil, withS. praecaptivus MC1 in epithelial cells, developing oocytes and the tropharium/vitellarium transition zone containing pro-oocytes.

In order to confirm that adult injection does not facilitate establishment ofS. praecaptivus infection that is maternally transmitted, we performed an experiment in which weevils were injected at adult stage withS. praecaptivus MC1. Out of 33 weevils, 28 developed mCherry fluorescence, indicating infection. However, following mating, no offspring displayed mCherry fluorescence or yieldedS. praecaptivus colonies when their homogenates were plated (n=30), confirming that adult injection does not lead to vertical transmission. Imaging of adult-injected weevil ovaries revealedS. praecaptivus MC1 attached to the exterior of the tropharium (Figure S2C) and vitellarium (Figure S2D), but no infection inside these structures. This was also occurred with midgut mesenteric ceca, which demonstrated only surface colonization withS. praecaptivus MC1 following adult microinjection (Figure S2E).

Rational engineering of a functional mutualism

Knowing thatS. pierantonius produces tyrosine and phenylalanine that promotes cuticular sclerotization²⁰, we engineered strains ofS. praecaptivus with modified biosynthetic capabilities. These encompass a Tyr/Phe auxotroph (ΔpheA-tyrA) and numerous candidate Tyr/Phe overproducing strains that were identified during rational engineering approaches inE. coli⁴⁶. While several mutantS. praecaptivus strains (ΔtyrR, ΔnuoN, ΔcsrA, Δzwf and Δmdh) demonstrated Tyr/Phe cross-feeding (Figure 5A;Figure S3A), the ΔtyrR strain was selected for our experiments because TyrR functions specifically as a repressor for genes of aromatic amino acid biosynthesis⁴⁷, whereas the other mutants are anticipated to have broader impacts on metabolic processes, potentially impacting the symbiosis. Tyr/Phe secretion was then confirmed for the ΔtyrR strain, using a liquid assay (Figure 5B). ΔpheA-tyrA, ΔtyrR and WT strains were then introduced into aposymbiotic weevil eggs. These strains lacked mCherry, in order to avoid confounding subsequent cuticle color assays.

Figure 5: — (A) Plate-based assay on minimal medium, showing a Δ*tyrR* overproducer cross-feeding Δ*pheA-tyrA* auxotroph. (B) Growth of an auxotrophic Δ*pheA-tyrA* strain over seven days in minimal medium alone or in the presence of wild type or Δ*tyrR* strains following inoculation of cells at equal densities. The auxotrophic Δ*pheA-tyrA* strain shows significant growth increase only in the presence of the Δ*tyrR* overproducer, relative to the wild type strain (>10 fold;p < 0.01). See additional data presented inFigure S3B. (C) Thorax cuticular redness of two-week-old sym weevils and their apo derivatives with and without Δ*pheA-tyrA,* WT and Δ*tyrR* strains ofS. praecaptivus injected at egg stage. Boxes on left show the raw images associated with the highest and the lowest red values in the dataset. (D) Larval development time of sym weevils and apo counterparts with and without Δ*pheAtyrA,* WT and Δ*tyrR* strains injected at egg stage. Matrices show results of pairwise statistical analyses (t-test) indicating no significant difference and asterisks indicating significance ofp < 0.05,p < 0.01,p < 0.001 andp < 0.0001. See alsoFigure S3.

Following injection of ΔpheA-tyrA, ΔtyrR and WTS. praecaptivus into aposymbiotic eggs, two week old adults were collected for imaging along with uninjected aposymbiotic and symbiotic grain weevils of the same age. Following imaging, weevils were homogenized and plated to characterize their infections. Color analysis was performed on a common quadrant of the weevil cuticle under controlled lighting conditions to ensure consistency (Figure 5C). Lighter cuticle coloration (increased red pigmentation) indicates decreased cuticular sclerotization and reduced symbiont Tyr/Phe biosynthesis²⁰. Accordingly, aposymbiotic grain weevils had cuticles with significantly higher red coloration than symbiotic counterparts (p < 0.0001). Among aposymbiotic weevils harboringS. praecaptivus, those with auxotrophic ΔpheA-tyrA had the reddest cuticles. Weevils with WTS. praecaptivus were significantly darker than those with ΔpheA-tyrA (p < 0.01), but were significantly lighter than uninjected aposymbionts (p < 0.05). This suggest thatS. praecaptivus depletes host Tyr/Phe; an effect that is exacerbated with an auxotrophic strain that cannot synthesize Tyr/Phede novo. Strikingly, weevils harboring the ΔtyrR overproducer had cuticles at least as dark as those of aposymbionts (no significant difference). Notably, the WT and ΔtyrR S. praecaptivus strains maintained very similar densities in the weevil (t-test;p = 0.84), indicating that color differences could not be explained as a function of change in the burden of infection.

To further assess impact on weevil fitness we compared larval development times of symbiotic and aposymbiotic weevils, including aposymbionts maintaining WT, ΔpheA-tyrA or ΔtyrR strains. Results (Figure 5D) show that symbiotic weevils have the shortest larval development time, consistent withS. pierantonius providing the greatest fitness benefit. No significant time differences were observed between uninjected aposymbionts and either (i) aposymbionts injected with WT or (ii) aposymbionts injected with ΔpheA-tyrA (p > 0.05). However, aposymbionts injected with ΔtyrR showed accelerated larval development compared to non-injected aposymbionts (p < 0.001), indicating that the ΔtyrR strain yields a beneficial (mutualistic) outcome, implying that symbiont Tyr/Phe production is also beneficial prior to adulthood.

DISCUSSION

Mutualistic inter-kingdom interactions involving microorganisms and animals/ plants are common and have facilitated many important innovations including aerobic energy generation, photosynthesis and nitrogen fixation⁴⁸. They create new biology from components with exclusive functions, catalyzing exploitation of novel niches, reducing the burden of competition⁴⁹. Insects have served as important models for study because symbiosis has made an exceptional contribution to their ecological success⁵⁰. However, the origin of these associations remains poorly understood⁵¹. This is partly due to the fact that certain mutualistic adaptations are anticipated to be maladaptive in a free-living state, leading to a causality dilemma. For example, the sharing of nutritional resources in mutualism is contraindicated in the free-living state where individuals must compete to acquire resources for growth. Further, mutualists must overcome natural antagonistic interactions (immunity) to forge an intimate association that mediates partner fidelity feedback necessary for selection to optimize mutualistic functionality^52,53.

