A species can benefit or be hurt by other species. For example, honeybees and flowering plants help each other to flourish, while lions and gazelles behave in ways that decrease each other’s populations. Understanding these relationships is important for controlling pests and diseases.

Sometimes it is easiest to study such interactions by looking at simple ones that happen on a small scale. Amoebas are common soil organisms that have the same basic organization as human cells. They are much larger and more complex than the bacteria that also live in the soil. How exactly the amoebas and bacteria interact in the soil is an important question, particularly as some of the bacteria can also live inside amoebas. Does this intimate relationship help or harm the amoeba?

Shu, Brock, Geist et al. studied the relationship between a widely studied species of social amoeba and two species of bacteria that can live inside it. Some of the amoebas naturally contained one of the bacteria species, and others were infected with the bacteria in experiments.

Throughout the entire life cycle of the amoebas, the bacteria lived inside them. During one part of the life cycle, amoebas form so-called fruiting bodies, which release spores that can develop into new amoebas. Shu et al. found that both types of bacteria alter the structure of the fruiting bodies in ways that reduce how well the spores disperse.

One of the bacteria species, called Burkholderia hayleyella, harmed the amoebas a lot. It caused most harm to amoebas that do not naturally host the bacteria. This indicates that the amoebas that do host this species may have evolved to avoid its worst effects.

The amoebas have many similarities to the white blood cells that clear bacteria from the human body. Certain bacteria can get inside white blood cells, causing diseases such as tuberculosis. Understanding how bacteria harm amoebas might be useful for understanding such diseases, and developing treatments for them. Though the bacteria Shu et al. studied are not toxic to humans, they are closely related to bacteria that are harmful. It is therefore possible that some bacteria that infect humans first evolve to infect amoebas.

Relationships are complicated because each party has evolved to maximize its own interests. Mutualisms arise when different parties can contribute unique abilities or resources to one another, under conditions where exploitation is controlled (Bronstein, 2015). Mutualisms where one or both parties are microbial fall under the general category of symbiosis. Symbioses are rife with potential conflict (Dale and Moran, 2006; Estrela et al., 2016; Garcia and Gerardo, 2014; Moran, 2007; Oliver et al., 2005). Despite this, symbiotic relationships are pervasive and persistent (Douglas, 2008; Moran et al., 1993; Wernegreen, 2017; Werner et al., 2015). The stability and ubiquity of these interactions implies that conflict can be managed or minimized by partners over multiple generations. However, stability does not imply stagnation. Recent studies suggest that examining extant symbiotic associations may not reveal the underlying mutualism to parasitism continuum that drives evolutionary change (Moran, 2007; Queller and Strassmann, 2018). Indeed, the view that symbiotic associations fluctuate along a mutualism-to-parasitism continuum is increasingly appreciated (McFall-Ngai et al., 2013).

Intracellular endosymbiosis involves a particularly intricate dance between partners. Intracellular endosymbionts must invade, survive, and replicate within host cells and move to new hosts. To exploit their host niche, mutualistic endosymbionts evolve specialized lifestyles that parallel those of intracellular pathogens (Casadevall, 2008; McCutcheon and Moran, 2011; Soto et al., 2009). Despite initial exploitative strategies, some endosymbionts become beneficial or even obligate for host survival, a situation common for many insect nutritional endosymbioses (Douglas, 2009). Strict vertical transmission of endosymbionts typically promotes a more tranquil relationship as the evolutionary fates of the two parties become increasingly interdependent (Ferdy and Godelle, 2005; Hosokawa et al., 2016). Host dependency also often leads to the reduction in size of symbiont genomes as they become more streamlined for life within their host, something true for both beneficial and pathogenic endosymbionts (Dale and Moran, 2006). Horizontally acquired facultative symbionts are more likely to retain active conflicts. This has been experimentally demonstrated by manipulating the jellyfish symbiont Symbiodinium microadriaticum towards solely vertical or horizontal transmission modes, leading to the evolution of more mutualistic or parasitic lineages respectively (Sachs and Wilcox, 2006). Though horizontal transmission can favor symbionts that behave more parasitically, selection can favor hosts that employ more severe countermeasures to limit symbiont entry or growth (Nyholm and McFall-Ngai, 2004; Ratzka et al., 2012; Reynolds and Rolff, 2008). Indeed, host-driven control can be extreme, as demonstrated by the ability of Paramecium bursaria to manipulate its facultative nutritional symbiont (Chlorella sp.) in a relationship that provides no apparent benefit for the imprisoned symbiont (Lowe et al., 2016).

