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Abstract This study establishes the copper tolerance range of the colonial marine tunicateBotryllus schlosseri. Furthermore, quantitative organismal phenotyping and quantitative proteomics were combined to characterize theB. schlosseriresponse to, and recovery from, acute copper exposure stress. Changes in the area ofB. schlossericolony systems and pigmentation provided sensitive, dose-dependent markers of exposure to, and recovery from, copper stress. Comprehensive quantitative proteomics using consistent data-independent acquisition (DIA) assay libraries revealed activation of detoxification, oxidative stress, and immune pathways during exposure to copper stress. In addition, quantitative proteomics uncovered enrichment of tissue remodeling and proliferative signaling pathways during recovery from copper stress. We identified 35 proteins whose expression closely mirrored phenotypic changes observed at the colonial system level. This specific proteome signature represents a comprehensive molecular underpinning of the organismal response ofB. schlosserito copper stress. In conclusion, this study establishes copper tolerance ranges of the invasive colonial tunicateB. schlosseriand explains the molecular underpinnings of stress-induced organismal phenotypes by identifying corresponding proteome signatures and their associated functional enrichments. Moreover, identification of copper concentrations that are stressful and highly disruptive on the molecular phenotype, yet readily recoverable from, lays a critical foundation for future studies directed at stress-induced adaptation and evolutionary trajectories of marine invertebrates in changing and novel environments. 

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. 

ABSTRACT Fluctuating salinity is symptomatic of climate change challenging aquatic species. The melting of polar ice, rising sea levels, coastal surface and groundwater salinization, and increased evaporation in arid habitats alter salinity worldwide. Moreover, the frequency and intensity of extreme weather events such as rainstorms and floods increase, causing rapid shifts in brackish and coastal habitat salinity. Such salinity alterations disrupt homeostasis and ultimately diminish the fitness, of aquatic organisms by interfering with metabolism, reproduction, immunity, and other critical aspects of physiology. Proteins are central to these physiological mechanisms. They represent the molecular building blocks of phenotypes that govern organismal responses to environmental challenges. Environmental cues regulate proteins in a concerted fashion, necessitating holistic analyses of proteomes for comprehending salinity stress responses. Proteomics approaches reveal molecular causes of population declines and enable holistic bioindication geared toward timely interventions to prevent local extinctions. Proteomics analyses of salinity effects on aquatic organisms have been performed since the mid‐1990s, propelled by the invention of two‐dimensional protein gels, soft ionization techniques for mass spectrometry (MS), and nano‐liquid chromatography in the 1970s and 1980s. This review summarizes the current knowledge on salinity regulation of proteomes from aquatic organisms, including key methodological advances over the past decades.