Dictyostelium discoideum: An Alternative Nonanimal Model for Developmental Toxicity Testing

A critical aspect of toxicity evaluation is developmental and reproductive toxicity (DART) testing. Traditionally, DART testing has been conducted in vivo in mammalian model systems. New legislation aimed at reducing animal use and the prohibitive costs associated with DART testing, together with a need to understand the genetic pathways underlying developmental toxicity means there is a growing demand for alternative model systems for toxicity evaluation. Here we explore the potential of the eukaryotic social amoeba Dictyostelium discoideum, which is already widely used as a simple model system for cell and developmental biology, as a potential nonanimal model for DART testing. We developed assays for high-throughput screening of toxicity during D. discoideum growth and development. This allowed the toxicity of a broad range of test compounds to be characterized, which revealed that D. discoideum can broadly predict mammalian toxicity. In addition, we show that this system can be used to perform functional genomic screens to compare the molecular modes of action of different compounds. For example, genome-wide screens for mutations that affect lithium and valproic acid toxicity allowed common and unique biological targets and molecular processes mediating their toxicity to be identified. These studies illustrate that D. discoideum could represent a predictive nonanimal model for DART testing due to its amenability to high-throughput approaches and molecular genetic tractability

A Simple Mechanism for Complex Social Behavior

The evolution of cooperation is a paradox because natural selection should favor exploitative individuals that avoid paying their fair share of any costs. Such conflict between the self-interests of cooperating individuals often results in the evolution of complex, opponent-specific, social strategies and counterstrategies. However, the genetic and biological mechanisms underlying complex social strategies, and therefore the evolution of cooperative behavior, are largely unknown. To address this dearth of empirical data, we combine mathematical modeling, molecular genetic, and developmental approaches to test whether variation in the production of and response to social signals is sufficient to generate the complex partner-specific social success seen in the social amoeba Dictyostelium discoideum. Firstly, we find that the simple model of production of and response to social signals can generate the sort of apparent complex changes in social behavior seen in this system, without the need for partner recognition. Secondly, measurements of signal production and response in a mutant with a change in a single gene that leads to a shift in social behavior provide support for this model. Finally, these simple measurements of social signaling can also explain complex patterns of variation in social behavior generated by the natural genetic diversity found in isolates collected from the wild. Our studies therefore demonstrate a novel and elegantly simple underlying mechanistic basis for natural variation in complex social strategies in D. discoideum. More generally, they suggest that simple rules governing interactions between individuals can be sufficient to generate a diverse array of outcomes that appear complex and unpredictable when those rules are unknown

A polychromatic 'greenbeard' locus determines patterns of cooperation in a social amoeba

Cheaters disrupt cooperation by reaping the benefits without paying their fair share of associated costs. Cheater impact can be diminished if cooperators display a tag (‘greenbeard’) and recognise and preferentially direct cooperation towards other tag carriers. Despite its popular appeal, the feasibility of such greenbeards has been questioned because the complex patterns of partner-specific cooperative behaviours seen in nature require greenbeards to come in different colours. Here we show that a locus (‘Tgr’) of a social amoeba represents a polychromatic greenbeard. Patterns of natural Tgr locus sequence polymorphisms predict partner-specific patterns of cooperation by underlying variation in partner-specific protein–protein binding strength and recognition specificity. Finally, Tgr locus polymorphisms increase fitness because they help avoid potential costs of cooperating with incompatible partners. These results suggest that a polychromatic greenbeard can provide a key mechanism for the evolutionary maintenance of cooperation

Inferring adaptive codon preference to understand sources of selection shaping codon usage bias.

