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We studied the function, development and aging of the adult nervous system in the colonial tunicate Botryllus schlosseri. Adults, termed zooids, are filter-feeding individuals. Sister zooids group together to form modules, and modules, in turn, are linked by a shared vascular network to form a well-integrated colony. Zooids undergo a weekly cycle of regression and renewal during which mature zooids are replaced by developing buds. The zooid brain matures and degenerates on this 7-day cycle. We used focal extracellular recording and video imaging to explore brain activity in the context of development and degeneration and to examine the contributions of the nervous system and vascular network to behavior. Recordings from the brain revealed complex firing patterns arising both spontaneously and in response to stimulation. Neural activity increases as the brain matures and declines thereafter. Motor behavior follows the identical time course. The behavior of each zooid is guided predominantly by its individual brain, but sister zooids can also exhibit synchronous motor behavior. The vascular network also generates action potentials that are largely independent of neural activity. In addition, the entire vascular network undergoes slow rhythmic contractions that appear to arise from processes endogenous to vascular epithelial cells. We found that neurons in the brain and cells of the vascular network both express multiple genes for voltage-gated Na+ and Ca2+ ion channels homologous (based on sequence) to mammalian ion channel genes., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The colonial tunicate Botrylloides leachii is exceptional at regenerating from a piece of vascular tunic after loss of all adults from the colony. Previous transcriptome analyses indicate a brief period of healing before regeneration of a new adult (zooid) in as little as 8-10 days. However, there is little understanding of how the resulting changes to gene expression, required to drive regeneration, are initiated and how the overall process is regulated. Rapid changes to transcription often occur in response to chromatin changes, mediated by histone modifications such as histone acetylation. Here, we investigated a group of key epigenetic modifiers, histone deacetylases (HDAC), which are known to play an important role in many biological processes such as development, healing and regeneration. Through our transcriptome data, we identified and quantified the expression levels of HDAC and histone acetyltransferase enzymes during whole-body regeneration (WBR). To determine whether HDAC activity is required for WBR, we inhibited its action using valproic acid and trichostatin A. HDAC inhibition prevented the final morphological changes normally associated with WBR and resulted in aberrant gene expression. Botrylloides leachii genes including Slit2 , TGF-β , Piwi and Fzd4 all showed altered mRNA levels upon HDAC inhibition in comparison with the control samples. Additionally, atypical expression of Bl_Piwi was found in immunocytes upon HDAC inhibition. Together, these results show that HDAC function, specifically HDAC I/IIa class enzymes, are vital for B. leachii to undergo WBR successfully., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The mechanisms of action potential (AP) generation in the myoepithelial cells of the tunicate heart are not yet well understood. Here, an attempt was made to elucidate these mechanisms by analyzing the effects of specific blockers of K + , Na + and Ca 2+ currents on the configuration of transmembrane APs and their frequency in the spontaneously beating ascidian heart. In addition, an immunocytochemical analysis of heart myoepithelial cells was performed. Staining with anti-FMRF-amide and anti-tubulin antibodies did not reveal any nerve elements within the heart tube. Treatment with 1 mmol l -1 TEA ( I K blocker) resulted in depolarization of heart cell sarcolemma by 10 mV, and inhibition of APs generation was recorded after 3 min of exposure. Prior