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Abstract The colonial tunicateBotryllus schlosseriregenerates weekly through a cyclical process in which adult zooids are replaced by a new generation of buds. While this dynamic asexual development is a hallmark of the species, its molecular regulation remains poorly understood. This study presents the first comprehensive proteomic analysis ofB. schlosseriblastogenesis at the individual zooid level, using data-independent acquisition mass spectrometry to quantify protein abundance across developmental stages. The results reveal extensive proteome remodeling between proliferating buds and degenerating zooids. Co-expression analysis identified stage-specific protein modules enriched for biosynthesis and cell cycle pathways in buds, and for apoptosis, catabolism, and metabolic remodeling in zooids. A focused comparison between takeover buds and takeover zooids uncovered distinct regulatory programs controlling proliferation and senescence. Key proteins, including CDK1, CDK2, HDAC2, and PCNA, were identified as candidate regulators of cell cycle progression. These findings provide a molecular framework for understanding regeneration in a basal chordate and offer protein targets that may enable cell cycle re-entry and long-term culture of tunicate primary cells. Summary StatementThis study maps proteome dynamics during the blastogenic cycle inBotryllus schlosseri, identifying candidate proteins that regulate cell proliferation and offer targets for tunicate cell line development. 

Advanced methodologies forBotryllus schlosseriartificial seawater systems are needed to decrease dependency of large-scale culture on natural seawater and expand use of this important new model organism to more inland laboratories. We constructed two botryllid tunicate customized closed aquaculture systems, a static system consisting of lightly aerated jars fed with commercial filter feeder diet, and a recirculating aquaculture system (RAS) consisting of standard marine RAS components fed live microalgae and zooplankton diets. Initially, static tunicate culture yielded exponential growth in contrast to the RAS system, which yielded poor survival and negligible growth. Modifications were made to the RAS system to improve water treatment proficiency that greatly improved tunicate survival and growth. Experiments were performed isolating feed and water type as variables that differed between the static and RAS systems to evaluate their effects. A live feed combination achieved five-fold greater growth relative to a commercial concentrate diet.B. schlosserimaintained in optimized RAS water achieved two-fold faster growth relative to animals maintained with freshly prepared artificial seawater indicating that the RAS water was beneficial to the animals. Feeding frequency of the RAS system was increased from three times per week to daily. The RAS system and procedural modifications resulted in comparable growth rates in the static and RAS systems. Both optimized systems are suitable for long-term propagation and sustenance of botryllid tunicate populations supporting both sexual and asexual modes of reproduction with a current residence time of over 24 months. 

Botryllus schlosseri is a colonial chordate species that exhibits robust stem cell-mediated regeneration capacities throughout life. It grows through a process of colonial budding known as blastogenesis, in addition to its sexual reproduction mode, embryogenesis. In natural conditions, colony growth peaks in the summer and drastically decreases in the winter due to various seasonal factors. We have established and optimized a method to rear B. schlosseri animals in landlocked laboratories where the controlled conditions allow colonies to grow continuously and exponentially through asexual reproduction under constant temperature, salinity, light, and nutrient conditions. During the weekly blastogenic cycle, all asexually derived parental zooids synchronously regress within one day and are replaced by a new generation. To enable comprehensive molecular phenotyping of this species we established a quantitative proteomics workflow and spectral library for Liquid chromatography/Tandem mass spectrometry (LCMS) data-independent acquisition (DIA). In this study, we quantified the relative abundances of several thousand proteins representing molecular phenotypes of B. schlosseri. This workflow enables us to use proteomics to characterize zooid development during its regenerative cycle, including the short critical period of the takeover stage, during which degenerating zooids undergo massive apoptosis while succeeding buds quickly mature and migrate into place. From this data, protein networks associated with regeneration and degeneration can be created, offering insights into tissue developmental pathways in vivo and future cell immortalization strategies in vitro. Furthermore, the establishment of quantitative proteomics workflows for B. schlosseri coupled with its unique life cycle features promotes the use of this model organism for the study of phenotypic plasticity and evolution of aging, stem cells, and mechanisms of regeneration and cell differentiation. This project is funded by NSF Grant MCB — 2127516. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process. 

Abstract 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 onB. schlosseriwith 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.