Solitary Tunicate Research Articles

Unique Aggregations of a Large Undescribed Solitary Tunicate in the Arabian Sea

We document aggregations of an undescribed benthic solitary tunicate of the family Pyuridae from the Arabian Sea. This new genus was found forming dense thickets in shallow rocky substrates around Masirah Island and the Dhofar area in Oman. Such aggregations of tunicates have not been reported before from coral reefs in the Indo-West Pacific region and the Atlantic. This observation contributes to our understanding of the ecology and biogeography of ascidians, setting the stage for a comprehensive species description and in-depth analysis of this species.

Filtration Rate of the Solitary, Pleated Tunicate Styela plicata on the Brown Tide-Forming Pelagophytes Aureoumbra lagunensis and Aureococcus anophagefferens

Recurring brown tide algal blooms of the pelagophyte Aureoumbra lagunensis in the northern Indian River, Mosquito, and Banana River lagoons on the Atlantic coast of central Florida, USA, have resulted in marked ecological impacts. Recent studies have investigated whether suspension feeding invertebrates could contribute to top-down control of these and other bloom-forming phytoplankton. While most studies focused on the effects A. lagunensis has on on bivalve filter feeding in the Indian River Lagoon, this study focused on feeding by the solitary pleated tunicate Styela plicata. Hourly filtration rates (FR) of individual S. plicata were measured after exposure to 5.0 x 105 cells mL–1 of either A. lagunensis or another pelagophyte Aureococcus anophagefferens. The latter species has not been observed in Florida estuaries but can also inhibit feeding. Tisochrysis lutea, an alga commonly used as feed in aquaculture was used as a control. Differences in hourly FR of either brown tide species compared to the control alga were statistically significant (p < 0.0001). Mean hourly FR of tunicates exposed to Aureoumbra lagunensis ranged from -282 mL hr−1 to 542 mL hr−1, whereas tunicates exposed to the control species Tisochrysis lutea had FR from 285 mL hr−1 to 2648 mL hr−1. Mean hourly FR of tunicates exposed to Aureococcus anophagefferens ranged from 343 mL hr−1 to 593 mL hr−1 compared to tunicates exposed to T. lutea which ranged from 600 mL hr−1 to 2482 mL hr−1. Further, to test whether cell density and a longer exposure time affected FR, individual S. plicata were initially exposed to cell densities of 103, 104, or 105 cells mL–1 of either A. lagunensis or T. lutea (control) for 24 hours. Subsequent measurement of hourly FR and showed cell density removal was not significantly different (p = 0.28 and 0.77, respectively). These results add to the body of literature on how organisms in the Indian River Lagoon filter-feeder community respond to brown tide exposure. The pleated tunicate may be an additional species contributing to top-down control of these bloom-forming microalgae.

Engaging Online Citizen Scientists and the Consensus Method to Monitor the Marine Biofouling Community

Citizen science is an important and useful approach to research that broadens public science engagement and expands the scale at which science can be conducted. Monitoring for marine non-native species has been repeatedly identified as a venue for citizen scientists to make substantial contributions. In this study, we evaluated the accuracy of identifications made by volunteers of marine invertebrates on the project Invader ID, hosted on the online citizen science portal Zooniverse. We tested the efficiency and accuracy of invertebrate identifications made through consensus, where more than one volunteer must agree on a final identification before it is added to the database. Using the Matthew’s Correlation Coefficient, we found that four volunteers in consensus balanced efficiency and accuracy when compared to gold standard scientist identifications. Common, large taxa were identified most accurately; Branching Bryozoa identifications made by four volunteers were 85% accurate, Solitary Tunicates 91% accurate, and Colonial Tunicates 64%. In community-based comparisons, the identity of the taxonomist (volunteer or scientist) had a small impact on overall community composition, while site and salinity gradients had a strong impact on composition. We suggest that citizen science monitoring programs focused on highly recognizable taxonomic groups, or on a few species within those groups that can provide crucial information for managers aiming to quickly identify new invasions. Moreover, long-term assessments of composition could be successfully monitored by volunteers from photographs, helping to bolster understanding of ongoing impacts of invasive species or climate change.

The first record of egg masses in tunicates deposited by the snubnose sculpin, Orthonopias triacis, from the Northeastern Pacific: evidence for convergent evolution of an unusual reproductive strategy.

