Botrylloides Violaceus Research Articles

Variation in Whole‐Body Regeneration Between Botrylloides Morphs and Species

Marine invertebrates exhibit an impressive regenerative capacity. Among these lineages, colonial ascidians possess the unique ability of whole‐body regeneration (WBR), in which a new adult (zooid) body is restored. Botrylloides species exemplify this regenerative capacity by regenerating new adults solely from vascular tissue within approximately 2 weeks after the complete loss of adults from the colony. Although WBR has been reported in some species of the Botrylloides genus, such as Botrylloides diegensis, Botrylloides violaceus, and Botrylloides anceps, this study provides the first insights into the WBR of Botrylloides jacksonianum.In this study, we examined and compared the WBR processes of B. jacksonianum, B. affinis anceps (hereafter referred to as B. anceps here), and B. diegensis. B. anceps displayed twin‐body regeneration, whereas B. jacksonianum and B. diegensis regenerated only one zooid each. Histological examination confirmed the presence of multiple vesicles formed from hemoblast aggregates during WBR, a phenomenon observed in other Botrylloides spp. The investigated species exhibited strong similarities in their WBR, with some variations in the overall process, indicating the presence of similar mechanisms underlying WBR. Understanding the intricacies of tunicate WBR contributes to our knowledge of regenerative biology and may offer valuable insights for regenerative medicine research in vertebrates.

De novo genome assembly and comparative genomics for the colonial ascidian Botrylloides violaceus.

Ascidians have the potential to reveal fundamental biological insights related to coloniality, regeneration, immune function, and the evolution of these traits. This study implements a hybrid assembly technique to produce a genome assembly and annotation for the botryllid ascidian, Botrylloides violaceus. A hybrid genome assembly was produced using Illumina, Inc. short and Oxford Nanopore Technologies long-read sequencing technologies. The resulting assembly is comprised of 831 contigs, has a total length of 121 Mbp, N50 of 1 Mbp, and a BUSCO score of 96.1%. Genome annotation identified 13 K protein-coding genes. Comparative genomic analysis with other tunicates reveals patterns of conservation and divergence within orthologous gene families even among closely related species. Characterization of the Wnt gene family, encoding signaling ligands involved in development and regeneration, reveals conserved patterns of subfamily presence and gene copy number among botryllids. This supports the use of genomic data from nonmodel organisms in the investigation of biological phenomena.

Medetomidine promotes settlement but inhibits recruitment of the introduced colonial ascidian (Botrylloides violaceus)

Colonial ascidians are common occupants of hard surfaces in coastal marinas as fouling organisms on pier pilings and ship hulls and have significant financial impact. Methods to inhibit fouling often are time consuming and labor intensive, thus treatments that prevent settlement for long time periods are warranted. Trybutyltin (TBT) appeared to be an effective antifouling agent, but is highly toxic to non-target species and now globally prohibited. Medetomidine is the active ingredient in the anti-fouling paint, Selektope, that shows promise to prevent attachment of marine fouling species. Here, we assessed the potential of medetomidine on larval metamorphosis of the invasive ascidian Botrylloides violaceus in static bioassays. We exposed newly released tadpole larvae to eight concentrations (0.5 mg/L, 1 mg/L, 5 mg/L, 10 mg/L, 20 mg/L, 40 mg/L, 50 mg/L) of medetomidine and recorded the number of larvae that were mobile, attached, metamorphosed or deceased after 1 h, 2 h, 4 h, 6 h, 24 h and 48 h. Larvae exposed to the highest concentrations of medetomidine quickly attached to the surface of Petri dishes, but failed to metamorphose. Larvae were mobile over the first 2 to 4 h of the experiment in the filtered sea water controls and low medetomidine concentrations. Larvae metamorphosed in all treatments except the two highest concentrations of medetomidine (40 and 50 mg/L), but ultimately all larvae exposed to medetomidine died. Medetomidine appears to have potential as a mechanism to control populations of B. violaceus, but we caution that further testing of non-target species is warranted.

Prokaryotic symbiont communities in three ascidian species introduced in both Ireland and New Zealand.

