Symbiotic Escherichia coli strains can better colonize host stinkbugs and outcompete natural symbiotic bacteria, but confer less fitness benefits

This study demonstrates that experimentally evolved *Escherichia coli* strains can rapidly acquire superior colonization abilities to outcompete native *Pantoea* symbionts in the stinkbug *Plautia stali*, yet fail to provide equivalent fitness benefits, thereby revealing a decoupling between infection success and mutualism that highlights the potential for "cheater-like" microbes to destabilize coevolved symbiotic partnerships.

The Gist

The Big Picture: A Bug, a Bacteria, and an Imposter

Imagine a stinkbug (specifically the brown-winged green stinkbug, Plautia stali) as a high-end restaurant. For this restaurant to stay open and serve delicious food, it needs a specific, trusted head chef (a native bacteria called Pantoea). This chef has worked there for millions of years. The restaurant cannot survive without him; he provides the nutrients the bug needs to grow and lay eggs.

Usually, this chef is so good at his job that no one else can take his place. But in this study, scientists decided to test what happens if they hire a temporary substitute chef from a completely different kitchen (a lab-evolved E. coli bacteria) and see if he can kick the original chef out.

The Experiment: The "Chef" Competition

The scientists set up three scenarios to see how these different bacteria performed:

1. The Native Chef (Sym A): The original, uncultivable Pantoea bacteria. It's the gold standard.
2. The Local Chefs (Sym C–F): Other versions of the native bacteria found in different parts of Japan. They are good, but not quite as perfect as the original.
3. The Substitute Chefs (The E. coli):
   • The "Rookie" (ΔintS): A normal E. coli with no special skills.
   • The "Trained" Substitute (CmL05G13): An E. coli that was forced to evolve in a lab for a year to become good at helping the bug.
   • The "Engineered" Substitute (ΔcyaA): An E. coli that was genetically tweaked to help the bug.

The Results: Two Different Types of Success

The study revealed a fascinating twist: Being good at getting hired is different from being good at doing the job.

1. The "Hiring" Phase (Colonization)

When the scientists put the bugs in a room with both the Native Chef and a Substitute Chef, they wanted to see who would take over the kitchen.

• The Native Chef vs. The Rookie: The Native Chef easily kicked the Rookie out. The Native Chef is the boss here.
• The Native Chef vs. The Trained Substitute: This was the shocker. The Trained Substitute (CmL05G13) was incredibly aggressive. It didn't just share the kitchen; it kicked the Native Chef out completely and took over the whole symbiotic organ. It was better at "hijacking" the host than the native bacteria that had co-evolved with it for millions of years.
• The Engineered Substitute: This one was good at getting in initially, but the Native Chef eventually pushed it out.

The Analogy: Imagine a new employee who is incredibly aggressive at networking and getting into the office. They manage to fire the long-time, loyal manager and take their desk. That's what the Trained Substitute did.

2. The "Performance" Phase (Fitness Benefits)

Once the Substitute Chef was in charge, did the restaurant run better?

• Growth (Survival): The Trained Substitute was actually pretty good at keeping the restaurant open. The bugs survived just fine, almost as well as with the Native Chef.
• Reproduction (Eggs): Here is where the Substitute failed. While the Native Chef helped the bugs lay 300 eggs, the Trained Substitute only helped them lay less than 100 eggs.

The Analogy: The Trained Substitute Chef is great at keeping the lights on and the building standing (survival), but he is terrible at cooking the special dishes that make the customers happy and bring in the money (reproduction). He is a "Cheater." He took the spot, got the benefits of living inside the bug, but didn't give back the full value of the original partnership.

Why Did This Happen?

The scientists found two main reasons for this strange outcome:

1. The "Cheater" Strategy: The E. coli evolved very quickly (in just a year) to be a master at invading and staying inside the bug. It learned how to win the competition for space. However, because it was only evolving for a short time, it didn't learn how to be a perfect partner for the bug's long-term reproduction.
2. Vertical Transmission: In this specific bug, the mother smears the bacteria onto the eggs, and the baby eats it. Because the bacteria are passed down directly from parent to child, the "Native Chef" didn't need to be a master fighter to keep his job; he just needed to be a good partner. The "Substitute," however, had to fight hard to get in, so it evolved to be a fighter, not a perfect partner.

