In the social amoeba Dictyostelium discoideum, shortened stalks may limit obligate cheater success even when exploitable partners are available.

Cooperation is widespread across life, but its existence can be threatened by exploitation. The rise of obligate social cheaters that are incapable of contributing to a necessary cooperative function can lead to the loss of that function. In the social amoeba Dictyostelium discoideum, obligate social cheaters cannot form dead stalk cells and in chimeras instead form living spore cells. This gives them a competitive advantage within chimeras. However, obligate cheaters of this kind have thus far not been found in nature, probably because they are often enough in clonal populations that they need to retain the ability to produce stalks. In this study we discovered an additional cost to obligate cheaters. Even when there are wild-type cells to parasitize, the chimeric fruiting bodies that result have shorter stalks and these are disadvantaged in spore dispersal. The inability of obligate cheaters to form fruiting bodies when they are on their own combined with the lower functionality of fruiting bodies when they are not represent limits on obligate social cheating as a strategy.

Costs of being a diet generalist for the protist predator Dictyostelium discoideum.

Consumers range from specialists that feed on few resources to generalists that feed on many. Generalism has the clear advantage of having more resources to exploit, but the costs that limit generalism are less clear. We explore two understudied costs of generalism in a generalist amoeba predator, Dictyostelium discoideum, feeding on naturally co-occurring bacterial prey. Both involve costs of combining prey that are suitable on their own. First, amoebas exhibit a reduction in growth rate when they switched to one species of prey bacteria from another compared to controls that experience only the second prey. The effect was consistent across all six tested species of bacteria. These switching costs typically disappear within a day, indicating adjustment to new prey bacteria. This suggests that these costs are physiological. Second, amoebas usually grow more slowly on mixtures of prey bacteria compared to the expectation based on their growth on single prey. There were clear mixing costs in three of the six tested prey mixtures, and none showed significant mixing benefits. These results support the idea that, although amoebas can consume a variety of prey, they must use partially different methods and thus must pay costs to handle multiple prey, either sequentially or simultaneously.

Reduced social function in experimentally evolved Dictyostelium discoideum implies selection for social conflict in nature.

Many microbes interact with one another, but the difficulty of directly observing these interactions in nature makes interpreting their adaptive value complicated. The social amoeba Dictyostelium discoideum forms aggregates wherein some cells are sacrificed for the benefit of others. Within chimaeric aggregates containing multiple unrelated lineages, cheaters can gain an advantage by undercontributing, but the extent to which wild D. discoideum has adapted to cheat is not fully clear. In this study, we experimentally evolved D. discoideum in an environment where there were no selective pressures to cheat or resist cheating in chimaeras. Dictyostelium discoideum lines grown in this environment evolved reduced competitiveness within chimaeric aggregates and reduced ability to migrate during the slug stage. By contrast, we did not observe a reduction in cell number, a trait for which selection was not relaxed. The observed loss of traits that our laboratory conditions had made irrelevant suggests that these traits were adaptations driven and maintained by selective pressures D. discoideum faces in its natural environment. Our results suggest that D. discoideum faces social conflict in nature, and illustrate a general approach that could be applied to searching for social or non-social adaptations in other microbes.

Parallel evolution of the G protein-coupled receptor GrlG and the loss of fruiting body formation in the social amoeba Dictyostelium discoideum evolved under low relatedness.

Aggregative multicellularity relies on cooperation among formerly independent cells to form a multicellular body. Previous work with Dictyostelium discoideum showed that experimental evolution under low relatedness profoundly decreased cooperation, as indicated by the loss of fruiting body formation in many clones and an increase of cheaters that contribute proportionally more to spores than to the dead stalk. Using whole-genome sequencing and variant analysis of these lines, we identified 38 single nucleotide polymorphisms in 29 genes. Each gene had 1 variant except for grlG (encoding a G protein-coupled receptor), which had 10 unique SNPs and 5 structural variants. Variants in the 5' half of grlG-the region encoding the signal peptide and the extracellular binding domain-were significantly associated with the loss of fruiting body formation; the association was not significant in the 3' half of the gene. These results suggest that the loss of grlG was adaptive under low relatedness and that at least the 5' half of the gene is important for cooperation and multicellular development. This is surprising given some previous evidence that grlG encodes a folate receptor involved in predation, which occurs only during the single-celled stage. However, non-fruiting mutants showed little increase in a parallel evolution experiment where the multicellular stage was prevented from happening. This shows that non-fruiting mutants are not generally selected by any predation advantage but rather by something-likely cheating-during the multicellular stage.