Ascidians or sea squirts are among the marine taxa with the most introduced species worldwide. These animals have a suite of biological characteristics that contribute to their successful establishment, including long reproductive seasons, rapid growth rates, and resistance to pollution. Here, we sequenced a fragment of the 16S ribosomal RNA gene to characterize symbiont diversity and host-specificity in the solitary species Syela clava and Ascidiella aspersa, and the colonial species Didemnum vexillum. Samples were collected from introduced populations in several marinas and mussel facilities around Ireland, and a marina in New Zealand. Two additional colonial species Botrylloides violaceus and Didemnum sp. were collected in Ireland, and ambient seawater was sampled from both countries for comparison. Data revealed a strong effect of host species and location on prokaryote symbiont composition, consistent with recent ascidian microbiome literature. However, a location effect did not manifest in alpha diversity metrics (e.g., the same ascidian species at different locations exhibited similar diversity) but was evident in beta diversity metrics (greater intra-specific differences across locations than within locations). Location effects were stronger than species effects only for the solitary species (i.e., A. aspersa from New Zealand was more similar to S. clava from New Zealand than to A. aspersa from Ireland). D. vexillum and A. aspersa hosted a high abundance of prokaryotic symbionts that werepreviously found in other ascidian species, while S. clava symbiotic community was more closely related to bacteria common in the marine environment. Further studies should aim to unravel host-microbe coevolutionary patterns and the microbial role in facilitating host establishment in different habitats.

The Influence of Marina Characteristics on Non-native Colonisation

The marina properties, such as; salinity range, tidal fluctuations, depth, and ascidian presence in-fluence the distribution of invasive non-natives. Transport vectors, aquaculture and fishery practic-es also have an important role in the distribution of invasive non-native species during the bioinva-sion process. We focused on 10 invasive non-native species of marine invertebrates in the UK. This study explained the distribution and ecology of five invasive non-native species (Styela clava (As-cidia), Didemnum vexillum (Ascidia), Caprella mutica (Crustacea), Crepidula fornicata (Gastropo-da), Watersipora subtorquata (Bryozoa)) and the marina characteristics of five invasive non-native species colonization (Austrominius modestus (Arthropoda), Ciona intestinalis (Ascidia), Botrylloides violaceus (Ascidia), Tricellaria inopinata (Bryozoa), Bugula neritina (Bryozoa)) in Wales coasts. The study site deployed settlement tiles in the inner and the outer tile as two positions in each marina: An equal number of vertical and horizontal tiles was deployed at each site with half-sampled at two weeks (very early colonization) and at eight weeks (later colonization). The results showed that some variations in marina characteristics affected the distribution of invasive non-native species. Invasive non-native species abundance, diversity and multivariate structure of the assemblages were very high. The colonization of tiles varied between locations at the entrance and within the marina, but not in any way, and the effect of tile orientation was surprisingly low.

Not all prey are created equal: Invasive ascidian diet mediates sea star wasting in Henricia sanguinolenta

Disease outbreaks and mass mortality events in terrestrial and marine taxa are increasing as a result of human influence, pollution, and climate change. Sea Star Wasting has become more common on the west coast of the United States, with increasing numbers of asteroids affected since 2013. Rising temperatures have traditionally been linked to mass mortality events, but other factors such as the effect of diet on individual susceptibility to Sea Star Wasting have not been examined. Here, we use laboratory experiments to test the frequencies of SSW sign onset and death of a native sea star when exposed to diets of two invasive ascidians. We show that prey identity affects mortality rates of affected predators. Sea stars that consumed Diplosoma listerianum experienced SSW signs, but 0% mortality, whereas animals that consumed Botrylloides violaceus, or no prey experienced 41% mortality. We expect that invader induced shifts in community species composition will increase the frequency of these mass die offs on both the west and east coasts.