Here, we established, characterized and engineered a synthetic insect-bacterial symbiosis to gain insight into the nature and complexity of adaptations facilitating mutualism. Following development of a protocol to introduce a nascent candidate symbiont,S. praecaptivus, into the eggs of aposymbiotic grain weevils (Sitophilus zeamais), we monitored weevils that maintained the association over ten generations through maternal transmission with ~50% efficiency per generation. Our results demonstrate thatS. praecaptivus undergoes sustained vertical transmission in a novel host, providing a model for long-term study of symbiotic interactions and evolutionary processes in symbiosis. Notably, the association can be maintained by selection of insects that display mCherry fluorescence at generational intervals. Our results show that cyclical vertical transmission mandates introduction ofS. praecaptivus into eggs, mimicking natural processes of transovarial transmission, as documented for native,S. pierantonius, in weevils⁵⁴. This accords withS. praecaptivus establishing infection in germ and/or stem cells such that subsequent differentiation processes propel infection into mature larval and adult tissues, including ovarioles. Although our experiment focused on introduction of bacteria into eggs, it is possible that establishment could occur later in development when, for example, larvae commence movement/feeding and may encounter injuries that provide opportunities for bacterial entry.

Our work also shows thatS. praecaptivus can establish infection in weevils that maintain their native symbiont,S. pierantonius. In those weevils, both bacteria reside in somatic and germinal bacteriomes in the gut and ovary²⁰. Previous work demonstrated production of specialized antimicrobial peptides (coleoptericins) that (i) prevent growth of symbionts outside of bacteriomes and (ii) control their proliferation inside bacteriomes by inducing bacterial cell filamentation⁵⁵. However, in our study,S. praecaptivus was observed infecting a range of weevil tissues, displaying no evidence of filamentation, indicating lack of susceptibility to these effects.

We found thatS. praecaptivus andS. pierantonius co-exist in germinal apical bacteriomes, transmitting together, albeit at lower efficiency forS. praecaptivus. Localization ofS. praecaptivus in ovarian tissues of aposymbiotic weevils revealed colonization of multiple cell types within ovarioles, including pro-oocytes and prefollicular cells assembling during oogenesis, along with nurse cells that sustain developing oocytes. This provides several, redundant, potential opportunities for transmission, possibly enabling members of the genusSodalis to undergo transmission in insects with diverse (panoistic, polytrophic and telotrophic) reproductive systems. It likely represents another factor explaining the success ofSodalis spp. in the board colonization of insects in nature¹⁵. Notably,S. praecaptivus is transmitted to eggs at a very early stage of oogenesis, in contrast to several other insect symbionts that are transmitted at later stages. For example, the aphid symbiont (Buchnera) is transmitted from maternal bacteriocytes to blastulae in the ovariole tips of pathenogenetically-reproducing aphids at oogenesis stage seven⁴³, or when eggs reach a size of 500μm in aphids reproducing oviparously⁵⁶. Further,Spiroplasma entersDrosophila oocytes when lipid transport channels open at oogenesis stage ten⁵⁷, andWolbachia transmission takes place in the telotrophic planthopper,Laodelphax striatellus, during vitellogenin transovarial transportation, which also takes place at a later stage of oogenesis⁵⁸.

The native symbiont of grain weevils,S. pierantonius, produces tyrosine and phenylalanine that facilitate cuticle sclerotization, yielding adult weevils that have a tough, dark exoskeleton^20,59,60. However, our work shows that wild typeS. praecaptivus does not engage in Tyr/Phe secretion as demonstrated by laboratory cross-feeding assays. Correspondingly, adult weevils injected with WT or auxotrophic strains ofS. praecaptivus have cuticles that are lighter in color than those of aposymbiotic counterparts, indicating reduced sclerotization, consistent with the notion that host Tyr/Phe levels are depleted by this bacterium. In order to generate a mutualistic strain ofS. praecaptivus, we employed rational engineering⁶¹ to identify a mutant strain ofS. praecaptivus (ΔtyrR) that overproduces and cross-feeds Tyr/Phe to an auxotroph^46,47. Introduction of this strain into weevil eggs resulted in the production of adults whose cuticle color was restored to that of uninfected (aposymbiotic) counterparts. Further, weevils maintaining the ΔtyrR strain had significantly reduced larval development time relative to their aposymbiotic (uninfected) counterparts, implying that Tyr/Phe production is also beneficial in the context of larval development and seems to present a more sensitive signal for symbiont Tyr/Phe provisioning in our synthetic system. Of course it is possible thatS. praecaptivus simply produces more Tyr/Phe in the larval stage or that it provides additional beneficial metabolites that selectively impact larval development. Together, this provides a genetic validation of the role of symbionts in Tyr/Phe production and cuticular sclerotization, which is thought to have played an important role in the radiation of beetles⁶² and ants⁶³ by enhancing strength, desiccation tolerance and predator/pathogen resistance^64,65.