In this study, the authors report the first record of egg masses deposited in solitary tunicates by the snubnose sculpin, Orthonopias triacis, from the Northeastern Pacific. Four egg masses were discovered in the tunicate Ascidia ceratodes that were genetically determined to be O. triacis. Female O. triacis had long ovipositors that allow deposition of their eggs inside the atrium of the tunicates. A comparison of host-tunicate size with ovipositor length of sculpins from the Northwestern Pacific, including the genera Furcina and Pseudoblennius, revealed that O. triacis had shorter ovipositors and spawned in the atrium of smaller species of tunicates. Ancestral state reconstruction of egg deposition in solitary tunicates using 1.86Mbp RNAseq data of 20 sculpin species from Northeastern and Northwestern Pacific revealed that this unusual spawning behaviour may have evolved convergently in different species occurring in the Northeastern vs. the Northwestern Pacific.

An introgression breakthrough left by an anthropogenic contact between two ascidians.

Human-driven translocations of species have diverse evolutionary consequences such as promoting hybridization between previously geographically isolated taxa. This is well illustrated by the solitary tunicate, Ciona robusta, native to the North East Pacific and introduced in the North East Atlantic. It is now co-occurring with its congener Ciona intestinalis in the English Channel, and C. roulei in the Mediterranean Sea. Despite their long allopatric divergence, first and second generation crosses showed a high hybridization success between the introduced and native taxa in the laboratory. However, previous genetic studies failed to provide evidence of recent hybridization between C. robusta and C. intestinalis in the wild. Using SNPs obtained from ddRAD-sequencing of 397 individuals from 26 populations, we further explored the genome-wide population structure of the native Ciona taxa. We first confirmed results documented in previous studies, notably (i) a chaotic genetic structure at regional scale, and (ii) a high genetic similarity between C. roulei and C. intestinalis, which is calling for further taxonomic investigation. More importantly, and unexpectedly, we also observed a genomic hotspot of long introgressed C. robusta tracts into C. intestinalis genomes at several locations of their contact zone. Both the genomic architecture of introgression, restricted to a 1.5Mb region of chromosome 5, and its absence in allopatric populations suggest introgression is recent and occurred after the introduction of the non-native species. Overall, our study shows that anthropogenic hybridization can be effective in promoting introgression breakthroughs between species at a late stage of the speciation continuum.

The comparison of the properties of nanocellulose isolated from colonial and solitary marine tunicates

This is the first comparative of tunicate cellulose nanocrystalline (t-CNC) from colonial and solitary tunicates. The t-CNC from the colonial tunicate Eudistoma sp. (CL1) was compared with solitary tunicates Polycarpa reniformis (CL2) and Phallusia nigra (CL3). Tunicate samples were extracted by methanol. Residues from the methanol extraction were then subjected to further cellulose purification using pre-hydrolysis, kraft-cooking, bleaching, and sulfuric acid hydrolysis to yield t-CNC. The solitary tunicates yielded higher microfibril contents after the bleaching step but obtained similar t-CNC content to the colonial one after acid hydrolysis. The isolated t-CNC were characterized using Fourier transform infrared spectroscopy, X-ray diffraction, thermalgravimetric analysis, and transmission electron microscopy. Both colonial and solitary tunicates yielded cellulose type I. The pure cellulose type I was successfully isolated from solitary tunicates whereas high inorganic impurities were observed in colonial tunicates. The isolate t-CNC showed high aspect ratios. The solitary and colonial tunicates provided t-CNC with crystallinity indexes over 97% and 35%, respectively. The crystalline size of t-CNCs ranged from 55-124 Å. The thermal stability of all isolated t-CNC was slightly decreased due to the sulfate functional groups gained after acid hydrolysis. We concluded that solitary tunicates were better than colonial tunicates as a source of t-CNC preparation.