Predation-resistant Pseudomonas bacteria engage in symbiont-like behavior with the social amoeba Dictyostelium discoideum.

The soil amoeba Dictyostelium discoideum acts as both a predator and potential host for diverse bacteria. We tested fifteen Pseudomonas strains that were isolated from transiently infected wild D. discoideum for ability to escape predation and infect D. discoideum fruiting bodies. Three predation-resistant strains frequently caused extracellular infections of fruiting bodies but were not found within spores. Furthermore, infection by one of these species induces secondary infections and suppresses predation of otherwise edible bacteria. Another strain can persist inside of amoebae after being phagocytosed but is rarely taken up. We sequenced isolate genomes and discovered that predation-resistant isolates are not monophyletic. Many Pseudomonas isolates encode secretion systems and toxins known to improve resistance to phagocytosis in other species, as well as diverse secondary metabolite biosynthetic gene clusters that may contribute to predation resistance. However, the distribution of these genes alone cannot explain why some strains are edible and others are not. Each lineage may employ a unique mechanism for resistance.

Symbiotic bacteria, immune-like sentinel cells, and the response to pathogens in a social amoeba.

Some endosymbionts living within a host must modulate their hosts' immune systems in order to infect and persist. We studied the effect of a bacterial endosymbiont on a facultatively multicellular social amoeba host. Aggregates of the amoeba Dictyostelium discoideum contain a subpopulation of sentinel cells that function akin to the immune systems of more conventional multicellular organisms. Sentinel cells sequester and discard toxins from D. discoideum aggregates and may play a central role in defence against pathogens. We measured the number and functionality of sentinel cells in aggregates of D. discoideum infected by bacterial endosymbionts in the genus Paraburkholderia. Infected D. discoideum produced fewer and less functional sentinel cells, suggesting that Paraburkholderia may interfere with its host's immune system. Despite impaired sentinel cells, however, infected D. discoideum were less sensitive to ethidium bromide toxicity, suggesting that Paraburkholderia may also have a protective effect on its host. By contrast, D. discoideum infected by Paraburkholderia did not show differences in their sensitivity to two non-symbiotic pathogens. Our results expand previous work on yet another aspect of the complicated relationship between D. discoideum and Paraburkholderia, which has considerable potential as a model for the study of symbiosis.

Paraburkholderia symbionts isolated from Dictyostelium discoideum induce bacterial carriage in other Dictyostelium species.

The social amoeba Dictyostelium discoideum engages in a complex relationship with bacterial endosymbionts in the genus Paraburkholderia, which can benefit their host by imbuing it with the ability to carry prey bacteria throughout its life cycle. The relationship between D. discoideum and Paraburkholderia has been shown to take place across many strains and a large geographical area, but little is known about Paraburkholderia's potential interaction with other dictyostelid species. We explore the ability of three Paraburkholderia species to stably infect and induce bacterial carriage in other dictyostelid hosts. We found that all three Paraburkholderia species successfully infected and induced carriage in seven species of Dictyostelium hosts. While the overall behaviour was qualitatively similar to that previously observed in infections of D. discoideum, differences in the outcomes of different host/symbiont combinations suggest a degree of specialization between partners. Paraburkholderia was unable to maintain a stable association with the more distantly related host Polysphondylium violaceum. Our results suggest that the mechanisms and evolutionary history of Paraburkholderia's symbiotic relationships may be general within Dictyostelium hosts, but not so general that it can associate with hosts of other genera. Our work further develops an emerging model system for the study of symbiosis in microbes.

The social amoeba Dictyostelium discoideum rescues Paraburkholderia hayleyella, but not P. agricolaris, from interspecific competition.