to this moment, the frequency of AP generation in a burst decreased from 16-18 to 2 beats min -1 owing to prolongation of the diastole. After application of ivabradine (3 or 10 µmol l -1 ), the spontaneous APs generation frequency decreased by 24%. Based on these results and published data, it is concluded that the key role in the automaticity of the ascidian heart is played by the outward K + currents, Na + currents, activated hyperpolarization current I f and a current of unknown nature I X ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Benthic marine suspension feeders provide an important link between benthic and pelagic ecosystems. The strength of this link is determined by suspension-feeding rates. Many studies have measured suspension-feeding rates using indirect clearance-rate methods, which are based on the depletion of suspended particles. Direct methods that measure the flow of water itself are less common, but they can be more broadly applied because, unlike indirect methods, direct methods are not affected by properties of the cleared particles. We present pumping rates for three species of suspension feeders, the clams Mya arenaria and Mercenaria mercenaria and the tunicate Ciona intestinalis , measured using a direct method based on particle image velocimetry (PIV). Past uses of PIV in suspension-feeding studies have been limited by strong laser reflections that interfere with velocity measurements proximate to the siphon. We used a new approach based on fitting PIV-based velocity profile measurements to theoretical profiles from computational fluid dynamic (CFD) models, which allowed us to calculate inhalant siphon Reynolds numbers ( Re ). We used these inhalant Re and measurements of siphon diameters to calculate exhalant Re , pumping rates, and mean inlet and outlet velocities. For the three species studied, inhalant Re ranged from 8 to 520, and exhalant Re ranged from 15 to 1073. Volumetric pumping rates ranged from 1.7 to 7.4 l h -1 for M . arenaria , 0.3 to 3.6 l h -1 for M . m ercenaria and 0.07 to 0.97 l h -1 for C . intestinalis We also used CFD models based on measured pumping rates to calculate capture regions, which reveal the spatial extent of pumped water. Combining PIV data with CFD models may be a valuable approach for future suspension-feeding studies., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Tunicates, small invertebrates within the phylum Chordata, possess a robust tubular heart which pumps blood through their open circulatory systems without the use of valves. This heart consists of two major components: the tubular myocardium, a flexible layer of myocardial cells that actively contracts to drive fluid down the length of the tube; and the pericardium, a stiff, outer layer of cells that surrounds the myocardium and creates a fluid-filled space between the myocardium and the pericardium. We investigated the role of the pericardium through in vivo manipulations on tunicate hearts and computational simulations of the myocardium and pericardium using the immersed boundary method. Experimental manipulations reveal that damage to the pericardium results in aneurysm-like bulging of the myocardium and major reductions in the net blood flow and percentage closure of the heart's lumen during contraction. In addition, varying the pericardium-to-myocardium (PM) diameter ratio by increasing damage severity was positively correlated with peak dye flow in the heart. Computational simulations mirror the results of varying the PM ratio experimentally. Reducing the stiffness of the myocardium in the simulations reduced mean blood flow only for simulations without a pericardium. These results indicate that the pericardium has the ability to functionally increase the stiffness of the myocardium and limit myocardial aneurysms. The pericardium's function is likely to enhance flow through the highly resistive circulatory system by acting as a support structure in the absence of connective tissue within the myocardium., (© 2015. Published by The Company of Biologists Ltd.)

