Whole body regeneration and developmental competition in two botryllid ascidians

Background Botryllid ascidians are a group of marine invertebrate chordates that are colonial and grow by repeated rounds of asexual reproduction to form a colony of individual bodies, called zooids, linked by a common vascular network. Two distinct processes are responsible for zooid regeneration. In the first, called blastogenesis, new zooids arise from a region of multipotent epithelium from a pre-existing zooid. In the second, called whole body regeneration (WBR), mobile cells in the vasculature coalesce and are the source of the new zooid. In some botryllid species, blastogenesis and WBR occur concurrently, while in others, blastogenesis is used exclusively for growth, while WBR only occurs following injury or exiting periods of dormancy. In species such as Botrylloides diegensis, injury induced WBR is triggered by the surgical isolation of a small piece of vasculature. However, Botryllus schlosseri has unique requirements that must be met for successful injury induced WBR. Our goal was to understand why there would be different requirements between these two species. Results While WBR in B. diegensis was robust, we found that in B. schlosseri, new zooid growth following injury is unlikely due to circulatory cells, but instead a result of ectopic development of tissues leftover from the blastogenic process. These tissues could be whole, damaged, or partially resorbed developing zooids, and we defined the minimal amount of vascular biomass to support ectopic regeneration. We did find a common theme between the two species: a competitive process exists which results in only a single zooid reaching maturity following injury. We utilized this phenomenon and found that competition is reversible and mediated by circulating factors and/or cells. Conclusions We propose that WBR does not occur in B. schlosseri and that the unique requirements defined in other studies only serve to increase the chances of ectopic development. This is likely a response to injury as we have discovered a vascular-based reversible competitive mechanism which ensures that only a single zooid completes development. This competition has been described in other species, but the unique response of B. schlosseri to injury provides a new model to study resource allocation and competition within an individual.

Identification and characterisation of Botrylloides species from Aotearoa New Zealand coasts

ABSTRACTBotryllid ascidians possess diverse biological abilities like whole-body regeneration (WBR), hibernation/aestivation, blastogenesis, metamorphosis, and natural chimerism. However, the absence of distinctive morphological features often makes identification problematic. Botrylloides diegensis is a botryllid ascidian that has been misidentified in previous studies and is recorded in GenBank as Botrylloides leachii due to the high morphological similarity between the sister species. More available sequences and strategies around identification would help resolve some of the confusion currently surrounding its ambiguous nature. We collected several Botrylloides samples from 7 locations around New Zealand and barcoded the species based on Cytochrome Oxidase I, Histone 3, 18S, and 28S ribosomal RNA markers. Network and Bayesian trees confirmed three Botrylloides species: B. diegensis, B. jacksonianum, and B. aff. anceps. Additionally, recognition assays were applied to analyse the histocompatibility between distinct morphs qualitatively.

First Record of Colonial Ascidian, Botrylloides diegensis Ritter and Forsyth, 1917 (Ascidiacea, Stolidobranchia, Styelidae), in South Korea

Botrylloides species are important members of the fouling community colonizing artificial substrates in harbors and marinas. During monitoring in 2017–2020 of non-indigenous species in Korea, one colonial ascidian species was distinctly different from other native colonial ascidians, such as B. violaceus and Botryllus schlosseri, in South Korea. This species was identified as B. diegensis. DNA barcodes with mitochondrial COI were used to identify one-toned and two-toned colonies of B. diegensis. Intraspecific variations between Korean and other regions of B. diegensis from the NCBI ranged from 0.0% to 1.3%. The Korean B. diegensis was clearly distinct from other species of Botrylloides at 15.8–24.2%. In phylogenetic analysis results, Korean B. diegensis was established as a single clade with other regions of B. diegensis and was clearly distinct from Korean B. violaceus. After reviewing previous monitoring data, it was found that two-toned B. diegensis was already found in six harbors by July 2017. It has now spread into 14 harbors along the coastal line of South Korea. This means that B. diegensis might have been introduced to South Korea between 1999 and 2016.