The Takeaway

This paper teaches us a valuable lesson about evolution and relationships:

• Winning the job isn't the same as doing the job well. A microbe can evolve to be incredibly good at taking over a host's body (colonization) but still be a "bad partner" that doesn't help the host reproduce as much as the original partner would.
• Evolution is fast. A bacteria that has never met a stinkbug before can evolve to outcompete a native bacteria in less than a year.
• The "Cheater" Risk: In nature, there is a constant risk that "cheater" microbes will invade a symbiotic relationship, take over the host, and lower the host's fitness, even if they keep the host alive.

In short, the scientists created a "super-invader" bacteria that could kick out the native symbiont, only to discover that while it was a great invader, it was a mediocre partner. It's like hiring a bodyguard who is great at fighting off intruders but terrible at taking care of your children.

Technical Summary

1. Problem Statement

The study addresses a central question in evolutionary biology: How do novel, non-native microbes invade and displace long-term, co-evolved mutualistic symbionts? Specifically, the researchers investigated whether laboratory-evolved or genetically engineered strains of Escherichia coli (originally a vertebrate gut bacterium with no natural association with the host) could compete against the native, essential gut symbionts (Pantoea spp.) of the brown-winged green stinkbug, Plautia stali.

Key questions included:

• Can E. coli rapidly evolve symbiotic capabilities sufficient to colonize the host?
• How do these evolved E. coli strains compete with native Pantoea strains (both cultivable and uncultivable) in co-infection scenarios?
• Does successful colonization by E. coli translate to equivalent host fitness benefits, or do these strains act as "cheaters" (exploiting the host without providing full mutualistic benefits)?

2. Methodology

The researchers utilized the Plautia stali–Pantoea system, a well-established model for vertical symbiont transmission.

• Host System: An inbred strain of P. stali (originally from Tsukuba, Japan) was used. Newborn nymphs were rendered aseptic (symbiont-free) via surface sterilization of eggs.
• Bacterial Strains:
  • Native Symbionts: Pantoea sp. A (Sym A, uncultivable, fixed in mainland populations) and Pantoea spp. C, D, E, F (Sym C–F, cultivable, found in island populations).
  • Control: Non-symbiotic E. coli $\Delta$intS.
  • Experimental E. coli:
    • CmL05G13: An evolved strain obtained via experimental evolution (13 host generations) selecting for improved host body color. It carries multiple mutations, including a disruptive mutation in cyaA.
    • $\Delta$cyaA: An artificial strain created by knocking out the cyaA gene in a standard E. coli background.
• Experimental Design:
  • Single Infection Assays: Aseptic nymphs were inoculated with a single bacterial strain to measure adult emergence rates, time to reproductive maturity, fecundity (egg count), and bacterial titers (quantified via CFU for cultivable strains and qPCR for uncultivable Sym A).
  • Competitive Co-infection Assays: Nymphs were inoculated with a 1:1 mixture of two strains (e.g., Sym A vs. CmL05G13). The researchers tracked which strain dominated the symbiotic organ at the second instar nymph stage and the adult stage.
• Quantification: Bacterial loads were measured at the 2nd instar and adult stages using Colony Forming Units (CFU) for cultivable strains and quantitative PCR (qPCR) targeting the groEL gene for uncultivable Sym A and E. coli.