Symbiotic relationships may be positive or negative under different environmental conditions (Leung and Poulin, 2008; Pérez-Brocal et al., 2011). An interesting example of context dependency occurs in the Acyrthosiphon pisum-Hamiltonella defensa symbiosis (Oliver et al., 2003; Oliver et al., 2005). H. defensa infection confers host resistance to parasitoid attack, with resistance being greater for hosts co-infected with Serratia symbiotica (Oliver et al., 2006). However, co-infection comes at a high fecundity cost, such that in parasitoid free environments, host fitness is reduced compared to uninfected counterparts (Oliver et al., 2006). This interplay between reproductive strategy, context, and evolutionary history illustrates the complexity and fluidity of symbiosis.

The facultative endosymbiosis between the social amoeba Dictyostelium discoideum and Burkholderia bacteria provides a promising system for revealing insights into symbiosis dynamics (Brock et al., 2011; DiSalvo et al., 2015). D. discoideum is a soil dwelling amoeba with an interesting life cycle involving unicellular and multicellular stages. During the unicellular stage, amoebae consume bacteria by phagocytosis and divide. When prey are scarce, amoebae aggregate by tens of thousands to form multicellular slugs that move towards heat and light, seeking out a location to form fruiting bodies. These fruiting bodies consist of a stalk of sacrificial dead cells which support a globular sorus containing hardy spore cells (Kessin, 2001). When spores are dispersed, they germinate into vegetative amoebae and the cycle continues. Processes employed by specialized immune-like cells (sentinel cells) during slug migration and fruiting body formation remove any remaining bacteria, typically producing bacteria-free sori (Brock et al., 2011; Chen et al., 2007; Cosson and Lima, 2014).

However, some D. discoideum isolates harvested from the wild are infected with Burkholderia symbionts (Brock et al., 2011). Infection persists in the lab throughout the social cycle, where intracellular bacteria can be visualized within spores and sori (DiSalvo et al., 2015). Infection can be terminated by treating hosts with antibiotics and induced by exposing naïve hosts to Burkholderia, thereby allowing us to easily mix and match partners and study subsequent fitness consequences (Brock et al., 2016a; DiSalvo et al., 2015).

D. discoideum also has a meiotic sexual process, that occurs much less frequently than the asexual proliferation process of binary fission that amoebae go through every few hours (Bloomfield et al., 2010). The asexual binary fission process results in lineages that can be quite different, though all of the same species. Thus, a lineage that has acquired a bacterial endosymbiont can evolve to tolerate it independent of uninfected lineages at least for the thousands of generations that might pass before sexual recombination occurs. Comparing such lineages can illuminate how natural selection operates in early stages of symbiosis.

The Dictyostelium-Burkholderia association is particularly compelling for studying the parasitism to mutualism continuum in endosymbiosis because the fitness consequences of infection are context dependent. Under standard laboratory growth conditions, Burkholderia infection is detrimental for hosts because it decreases slug migration and spore production (Brock et al., 2016a; DiSalvo et al., 2015). However, infection can be beneficial. Infection induces secondary carriage of edible bacteria (Burkholderia is typically inedible), which can increase host fitness in food scarce conditions when the carried food bacteria reseed new environments with a food source (Brock et al., 2011; DiSalvo et al., 2015). For this reason, field-infected host amoebae are also called ‘farmers’ and uninfected host amoebae are called ‘non-farmers.’ Additionally, infected hosts are less harmed by ethidium bromide exposure, possibly mediated by bacterial degradation of the toxin (Brock et al., 2016a).

We have identified three Burkholderia species associated with Dictyostelium: B. agricolaris, B. hayleyella, and B. bonniea (Brock et al., 2018). Most of our work has been conducted with the first two species, which differentially impact host fitness. Here we probe the interaction between host and symbiont genotypes (with regards to their association history) with infection outcomes using a set of previously field-collected and characterized amoebae isolates and their associated Burkholderia symbionts (Brock et al., 2011; DiSalvo et al., 2015). We use standard laboratory conditions in which symbiont infection is shifted towards host detrimental outcomes. Since all three Burkholderia species can be cultured on Petri plates with standard media, none of them are entirely dependent on Dictyostelium for survival.

We find that B. hayleyella is most detrimental for D. discoideum hosts in general, but is most costly to those first exposed to it in the lab. We also document symbiont localization and morphological symptoms in hosts throughout development. Although both species of bacteria can be observed within phagocytic vacuoles, B. hayleyella infects more cells and damages fruiting body structures. These morphological aberrations are also less severe in native-hayleyella hosts, suggesting that native hosts have evolved mechanisms to withstand symbiont colonization.