Tyrosine and phenylalanine overproduction and secretion were observed to result from several single gene knockouts inS. praecaptivus (ΔtyrR, ΔnuoN, ΔcsrA, Δzwf and Δmdh). Since null mutants are anticipated to arise spontaneously in natural populations of bacteria in the environment, this suggests that insects can readily acquire bacterial strains capable of secreting specific nutrients as a consequence of spontaneous mutations. In support of this, many examples of nutrient cross-feeding have been identified in natural microbial communities that increase the collective efficiency of resource utilization^66,67. Taken together, these results suggest that adaptation to nutrient secretion is not a significant bottleneck in the evolution of mutualistic associations that focus on nutrient provisioning. Further support for this notion was obtained in a recent study showing that mutualism could be established between a stinkbug and anE. coli strain that was experimentally evolved to facilitate mutualism in this host⁶⁸. However, in the case of this synthetic symbiosis, theE. coli are not transmitted transovarially, but are instead inoculated onto the surface of host eggs, facilitating vertical transmission. We reason that bacterial adaptation to transovarial transmission likely requires more complex genetic underpinnings, conferring an ability to infect ovarioles and eggs. Critically, our work shows that a (non-engineered/wild-type) free-living relative of a widely distributed group of insect symbionts has an intrinsic capability to establish and sustain vertical transmission in a novel insect host (albeit one that naturally harbors aSodalis symbiont) with no obvious detrimental effects. However, injection of a quorum-sensing mutant (ΔypeI), demonstrating constitutive expression of virulence factors^36,37 was observed to kill weevil eggs with striking efficiency, highlighting the lability and complexity of interactions facilitating maintenance and vertical transmission of a symbiont.

Given that natural selection lacks foresight, it is important to recognize that the ability ofS. praecaptivus to associate with an insect host might be a function of selection pressures mediated by a biphasic lifestyle comprising a free-living state in addition to host association⁶⁹. Indeed, it has been proposed thatS. praecaptivus might use insects as vectors to facilitate transmission between animal and/or plant hosts in the environment²⁴. Alternatively, the ability ofS. praecaptivus to associate with insects might simply be a side effect of its ability to associate with plant and/or mammalian hosts^70–72, although it is notable thatS. praecaptivus maintains virulence factors characterized as insect-specific³⁶. Interestingly, recent work indicates that free-livingSodalis spp. maintain a substantial presence in decaying wood³³ andS. praecaptivus was in fact isolated from a human following impalement with a dead tree branch²⁴. Since insects are also known to frequently associate with decaying wood, it is possible that free-living members of the genusSodalis use insects as vectors for transmission among decaying trees in the environment⁷³.

Collectively, our work shows that bacterial genera such asSodalis, that frequently develop symbiotic associations with a wide range of insect taxa, have extensive adaptations that facilitate infection, benign persistence and vertical transmission in insect hosts. Vertical transmission, in particular, lays the foundation for the evolution of mutualism by facilitating strong partner-fidelity feedback. Metabolic adaptations leading to nutrient secretion can have relatively simple genetic etiologies that can be honed by subsequent degenerative changes that mimic strategies utilized in microbial rational engineering to eliminate competing metabolic activities, favoring production of selected resources⁷⁴. While our work shows that vertical transmission occurs initially with sub-optimal efficiency, it should be noted that in nature the acquisition of a new biological function often facilitates ecological diversification, providing a unique niche for partners to exploit, replete with strong selection pressure to maintain functionality of the association and thereby increase the efficiency of vertical transmission. Further, our work demonstrates the autocatalytic quality of symbiosis, in which an existing symbiont creates favorable host conditions for the acquisition of a nascent symbiont, leading to functional augmentation, symbiont replacement and metabolic integration⁷⁵. Numerous studies have revealed evidence of these events in nature, rationalized as a consequence of loss of fitness of an existing symbiont^58,76–78 or acquisition of new functionality in response to environmental change or niche expansion^79,80. In simple terms, mutualism can be described as a state of coexistence in which the benefits of a partnership outweigh the inherent costs⁸¹. The ability ofS. praecaptivus to maintain a benign infection, combined with pre-existing host adaptations that facilitate bacterial maintenance, likely contribute significantly towards a reduction in those costs.

The development of a synthetic, transovarially-transmitted symbiosis provides new opportunities to advance knowledge in symbiosis. First, becauseS. praecaptivus is amenable to culture and manipulation, this system can be used to investigate mechanistic adaptations underlying symbiosis and mutualism, throughout the spectrum of insect development. Second, this system can be maintained for long-term study of host-symbiont adaptation and degenerative evolution. Finally, many insects, including certain disease vectors, are not amenable to germ line genetic modification and symbionts could be used as a platform to express transgenes, either to investigate molecular processes or interfere with processes of disease transmission in natural insect populations^37,82,83.

STAR METHOD

RESOURCE AVAILIABILITY

Lead contact

Further information and request for resources and reagents should be directed to and will be fulfilled by the lead contact, Colin Dale (colin.dale@utah.edu)

Materials availability

Mutant strains generated in this study are available upon request from the lead contact, Colin Dale (colin.dale@utah.edu).

Data and code availability

All sequence reads derived from genomic sequencing were deposited in the NCBI sequence read archive (SRA) under accession SAMN26947704, SAMN26947705 and SAMN26947706 for the MC1, ΔpheA-tyrA and ΔtyrR strains, respectively.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Insects:

Grain weevils (Sitophilus zeamais), originally obtained from USDA, Manhattan, KS, U.S.A, were reared on organic whole yellow maize (Purcell Mountain Farms) in an Darwin insect chamber at 25 °C and 62% relative humidity (RH). Symbiont-free (aposymbiotic) weevils were generated by rearing on rifampicin treated corn prepared by hydrating dried corn with a 3% (w/v) solution of rifampicin (1 mg/ml)³⁶. Following treatment for one generational interval, the resulting aposymbiotic weevils were maintained on untreated grain and are checked periodically to confirm the absence of bacteriomes.

Bacterial strains

This study involved the use ofSodalis praecaptivus strain HS, which is a close relative of insect-associatedSodalis spp. symbionts that was isolated from an infected human^24,34, and has been previously deposited in the American Type Culture Collection (ATCC) as product BAA-2554. For all experiments outlined in the study, it was cultured in LB liquid media and plated on LB agar with appropriate antibiotics as outlined in method details, presented below, at 30 °C, under atmospheric air. Strains were preserved at −80 °C in LB media with 15% (w/v) glycerol.

All mutant strains ofS. praecaptivus utilized throughout this study, can be obtained from the lead contact Colin Dale (colin.dale@utah.edu).

