ABSTRACT: Harsh environments enforce the expression of behavioural, morphological, physiological, and reproductive rejoinders, including torpor. Here we study the morphological, cellular, and molecular alterations in torpor architype in the colonial urochordate Botrylloides aff. leachii by employing whole organism Transmission electron (TEM) and light microscope observations, RNA sequencing, real-time polymerase chain reaction (qPCR) quantification of selected genes, and immunoloc- alization of WNT, SMAD and SOX2 gene expressions. On the morphological level, torpor starts with gradual regression of all zooids and buds which leaves the colony surviving as condensed vasculature remnants that may be ‘aroused’ to regenerate fully functional colonies upon changes in the environment. Simultaneously, we observed altered distributions of hemolymph cell types. Phagocytes doubled in number, while the number of morula cells declined by half. In addition, two new circulating cell types were observed, multi-nucleated and bacteria-bearing cells. RNA sequencing technology revealed marked differences in gene expression between different organism compartments and states: active zooids and ampullae, and between mid-torpor and naive colonies, or naive and torpid colonies. Gene Ontology term enrichment analyses further showed disparate biological processes. In torpid colonies, we observed overall 233 up regulated genes. These genes included NR4A2, EGR1, MUC5AC, HMCN2 and. Also, 27 transcription factors were upregulated in torpid colonies including ELK1, HDAC3, RBMX, MAZ, STAT1, STAT4 and STAT6. Interestingly, genes involved in developmental processes such as SPIRE1, RHOA, SOX11, WNT5A and SNX18 were also upregulated in torpid colonies. We further validated the dysregulation of 22 genes during torpor by utilizing qPCR. Immunohistochemistry of representative genes from three signaling pathways revealed high expression of these genes in circulated cells along torpor. WNT agonist administration resulted in early arousal from torpor in 80% of the torpid colonies while in active colonies WNT agonist triggered the torpor state. Abovementioned results thus connote unique transcriptome landscapes associated with Botrylloides leachii torpor.