Early detection of marine non-indigenous species on Svalbard by DNA metabarcoding of sediment

Non-indigenous species (NIS) in the Arctic have an increased likelihood of arrival from ship traffic in the region, while the survival potential of the species becomes more likely in a warming environment. Monitoring is essential to detect the rate and magnitude of the establishment of NIS. In this study, a list of 123 potential marine NIS for Svalbard was drafted and the presence of marine NIS in soft sediment of Kongsfjorden in Svalbard was assessed using molecular metabarcoding techniques. For 37 species, including eight potential Arctic NIS, we generated new 18S and/or COI barcode sequences to improve the available online reference databases. In total, 299 species were identified in the sediment samples, including seven potential NIS. Three of these potential NIS have not been reported before in Svalbard: the harpacticoid copepod Euterpina acutifrons, and the ascidians Botrylloides violaceus and Molgula manhattensis. Another novel observation for Svalbard was the polychaete Chone mollis. Additional studies are needed to assess whether the NIS have been established on Svalbard and what their potential impact on the local system may be. Metabarcoding proved to be an effective monitoring tool to detect the presence of new species in Svalbard marine waters. We advise its use to set up a baseline record for the presence of NIS at points of entry, especially harbours. This approach is also valuable for biodiversity monitoring, in particular the detection of small organisms and life stages that are hard to identify using current visual techniques.

The influence of invasive ascidian diets on the growth of the sea star Henricia sanguinolenta

AbstractInvasive species can disrupt food webs by altering the abundance of prey species or integrating into the food web themselves. In the Gulf of Maine, there have been a suite of invasions that have altered the composition of the benthic ecosystem. These novel prey species can potentially benefit native predators depending on their nutritional value and relative abundance. We measured feeding instances of the native blood star, Henricia sanguinolenta, and changes in the seasonal abundances of invasive ascidian prey species. Results indicate that H. sanguinolenta forages optimally, as the blood star will prey on invasive ascidians when in high abundance, but feed on other species during periods of scarcity. Further, our study shows that blood stars prey on a wider variety of species than was previously known, such as small bivalves and barnacles. Additionally, we compared growth and reproduction of sea stars fed different combinations of invasive ascidians (Diplosoma listerianum or Botrylloides violaceus) or a native sponge (Haliclona oculata). Sea stars grew more on the native diet when compared with the invasive ascidian species, and D. listerianum appeared to be a superior quality food source when compared with B. violaceus. By comparing our data with historical data, we determined that there was a dramatic increase in sea star populations between 1980 and 2011, but then populations decreased by almost half from 2011 to 2016–2017. These data suggest that while invasive ascidians may have helped sea star populations at one point, sea stars are declining without their native food source.

Behavioural Responses of the Colonial Sea Squirt Botrylloides violaceus Oka to Suspended Food Micro-Particles in Laboratory Cultures

Violet sea squirts are noteworthy model organisms, because they provide insights into various physiologic processes, including cell senescence, ageing, apoptosis and allorecognition. Consequently, their culture is critical to permit experimental studies. Most papers refer to short periods of rearing using various feeds, both living and conserved, missing a formal justification for their use or indications of their actual nutritional value. Here, we use two behavioural responses—the percentage of open siphons and the frequency of zooid contractions—as compared to the abundance of suspended microparticles during feeding tests, to identify feeds able to promote filter-feeding. The results will enable to formulate compound diets that maximise positive physiological responses. Our tests demonstrated that plant items, such as dry microalgae and cyanobacteria (Arthrospira platensis, commercially known as Spirulina), along with living planktonic Haptophyta (Isochrysis galbana), trigger clear positive reactions, represented by a higher frequency of zooid contractions and larger proportions of open siphons. These responses correspond to decreases in the concentrations of suspended microparticles during the experiment and indicate higher filter-feeding activity. In contrast, feeds commonly administered to colonies, such as milk powder, dried eggs and artificial plankton, triggered negative behavioural responses, and their intake was lower during the feeding trials.