METHOD DETAILS

Genetic modification ofSodalis praecaptivus

Lambda Red recombineering was utilized to generate recombinant strains ofS. praecaptivus maintaining plasmid pRed/Gamm (CAT) using methodologies developed and outlined in previous studies⁸⁴. For the work outlined in this study, we engineered a strain that expresses the fluorescent mCherry protein, in order to visualizeS. praecaptivus in grain weevils. In addition, we engineeredS. praecaptivus strains that are (1) auxotrophic for phenylalanine and tyrosine and (2) overproduce these aromatic amino acids. The auxotroph was generated by knocking out both thetyrA andpheA genes, encoding enzymes involved in the terminal steps of tyrosine and phenylalanine biosynthesis, respectively, yielding a strain that is incapable of biosynthesizing either amino acid⁸⁵. Candidate Tyr/Phe overproducing strains were generated in accordance with a rational engineering strategy previously developed to facilitate overproduction of L-DOPA inE. coli^46,47. This encompassed generation ofS. praecaptivus mutants lackingtyrR,nuoN, ppc, ptsHIcrr, csrA, zwf,mdh genes.

Preparation of an mCherry-expression cassette.

An mCherry and zeocin resistance cassette (1.5 kbp), codon-optimized for efficient expression in gamma Proteobacteria^86,87, was amplified from bacterial DNA by PCR in a reaction comprising 10 μl of 5X PCR buffer, 4 μl of 25 mM dNTPs, 3 μl of 25 mM MgCl₂, 1.25 μl of 20 μM forward primer (#2272), 1.25 μl of 20 μM reverse primer (#2273), 0.5 μl of Phusion High Fidelity DNA Polymerase (Thermo Fisher Scientific) and 1 μl of template DNA. The cycling conditions involved initial denaturation at 98 °C for 2 min, followed by 29 cycles of denaturation (98 °C; 30 s), annealing (56 °C; 30 s) and extension (72 °C; 1 min), followed by a final extension at 72 °C for 2 min. This yielded a single amplicon of expected size (1.5-kbp), as determined by gel electrophoresis.

We elected to insert the mCherry-zeocin cassette into thelacZ gene ofS. praecaptivus based on the notion that lactose is not present in insects and therefore, disruption of this gene should not negatively impact the interaction betweenS. praecaptivus and weevils. Furthermore, insertional inactivation oflacZ can be detected by plating bacteria on media with IPTG and X-Gal⁸⁸, facilitating selection of recombinants. Three consecutive PCR reactions were employed to generate a construct that could be integrated into thelacZ gene ofS. praecaptivus strain 101³⁶ using lambda Red recombineering⁸⁴. DNA from wild-typeS. praecaptivus was isolated from cultured cells by heating at 98°C for 5 min to provide template for PCR reactions. In the first PCR, 212 bp of the 5’ end (primer #2286/#2287) and 278 bp of the 3’ end (#2289/#2290) of thelacZ gene were amplified with a flanking tail using the following a PCR reaction composed of 12.5 μl of 2X Phusion, 6.5 μl of nuclease free water, 2.5 μl of 2.5 μM forward primer, 2.5 μl of 2.5 μM reverse primer and 1 μl of DNA template. The PCR was performed with an initial denaturation at 98°C for 30 s, followed by 35 cycles of denaturation (98 °C; 10 s), annealing (58 °C; 30 s) and extension (72 °C; 2 min). The resulting PCR product was then purified using Agencourt AMPure XP magnetic beads, in accordance with manufacturer’s protocol. In the second PCR, 4 μl of 5’ and 3’lacZ PCR products were amplified with 4 μl of the mCherry-zeocin cassette to generate a chimeric product with 12 μl of 2X Taq Polymerase MasterMix (Thermo Fisher Scientific) with initial denaturation at 95°C for 30 s, followed by 10 cycles of denaturation (94 °C; 15 s), annealing (45 °C; 30 s) and extension (72 °C; 1min). The third PCR step was used to amplify the final 2 kbp disruption fragment from the second PCR product using 1 μl each of primers #2287 and #2290, which anneal to the 5’ and 3’ ends oflacZ, respectively. This reaction was conducted with 13 μl of 2X Taq Polymerase MasterMix (Thermo Fisher Scientific), 11 μl of nuclease free water and 24 μl of the 2^nd PCR product. The PCR conditions involved an initial denaturation at 95 °C for 30 sec, followed by 35 cycles of denaturation (94 °C; 15 s), annealing (58 °C; 30 s) and extension (72 °C; 1.5 min). The third PCR product was again purified using AMPure XP beads to generate template for recombineering.

Preparation of ΔpheA-tyrA and Phe and Tyr overproductionrecombineering constructs.

Genetic constructs to generate these mutant strains were prepared using a similar three-step PCR procedure, as detailed above for the mCherry-zeocin cassette, to generate chimeric PCR products with gentamycin, spectinomycin or kanamycin resistance cassettes for ΔpheA-tyrA and all candidate gene knockouts yielding Phe and Tyr overproduction (ΔtyrR, ΔnuoN, Δppc, ΔptsHIcrr, ΔcsrA, Δzwf and Δmdh). The PCRs and clean up steps were conducted using reagents and conditions outlined for preparation of the mCherry-zeocin construct, using primers listed inTable S1.

Lambda Red recombineering.

Wild-typeS. praecaptivus strain 101 culture maintaining the plasmid pRed/Gamm (CAT)³⁶ was cultured overnight in 3 ml LB with 30 μg/ml chloramphenicol. The resulting cells were then inoculated into 25 ml 2YT medium (20 mg/ml Tryptone, 8 mg/ml Yeast Extract, 10 mg/ml NaCl, pH 5.8) with 30 μg/ml chloramphenicol and permitted to grow for 3 hours in a 30 °C shaking incubator (200 rpm). The expression of the lambda Red functions was induced by adding arabinose at 4 mg/ml and the culture was allowed to grow for another 30 min under the same conditions.S. praecaptivus cells were then pelleted by centrifugation at 9000 × g. for 20 min at 4 °C, washed twice in cold sterile de-ionized water and resuspended in a fresh aliquot of 25 ml ice cold sterile de-ionized water. Two additional rounds of washing and resuspension were performed, first using a resuspension volume of 25 ml and second using a resuspension volume of 1 ml. This yields high efficiency electro-competentS. praecaptivus cells that can be transformed with recombineering constructs (as outlined here) or plasmids. The prepared PCR products were then combined with 80 μl of competent cells and electroporated at 1600 V/s using an Eppendorf electroporator model 2510. The cells were permitted to recover for 16 hours by plating on L agar without antibiotic selection before replica plating onto L agar with IPTG (100 mM), X-gal (100 mg/ml) and appropriate antibiotic (15 μg/ml zeocin, 40 μg/ml spectinomycin, 5 μg/ml gentamicin or 30 μg/ml kanamycin).