Sea-trial verification of a novel system for monitoring biofouling and testing anti-fouling coatings in highly energetic environments targeted by the marine renewable energy industry

A novel system was developed to deploy settlement panels to monitor biofouling growth in situ and evaluate antifouling coatings at depths representative of operational conditions of full-scale marine renewable energy devices. Biofouling loading, species diversity, and succession were assessed at depths ranging from 25-40 m at four tests sites in Orkney (UK) featuring extreme wave and tidal current exposure to more sheltered conditions. Evaluations were carried out over a period of 8 months with intermediate retrieval of samples after 3 months. Early pioneer fouling communities, comprised of colonial hydroids, were succeeded by tube-forming amphipods across sites while solitary tunicates dominated in greater shelter. The highest biofouling loading was observed on high-density polyethylene (HDPE) panels (6.17 kg m−2) compared with coated steel (3.34 kg m−2) panels after 8 months. Distinct assemblages were present at exposed vs sheltered sites. Better understanding of fouling and antifouling strategies may provide guidance to more effectively manage biofouling impacts in this sector.

Oocyte Differentiation and Reproductive Health of Solitary Tunicate (Styela plicata) from Eastern Coast of Thailand

Histopathological examination is a widely acknowledged technique to assess the reproductive health of aquatic organisms, but it has never been applied to the tunicate Styela plicata, a potential indicator species of water quality. In this study, we examined the oocyte differentiation of S. plicata obtained from the eastern coast of the Gulf of Thailand in order to provide basic information for future assessment of its reproductive health. The mature gonad of S. plicata comprised several ovo-testicular convoluted tubes, in which each tube was divided into apical and terminal portions. The ovarian tissue is located in the apical part of the tunicate body and contained oocytes of various differentiation stages (asynchronous development type) consisting of the four phases namely oogonial proliferation phase, primary growth phase, secondary growth phase (secondary growth and full-growth stages), and post-ovulatory phase. Changes in the morphology of oocytes and follicular cells were described for each differentiation stage. In addition, we unexpectedly observed a high prevalence of atretic follicles (24.5%), which might indicate the oocyte damage by environmental stresses. These findings would be useful for future assessment of reproductive histology of S. plicata and other tunicate species from environmental perspectives.

Diversity, Bioactivity Profiling and Untargeted Metabolomics of the Cultivable Gut Microbiota of Ciona intestinalis.

It is widely accepted that the commensal gut microbiota contributes to the health and well-being of its host. The solitary tunicate Ciona intestinalis emerges as a model organism for studying host–microbe interactions taking place in the gut, however, the potential of its gut-associated microbiota for marine biodiscovery remains unexploited. In this study, we set out to investigate the diversity, chemical space, and pharmacological potential of the gut-associated microbiota of C. intestinalis collected from the Baltic and North Seas. In a culture-based approach, we isolated 61 bacterial and 40 fungal strains affiliated to 33 different microbial genera, indicating a rich and diverse gut microbiota dominated by Gammaproteobacteria. In vitro screening of the crude microbial extracts indicated their antibacterial (64% of extracts), anticancer (22%), and/or antifungal (11%) potential. Nine microbial crude extracts were prioritized for in-depth metabolome mining by a bioactivity- and chemical diversity-based selection procedure. UPLC-MS/MS-based metabolomics combining automated (feature-based molecular networking and in silico dereplication) and manual approaches significantly improved the annotation rates. A high chemical diversity was detected where peptides and polyketides were the predominant classes. Many compounds remained unknown, including two putatively novel lipopeptides produced by a Trichoderma sp. strain. This is the first study assessing the chemical and pharmacological profile of the cultivable gut microbiota of C. intestinalis.

Diverse Aromatic Metabolites in the Solitary Tunicate Cnemidocarpa irene.

Fourteen aromatic metabolites (6-19) were isolated from an aqueous extract of the solitary tunicate Cnemidocarpa irene collected in Hokkaido, Japan. The structures of the metabolites were determined based on the spectroscopic interpretations, including one- and two-dimensional NMR, mass spectra, UV, and circular dichroism data. The biopterin analogue 10 modulated the behavior of mice after intracerebroventricular injection and showed a weak affinity to ionotropic glutamate receptor subtypes. Analyses of fluorescent coelomic fluid of the tunicate revealed that pterin 12 was responsible for the fluorescence of the blood cells, while β-carbolines 1 and 3 were fluorescent compounds in the serum. The metabolic profiles in adults, juveniles, larvae, and eggs of the animal differed substantially, suggesting that the metabolism of the animal, especially biosynthesis of aromatic secondary metabolites, changes over different life stages.