Bacterial endosymbionts can provide benefits for their eukaryotic hosts, but it is often unclear if endosymbionts benefit from these relationships. The social amoeba Dictyostelium discoideum associates with three species of Paraburkholderia endosymbionts, including P. agricolaris and P. hayleyella. These endosymbionts can be costly to the host but are beneficial in certain contexts because they allow D. discoideum to carry prey bacteria through the dispersal stage. In experiments where no other species are present, P. hayleyella benefits from D. discoideum while P. agricolaris does not. However, the presence of other species may influence this symbiosis. We tested if P. agricolaris and P. hayleyella benefit from D. discoideum in the context of resource competition with Klebsiella pneumoniae, the typical laboratory prey of D. discoideum. Without D. discoideum, K. pneumoniae depressed the growth of both Paraburkholderia symbionts, consistent with competition. P. hayleyella was more harmed by interspecific competition than P. agricolaris. We found that P. hayleyella was rescued from competition by D. discoideum, while P. agricolaris was not. This may be because P. hayleyella is more specialized as an endosymbiont; it has a highly reduced genome compared to P. agricolaris and may have lost genes relevant for resource competition outside of its host.

Reduced and Nonreduced Genomes in Paraburkholderia Symbionts of Social Amoebas.

The social amoeba Dictyostelium discoideum is a predatory soil protist frequently used for studying host-pathogen interactions. A subset of D. discoideum strains isolated from soil persistently carry symbiotic Paraburkholderia, recently formally described as P. agricolaris, P. bonniea, and P. hayleyella. The three facultative symbiont species of D. discoideum present a unique opportunity to study a naturally occurring symbiosis in a laboratory model protist. There is a large difference in genome size between P. agricolaris (8.7 million base pairs [Mbp]) versus P. hayleyella and P. bonniea (4.1 Mbp). We took a comparative genomics approach and compared the three genomes of D. discoideum symbionts to 12 additional Paraburkholderia genomes to test for genome evolution patterns that frequently accompany host adaptation. Overall, P. agricolaris is difficult to distinguish from other Paraburkholderia based on its genome size and content, but the reduced genomes of P. bonniea and P. hayleyella display characteristics indicative of genome streamlining rather than deterioration during adaptation to their protist hosts. In addition, D. discoideum-symbiont genomes have increased secretion system and motility genes that may mediate interactions with their host. Specifically, adjacent BurBor-like type 3 and T6SS-5-like type 6 secretion system operons shared among all three D. discoideum-symbiont genomes may be important for host interaction. Horizontal transfer of these secretion system operons within the amoeba host environment may have contributed to the unique ability of these symbionts to establish and maintain a symbiotic relationship with D. discoideum. IMPORTANCE Protists are a diverse group of typically single cell eukaryotes. Bacteria and archaea that form long-term symbiotic relationships with protists may evolve in additional ways than those in relationships with multicellular eukaryotes such as plants, animals, or fungi. Social amoebas are a predatory soil protist sometimes found with symbiotic bacteria living inside their cells. They present a unique opportunity to explore a naturally occurring symbiosis in a protist frequently used for studying host-pathogen interactions. We show that one amoeba-symbiont species is similar to other related bacteria in genome size and content, while the two reduced-genome-symbiont species show characteristics of genome streamlining rather than deterioration during adaptation to their host. We also identify sets of genes present in all three amoeba-symbiont genomes that are potentially used for host-symbiont interactions. Because the amoeba symbionts are distantly related, the amoeba host environment may be where these genes were shared among symbionts.

Context dependence in the symbiosis between Dictyostelium discoideum and Paraburkholderia.

Symbiotic interactions change with environmental context. Measuring these context-dependent effects in hosts and symbionts is critical to determining the nature of symbiotic interactions. We investigated context dependence in the symbiosis between social amoeba hosts and their inedible Paraburkholderia bacterial symbionts, where the context is the abundance of host food bacteria. Paraburkholderia have been shown to harm hosts dispersed to food-rich environments, but aid hosts dispersed to food-poor environments by allowing hosts to carry food bacteria. Through measuring symbiont density and host spore production, we show that this food context matters in three other ways. First, it matters for symbionts, who suffer a greater cost from competition with food bacteria in the food-rich context. Second, it matters for host-symbiont conflict, changing how symbiont density negatively impacts host spore production. Third, data-based simulations show that symbiosis often provides a long-term fitness advantage for hosts after rounds of growth and dispersal in variable food contexts, especially when conditions are harsh with little food. These results show how food context can have many consequences for the Dictyostelium-Paraburkholderia symbiosis and that both sides can frequently benefit.