Here, we characterized D. discoideum infection by two symbiotic Burkholderia species, B. hayleyella and B. agricolaris (Brock et al., 2018). We looked at their impact on D. discoideum by comparing wild type, cured, and re-infected hosts. We assessed fitness measured as production of viable spores, and also evaluated morphological changes in amoebae, slugs, and fruiting bodies with numerical and microscopic data. We found that both Burkholderia species are a burden to D. discoideum under our experimental conditions. However, wild collected hosts did not differ in viable spore production according to whether or not they carried either species of Burkholderia. Even so, D. discoideum with their field-collected state of infection did differ in fruiting body dimensions, with uninfected hosts generally having taller, larger stalks, and larger sori. What explains the differences from experimental infection is unclear. Infection in the wild may be at a lower level than we used experimentally or may have initiated at a lower level that slowly amplified over time, allowing host acclimation to the metabolic costs of infection.

Once D. discoideum hosts are cured with antibiotics, so all comparisons can start from the same baseline, we found that there were few within treatment differences according to host type. The only exception to this is that native-hayleyella hosts produced more viable spores than did naïve or native-agricolaris hosts. This fitness difference is an indication of co-adaptation.

When we map naïve, native-agricolaris, and native-hayleyella host types from Haselkorn et al. (2018) on a previously generated phylogeny, we see no phylogenetic differentiation whatsoever between host types (Douglas et al., 2011). It is most likely that diverse amoebae isolates get infected with Burkholderia in nature, and then adaptations that provide these hosts with increased resilience are selected for over time. Perhaps the general lack of difference among host types with infection is due to sex, which not only recombines genes, but also exposes new clones horizontally to endosymbionts like Burkholderia. Dictyostelium recombination rates are high in natural populations (Flowers et al., 2010). The differences reported here among clones of D. discoideum indicate that specific co-adaptation in clones infected with B. hayleyella can occur during the long periods between sexual reproduction. Thus, reproduction by binary fission and vertical transmission of B. hayleyella is likely to occur much more often than sexual reproduction which could result in horizontal transmission.

Our previous work demonstrated that Burkholderia infections have contextually dependent costs and benefits for their hosts. In food abundant conditions (which we used here) Burkholderia infections are generally detrimental to host fitness (Brock et al., 2011). However, when dispersed to food scarce conditions, Burkholderia infected hosts are able to transport food bacteria with them which restocks their food source and results in higher host fitness (‘farming’). These fitness outcomes may result from the ongoing dynamics underlying long-term symbiosis.

A compelling question is what mediates this tolerance to B. hayleyella infection in its native host? It is possible that native hosts better inhibit infection events or better control intracellular replication of symbiont cells. The percent of spore cells infected in the population post symbiont exposure are not significantly different between a native-hayleyella host and naïve hosts, which does not support the idea that infection events are inhibited by hosts conditioned to that species. Further, we did not see a significantly different intracellular density of B. hayleyella-RFP across hosts from confocal micrographs, suggesting that intact spores from different hosts can accommodate similar numbers of bacterial cells. However, our TEM analysis qualitatively points to the idea that after infection, intracellular replication rates may differ between naïve and native-hayleyella hosts. TEM images of native and naïve B. hayleyella hosts consistently suggest a higher load of intracellular bacterial cells at each stage. For instance, naïve B. hayleyella hosts produce paltry stalks that appear to be overwhelmed by bacterial cells and infected spore cells that look on the verge of deteriorating. Neither of these extreme states were observed in the native host. This could be mediated by host countermeasures that control intracellular symbiont growth or disarm potential toxic symbiont byproducts.

Compared to B. hayleyella, B. agricolaris infections result in more modest (and statistically insignificant) drops in D. discoideum viable spore production for naïve and cured re-infected native-agricolaris hosts. B. agricolaris appears to be less invasive to the host population as it is found in only about a quarter of spore population after exposure. Despite this, it is maintained in infected populations throughout the social stage and over multiple social cycles (DiSalvo et al., 2015). However, B. agricolaris establishes a higher intracellular density within infected spores than B. hayleyella. These observations may be explained by reduced intracellular entrance efficiency coupled with higher intracellular replication rates. Alternatively, B. agricolaris could be more efficiently cleared by host mechanisms and requires a higher multiplicity of infection to overcome clearance, something that might help explain its more modest detrimental effect on host populations.

