Physiological changes during torpor favor association with Endozoicomonas endosymbionts in the urochordate Botrylloides leachii.

Environmental perturbations evoke down-regulation of metabolism in some multicellular organisms, leading to dormancy, or torpor. Colonies of the urochordate Botrylloides leachii enter torpor in response to changes in seawater temperature and may survive for months as small vasculature remnants that lack feeding and reproductive organs but possess torpor-specific microbiota. Upon returning to milder conditions, the colonies rapidly restore their original morphology, cytology and functionality while harboring re-occurring microbiota, a phenomenon that has not been described in detail to date. Here we investigated the stability of B. leachii microbiome and its functionality in active and dormant colonies, using microscopy, qPCR, in situ hybridization, genomics and transcriptomics. A novel lineage of Endozoicomonas, proposed here as Candidatus Endozoicomonas endoleachii, was dominant in torpor animals (53-79% read abundance), and potentially occupied specific hemocytes found only in torpid animals. Functional analysis of the metagenome-assembled genome and genome-targeted transcriptomics revealed that Endozoicomonas can use various cellular substrates, like amino acids and sugars, potentially producing biotin and thiamine, but also expressing various features involved in autocatalytic symbiosis. Our study suggests that the microbiome can be linked to the metabolic and physiological states of the host, B. leachii, introducing a model organism for the study of symbioses during drastic physiological changes, such as torpor.

Transcriptome landscapes that signify Botrylloides leachi (Ascidiacea) torpor states.

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 immunolocalization 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.

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. At the outset of hibernation we documented high expression levels of PIWI, PL-10 and PCNA in cells residing in cell islands (CIs), which are the specific stem cell niches found along the endostyle at the ventral side of the zooids. During hibernation, most of the PIWI+/ PL-10+/PCNA+ cells were the MNCs, now located in the newly shaped and dilated vasculature, where they increased in numbers. Also, most of the PCNA+ cells were identified as MNCs. We further documented that the Bl-PIWI RNA (in situ hybridization) and protein (immunohistochemistry) expressions documented during the hibernation/arousal processes diverged significantly from normal blastogenesis expressions. Counting PIWI+ blood cells at various blastogenic stages revealed a significant increase as the hibernation progressed, peaking in aroused colonies at an average of 30 PIWI+ cells/ampulla. The Pl-10 protein expression patterns in the zooids and buds changed as the hibernation progressed, similar to the PIWI and PCNA expressions. Considering the evolutionary perspectives to hibernation we propose linkages to the disposable-soma theory.