Host selection and ovipositor length in eight sympatric species of sculpins that deposit their eggs into tunicates or sponges

Interspecific interactions between parasites and hosts can influence the evolution of behavioural and morphological adaptations of both parasites and their hosts. There is, however, little empirical evidence available regarding the evolution of reproductive traits driven by these interactions. In this paper, we investigated host selection and ovipositor length in nine sympatric marine sculpins that oviposit into tunicates or sponges. Field and genetic studies have revealed host use for eight out of nine species of sculpins investigated here: five species of Pseudoblennius, two species of Furcina and one species of Vellitor. For one species studied (V. minutus), no egg masses could be found. Ovipositor length reflects morphology of host species utilised: six sculpin species had extremely long ovipositors allowing females to attach eggs to the deep atrium of solitary tunicates, whereas the two species that attached their eggs to the small space of atrial siphon of colonial tunicates and the spongocoel of sponges had short ovipositors. Ovipositor length varied between solitary-tunicate spawners and species with longer ovipositors selected larger tunicates. Since the ancestral form is non-parasitic, the ovipositor evolved as an adaptation to utilise sponges and tunicates as hosts. Sculpins found sympatrically may show host specificity to avoid interspecific competition for spawning niches and ovipositors may have evolved depending on the species and size of host invertebrates.

Assessment of tunicates found in the northern waters of Ambon Island and its rearing requirements in a laboratory setting

The Banda Sea region is situated in the Wallacea biogeographical area, known as one of the biodiversity hotspots of the world. The region harbours various types of ecosystems, ranging from mangroves and coral reefs to seaweeds, indicating its potential for harbouring organisms that produce bioactive compounds, such as sea cucumbers, soft corals, and tunicates. While many studies have been carried out on the first two organisms, much lesser is known about the latter one, notably in this region. The assessment is our first attempt to explore the tunicates found in northern coastal waters of Ambon Island, an area that is a part of the Banda Sea. Two types of tunicates were obtained during the survey, namely the violet-green and white-yellow variants of solitary tunicates Polycarpa aurata (15 specimens) and social ones of Didemnum molle (3 specimens). The Polycarpa specimens’ heights ranged from 2.5 to 4.8 cm with wet weights from 9.4 to 28.03 g, and the Didemnum ones were of 1.2 - 2 cm and wet weights from 1.2 to 5 g. Feeding experiment indicated that low volume (5-10 ml) of pure cultures such as Dunaliella sp., Nannochloropsis sp. and Tetraselmis sp. in small aquarium (40 x 60 cm) resulted in good survival rate for Polycarpa aurata collected from northern Ambon Island waters. Rearing experiments in a discontinuous aquarium setting using solitary tunicates indicated the following requirements: thorough cleaning of the tanks, regular feeding intervals, and regular cleaning of each tunicate. The regular cleaning of mucous at the external part of tunicates is indispensable to ensure the wellbeing of the organisms.

Developmental system drift in motor ganglion patterning between distantly related tunicates

BackgroundThe larval nervous system of the solitary tunicate Ciona is a simple model for the study of chordate neurodevelopment. The development and connectivity of the Ciona motor ganglion have been studied in fine detail, but how this important structure develops in other tunicates is not well known.Methods and ResultsBy comparing gene expression patterns in the developing MG of the distantly related tunicate Molgula occidentalis, we found that its patterning is highly conserved compared to the Ciona MG. MG neuronal subtypes in Molgula were specified in the exact same positions as in Ciona, though the timing of subtype-specific gene expression onset was slightly shifted to begin earlier, relative to mitotic exit and differentiation. In transgenic Molgula embryos electroporated with Dmbx reporter plasmids, we were also able to characterize the morphology of the lone pair of descending decussating neurons (ddNs) in Molgula, revealing the same unique contralateral projection seen in Ciona ddNs and their putative vertebrate homologs the Mauthner cells. Although Dmbx expression labels the ddNs in both species, cross-species transgenic assays revealed significant changes to the regulatory logic underlying Dmbx transcription. We found that Dmbx cis-regulatory DNAs from Ciona can drive highly specific reporter gene expression in Molgula ddNs, but Molgula sequences are not active in Ciona ddNs.ConclusionsThis acute divergence in the molecular mechanisms that underlie otherwise functionally conserved cis-regulatory DNAs supports the recently proposed idea that the extreme genetic plasticity observed in tunicates may be attributed to the extreme rigidity of the spatial organization of their embryonic cell lineages.

