Deep quantitative proteomics of North American Pacific coast star tunicate (Botryllus schlosseri).

Botryllus schlosseri, is a model marine invertebrate for studying immunity, regeneration, and stress-induced evolution. Conditions for validating its predicted proteome were optimized using nanoElute® 2 deep-coverage LCMS, revealing up to 4930 protein groups and 20,984 unique peptides per sample. Spectral libraries were generated and filtered to remove interferences, low-quality transitions, and only retain proteins with >3 unique peptides. The resulting DIA assay library enabled label-free quantitation of 3426 protein groups represented by 22,593 unique peptides. Quantitative comparisons of single systems from a laboratory-raised with two field-collected populations revealed (1) a more unique proteome in the laboratory-raised population, and (2) proteins with high/low individual variabilities in each population. DNA repair/replication, ion transport, and intracellular signaling processes were distinct in laboratory-cultured colonies. Spliceosome and Wnt signaling proteins were the least variable (highly functionally constrained) in all populations. In conclusion, we present the first colonial tunicate's deep quantitative proteome analysis, identifying functional protein clusters associated with laboratory conditions, different habitats, and strong versus relaxed abundance constraints. These results empower research on B. schlosseri with proteomics resources and enable quantitative molecular phenotyping of changes associated with transfer from in situ to ex situ and from in vivo to in vitro culture conditions.

Transient impacts of UV-B irradiation on whole body regeneration in a colonial urochordate.

Within the chordates, only some colonial ascidians experience whole body regeneration (WBR), where amputated small colonial fragments containing blood-vessels have the capability to regenerate the entire functional adult zooid within 1-3 weeks. Studying WBR in small colonial fragments taken at different blastogenic stages (the weekly developmental process characteristic to botryllid ascidians) from the ascidian Botrylloides leachii, about half of the fragments were able to complete regeneration (cWBR) three weeks following separation, about half were still in uncomplete, running regeneration (rWBR), and only a small percentage died. cWBR significantly increased in fragments that originated from a late blastogenic stage compared to an early stage. Most B. leachii populations reside in shallow waters, under variable daily natural UV irradiation, and it is of interest to elucidate irradiation effects on development and regeneration. Here, we show that UV-B irradiation resulted in enhanced mortality, with abnormal morphological changes in surviving fragments, yet with non-significant cWBR vs. rWBRs. Further, UV-B irradiation influenced the proportion of blood cells (morula cells, hemoblasts) and of multinucleated cells, a new WBR-associated cell type. At 24-h post-amputation we observed enhanced expression of ß-catenin (a signaling pathway that plays indispensable roles in cell renewal and regeneration), H3 and PCNA in all cell types of non-irradiated as compared to irradiated fragments. These elevated levels were considerably reduced 9-days later. Since WBR is a highly complex phenomenon, the employment of specific experimental conditions, as UV-B irradiation, alongside blastogenesis (the weekly developmental process), elucidates undisclosed facets of this unique biological occurrence such as transient expression of signature genes.

Improved Media Formulations for Primary Cell Cultures Derived from a Colonial Urochordate.

The cultivation of marine invertebrate cells in vitro has garnered significant attention due to the availability of diverse cell types and cellular potentialities in comparison to vertebrates and particularly in response to the demand for a multitude of applications. While cells in the colonial urochordate Botryllus schlosseri have a very high potential for omnipotent differentiation, no proliferating cell line has been established in Botryllus, with results indicating that cell divisions cease 24-72 h post initiation. This research assessed how various Botryllus blood cell types respond to in vitro conditions by utilizing five different refinements of cell culture media (TGM1-TGM5). During the initial week of culture, there was a noticeable medium-dependent increase in the proliferation and viability of distinct blood cell types. Within less than one month from initiation, we developed medium-specific primary cultures, a discovery that supports larger efforts to develop cell type-specific cultures. Specific cell types were easily distinguished and classified based on their natural fluorescence properties using confocal microscopy. These results are in agreement with recent advances in marine invertebrate cell cultures, demonstrating the significance of optimized nutrient media for cell culture development and for cell selection.

UV-B radiation bearings on ephemeral soma in the shallow water tunicate Botryllus schlosseri.

Sedentary shallow water marine organisms acquire numerous protective mechanisms to mitigate the detrimental effects of UV radiation (UV-R). Here we investigated morphological and gene expression outcomes in colonies of the cosmopolitan ascidian Botryllus schlosseri, up to 15-days post UV-B irradiation. Astogeny in Botryllus is characterized by weekly repeating sets of asexual budding, coinciding with apoptotic elimination of functional zooids (blastogenesis). Ten UV-B doses were administered to three clusters: sublethal, enhanced-mortality, lethal (LD50 = 6.048 kJ/m2) which differed in mortality rates, yet reflected similar distorted morphotypes, and arrested blastogenesis, all intensified in the enhanced-mortality/lethal clusters. Even the sub-lethal doses inflicted expression modifications in 8 stress proteins (HSP 90/70 families and NIMA) as well as morphological blastogenesis. The morphological/gene-expression impacts in surviving colonies lasted for 15 days post irradiation (two blastogenic-cycles), where all damaged and arrested zooids/buds were absorbed, after which the colonies returned to their normal blastogenic-cycles and gene expression profiles, and initiated new buds. The above reflects a novel colonial maintenance strategy associated with the disposable-soma tenet, where the ephemeral soma in Botryllus is eliminated without engaging with the costs of repair, whereas other colonial components, primarily the pool of totipotent stem cells, are sustained under yet unknown colonial-level regulatory cues.