The role of invasive tunicates as reservoirs of molluscan pathogens

Ascidian tunicates frequently display rapid expansion when introduced beyond their native range and are considered successful invaders. This invasive potential may be exacerbated by a warming climate, allowing for the occupation of environmental niches previously held by native species. Research into tunicate invasion ecology is prevalent, but less is known about their role in pathogen maintenance. This study investigated the impact of invasive tunicates on the maintenance of pathogens that affect commercial bivalves, including the cultured species Ostrea edulis (European flat oyster) and Crassostrea gigas (Pacific oyster), and the fished species Cerastoderma edule (Common cockle). Focal pathogens included ostreid herpesvirus OsHV-1 μVar, Vibrio aestuarianus, Bonamia ostreae and Minchinia spp. The range of pathogens in their molluscan hosts was determined and the tunicates Botrylloides violaceus, Didemnum vexillum and Styela clava were then screened for these same pathogens, using both field samples from oyster culture sites and marinas and a series of laboratory cohabitation trials. Sample sites reflected areas close to and further away from known pathogen sources. PCR, Sanger sequencing and histology confirmed the presence of B. ostreae and Minchinia mercenariae-like in S. clava, and V. aestuarianus was confirmed by qPCR in B. violaceus and D. vexillum. Furthermore, histology confirmed Minchinia mercenariae-like sporonts in S. clava suggesting that the tunicate can facilitate replication of this species. S. clava also maintained B. ostreae in tanks with no oysters present. The results indicate that tunicates can act as reservoirs of infection in areas where disease occurs and potentially transport diseases to uninfected sites.

Predators Associated with Marinas Consume Indigenous over Non-indigenous Ascidians

The establishment of non-indigenous species is influenced both by species traits and the biotic interactions that create resistance to or facilitate invasion. Predation, for example, creates resistance to non-indigenous ascidians in benthic habitats. Daytime surveys of fouling communities on floating docks in the Charleston Marina, Oregon, USA in August 2014 revealed that the indigenous ascidian Distapla occidentalis was present at all sites and was being consumed by the flatworm Eurylepta leoparda and the nudibranch Hermissenda crassicornis. In contrast, the non-indigenous Botrylloides violaceus and Botryllus schlosseri were present at fewer sites and lacked signs of predation. Parallel surveys in July 2015 again revealed predators found on docks were eating the indigenous ascidian. Examination of surrounding intertidal habitats revealed the dominant predators were the hermit crab Pagurus granosimanus and shore crab Hemigrapsus oregonensis, species not found in the marina fouling community. Feeding assays with the four predators and the ascidians showed that the flatworm specialized on the indigenous D. occidentalis, eating it almost 50 times faster than the NIS, which it did not consume. The nudibranch and shore crab mainly fed on D. occidentalis as well, consuming it two to four times faster than the NIS. In contrast, the benthic hermit crab mainly fed on the non-indigenous ascidian, feeding on it almost three times faster than it ate D. occidentalis. Hence, the non-indigenous ascidian likely faced reduced predation pressure in the marina fouling communities, increasing invasion risk. The study also examined several aspects of ascidian palatability, which showed that predator feeding was affected by differences in tunic structure. The study suggests that the different predators in the fouling and benthic communities in this marina have a role in the invasion of ascidians.

Investigation of Wnt Pathway Genes in a Highly Regenerative Marine Invertebrate

Colonial ascidians are the closest known relatives to vertebrates that retain whole body regeneration. Understanding molecular pathways involved in regeneration in a diverse range of organisms will provide insights into evolutionary changes in regeneration. Specifically, our research explores Wnt signaling in the colonial ascidian Botrylloides violaceus; this highly conserved pathway is universally involved in studied animals with any regenerative ability. A recent rough draft genome for B. violaceus reveals multiple putative ligands, receptors, and downstream effectors from the Wnt pathway. Primers designed from the draft genome are being used in PCR to validate these predicted genes. Future studies will use qPCR to track gene expression of different Wnt pathway members through regeneration.Support or Funding InformationThe Frost Fund, College of Science and Mathematics at California Polytechnic State University, San Luis Obispo

Hybrid Genome Assembly of a Regenerative Chordate

Modern genome sequencing technologies open new avenues for research on biological processes in non‐model organisms. Colonial ascidians are a diverse group of invertebrate chordates capable of whole body regeneration, an unusual trait that is absent from vertebrates. Although genomic data are available for a few related species, there exist no such data for the highly regenerative species Botrylloides violaceus. Using a de novo hybrid assembly approach, we present the first draft of the B. violaceus genome sequence. By integrating short read and long read sequencing technologies, genome assembly becomes cost‐effective and easily accessible. Comparative genomic analysis between B. violaceus and other chordate genomes reveals insights into the evolution of gene families implicated in regeneration and development.Support or Funding InformationWarren J. Baker and Robert D. Koob Endowments, California Polytechnic State University; The Frost Fund, College of Science and Mathematics, California Polytechnic State University