Genetic and phenotypic verification.

All resulting transformants were found to be resistant to appropriate antibiotics following recombineering. PCR assays were performed using primers flanking the insertion site in target genes to confirm that the constructs were inserted in the anticipated fashion in theS. praecaptivus genome. The amplification of the insertion region (PCR product sizes listed inTable S1) was achieved by PCR with a reaction mixture composed of 0.5 μl of 2.5 μM forward and 2.5 μM reverse primer (Table S1), 12.5 μl of 2X Taq Polymerase MasterMix (Thermo Fisher Scientific), 10.25 μl of nuclease free water, 1 μl of DNA template and 1.25 μl DMSO. The cycling condition comprised an initial denaturation at 95 °C for 1 min, followed by 35 cycles of denaturation (94 °C; 30 sec), annealing (58 °C; 30 sec) and extension (72 °C; 4 min). In addition, a PCR assay for the presence of thetam gene (440 bp) (GenBank:AHF76984.1) was performed to verify that the transformant wasS. praecaptivus³⁶. This PCR used 12.5 μl 2X Taq Polymerase MasterMix (Thermo Fisher Scientific), 1 μl of forward (#127) and reverse (#128) primer, 11.5 μl nuclease free water and 1 μl of template DNA from the transformant. The thermocycler conditions included an initial denaturation at 95 °C for 1 min, followed by 25 cycles of denaturation (94 °C; 30 s), annealing (48 °C; 30 s) and extension (72 °C; 3 min).

Phenotypic tests were performed to validate the recombinant strains. MCherry fluorescence was confirmed by fluorescent microscopy and the resulting recombinant was designatedS. praecaptivus strain MC1. The double auxotrophic phenotype of the ΔpheA-tyrA strain was confirmed by replica plating on minimal media with and without tyrosine and phenylalanine supplementation. The functions of the candidate Phe and Tyr overproducing strains were assessed using a cross-feeding assay in which putative overproducers and the wild type strain (control) were streaked adjacent to the ΔpheA-tyrA auxotroph. In addition, tyrosine production was validated for the ΔtyrR mutant during growth of the putative overproducer and wild type strain (control) in minimal media using a colorimetric tyrosine assay (Sigma-Aldrich). These assays were performed in triplicate for the wild type and ΔtyrR strains, following 5 days of growth in minimal media at 30 °C with shaking (200 rpm), according to the manufacture’s’ protocol and measured using a POLARstar OPTIMA spectrophotometer.

Genome sequencing of recombinant strains.

Prior to injection in weevils,S. praecaptivus MC1,ΔpheA-tyrA and ΔtyrR strains were sequenced to confirm that (1) the inserted cassette was integrated into the anticipated genomic location, and (2) the lambda Red recombination event did not induce any extraneous mutations inS. praecaptivus. Genomic DNA for each strain was extracted from cells that were isolated as single colonies and cultured on L plates with IPTG (100 mM) and X-gal (100 mg/ml) for 48 hours at 30 °C. Bacterial cells were collected and transferred into 180 μl Buffer ATL, and DNA was extracted using a Qiagen Blood and Tissue Kit protocol (Qiagen, Germany), following the manufacturer’s protocol for Gram negative bacteria. The resulting genomic DNA was treated with 1 μl RNaseA for 15 minutes at room temperature and purified with Ampure XP purification beads (Axygen) prior to final elution in 50 μl nuclease-free water.

Library construction was performed using NEBNext Ultra II DNA Library Prep Kit (New England BioLabs, USA) and NovaSeq S4 Reagent Kit v1.5 (2 ×150 bp). Whole-genome sequencing was performed on a NovaSeq 600 system (Illumina) at the University of Utah Huntsman Cancer Institute High-Throughput Genomics Core Facility, yielding 34.2 Gb, 39.2 Gb and 41.2 Gb of raw sequence reads, respectively, for the MC1, ΔpheA-tyrA and ΔtyrR strains. Reads were quality trimmed in BBDuk and aligned back to theS. praecaptivus wild type reference sequence (CP006569) using Geneious Prime 2022.0.2 with default parameters. The resulting alignments were then inspected manually for mismatches. Trimmed reads were also assembled using the SPAdes assembler with default parameters. The resulting contigs were then aligned to sequences comprising the resistance cassettes used for lambda Red recombination to identify those contigs representing regions of the chromosome that were genetically modified. All three mutant strains were confirmed to have the correct genetic modifications with no gene duplications or other rearrangement in theS. praecaptivus genome. Further, no extraneous mutations were identified in any of the recombinant strains. All sequence reads derived from genomic sequencing were deposited in the NCBI sequence read archive (SRA) under accession SAMN26947704, SAMN26947705 and SAMN26947706 for the MC1, ΔpheA-tyrA and ΔtyrR strains, respectively.