Many organisms form symbiotic relationships with other species. These symbioses often exhibit context dependence, where the sign or magnitude of one partner's effect on the other will change in different environments. Context­dependent effects make it difficult to assign interactions to categories like mutualisms or antagonisms because they involve both benefits and costs depending on the environment. However, in some cases, accounting for context dependence can clarify an interaction so that it more easily fits a mutualism or antagonism. We investigated context dependence using the symbiosis between Dictyostelium discoideum and two symbiotic Paraburkholderia species. In this symbiosis, Paraburkholderia bacteria allow hosts to carry food bacteria to food­poor contexts, where hosts rarely survive without food, but reduce host fitness in the more hospitable food­rich contexts. The effect of food context on Paraburkholderia symbionts is unknown. We show that Paraburkholderia symbionts are also affected by this context, through facing reduced competition after being dispersed by hosts to food­poor contexts. We also identify a new way that symbionts affect hosts, where symbiont density reduces host fitness, but less so in food­poor contexts. Finally, we use simulations to show that infected hosts benefit in the long term across variable food contexts, especially in the harshest environments with little food. These results show that context dependence in symbiosis can have many consequences for hosts and symbionts, although in general for D. discoideum and Paraburkholderia, both are likely to benefit.

Novel Chlamydiae and Amoebophilus endosymbionts are prevalent in wild isolates of the model social amoeba Dictyostelium discoideum.

Amoebae interact with bacteria in multifaceted ways. Amoeba predation can serve as a selective pressure for the development of bacterial virulence traits. Bacteria may also adapt to life inside amoebae, resulting in symbiotic relationships. Indeed, particular lineages of obligate bacterial endosymbionts have been found in different amoebae. Here, we screened an extensive collection of Dictyostelium discoideum wild isolates for the presence of these bacterial symbionts using endosymbiont specific PCR primers. We find that these symbionts are surprisingly common, identified in 42% of screened isolates (N = 730). Members of the Chlamydiae phylum are particularly prevalent, occurring in 27% of the amoeba isolated. They are novel and phylogenetically distinct from other Chlamydiae. We also found Amoebophilus symbionts in 8% of screened isolates (N = 730). Antibiotic-cured amoebae behave similarly to their Chlamydiae or Amoebophilus-infected counterparts, suggesting that these endosymbionts do not significantly impact host fitness, at least in the laboratory. We found several natural isolates were co-infected with multiple endosymbionts, with no obvious fitness effect of co-infection under laboratory conditions. The high prevalence and novelty of amoeba endosymbiont clades in the model organism D. discoideum opens the door to future research on the significance and mechanisms of amoeba-symbiont interactions.

Inference of symbiotic adaptations in nature using experimental evolution.

Microbes must adapt to the presence of other species, but it can be difficult to recreate the natural context for these interactions in the laboratory. We describe a method for inferring the existence of symbiotic adaptations by experimentally evolving microbes that would normally interact in an artificial environment without access to other species. By looking for changes in the fitness effects microbes adapted to isolation have on their partners, we can infer the existence of ancestral adaptations that were lost during experimental evolution. The direction and magnitude of trait changes can offer useful insight as to whether the microbes have historically been selected to help or harm one another in nature. We apply our method to the complex symbiosis between the social amoeba Dictyostelium discoideum and two intracellular bacterial endosymbionts, Paraburkholderia agricolaris and Paraburkholderia hayleyella. Our results suggest P. hayleyella-but not P. agricolaris-has generally been selected to attenuate its virulence in nature, and that D. discoideum has evolved to antagonistically limit the growth of Paraburkholderia. The approach demonstrated here can be a powerful tool for studying adaptations in microbes, particularly when the specific natural context in which the adaptations evolved is unknown or hard to reproduce.

The Ecology and Evolution of Amoeba-Bacterium Interactions.

Amoebae are protists that have complicated relationships with bacteria, covering the whole spectrum of symbiosis. Amoeba-bacterium interactions contribute to the study of predation, symbiosis, pathogenesis, and human health. Given the complexity of their relationships, it is necessary to understand the ecology and evolution of their interactions. In this paper, we provide an updated review of the current understanding of amoeba-bacterium interactions. We start by discussing the diversity of amoebae and their bacterial partners. We also define three types of ecological interactions between amoebae and bacteria and discuss their different outcomes. Finally, we focus on the implications of amoeba-bacterium interactions on human health, horizontal gene transfer, drinking water safety, and the evolution of symbiosis. In conclusion, amoeba-bacterium interactions are excellent model systems to investigate a wide range of scientific questions. Future studies should utilize advanced techniques to address research gaps, such as detecting hidden diversity, lack of amoeba genomes, and the impacts of amoeba predation on the microbiome.