Larval exposure to low salinity compromises metamorphosis and growth in the colonial ascidian Botrylloides violaceus

AbstractCoastal and estuarine ecosystems exhibit extreme fluctuations in abiotic factors that influence the success and distribution of many introduced marine invertebrates. The frequency of these fluctuations may worsen under the scenario of climate change due to more intense storm events or greater precipitation. While many studies have focused on the response of adult ascidians to salinity, few have examined the response of larvae to low salinity events or post‐metamorphic asexual reproduction after larval exposure to low salinity. Here we tested the effect of low salinity on larval metamorphosis and post‐metamorphic growth of the introduced ascidian Botrylloides violaceus. We exposed tadpole larvae to salinities ranging 0–30 ppt. Tadpoles metamorphosed in salinities as low as 16 ppt and percent metamorphosis of larvae was not significantly different at salinities >20 ppt. A few tadpoles metamorphosed within 2–4 hr after release, and larval metamorphosis continued for 3–4 days, suggesting that larvae could disperse considerable distances. We then tested post‐metamorphic survival, growth, and asexual reproduction of larvae exposed (5–7 days) to varying salinities (18, 20, 25, and 30 ppt), and placed the resulting colonies in full‐salinity natural seawater; colony growth was monitored for 9 weeks post‐metamorphosis. Results indicated that colonies whose larvae were exposed to low salinity (<25 ppt) had fewer zooids than colonies from larvae exposed to seawater at 30 ppt, but total area occupied by each colony was similar among salinity treatments. Because each individual zooid within a colony filter‐feeds and reproduces through asexual budding, fewer feeding individuals within a colony will likely reduce future growth and reproduction. Due to the likelihood of more frequent heavy rain events and low‐salinity conditions for the northwest Atlantic Ocean, there is need for continued research to assess the response of ascidians to factors like salinity and to evaluate the spread of ascidians and their adaptation to estuarine and coastal habitats.

Wnt Pathway Involvement in Whole Body Regeneration in Botrylloides violaceus

Colonial ascidians, or sea squirts, are invertebrate urochordates noted for their remarkable regenerative potential. One such organism, Botrylloides violaceus, is capable of undergoing whole body regeneration (WBR) from its peripheral vasculature. Studying the cellular mechanisms of regeneration in B. violaceus could provide insight into regenerative mechanisms in vertebrates. Specifically, since the Wnt signaling pathway has been consistently linked with regeneration in other organisms, we investigated its possible involvement in WBR for B. violaceus. Our results demonstrate that B. violaceus completed WBR an average of 4.5 days faster than control regenerates when treated with a Wnt agonist. This suggests that the Wnt pathway is involved in activating cellular signals that facilitate WBR in B. violaceus. We continue to investigate effects of other pharmacological manipulations of the Wnt pathway. We are also attempting to amplify possible Wnt homologs using PCR with degenerate primers derived from conserved Wnt sequences. Obtaining DNA sequence for B. violaceus Wnts will allow future experiments on Wnt gene expression during regeneration.Support or Funding InformationCalifornia Polytechnic State University, Biological Sciences Department; Frost Summer 2017 Undergraduate Research ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Predicting global ascidian invasions