Alternative synonymous codons are often used at unequal frequencies. Classically, studies of such codon usage bias (CUB) attempted to separate the impact of neutral from selective forces by assuming that deviations from a predicted neutral equilibrium capture selection. However, GC-biased gene conversion (gBGC) can also cause deviation from a neutral null. Alternatively, selection has been inferred from CUB in highly expressed genes, but the accuracy of this approach has not been extensively tested, and gBGC can interfere with such extrapolations (e.g., if expression and gene conversion rates covary). It is therefore critical to examine deviations from a mutational null in a species with no gBGC. To achieve this goal, we implement such an analysis in the highly AT rich genome of Dictyostelium discoideum, where we find no evidence of gBGC. We infer neutral CUB under mutational equilibrium to quantify “adaptive codon preference,” a nontautologous genome wide quantitative measure of the relative selection strength driving CUB. We observe signatures of purifying selection consistent with selection favoring adaptive codon preference. Preferred codons are not GC rich, underscoring the independence from gBGC. Expression-associated “preference” largely matches adaptive codon preference but does not wholly capture the influence of selection shaping patterns across all genes, suggesting selective constraints associated specifically with high expression. We observe patterns consistent with effects on mRNA translation and stability shaping adaptive codon preference. Thus, our approach to quantifying adaptive codon preference provides a framework for inferring the sources of selection that shape CUB across different contexts within the genome

The genetic architecture underlying prey-dependent performance in a microbial predator

Natural selection should favour generalist predators that outperform specialists across all prey types. Two genetic solutions could explain why intraspecific variation in predatory performance is, nonetheless, widespread: mutations beneficial on one prey type are costly on another (antagonistic pleiotropy), or mutational effects are prey-specific, which weakens selection, allowing variation to persist (relaxed selection). To understand the relative importance of these alternatives, we characterised natural variation in predatory performance in the microbial predator Dictyostelium discoideum. We found widespread nontransitive differences among strains in predatory success across different bacterial prey, which can facilitate stain coexistence in multi-prey environments. To understand the genetic basis, we developed methods for high throughput experimental evolution on different prey (REMI-seq). Most mutations (~77%) had prey-specific effects, with very few (~4%) showing antagonistic pleiotropy. This highlights the potential for prey-specific effects to dilute selection, which would inhibit the purging of variation and prevent the emergence of an optimal generalist predator

Fitness Trade-offs Result in the Illusion of Social Success

Cooperation is ubiquitous across the tree of life, from simple microbes to the complex social systems of animals. Individuals cooperate by engaging in costly behaviors that can be exploited by other individuals who benefit by avoiding these associated costs. Thus, if successful exploitation of social partners during cooperative interactions increases relative fitness, then we expect selection to lead to the emergence of a single optimal winning strategy in which individuals maximize their gain from cooperation while minimizing their associated costs. Such social “cheating” appears to be widespread in nature, including in several microbial systems, but despite the fitness advantages favoring social cheating, populations tend to harbor significant variation in social success rather than a single optimal winning strategy. Using the social amoeba Dictyostelium discoideum, we provide a possible explanation for the coexistence of such variation. We find that genotypes typically designated as “cheaters” because they produce a disproportionate number of spores in chimeric fruiting bodies do not actually gain higher fitness as a result of this apparent advantage because they produce smaller, less viable spores than putative “losers.” As a consequence of this trade-off between spore number and viability, genotypes with different spore production strategies, which give the appearance of differential social success, ultimately have similar realized fitness. These findings highlight the limitations of using single fitness proxies in evolutionary studies and suggest that interpreting social trait variation in terms of strategies like cheating or cooperating may be misleading unless these behaviors are considered in the context of the true multidimensional nature of fitness

Migration in the social stage of Dictyostelium discoideum amoebae impacts competition

Interaction conditions can change the balance of cooperation and conflict in multicellular groups. After aggregating together, cells of the social amoeba Dictyostelium discoideum may migrate as a group (known as a slug) to a new location. We consider this migration stage as an arena for social competition and conflict because the cells in the slug may not be from a genetically homogeneous population. In this study, we examined the interplay of two seemingly diametric actions, the solitary action of kin recognition and the collective action of slug migration in D. discoideum, to more fully understand the effects of social competition on fitness over the entire lifecycle. We compare slugs composed of either genetically homogenous or heterogeneous cells that have migrated or remained stationary in the social stage of the social amoeba Dictyostelium discoideum. After migration of chimeric slugs, we found that facultative cheating is reduced, where facultative cheating is defined as greater contribution to spore relative to stalk than found for that clone in the clonal state. In addition our results support previous findings that competitive interactions in chimeras diminish slug migration distance. Furthermore, fruiting bodies have shorter stalks after migration, even accounting for cell numbers at that time. Taken together, these results show that migration can alleviate the conflict of interests in heterogeneous slugs. It aligns their interest in finding a more advantageous place for dispersal, where shorter stalks suffice, which leads to a decrease in cheating behavior

