Insights into the unique torpor of Botrylloides leachi, a colonial urochordate

Rough environmental conditions make the survival of many multi-cellular organisms almost impossible, enforcing behavioral, morphological, physiological and reproductive rejoinders that can cope with harsh times and hostile environments, frequently through down-regulation of metabolism into basal states of dormancy, or torpor. This study examines one of the most unique torpor strategies seen within the phylum Chordata, exhibited by the colonial urochordate Botrylloides leachi, which enters a state of hibernation or aestivation in response to thermal stress, during which all of its functional colonial units (zooids) are entirely absorbed and the colony survives as small remnants of the vasculature, lacking both feeding and reproduction organs. Tissue vestiges then regenerate fully functional colony when re-exposed to milder environmental conditions. The whole metamorphic cycle of hibernation and arousal was studied here and divided into seven major stages, during which the anatomical characteristics of the zooids, the blood cell populations and the expression patterns of some “stem cell” markers were monitored. The first two phases are associated with the shortening of the blastogenic cycles from the typical 7-day cycle to 3–5day long cycles and with the significant diminution of zooids, leaving a carpet of vasculature. During hibernation this colonial carpet is made of a twisted, opaque and condensed mass of vasculature, loaded with condensed masses of blood cells that possess two types of multicellular structures, the 20–50µm “morula-like” opaque balls of cells, and small single-layer epithelial spheres, “blastula-like” structures (50–80µm). Arousal from hibernation starts with the emergence of several clear tunic areas among the vasculature lacunae, which then turn into transparent buds that become progressively larger and opaque. This is followed by sluggish, newfangled cell movement within the vasculature, which increases in intensity and rate over time. A closer examination of the vasculature revealed dramatic vicissitudes in the blood cell constituency as hibernation progressed, which is manifested by the appearance of two novel cell types not recorded in regular colonies, the multinucleate cells (MNC) and storage cells, each with 2–3 distinct cell morphs. Using mixtures of pre-labeled where half stained with a florescent marker for membranes and half stained for DNA we recorded within 2–3 days from onset new MNC stained by both staining, attesting for the de novo formation of MNC through cells fusion.