High above the rest: standing behaviors in the amoebae of Sappinia and Thecamoeba.

Many terrestrial microbes have evolved cell behaviors that help them rise above their substrate, often to facilitate dispersal. One example of these behaviors is found in the amoebae of Sappinia pedata, which actively lift most of their cell mass above the substrate, known as standing. This standing behavior was first described in S. pedata in the 1890s from horse dung isolates but never molecularly characterized from dung. Our study expands this understanding, revealing the first molecularly confirmed S. pedata from herbivore dung in Mississippi, USA, and describing a new species, Sappinia dangeardi n. sp., with larger trophozoite cells. Additionally, we isolated another standing amoeba, Thecamoeba homeri n. sp., from soil, exhibiting a previously unreported "doughnut shape" transient behavior. In S. dangeardi n. sp., we discovered that standing is likely triggered by substrate drying, and that actin filaments actively localize in the "stalk" to support the standing cells, as observed through confocal microscopy. While the purpose of standing behaviors has not been investigated, we hypothesize it is energetically expensive and therefore a significant evolutionary strategy in these organisms. Overall, this study emphasizes behavioral adaptations to terrestrial environments within Amoebozoa, stressing the importance of diverse laboratory conditions that replicate natural habitats.