Oceanographic barriers to gene flow promote genetic subdivision of the tunicate Ciona intestinalis in a North Sea archipelago

Pelagic larval development has the potential to connect populations over large geographic distances and prevent genetic structuring. The solitary tunicate Ciona intestinalis has pelagic eggs and a swimming larval stage lasting for maximum a few days, with the potential for a homogenizing gene flow over relatively large areas. In the eastern North Sea, it is found in a geomorphologically complex archipelago with a mix of fjords and open costal habitats. Here, the coastal waters are also stratified with a marked pycnocline driven by salinity and temperature differences between shallow and deep waters. We investigated the genetic structure of C. intestinalis in this area and compared it with oceanographic barriers to dispersal that would potentially reduce connectivity among local populations. Genetic data from 240 individuals, sampled in 2 shallow, and 4 deep-water sites, showed varying degrees of differentiation among samples (FST = 0.0–0.11). We found no evidence for genetic isolation by distance, but two distant deep-water sites from the open coast were genetically very similar indicating a potential for long-distance gene flow. However, samples from different depths from the same areas were clearly differentiated, and fjord samples were different from open-coast sites. A biophysical model estimating multi-generation, stepping-stone larval connectivity, and empirical data on fjord water mass retention time showed the presence of oceanographic barriers that explained the genetic structure observed. We conclude that the local pattern of oceanographic connectivity will impact on the genetic structure of C. intestinalis in this region.

Correction: Proceraea exoryxae sp. nov. (Annelida, Syllidae, Autolytinae), the first known polychaete miner tunneling into the tunic of an ascidian.

[This corrects the article DOI: 10.7717/peerj.3374.].

De novo draft assembly of the Botrylloides leachii genome provides further insight into tunicate evolution

Tunicates are marine invertebrates that compose the closest phylogenetic group to the vertebrates. These chordates present a particularly diverse range of regenerative abilities and life-history strategies. Consequently, tunicates provide an extraordinary perspective into the emergence and diversity of these traits. Here we describe the genome sequencing, annotation and analysis of the Stolidobranchian Botrylloides leachii. We have produced a high-quality 159 Mb assembly, 82% of the predicted 194 Mb genome. Analysing genome size, gene number, repetitive elements, orthologs clustering and gene ontology terms show that B. leachii has a genomic architecture similar to that of most solitary tunicates, while other recently sequenced colonial ascidians have undergone genome expansion. In addition, ortholog clustering has identified groups of candidate genes for the study of colonialism and whole-body regeneration. By analysing the structure and composition of conserved gene linkages, we observed examples of cluster breaks and gene dispersions, suggesting that several lineage-specific genome rearrangements occurred during tunicate evolution. We also found lineage-specific gene gain and loss within conserved cell-signalling pathways. Such examples of genetic changes within conserved cell-signalling pathways commonly associated with regeneration and development that may underlie some of the diverse regenerative abilities observed in tunicates. Overall, these results provide a novel resource for the study of tunicates and of colonial ascidians.

Population genetic patterns of the solitary tunicate, Molgula manhattensis, in invaded Chinese coasts: large-scale homogeneity but fine-scale heterogeneity

Frequent anthropogenic activities and rapid microevolution associated with rapidly changing environments have complicated the population genetic patterns of invasive species in invaded ranges. In order to deeply understand the mechanisms of invasion success, it is crucial to illuminate population genetic patterns at varied geographical scales in invaded ranges, as well as potential influential factors contributing to observed patterns. In this study, we used both the mitochondrial cytochrome c oxidase subunit I (COI) gene and nuclear microsatellites to investigate the population genetic patterns of a hermaphroditic solitary tunicate, Molgula manhattensis, in invaded Chinese coasts. Our results showed a low level of genetic diversity based on both types of genetic markers. Multiple analyses exhibited a high level of population genetic homogeneity across a wide geographical range. Interestingly, we detected significant population genetic structure at fine geographical scales, particularly for two populations sampled from the Bohai Sea (Laoting and Laizhou). The complex genetic patterns observed in this study, together with multiple putative factors responsible for such patterns, are expected to help understand the invasion success and dispersal dynamics of M. manhattensis along Chinese coasts.