Coenzyme A and protein CoAlation levels are regulated in response to oxidative stress and during morphogenesis in Dictyostelium discoideum

Dictyostelium discoideum (D. discoideum) is a simple eukaryote with a unique life cycle in which it differentiates from unicellular amoebae into a fruiting body upon starvation. Reactive oxygen species (ROS) have been associated with bacterial predation, as well as regulatory events during D. discoideum development and differentiation. Coenzyme A (CoA) is a key metabolic integrator in all living cells. A novel function of CoA in redox regulation, mediated by covalent attachment of CoA to cellular proteins in response to oxidative or metabolic stress, has been recently discovered and termed protein CoAlation. In this study, we report that the level of CoA and protein CoAlation in D. discoideum are developmentally regulated, and correlate with the temporal expression pattern of genes implicated in CoA biosynthesis during morphogenesis. Furthermore, treatment of growing D. discoideum cells with oxidising agents results in a dose-dependent increase of protein CoAlation. However, much higher concentrations were required when compared to mammalian cells and bacteria. Increased resistance of D. discoideum to oxidative stress induced by H2O2 has previously been attributed to high levels of catalase activity. In support of this notion, we found that H2O2-induced protein CoAlation is significantly increased in CatA-deficient D. discoideum cells. Collectively, this study provides insights into the role of CoA and protein CoAlation in the maintenance of redox homeostasis in amoeba and during D. discoideum morphogenesis

Conditional expression explains molecular evolution of social genes in a microbe

Conflict is thought to play a critical role in the evolution of social interactions by promoting diversity or driving accelerated evolution. However, despite our sophisticated understanding of how conflict shapes social traits, we have limited knowledge of how it impacts molecular evolution across the underlying social genes. Here we address this problem by analyzing the genome-wide impact of social interactions using genome sequences from 67 Dictyostelium discoideum strains. We find that social genes tend to exhibit enhanced polymorphism and accelerated evolution. However, these patterns are not consistent with conflict driven processes, but instead reflect relaxed purifying selection. This pattern is most likely explained by the conditional nature of social interactions, whereby selection on genes expressed only in social interactions is diluted by generations of inactivity. This dilution of selection by inactivity enhances the role of drift, leading to increased polymorphism and accelerated evolution, which we call the Red King process

Differentiation-Inducing Factor-1 and -2 Function also as Modulators for Dictyostelium Chemotaxis

BackgroundIn the early stages of development of the cellular slime mold Dictyostelium discoideum, chemotaxis toward cAMP plays a pivotal role in organizing discrete cells into a multicellular structure. In this process, a series of signaling molecules, such as G-protein-coupled cell surface receptors for cAMP, phosphatidylinositol metabolites, and cyclic nucleotides, function as the signal transducers for controlling dynamics of cytoskeleton. Differentiation-inducing factor-1 and -2 (DIF-1 and DIF-2) were originally identified as the factors (chlorinated alkylphenones) that induce Dictyostelium stalk cell differentiation, but it remained unknown whether the DIFs had any other physiologic functions.Methodology/Principal FindingsTo further elucidate the functions of DIFs, in the present study we investigated their effects on chemotaxis under various conditions. Quite interestingly, in shallow cAMP gradients, DIF-1 suppressed chemotaxis whereas DIF-2 promoted it greatly. Analyses with various mutants revealed that DIF-1 may inhibit chemotaxis, at least in part, via GbpB (a phosphodiesterase) and a decrease in the intracellular cGMP concentration ([cGMP]i). DIF-2, by contrast, may enhance chemotaxis, at least in part, via RegA (another phosphodiesterase) and an increase in [cGMP]i. Using null mutants for DimA and DimB, the transcription factors that are required for DIF-dependent prestalk differentiation, we also showed that the mechanisms for the modulation of chemotaxis by DIFs differ from those for the induction of cell differentiation by DIFs, at least in part.Conclusions/SignificanceOur findings indicate that DIF-1 and DIF-2 function as negative and positive modulators for Dictyostelium chemotaxis, respectively. To our knowledge, this is the first report in any organism of physiologic modulators (small molecules) for chemotaxis having differentiation-inducing activity