Loss and resiliency of social amoeba symbiosis under simulated warming.

Anthropogenic global change is increasingly raising concerns about collapses of symbiotic interactions worldwide. Therefore, understanding how climate change affects symbioses remains a challenge and demands more study. Here, we look at how simulated warming affects the social ameba Dictyostelium discoideum and its relationship with its facultative bacterial symbionts, Paraburkholderia hayleyella and Paraburkholderia agricolaris. We cured and cross-infected ameba hosts with different symbionts. We found that warming significantly decreased D. discoideum's fitness, and we found no sign of local adaptation in two wild populations. Experimental warming had complex effects on these symbioses with responses determined by both symbiont and host. Neither of these facultative symbionts increases its hosts' thermal tolerance. The nearly obligate symbiont with a reduced genome, P. hayleyella, actually decreases D. discoideum's thermal tolerance and even causes symbiosis breakdown. Our study shows how facultative symbioses may have complex responses to global change.

Low Base-Substitution Mutation Rate but High Rate of Slippage Mutations in the Sequence Repeat-Rich Genome of Dictyostelium discoideum.

We describe the rate and spectrum of spontaneous mutations for the social amoeba Dictyostelium discoideum, a key model organism in molecular, cellular, evolutionary and developmental biology. Whole-genome sequencing of 37 mutation accumulation lines of D. discoideum after an average of 1,500 cell divisions yields a base-substitution mutation rate of 2.47 × 10-11 per site per generation, substantially lower than that of most eukaryotic and prokaryotic organisms, and of the same order of magnitude as in the ciliates Paramecium tetraurelia and Tetrahymena thermophila Known for its high genomic AT content and abundance of simple sequence repeats, we observe that base-substitution mutations in D. discoideum are highly A/T biased. This bias likely contributes both to the high genomic AT content and to the formation of simple sequence repeats in the AT-rich genome of Dictyostelium discoideum In contrast to the situation in other surveyed unicellular eukaryotes, indel rates far exceed the base-substitution mutation rate in this organism with a high proportion of 3n indels, particularly in regions without simple sequence repeats. Like ciliates, D. discoideum has a large effective population size, reducing the power of random genetic drift, magnifying the effect of selection on replication fidelity, in principle allowing D. discoideum to evolve an extremely low base-substitution mutation rate.

Endosymbiotic adaptations in three new bacterial species associated with Dictyostelium discoideum: Paraburkholderia agricolaris sp. nov., Paraburkholderia hayleyella sp. nov., and Paraburkholderia bonniea sp. nov.

Here we give names to three new species of Paraburkholderia that can remain in symbiosis indefinitely in the spores of a soil dwelling eukaryote, Dictyostelium discoideum. The new species P. agricolaris sp. nov., P. hayleyella sp. nov., and P. bonniea sp. nov. are widespread across the eastern USA and were isolated as internal symbionts of wild-collected D. discoideum. We describe these sp. nov. using several approaches. Evidence that they are each a distinct new species comes from their phylogenetic position, average nucleotide identity, genome-genome distance, carbon usage, reduced length, cooler optimal growth temperature, metabolic tests, and their previously described ability to invade D. discoideum amoebae and form a symbiotic relationship. All three of these new species facilitate the prolonged carriage of food bacteria by D. discoideum, though they themselves are not food. Further studies of the interactions of these three new species with D. discoideum should be fruitful for understanding the ecology and evolution of symbioses.

The gene's eye view, the Gouldian knot, Fisherian swords and the causes of selection.