Free-living animals and their larvae utilize light and gravity as cues to navigate in open space. Detection and response to these environmental stimuli are important for the dispersal and settlement of ascidian larvae. Two pigmented structures in the brain of the ascidian larva, the ocellus and the otolith, have been shown to function as the photoreceptive and gravity sensitive organs, respectively. Here, we show that pigmentation is essential for proper phototactic and geotactic behavior in larvae of the ascidian species Ciona savignyi. Two recessive and complementing mutant lines of C. savignyi, immaculate and spotless, that specifically disrupt the pigmentation of the sensory organs during larval development are described. Homozygous mutant larvae are unable to respond properly to gravity and illumination cues while settling. Genetic analysis shows that spotless is caused by a point mutation within the tyrosinase gene that creates a premature stop codon, while the molecular nature of immaculate is unknown. Although the role of pigmentation in the ocellus of C. savignyi is similar to that in vertebrate visual systems, our results demonstrate a novel use of melanin in geotactic behavior.

Colonies of the urochordate Botryllus schlosseri may fuse upon contact if they share common alleles on the highly polymorphic fusibility/histocompatibility locus. While, in these chimeras, one of the partners is usually morphologically eliminated (resorbed), circulating totipotent cells of the inferior genotype on the resorption phenomenon may parasitize either the soma or the germ line of the winner. Here, we show an environmental split of the two stem cell lineages that may develop germ cell parasitism vs somatic cell cooperation. Each naturally formed Botryllus chimera can be a composite of component genotypes created through two unlinked parasitic germ and somatic cell lineage interactions. The germ line parasitism is inherited through a pedigree. Conversely, by using amplified fragment length polymorphism (AFLP) and microsatellite alleles as polymorphic genetic markers, and seawater temperature as the variable environmental factor, we documented that the somatic constituent of chimeric zooids was shifted from one genotype to another, in accordance with the changes in seawater temperatures. This variable somatic state of chimerism in the field may, thus, carry benefits to the chimeral entity, which presents synergistically, at any time, the best-fitted combination of its genetic components.

In the colonial growth of botryllid ascidians, blastogenesis (bud formation) is a cyclical and synchronized developmental process characterized by a weekly rhythm of budding and apoptotic events. Very little is known about this cycle regulation and its control. In this study, the in vitro fate of developing buds and regressing zooids extirpated from Botryllus schlosseri colonies at different blastogenic stages were examined, revealing that stages 'B' to 'D' buds (but not stage 'A' buds) developed new structures under in vitro conditions. These were mainly spheres (up to 1 mm in diameter) and epithelial monolayers around the attached buds. We also found that: (1) when attached spheres and epithelial monolayers appeared, the life expectancy of an isolated bud in vitro reached 50-60 days, five times the life expectancy of intact, in vivo developing zooids; (2) the life expectancy of in vitro buds that remained unattached to the substrates was at least 150 days; (3) after attaching to the substrates, buds obeyed a newly imposed developmental clock dictating up to 35 survival days for spheres and up to 14 days for epithelial monolayers; (4) the prevailing mode of death in vitro was necrotic, in contrast to the apoptotic mode of zooidal deterioration at the takeover phase of blastogenesis; (5) under in vitro conditions, degenerating zooids surprisingly produced epithelial monolayers within 3 weeks of culturing. Monolayers survived for up to 10 additional days, extending the lifespan of the degenerating zooids from a few hours to up to 1 month. We conclude that under in vitro conditions, not only are the underlying colonial growth mechanisms replaced by different developmental pathways, but also the internal colonial-level clocks programming death, are replaced by a new biological mechanism with different timetables.

Understanding how the shape and motion of an aquatic animal affects the performance of swimming requires knowledge of the fluid forces that generate thrust and drag. These forces are poorly understood for the large diversity of animals that swim at Reynolds numbers (Re) between 10(0) and 10(2). We experimentally tested quasi-steady and unsteady blade-element models of the hydrodynamics of undulatory swimming in the larvae of the ascidian Botrylloides sp. by comparing the forces predicted by these models with measured forces generated by tethered larvae and by comparing the swimming speeds predicted with measurements of the speed of freely swimming larvae. Although both models predicted mean forces that were statistically indistinguishable from measurements, the quasi-steady model predicted the timing of force production and mean swimming speed more accurately than the unsteady model. This suggests that unsteady force (i.e. the acceleration reaction) does not play a role in the dynamics of steady undulatory swimming at Re approximately 10(2). We explored the relative contribution of viscous and inertial force to the generation of thrust and drag at 10(0)10(2)) and low (<10(0)) Re, the fluid forces that generate thrust cannot be assumed to be the same as those that generate drag at intermediate Re.