Generalized transduction procedure forS. praecaptivus

In order to introduce the mCherry allele into aS. praecaptivus ΔypeI strain, constructed and validated in a previous study³⁶, we took advantage of an endogenous phage transduction system. Phage induction was achieved by growing a 1:20 dilution of an overnight culture ofS. praecaptivus MC1 in LB media at 30 °C for 8 hours with shaking at 200 rpm and then exposing the resulting culture in an open petri dish to UV light from a germicidal lamp in a Labconco model 36208/36209 TYPE A2 laminar flow hood for 30 sec. Following exposure, the culture was maintained at 30 °C for 12 hours. Chloroform was then added to 1% (v/v) and mixed thoroughly by vortexing. Cells were then pelleted by centrifugation at 8000 rpm for 20 min and the supernatant (containing phage) was stored at 4 °C and plated on LB media to ensure that it did not contain any viableS. praecaptivus cells. Transduction was performed by mixing 200 μl of phage suspension with 100 μl of an overnight culture ofS. praecaptivus ΔypeI³⁶ and 900 μl of LB media. Following growth for one hour at 30 °C without shaking, the mixture was plated on LB agar with 15 μg/ml zeocin, 40 mg/ml spectinomycin, 100 mM IPTG and 100 mg/ml X-gal, and incubated for 3 days at 30 °C. A single colony demonstrating spectinomycin resistance (indicative of ΔypeI) and zeocin resistance (indicative of mCherry-bleoR presence) was streaked onto a second plate and a single colony was isolated for microinjection into weevil eggs.

Microinjection ofS. praecaptivus MC1 into aposymbiotic and symbiotic weevil eggs:

Weevil egg isolation.

Weevil eggs that were deposited in grain were detected by staining gelatinous egg plugs using acid fuchsin^89,90 and destaining in DI water until only the egg plugs remain stained. The egg plugs were then removed using forceps, and the egg inside the cavity was carefully removed for use in the microinjection procedure. Only eggs that were deposited by weevils within the past 24 hours were used in this study.

Egg preparation for microinjection.

Isolated eggs were attached in a consistent polar orientation to a microscope slide with heptane glue to preclude the possibility of movement during the microinjection procedure. Following attachment, eggs were dehydrated for 5 min at 25 °C. Wrinkles on the egg surface were observed to be correlated with a poor outcome of microinjection procedure, perhaps indicating damage incurred during their isolation or excessive dehydration. After dehydration, a 2 μl drop of gas-permeable halocarbon oil 700 (Sigma-Aldrich) was placed on the surface of each egg to achieve complete immersion, inhibiting further dehydration and facilitating gas exchange.

Injection needle preparation.

S. praecaptivus MC1 strain was cultured in LB medium overnight in a 30 °C shaking incubator and concentrated to OD_600nm = 1 in 0.85% (w/v) NaCl. First, 2 μl of the prepared bacterial culture were drawn into one end of a 3.5” glass tube (Drummond #2–00-203-G/X) by capillary action. The tube was then pulled on a needle puller (Sutter Instrument Co Model P-97) with settings of heat = 270, pull = 20, velocity = 40, time = 150. Subsequently, a sterilized tweezer was used to break the pulled needle and expose the sharp end for injection.

Microinjection.

The prepared needle, replete with bacterial culture was then attached to an empty syringe held by a micromanipulator (Narishige, Model M-152) to facilitate accurate subsequent injection, and adjusted to be perpendicular to the line of eggs attached to the glass slide. Injections were performed into the posterior poles of eggs under phase contrast microscopic observation followingDrosophila egg microinjection procedures⁹¹. Approximately 0.005~0.02 μl of bacterial culture was then injected into each egg. Following injection, the glass slides with injected eggs were maintained in an incubator at 25 °C and 62% RH.

Egg hatching and transfer to grains.

After 4–6 days, the first instar larval stage was observed to emerge from microinjected eggs. Immediately following emergence, larvae were transferred back to corn grains to facilitate completion of their larval development. In some cases, eggs that were observed to contain developing larvae failed to hatch, likely due to injury, and were abandoned. Maize grains were soaked in sterile deionized water for 5 min to facilitate weevil transplantation and subsequent survival. Transplantation was achieved by first drilling a 1.5 mm diameter hole into the grain and then carefully implanting the larva. The hole was then gently packed with finely powdered cornmeal and a thin layer of glutinous rice-water cement was used to seal the hole to simulate the tough coating that is found on the grain surface. The glutinous rice-water cement was prepared by combing 0.5 g glutinous rice flour and 2 ml DI water and heated in a 1000 W microwave on full power for 30 sec, providing sufficient cement for 30 larval implantations. The transplanted grains were then maintained under standard conditions (25 °C, 62% RH) for one month to facilitate the completion of weevil development to adulthood.

Microinjection ofS. praecaptivus MC1 in aposymbiotic (apo) and symbiotic weevil adults

A suspension ofS. praecaptivus MC1 in 0.85% (w/v) NaCl (OD_600nm = 1) was prepared for injection into adult weevils using needles pulled from 3.5” glass capillary tubes (Drummond #2–00-203-G/X) at settings of heat = 292, pull = 100, velocity = 24, time = 250. Adult aposymbiotic maize weevils (less than 3 weeks following emergence) were then microinjected withS. praecaptivus MC1 using an established protocol³⁵ involving dipping the capillary needle into the bacterial suspension and then piercing the thoracic hemocoel of the adult weevils with the contaminated needle. All of the resulting adult weevils were maintained in the laboratory at 25 °C and 62% RH for 3 weeks to reproduce.

Live staining for confocal imaging

Weevils were processed for confocal microscopic imaging by careful dissection in 0.85% (w/v) NaCl. Dissected tissues (i.e. gut, ovaries) were washed in saline and placed on a freshly made 1.5 mm 0.5% agarose pad on a microscope slide. The tissues were stained with Hoechst 33342 (10 mg/ml) and CellMask Green (Thermo Fisher Scientific, 5 mg/ml) to stain nuclei and membranes, respectively, by adding 1 μl of each dye on to the top of the samples. The slide was then covered with a cover slip (No. 1.5: 0.175 mm +/− 0.015), sealed using Valap (1:1:1 mix of vaseline:lanolin:wax). Twenty minutes later, once the stains penetrated the tissues, confocal imaging was performed on a Zeiss LSM880 microscope equipped with an AiryScan detector, a 20X AIR objective and a 63X NA1.4 oil immersion objective. Imaging was performed using appropriate excitation and emission filters for Hoechst 33342 and CellMask Green and mCherry, and images were processed in ZEN Blue 2.1 (Zeiss) and Imaris Viewer 9.6.0 (Bitplane). Single plane images selected from the z stacks are presented in this manuscript.