The biological units-of-selection debate has centred on questions of which units experience selection and adaptation. Here, I use a causal framework and the Price equation to develop the gene's eye perspective. Genes are causally special in being both replicators and interactors. Gene effects are tied together in a complex Gouldian knot of interactions, but Fisher deployed three swords to try to cut the knot. The first, Fisher's average excess, is non-causal, so not fully satisfactory in that respect. The Price equation highlights Fisher's other two swords, choosing to model only selection, and only the part that is transmissible across generations. The models developed here show that many causes of organismal fitness do not cause Gouldian complications. Only two kinds of elements must be added to the focal gene for a causal explanation of its selective change: co-replicators that are associated with the focal gene and co-interactors that interact non-additively with the focal gene. Identical equations for co-replication and co-interaction describe interactions between gene copies at a single locus or at separate loci, and also for genes situated within the same individual or in different individuals. These results resolve some of the objections to the gene's eye view. This article is part of the theme issue 'Fifty years of the Price equation'.

Wild Dictyostelium discoideum social amoebae show plastic responses to the presence of nonrelatives during multicellular development.

When multiple strains of microbes form social groups, such as the multicellular fruiting bodies of Dictyostelium discoideum, conflict can arise regarding cell fate. Both fixed and plastic differences among strains can contribute to cell fate, and plastic responses may be particularly important if social environments frequently change. We used RNA-sequencing and photographic time series analysis to detect possible conflict-induced plastic differences between wild D. discoideum aggregates formed by single strains compared with mixed pairs of strains (chimeras). We found one hundred and two differentially expressed genes that were enriched for biological processes including cytoskeleton organization and cyclic AMP response (up-regulated in chimeras), and DNA replication and cell cycle (down-regulated in chimeras). In addition, our data indicate that in reference to a time series of multicellular development in the laboratory strain AX4, chimeras may be slightly behind clonal aggregates in their development. Finally, phenotypic analysis supported slower splitting of aggregates and a nonsignificant trend for larger group sizes in chimeras. The transcriptomic comparison and phenotypic analyses support discoordination among aggregate group members due to social conflict. These results are consistent with previously observed factors that affect cell fate decision in D. discoideum and provide evidence for plasticity in cAMP signaling and phenotypic coordination during development in response to social conflict in D. discoideum and similar microbial social groups.

Cooperation and conflict in the social amoeba Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum has provided considerable insight into the evolution of cooperation and conflict. Under starvation, D. discoideum amoebas cooperate to form a fruiting body comprised of hardy spores atop a stalk. The stalk development is altruistic because stalk cells die to aid spore dispersal. The high relatedness of cells in fruiting bodies in nature implies that this altruism often benefits relatives. However, since the fruiting body forms through aggregation there is potential for non-relatives to join the aggregate and create conflict over spore and stalk fates. Cheating is common in chimeras of social amoebas, where one genotype often takes advantage of the other and makes more spores. This social conflict is a significant force in nature as indicated by rapid rates of adaptive evolution in genes involved in cheating and its resistance. However, cheating can be prevented by high relatedness, allorecognition via tgr genes, pleiotropy and evolved resistance. Future avenues for the study of cooperation and conflict in D. discoideum include the sexual cycle as well as the relationship between D. discoideum and its bacterial symbionts. D. discoideum's tractability in the laboratory as well as its uncommon mode of aggregative multicellularity have established it as a promising model for future studies of cooperation and conflict.

Long-term evolutionary conflict, Sisyphean arms races, and power in Fisher's geometric model.

Evolutionary conflict and arms races are important drivers of evolution in nature. During arms races, new abilities in one party select for counterabilities in the second party. This process can repeat and lead to successive fixations of novel mutations, without a long-term increase in fitness. Models of co-evolution rarely address successive fixations, and one of the main models that use successive fixations-Fisher's geometric model-does not address co-evolution. We address this gap by expanding Fisher's geometric model to the evolution of joint phenotypes that are affected by two parties, such as probability of infection of a host by a pathogen. The model confirms important intuitions and offers some new insights. Conflict can lead to long-term Sisyphean arms races, where parties continue to climb toward their fitness peaks, but are dragged back down by their opponents. This results in far more adaptive evolution compared to the standard geometric model. It also results in fixation of mutations of larger effect, with the important implication that the common modeling assumption of small mutations will apply less often under conflict. Even in comparison with random abiotic change of the same magnitude, evolution under conflict results in greater distances from the optimum, lower fitness, and more fixations, but surprisingly, not larger fixed mutations. We also show how asymmetries in selection strength, mutation size, and mutation input allow one party to win over another. However, winning abilities come with diminishing returns, helping to keep weaker parties in the game.