Exploration of the Innate Immune System of Styela clava: Zn2+ Binding Enhances the Antimicrobial Activity of the Tunicate Peptide Clavanin A.

Tunicates have been used as primitive models for understanding cell-mediated and humoral immunity. Clavanin A (ClavA) is one member of a family of antimicrobial peptides produced by the solitary tunicate Styela clava. In this work, we demonstrate that ClavA utilizes Zn2+ ions to potentiate its antimicrobial activity not only by reducing the concentration at which the peptide inhibits the growth of bacteria but also by increasing the rate of killing. Membrane depolarization, β-galactosidase leakage, and potassium leakage assays indicate that ClavA is membrane active, forms small pores, but induces cell death by targeting an intracellular component. ClavA and ClavA-Zn2+ added to Escherichia coli and imaged by confocal microscopy translocate across the cell membrane. E. coli mutants lacking the functional Zn2+ import system are less susceptible to ClavA, suggesting that the synergistic activity between ClavA and Zn2+ has a cytoplasmic target, which is further supported by its nucleolytic activity. Overall, these studies identify a remarkable new mechanism by which zinc contributes to the immune response in the tunicate S. clava.

Chitin protects the gut epithelial barrier in a protochordate model of DSS-induced colitis.

ABSTRACTThe gastrointestinal tract of Ciona intestinalis, a solitary tunicate that siphon-filters water, shares similarities with its mammalian counterpart. The Ciona gut exhibits other features that are unique to protochordates, including certain immune molecules, and other characteristics, e.g. chitin-rich mucus, which appears to be more widespread than considered previously. Exposure of Ciona to dextran sulphate sodium (DSS) induces a colitis-like phenotype similar to that seen in other systems, and is characterized by alteration of epithelial morphology and infiltration of blood cells into lamina propria-like regions. DSS treatment also influences the production and localization of a secreted immune molecule shown previously to co-localize to chitin-rich mucus in the gut. Resistance to DSS is enhanced by exposure to exogenous chitin microparticles, suggesting that endogenous chitin is critical to barrier integrity. Protochordates, such as Ciona, retain basic characteristics found in other more advanced chordates and can inform us of uniquely conserved signals shaping host-microbiota interactions in the absence of adaptive immunity. These simpler model systems may also reveal factors and processes that modulate recovery from colitis, the role gut microbiota play in the onset of the disease, and the rules that help govern the reestablishment and maintenance of gut homeostasis.

Behavioural response of bivalve molluscs to calcium hydroxide

Blue mussels (Mytilus edulis) that are cultivated in the marine area around Prince Edward Island, Eastern Canada, are susceptible to the heavy biofouling of their shells by an invasive solitary tunicate, Styela clava, which rapidly proliferates. To mitigate this issue, mussel farmers periodically lift the longlines on which the mussels are suspended out of the water to spray a highly alkaline (~12.7pH units) calcium hydroxide solution onto fouled individuals. Here, we tested the hypothesis that calcium hydroxide exerts behavioural stress on mussels and other bivalves. Field surveys revealed that the alkalinity of the seawater in the vicinity of longlines increased (9.3–11.7pH units) immediately after treated mussel sleeves were returned into the water column. Thereafter, pH values declined rapidly, and met federal water quality guidelines (7.0–8.7pH units) within 3.1±0.5min (range 0.3–10.5min, n=31 sleeves). Cultivated mussels challenged to both emersion and calcium hydroxide closed their valves for 14.0±3.3min (n=18) compared to 6.5±1.6min (n=17) by control mussels (emersion only). We subsequently assessed how three benthic bivalve species (M. edulis, Crassostrea virginica (eastern oyster) and Argopecten irradians (bay scallop)) respond to weak (pH≤9.2) but sustained (3-h daily for 3days) alkalinity conditions. All three species consistently responded by completely or partially closing their valves. However, all behavioural responses were short-lived (0.2–4.7h), and were generally confined to the treatment period. In conclusion, spraying calcium hydroxide onto cultivated mussels has limited impact on seawater alkalinity and the behaviour of nearby bivalves.