Preparation ofS. pierantonius for electron microscopy

Fifth instarSitophilus zeamais larvae were isolated from maize grains and subjected to dissection to remove bacteriomes located at the anterior end of the midgut into 0.1 M phosphate buffer (pH 7.2). Bacteriomes were then homogenized in a Dounce glass sub-cellular homogenizer to release bacteria from insect cells. Cellular debris was removed by centrifugation at 500 × g for 1 min. The supernatant was then subjected to three rounds of centrifugation (2000 × g for 5 min) and washing in 0.1 M phosphate buffer (pH 7.2). After the final washing step, the bacterial cell pellets was resuspended in 1% osmium tetroxide for 40 minutes and dehydrated using a graded series of ethanol (30%, 50%, 70%, 90%, 100%) in 5 min steps. Cells were then filtered onto a 0.2 micron polycarbonate filter for critical point drying. Following mounting, specimens were sputter coated with 10 nM gold/platinum and then visualized using a FEI Nova NanoSEM^™ scanning microscope.

Measuring weevil cuticle color and larval development time

In order to compare weevil cuticle coloration, we collected aposymbiotic weevils that were injected with ΔpheA-tyrA, ΔtyrR and WTS. praecaptivus individually at egg stage along with non-injected aposymbiotic and symbiotic counterparts that were subject to the same egg isolation and larval implantation procedure. The larval development time for each injected and non-injected group was recorded. This represents the number of days from implantation of a first instar larva into corn to the subsequent emergence of the adult weevil. All weevils were collected at 14 days post adult emergence, based on the results of a pilot experiment demonstrating that the difference in cuticle color between symbiotic and aposymbiotic weevils was highest at that time point. Weevils were first washed in DI water, placed on a white background and a drop of glycerol was added to coat their exoskeleton²⁰. Images of each weevil were obtained under consistent lighting conditions with a single light source under a dissection microscope (Leica M205 FCA). A square in the center of the thorax was then cropped with its side length equals to half of the thorax width, and an average red value for the square was then computed using ImageJ software. Weevils were homogenized and plated to verify their infection status following imaging.

QUANTIFICATION AND STATISTICAL ANALYSIS

Chi squared tests were performed manually for the following purposes:

To determine if the adult weevil infection status is biased according to sex; results subsection “Dynamics ofS. praecaptivus transmission”; n = 100 (number of weevils); significant difference is defined asp < 0.05.

To determine the stability of infection in weevil larvae vs. adults; results subsection “Dynamics ofS. praecaptivus transmission”; n = 40 (number of weevils); significant difference is defined asp < 0.05.

To determine if female weevil reproductive age is correlated with offspring infection frequency; results subsection “Dynamics ofS. praecaptivus transmission” andFigure 2D; n = 43 (number of weevils); significant difference is defined asp < 0.05. Two samples were excluded due to an absence of infected offspring.

T tests were performed in Microsoft Excel for the following purposes:

To compare growth of theS. praecaptivus ΔpheA-tyrA mutant strain during co-culture with otherS. praecaptivus strains; results subsection “Rational engineering of a functional mutualism” andFigure 5B; n (number of biological replicates) = 4; Mean = 2.04 × 10⁷ CFU/ml (grow with ΔtyrR) and 3.84 × 10⁶ CFU/ml (grow with WT); SD = 2.13 × 10⁶ CFU/ml (grow with ΔtyrR) and 3.92 × 10⁵ CFU/ml (grow with WT); significant difference is defined asp < 0.05.

To compare infection densities in weevils; results subsection “Rational engineering of a functional mutualism” andFigure 5C andD; n (number of weevils) = 16 (for apo+ΔpheA-tyrA and apo+WT) and 17 (for apo+ΔtyrR); Mean = 2.22 × 10⁶ CFU/weevil (apo+ΔpheA-tyrA), 2.84 × 10⁶ CFU/weevil (apo+WT) and 2.67 × 10⁶ CFU/weevil (apo+ΔtyrR); SD = 2.36 × 10⁶ CFU/weevil (apo+ΔpheA-tyrA), 1.71 × 10⁶ CFU/weevil (apo+WT) and 1.94 × 10⁶ CFU/weevil (apo+ΔtyrR); significant difference is defined asp < 0.05.

To compare the cuticle color (red value) among weevils; results subsection “Rational engineering of a functional mutualism” andFigure 5C; n (number of weevils) = 16 (for apo, apo+ΔpheA-tyrA and apo+WT), 17 (for apo+ΔtyrR) and 19 for sym; Mean = 31.6 (apo), 52.9 (apo+ΔpheA-tyrA), 40 (apo+WT), 27.3 (apo+ΔtyrR) and 11.44 (sym); SD = 10.39 (apo), 13.77 (apo+ΔpheA-tyrA), 6.98 (apo+WT), 6.62 (apo+ΔtyrR) and 3.7 (sym); significant difference is defined asp < 0.05.

To compare the larval development time among weevils; results subsection “Rational engineering of a functional mutualism” andFigure 5D; n (number of weevils) = 16 (for apo, apo+ΔpheA-tyrA and apo+WT), 17 (for apo+ΔtyrR) and 19 for sym; Mean = 44.41 (apo), 45.35 (apo+ΔpheA-tyrA), 43.24 (apo+WT), 40.44 (apo+ΔtyrR) and 38.47 (sym); SD = 3.04 (apo), 3.84 (apo+ΔpheA-tyrA), 3.44 (apo+WT), 3.05 (apo+ΔtyrR) and 2.89 (sym); significant difference is defined asp < 0.05.

Kaplan-Meier analysis was performed using the R software package:

To determine if weevil development time is correlated with infection status; results subsection “Dynamics ofS. praecaptivus transmission” andFigure 2C; n = 29 (number of weevils); significant difference is defined asp < 0.05.

Supplementary Material

Supplemental Video

Video S1: Live F₁₀ descendant of the mainS. praecaptivus MC1 injected weevil line along with an uninfected control weevil visualized for two seconds under normal light then under mCherry fluorescent light, related toFigure 1.

