Experimental evolution of Dictyostelium discoideum cheating under relaxed selection

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 chimeric 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 chimeras. D. discoideum lines grown in this environment evolved reduced competitiveness within chimeric 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.

Methods
Data represent the results of assays measuring various phenotypes of D. discoideum strains before and after experimental evolution under relaxed selection.

See manuscript for full methods, but in short:

Cheating assays - combine strains of interest with labelled control strain at 50:50 ratio. Plate atop filter paper on non-nutrient plates, allow to form chimeric fruiting bodies.  Harvest fruiting bodies, suspend in buffer, and photograph using bright field and fluorescent microscopy. Calculate fraction of fluorescently labelled spores.

Slug migration assays - inoculate strains of interest along a secant line on one end of a non-nutrient plate. Cover in opaque foil except for a small hole that allows light from the opposite end of the plate. Wait 8 days, then photograph plates. Measure distance of each fruiting body from the starting line.

Cell count/spore count/sporulation efficiency assays - inoculate strains of interest on plates, wait for growth/development, and then quantify cells using a hemacytometer.

诸多微生物之间存在相互作用，但受限于难以在自然环境中直接观测此类相互作用，解读其适应价值始终颇具挑战。社会性变形虫盘基网柄菌（Dictyostelium discoideum）可形成聚集体，其中部分细胞会为其他细胞的生存与繁衍做出牺牲。在包含多个无关谱系的嵌合聚集体（chimeric aggregates）中，欺骗型菌株可通过减少自身投入来获取竞争优势，但目前学界尚未完全明确野生型盘基网柄菌在多大程度上演化出了欺骗行为的适应性。本研究中，我们在不存在嵌合聚集体内欺骗或抗欺骗选择压力的环境中，对盘基网柄菌进行了实验演化培养。在此环境中培养的盘基网柄菌品系，其在嵌合聚集体内的竞争力出现下降，且在蛞蝓阶段（slug stage）的迁移能力也有所降低。与之相对，我们未观察到细胞数量这一未经历选择松弛（relaxed selection）的性状出现下降。实验室条件下已变得无关紧要的性状出现了丢失，这提示此类性状原本是由盘基网柄菌在自然环境中面临的选择压力所驱动并维持的适应性特征。本研究结果表明，盘基网柄菌在自然环境中面临社会冲突，同时也展示了一种可用于探索其他微生物社会适应性或非社会适应性的通用研究方法。

方法
本数据集的数据为：在选择松弛条件下进行实验演化前后，对盘基网柄菌菌株的多种表型进行测定所得到的结果。
完整实验方法详见研究论文，简要概述如下：
1. 欺骗性测定（Cheating assays）：将目标菌株与标记好的对照菌株以50:50的比例混合，铺于非营养培养基的滤纸上，使其形成嵌合子实体（fruiting bodies）。收集子实体并重悬于缓冲液中，随后利用明场显微镜与荧光显微镜进行成像，计算荧光标记孢子的占比。
2. 蛞蝓迁移测定（Slug migration assays）：将目标菌株沿非营养平板一端的截线接种。用不透明铝箔覆盖平板，仅保留一个小孔以使光线从平板的另一端照射。培养8天后对平板进行成像，测量每个子实体与接种起始线之间的距离。
3. 细胞计数/孢子计数/孢子形成效率测定（Cell count/spore count/sporulation efficiency assays）：将目标菌株接种于平板上，待其完成生长与发育后，利用血细胞计数板（hemacytometer）对细胞进行定量。