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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. 

The colonial ascidianBoytryllus schlosseriis an invasive marine chordate that thrives under conditions of anthropogenic climate change. We show that theB. schlosseriexpressed proteome contains unusually high levels of proteins that are adducted with 4-hydroxy-2-nonenal (HNE). HNE represents a prominent posttranslational modification resulting from oxidative stress. Although numerous studies have assessed oxidative stress in marine organisms HNE protein modification has not previously been determined in any marine species. LC/MS proteomics was used to identify 1052 HNE adducted proteins inB. schlosserifield and laboratory populations. Adducted amino acid residues were ascertained for 1849 modified sites, of which 1195 had a maximum amino acid localization score. Most HNE modifications were at less reactive lysines (rather than more reactive cysteines). HNE prevelance on most sites was high. These observations suggest thatB. schlosseriexperiences and tolerates high intracellular reactive oxygen species levels, resulting in substantial lipid peroxidation. HNE adducted B. schlosseri proteins show enrichment in mitochondrial, proteostasis, and cytoskeletal functions. Based on these results we propose that redox signaling contributes to regulating energy metabolism, the blastogenic cycle, oxidative burst defenses, and cytoskeleton dynamics duringB. schlosseridevelopment and physiology. A DIA assay library was constructed to quantify HNE adduction at 72 sites across 60 proteins that represent a holistic network of functionally discernable oxidative stress bioindicators. We conclude that the vast amount of HNE protein adduction in this circumpolar tunicate is indicative of high oxidative stress tolerance contributing to its range expansion into diverse environments. 

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. 

Abstract Chimerism is a coalescence of conspecific genotypes. Although common in nature, fundamental knowledge, such as the spatial distribution of the genotypes within chimeras, is lacking. Hence, we investigated the spatial distribution of conspecific genotypes within the brooding coralStylophora pistillata, a common species throughout the Indo-Pacific and Red Sea. From eight gravid colonies, we collected planula larvae that settled in aggregates, forming 2–3 partner chimeras. Coral chimeras grew in situ for up to 25 months. Nine chimeras (8 kin, 1 non-related genotypes) were sectioned into 7–17 fragments (6–26 polyps/fragment), and genotyped using eight microsatellite loci. The discrimination power of each microsatellite-locus was evaluated with 330 ‘artificial chimeras,’ made by mixing DNA from three differentS. pistillatagenotypes in pairwise combinations. In 68% of ‘artificial chimeras,’ the second genotype was detected if it constituted 5–30% of the chimera. Analyses ofS. pistillatachimeras revealed that: (a) chimerism is a long-term state; (b) conspecifics were intermixed (not separate from one another); (c) disproportionate distribution of the conspecifics occurred; (d) cryptic chimerism (chimerism not detected via a given microsatellite) existed, alluding to the underestimation of chimerism in nature. Mixed chimerism may affect ecological/physiological outcomes for a chimera, especially in clonal organisms, and challenges the concept of individuality, affecting our understanding of the unit of selection. 

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.