In-land long-term culture of colonial ascidians

1.AbstractTunicates are highly diverse marine invertebrate filter-feeders that are vertebrates’ closest relatives. These organisms, despite a drastically different body plan during their adulthood, have a tissue complexity related to that of vertebrates. Ascidians, which compose most of the Tunicata, are benthic sessile hermaphrodites that reproduce sexually through a motile tadpole larval stage. Over half of the known ascidians species are able to reproduce asexually through budding, typically leading to the formation of colonies where animals, called zooids, are interconnected by an external vascular system. In addition, colonial ascidians are established models for important biological processes including allorecognition, immunobiology, aging, angiogenesis and whole-body regeneration. However, the current paucity in breeding infrastructures limits the study of these animals to coastal regions.To promote a wider scientific spreading and popularity of colonial ascidians, we have developed a flexible recirculating husbandry setup for their long-term in-lab culture. Our system is inspired both by the flow-through aquariums used by coastal ascidian labs, as well as by the recirculating in-lab systems used for zebrafish research. Our hybrid system thus combines colony breeding, water filtering and food culturing in a semi-automated system where specimens develop on hanging microscopy glass slides. Temperature, light/dark cycles, flow speed and feeding rates can be controlled independently in four different breeding environments to provide room for species-specific optimization as well as for running experiments. This setup is complemented with a quarantine for the acclimatization of wild isolates.We here present our success in breeding Botrylloides diegensis, a species of colonial ascidians, for more than 3 years in recirculating artificial seawater over 600 km from their natural habitat. We show that colonies adapt well to in-lab culturing and that a specific strain can be isolated, propagated and used for research efficiently over prolonged periods of time. The flexible and modular structure of our system can be scaled and adapted to the needs of specific species as well as of particular laboratory spaces. Overall, we show that Botrylloides diegensis can be proficiently bred in-land and suggest that our results can be extended to other species of colonial ascidians to promote research on these fascinating animals.HighlightsFirst in-land recirculating aquaculture for colonial ascidiansOver 3 years of continuous breedingSemi-automated setup with minimized maintenanceGood biomass production for strain propagation4 different culture conditions for optimized breeding for species of interest

Integrin-alpha-6+ Candidate stem cells are responsible for whole body regeneration in the invertebrate chordate Botrylloides diegensis

Colonial ascidians are the only chordates able to undergo whole body regeneration (WBR), during which entire new bodies can be regenerated from small fragments of blood vessels. Here, we show that during the early stages of WBR in Botrylloides diegensis, proliferation occurs only in small, blood-borne cells that express integrin-alpha-6 (IA6), pou3 and vasa. WBR cannot proceed when proliferating IA6+ cells are ablated with Mitomycin C, and injection of a single IA6+ Candidate stem cell can rescue WBR after ablation. Lineage tracing using EdU-labeling demonstrates that donor-derived IA6+ Candidate stem cells directly give rise to regenerating tissues. Inhibitors of either Notch or canonical Wnt signaling block WBR and reduce proliferation of IA6+ Candidate stem cells, indicating that these two pathways regulate their activation. In conclusion, we show that IA6+ Candidate stem cells are responsible for whole body regeneration and give rise to regenerating tissues.

Integrin-alpha-6+ Stem Cells (ISCs) are responsible for whole body regeneration in an invertebrate chordate

Colonial ascidians are the only chordates able to undergo whole body regeneration (WBR), during which entire new bodies can be regenerated from small fragments of blood vessels. Here, we show that during the early stages of WBR in Botrylloides diegensis, proliferation occurs only in small, blood-borne cells that express integrin-alpha-6 (IA6), pou3 and vasa. Ablation of proliferating cells using Mitomycin C (MMC) blocks WBR in vascular fragments, but can be rescued by injection of cycling cells isolated from an untreated individual. Using prospective isolation and limit dilution analyes, we found that FACS-isolated IA6+ stem cells (ISCs) could rescue WBR in MMC treated vascular fragments, even when injecting only a single cell. Lineage tracing using EdU-labeling further revealed that donor-derived ISCs directly give rise to regenerating tissues. Inhibitors of either Notch or canonical Wnt signaling block WBR and reduce proliferation of ISCs, indicating that these two pathways regulate ISC activation.