A great diversity of unicellular and invertebrate organisms swim along a helical path, but it is not well understood how asymmetries in the body shape or the movement of propulsive structures affect a swimmer's ability to perform the body rotation necessary to move helically. The present study found no significant asymmetries in the body shape of ascidian larvae (Distaplia occidentalis) that could operate to rotate the body during swimming. By recording the three-dimensional movement of free-swimming larvae, it was found that the tail possessed two bends, each with constant curvature along their length. As these bends traveled posteriorly, the amplitude of curvature changes was significantly greater in the concave-left direction than in the concave-right direction. In addition to this asymmetry, the tail oscillated at an oblique angle to the midline of the trunk. These asymmetries generated a yawing moment that rotated the body in the counterclockwise direction from a dorsal view, according to calculations from hydrodynamic theory. The tails of resting larvae were bent in the concave-left direction with a curvature statistically indistinguishable from the median value for tail curvature during swimming. The flexural stiffness of the tails of larvae, measured in three-point bending, may be great enough to allow the resting curvature of the tail to have an effect on the symmetry of kinematics. This work suggests that asymmetrical tail motion is an important mechanism for generating a yawing moment during swimming in ascidian larvae and that these asymmetries may be caused by the tail's bent shape. Since helical motion requires that moments also be generated in the pitching or rolling directions, other mechanisms are required to explain fully how ascidian larvae generate and control helical swimming.

Most biological motions are three-dimensional. This includes the trajectories of whole organisms and of their appendages. While recordings of three-dimensional trajectories are sometimes published, quantitative analysis of these trajectories is uncommon, primarily because there are no standard techniques or conventions in biology for the analysis of three-dimensional trajectories. This paper describes a new technique, finite helix fit (FHF), based on the geometry of three-dimensional curves, whereby a three-dimensional trajectory is completely described by its velocity, curvature and torsion. FHF estimates these parameters from discretely sampled points on a trajectory (i.e. from positional data such as x,y,z coordinates). Other measures of motion can be derived from these parameters, such as the translational and rotational (or angular) velocities of an organism. The performance of the algorithms is demonstrated using simulated trajectories and trajectories of freely swimming organisms (a flagellate, Chlamydomonas reinhardtii; a ciliate, Paramecium tetraurelia; spermatozoa of a sea urchin, Arbacia punctulata; larvae of an ascidian, Botrylloides sp.).

We can gain biomechanical insights if we couple knowledge of the environments, ecological roles and life history strategies of organisms with our laboratory analyses of their mechanical function or fluid dynamics, as illustrated by studies of the mechanical design of bottom-dwelling marine organisms. Obviously, measurements of the spatial and temporal distribution of loads on an organism in nature reveal the magnitudes and rates at which biomechanical tests should be performed in the laboratory. Furthermore, knowledge of the population biology and ecological interactions of the organisms being studied is crucial to determine when during the life of an individual particular aspects of mechanical performance should be measured; not only can the size, shape and material properties of an individual change during ontogeny, but so can its habitat, activities and ecological role. Such ecological information is also necessary to determine whether the aspects of mechanical performance being studied are biologically important, i.e. whether they affect the survivorship or fitness of the organisms. My point in raising these examples is to illustrate how ecological studies can enhance or change our understanding of biomechanical function.

Sessile, soft-bodied, compound ascidians are successful competitors for substrata in crowded benthic and epibiotic marine communities and can be effective colonists of new sites, through adult rafting and reattachment. Adhesion to the substratum is essential for these ecologically important functions; we therefore studied the material properties of colony attachment to various substrata in the rafting ascidians Botrylloides sp. We found that, compared with the strength of the colony tissues, the glue attaching Botrylloides sp. to the substratum is very weak. This relative weakness may protect the soft-bodied colonies from damage if they are ripped from their host. For sessile animals, such a weak-glue 'strategy' is only effective if the animals can later reattach to a substratum. By detaching Botrylloides sp. colonies from host eelgrass blades and allowing them to reattach, before measuring peel strengths, we learned that the initial reattachment of a colony depends upon rapid new growth of the colony rather than on fresh secretion of glue beneath old zooids. We also found that the propagation peel force necessary to remove Botrylloides sp. from different substrata (e.g. mussel shells, barnacle basal plates or eelgrass blades) depends upon the surface texture of the host. Thus, the overall tenacity of a colony is affected by the types of substrata that it overgrows.