Earlier studies have identified other differences between naïve D. discoideum and those carrying B. hayleyella (Brock et al., 2013; Brock et al., 2016b; Stallforth et al., 2013). D. discoideum hosts carrying B. hayleyella harmed symbiont-free D. discoideum clones, causing them to lose in social competition (Brock et al., 2013). In another study we compared cured and uncured clones of B. hayleyella and found that slugs from uncured clones move less far across a Petri plate, a cost of infection (Bates et al., 2015). Interestingly, in the current study we detected no visible differences between cured and uncured slug cells. The recent discovery of sentinel cells as innate immune cells (Chen et al., 2007) made us wonder about how they fare with Burkholderia infected hosts. We found that D. discoideum hosts with Burkholderia do not produce as many sentinel cells but even so seem as resistant to toxins as uninfected lines with normal levels of sentinel cells (Brock et al., 2013; Brock et al., 2016b; Stallforth et al., 2013).

Another interesting recent result on the interaction between D. discoideum and bacteria involves the role of the lectin discoidin 1 (Dinh et al., 2018). Clones infected with Burkholderia produced much greater quantities of lectins early in the social stage compared to uninfected clones. These lectins coated the food bacterium K. pneumoniae allowing it to avoid digestion. This is undoubtedly just a beginning, though a fascinating one, in our understanding of how Burkholderia take over D. discoideum cellular machinery to change relationships with bacteria.

D.D. discoideum is already a popular system for examining the molecular mechanisms of bacterial pathogenesis for a variety of important pathogens (Cosson and Soldati, 2008). However, this work demonstrates several properties of this interaction that are distinct from other Dictyostelium-bacterial associations, possibly owing to its natural prevalence. For instance, Bordetella bronchiseptica can intracellularly infect vegetative D. discoideum amoebae and persist in sorus contents. However, in contrast to Burkholderia, B. bronchiseptica is localized extracellularly in sori rather than inside spore cells (Taylor-Mulneix et al., 2017). Infections of D. discoideum with other intracellular pathogens such as Legionella pneumophila often produce secreted multilamellar bodies which harbor the bacterial pathogen (Denoncourt et al., 2014; Paquet and Charette, 2016), while in this study we found no evidence of Burkholderia excreted in multilamellar bodies. In addition, many bacteria resistant to amoebae are found packaged in multilamellar bodies, a process speculated to enhance their resistance to environmental stress (Denoncourt et al., 2014). Thus, Burkholderia symbionts are most likely employing alternative tactics to not only evade digestion, but to also evade expulsion.

Burkholderia is important in another model symbiosis system, that with the bean bug, Riptortus pedestris (Takeshita and Kikuchi, 2017). These bugs acquire Burkholderia insecticola horizontally. They reside in the bean bug gut where they are presumably active in nutrition. They are a good model because it is a facultative symbiosis and both partners can be cultured independently.

We are in a new age of symbiosis studies where we can apply Koch’s principles of curing, re-infecting, and looking for evidence of disease. We can use genomics, experimental evolution and many other methods to ever more systems. The D. discoideum-Burkholderia system holds unique potential for studying eukaryote-bacterial associations. Given its natural occurrence, we can perform long-term ecological surveys, easily isolate new host-symbiont pairs, investigate naturally derived vs newly induced associations using a variety of partner pairing, and we can do experiments in evolution allowing each partner to evolve together or separately. In time, B. hayleyella and B. agricolaris in D. discoideum may be added to the classic symbioses of squid-vibrio, aphid-Buchnera, tsetse fly-Wiggelsworthia, legume-Rhizobia and more (Bennett and Moran, 2015; Bing et al., 2017; Koehler et al., 2018; Werner et al., 2015).

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Author details

1. Longfei Shu

   1. Environmental Microbiomics Research Center and Guangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation Technology, School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou, China
   2. Department of Biology, Washington University, St Louis, United States

   Contribution

   Conceptualization, Investigation, Methodology

   Contributed equally with

   Debra A Brock

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9683-906X

2. Debra A Brock

   Department of Biology, Washington University, St Louis, United States

   Contribution

   Conceptualization, Validation, Investigation, Methodology

   Contributed equally with

   Longfei Shu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4349-5854

3. Katherine S Geist

   Department of Biology, Washington University, St Louis, United States

   Contribution

   Data curation, Formal analysis, Validation

   Competing interests

   No competing interests declared

4. Jacob W Miller

   Department of Biological Sciences, Southern Illinois University, Edwardsville, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. David C Queller

   Department of Biology, Washington University, St Louis, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

6. Joan E Strassmann

   Department of Biology, Washington University, St Louis, United States

   Contribution

   Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Methodology, Project administration, Writing—review and editing

   For correspondence

   strassmann@wustl.edu

   Competing interests

   No competing interests declared

7. Susanne DiSalvo

   Department of Biological Sciences, Southern Illinois University, Edwardsville, United States

   Contribution

   Conceptualization, Data curation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   sdisalv@siue.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4001-4672

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