Download video file^{(21.2MB, mp4)}

Supplement

NIHMS1884393-supplement-Supplement.pdf^{(7.7MB, pdf)}

KEY RESOURCES TABLE.

REAGENT or RESOURCE	SOURCE	IDENTIFIERRRRrRRR
Bacterial and virus strains
S. praecaptivus wild type	ATCC	BAA-2554
S. praecaptivus MC1	This paper	CD 2555
S. praecaptivus 101	³⁶	CD 101
S. praecaptivus ΔpheA-tyrA	This paper	CD 1663
S. praecaptivus ΔtyrR	This paper	CD 1987
S. praecaptivus ΔnuoN	This paper	CD 1728
S. praecaptivus Δmdh	This paper	CD 1993
S. praecaptivus Δppc	This paper	CD 2131
S. praecaptivus ΔptsHIcrr	This paper	CD 1991
S. praecaptivus ΔcsrA	This paper	CD 708
S. praecaptivus Δzwf	This paper	CD 1989
S. praecaptivus MC1 ΔypeI	This paper	CD 2565
Biological samples
Sitophilus zeamais	USDA, Manhattan, KS, U.S.A.https://www.ars.usda.gov/plains-area/mhk/cgahr/	N/A
Oligonucleotides
SeeTable S2, Oligonucleotides used in this study	N/A	N/A
Recombinant DNA
Plasmid pRed/Gamm (CAT)	⁸⁴	N/A
Software and Algorithms
Adobe Illustrator	N/A	https://www.adobe.com/products/illustrator.html
Imaris Viewer	N/A	https://imaris.oxinst.com/imaris-viewer
Geneious Prime 2022.0.2	N/A	https://www.geneious.com
BBDuk	N/A	https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/
SPAdes assembler	N/A	⁹²
Image J 1.53a	N/A	https://imagej.nih.gov/ij/
Deposited Data
S. praecaptivus MC1 sequence reads	This paper	SRA: SAMN26947704
S. praecaptivus ΔtyrR sequence reads	This paper	SRA: SAMN26947706
S. praecaptivus ΔpheA-tyrA sequence reads	This paper	SRA: SAMN26947705

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ACKNOWLEDGMENTS

We gratefully acknowledge funding provided by National Science Foundation award DEB1926738 (to C.D.) and National Institute of Health NIGMS award R35 GM136389 (to K.G.G.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Prof. Philippe Cluzel (Harvard Univ.) for provision of the mCherry cassette and Shinichiro Enomoto for help constructing strains. We also thank Sara Weinstein for assistance with statistics.

Footnotes

DECLARATION OF INTERESTS

The authors declare no competing interests.

INCLUSION AND DIVERSITY

We worked to ensure sex balance in the selection of non-human subjects. One or more of the authors of this paper self-identifies as a member of the LGBTQ+ community. One or more of the authors of this paper self-identifies as living with a disability.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Video

Download video file^{(21.2MB, mp4)}

Supplement

NIHMS1884393-supplement-Supplement.pdf^{(7.7MB, pdf)}

Data Availability Statement

All sequence reads derived from genomic sequencing were deposited in the NCBI sequence read archive (SRA) under accession SAMN26947704, SAMN26947705 and SAMN26947706 for the MC1, ΔpheA-tyrA and ΔtyrR strains, respectively.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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Rational engineering of a synthetic insect-bacterial mutualism

Yinghua Su

Ho-Chen Lin

Li Szhen Teh

Fabienne Chevance

Ian James

Clara Mayfield

Kent G Golic

James A Gagnon

Ofer Rog

Colin Dale

SUMMARY

Graphical Abstract

INTRODUCTION

RESULTS

Egg injection establishes a synthetic, insect-bacterial symbiosis

Figure 1: Establishment ofS. praecaptivus MC1 in aposymbiotic weevils following egg injection.

Egg injection yields sustained vertical transmission ofS. praecaptivus

Figure 2: Dynamics ofS. praecaptivus MC1 infection following egg injection.

Dynamics ofS. praecaptivus transmission

S. praecaptivus colonizes prototypical bacteriomes in grain weevils

Figure 3: Localization ofSodalis praecaptivus MC1 expressing mCherry (red) in offspring of aposymbiotic (apo) and symbiotic (sym) weevils infected by egg microinjection.

S. praecaptivus infects weevil eggs at early stage of oogenesis

Figure 4: Low (left) and high (right) magnification confocal images ofS. praecaptivus MC1 expressing mCherry (red) in ovaries of offspring from aposymbiotic (apo) and symbiotic (sym) weevils following egg microinjection.

Rational engineering of a functional mutualism

Figure 5: Characterization ofS. praecaptivus strains with modified tyrosine and phenylalanine biosynthesis.

DISCUSSION

STAR METHOD

RESOURCE AVAILIABILITY

Lead contact

Materials availability

Data and code availability

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Insects:

Bacterial strains

METHOD DETAILS

Genetic modification ofSodalis praecaptivus

Preparation of an mCherry-expression cassette.

Preparation of ΔpheA-tyrA and Phe and Tyr overproductionrecombineering constructs.

Lambda Red recombineering.

Genetic and phenotypic verification.

Genome sequencing of recombinant strains.

Generalized transduction procedure forS. praecaptivus

Microinjection ofS. praecaptivus MC1 into aposymbiotic and symbiotic weevil eggs:

Weevil egg isolation.

Egg preparation for microinjection.

Injection needle preparation.

Microinjection.

Egg hatching and transfer to grains.

Microinjection ofS. praecaptivus MC1 in aposymbiotic (apo) and symbiotic weevil adults

Live staining for confocal imaging

Preparation ofS. pierantonius for electron microscopy

Measuring weevil cuticle color and larval development time

QUANTIFICATION AND STATISTICAL ANALYSIS

Chi squared tests were performed manually for the following purposes:

T tests were performed in Microsoft Excel for the following purposes:

Kaplan-Meier analysis was performed using the R software package:

Supplementary Material

KEY RESOURCES TABLE.

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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