3. Key Results

A. Single Infection Performance (Host Fitness)

• Survival (Emergence Rate): The native uncultivable symbiont (Sym A) provided the highest survival (71.7%). The evolved E. coli (CmL05G13) and artificial E. coli ($\Delta$cyaA) supported survival rates (56%) comparable to the cultivable Pantoea strains (Sym D–F), but significantly lower than Sym A. The non-symbiotic control ($\Delta$intS) resulted in very low survival (4%).
• Reproduction (Fecundity): Sym A conferred the highest fecundity (300 eggs). Cultivable Pantoea strains provided moderate fecundity (200 eggs). Crucially, both evolved and artificial E. coli strains resulted in significantly low fecundity (<100 eggs), comparable to the non-symbiotic control.
• Bacterial Titers: High bacterial titers in the symbiotic organ correlated with higher host survival. Sym A, CmL05G13, and $\Delta$cyaA maintained high titers ($10^8$ gene copies in adults), whereas cultivable Pantoea and the control had lower titers.

B. Competitive Co-infection Outcomes

• Vs. Cultivable Pantoea (Sym C–F): The evolved E. coli strain (CmL05G13) outcompeted all cultivable Pantoea strains (Sym C, D, E, F), becoming the dominant symbiont in the adult stage.
• Vs. Native Uncultivable Pantoea (Sym A):
  • CmL05G13: Successfully invaded and displaced Sym A in the majority of hosts, maintaining dominance from the nymphal stage through adulthood.
  • $\Delta$cyaA: Initially dominated Sym A in nymphs but was outcompeted and displaced by Sym A by the adult stage.
  • $\Delta$intS: Initially dominated Sym A in nymphs but was completely displaced by Sym A in adults.
• Conclusion on Competition: The experimentally evolved E. coli (CmL05G13) possesses superior colonization competitiveness, surpassing even the co-evolved, genome-reduced native symbiont (Sym A).

C. The "Cheater" Phenotype

Despite their superior ability to colonize and displace native symbionts, the E. coli strains (CmL05G13 and $\Delta$cyaA) failed to provide the reproductive benefits associated with native symbionts. While they ensured host survival (emergence), they severely compromised host fecundity. This decoupling of infection success and host fitness benefit characterizes them as "cheater-like" associates.

4. Key Contributions

1. Rapid Evolution of Symbiosis: Demonstrated that a non-symbiotic bacterium (E. coli) can evolve mutualistic traits (improving host survival) and high colonization competitiveness within a short timeframe (approx. 1 year/13 generations) in the lab.
2. Displacement of Native Symbionts: Provided experimental evidence that an evolved non-native symbiont can outcompete and replace a native, vertically transmitted, essential symbiont (Pantoea sp. A) that has co-evolved with the host for millions of years.
3. Decoupling of Traits: Identified a clear evolutionary trade-off where the ability to colonize (competition) does not necessarily correlate with the ability to provide full mutualistic benefits (reproduction). The evolved E. coli strains optimize for persistence (high titers) rather than host reproductive output.
4. Mechanism of Transmission: Highlighted a potential difference between vertical transmission systems (P. stali) and environmental acquisition systems (e.g., Riptortus pedestris). In P. stali, the lack of strong selection for "colonization barriers" (since symbionts are smeared directly on eggs) may allow cheaters to invade more easily than in systems where nymphs must select symbionts from a diverse environment.

5. Significance

• Evolutionary Dynamics of Cheating: The study provides a robust experimental model for studying how "cheater" microbes invade mutualistic systems. It suggests that in vertically transmitted symbioses, selection may favor host fitness contributions over colonization competitiveness, leaving the system vulnerable to invasion by strains that are good at colonizing but poor at benefiting the host.
• Symbiont Replacement: It challenges the assumption that co-evolved native symbionts are invincible. It shows that evolutionary time is not the only factor determining symbiotic stability; rapid adaptation in a new host can lead to the displacement of ancient partners.
• Model for Symbiosis Research: The P. stali–E. coli system serves as a powerful tool for dissecting the genetic and molecular mechanisms underlying the transition from free-living to symbiotic lifestyles, and the evolutionary conflicts between symbiont transmission success and host fitness.

In summary, the paper reveals that while laboratory-evolved E. coli can rapidly acquire the "weapons" to invade and dominate a stinkbug's symbiotic organ, they lack the "cooperation" required to fully support the host's reproductive success, effectively acting as evolutionary cheaters.