AbstractAimMany species of ascidians are invasive and can cause both ecological and economic losses. Here, we describe risk assessment for nineteen ascidian species and predict coastal regions that are more vulnerable to arrival and expansion.LocationGlobal.MethodsWe used ensemble niche modelling with three algorithms (Random Forest, Support Vector Machine and MaxEnt) to predict ecologically suitable areas and evaluated our predictions using independent (area under the curve—AUC) and dependent thresholds (true skill statistics—TSS). Environmental variables were maximum and the range of sea surface temperature, mean salinity and maximum chlorophyll. We used our niche modelling results and a modified invasibility index to compare invasion risk among 15 coastal regions.ResultsCurrently, the most invaded regions are in temperate latitudes of the Northern Hemisphere and Temperate Australasia, which are regions most prone for new invasions. In the tropics, the West and Central Indo‐Pacific are two regions of strong concern, the former with high risk of primary invasion by Botryllus schlosseri and Didemnum perlucidum. In the Southern Hemisphere, the Southwest and Southeast Atlantic are most at risk, both subject to invasion by Botrylloides violaceus, Didemnum vexillum, Molgula manhattensis and Styela clava among others. Regions most at risk of expansion of established invasive species are the Central Indo‐Pacific, Northwest Pacific, Mediterranean and West Indo‐Pacific.Main conclusionsAll regions studied have areas that are suitable and connected to receive new ascidian introductions or that may permit the spread of already established species. Risk comparison of primary introductions and expansion of established introduced ascidians among regions will allow managers to prioritize species of concern for each region both for monitoring future introductions or to enforce control actions towards established species to decrease the risk of regional expansion.

Variation in dispersal and competitive ability mediate realized connectivity in two benthic marine invertebrates

For marine species with complex lifecycles, linking dispersal with local species interactions to predict spatial patterns of realized connectivity is an enduring challenge. So far, because the scale of dispersal in many species exceeds our ability to manipulate it, theoretical and modelling developments outpace empirical support, and reconciling theory with direct experimental evidence is understandably rare. With two species of marine invertebrate (Botrylloides violaceus and Bugula stolonifera), the goal of this study was to experimentally test whether within and among species variation in larval dispersal and post-settlement competitive interactions determine spatially explicit differences in colony performance and population structure for each species. For B. stolonifera, the results showed incorporating the origins of larvae with post-settlement competitive interactions can qualitatively predict the establishment and performance of individuals among spatially discrete habitat patches. However, dispersal and competition were poor predictors of spatial population structure and individual performance for B. violaceus indicating for some species relationships between dispersal and competition can be more complex than is assumed. The results highlight the often acknowledged but rarely tested need to predict realized connectivity we must follow individuals from spawning to reproductive maturity to encompass well studied, but implicit mechanisms (such as competition and settlement behaviour), that may mediate connectivity.

Projecting present and future habitat suitability of ship-mediated aquatic invasive species in the Canadian Arctic

A rise in Arctic shipping activity resulting from global warming and resource exploitation is expected to increase the likelihood of aquatic invasive species (AIS) introductions in the region. In this context, the potential threat of future AIS incursions at a Canadian Arctic regional scale was examined. Habitat suitability under current environmental conditions and future climate change scenarios was projected for a subset of eight potential invaders ranked as having a high risk of establishment in the Canadian Arctic based on dispersal pathways/donor regions, biological attributes and invasion history: (1) Amphibalanus improvisus, (2) Botrylloides violaceus, (3) Caprella mutica, (4) Carcinus maenas, (5) Littorina littorea, (6) Membranipora membranacea, (7) Mya arenaria and (8) Paralithodes camtschaticus. Habitat modelling was performed using MaxEnt based on globally known native and non-native occurrence records and environmental ranges for these species. Results showed that under current environmental conditions the habitat is suitable in certain regions of the Canadian Arctic such as the Hudson Complex and Beaufort Sea for L. littorea, M. arenaria and P. camtschaticus. Under a future climate change scenario, all species showed poleward gains in habitat suitability with at least some regions of the Canadian Arctic projected to be suitable for the complete suite of species modelled. The use of these models is helpful in understanding potential future AIS incursions as a result of climate change and shipping at large spatial scales. These approaches can aid in the identification of high risk regions and species to allow for more focused AIS monitoring and research efforts in response to climate change.

Inconsistency for the niche breadth invasion success hypothesis in aquatic invertebrates

AbstractComparison of highly successful and less successful invasive species can highlight traits that are associated with invasion success, and indicate the associated risk of further establishment or invasion from novel species. We compared variation in δ13C and δ15N, or isotopic niche, in the tissues of matched pairs of highly successful and less successful (respectively) freshwater and marine aquatic invasive species: violet tunicate Botrylloides violaceus and golden star tunicate Botryllus schlosseri from the northwest Atlantic coast; spiny waterflea Bythotrephes longimanus and fishhook waterflea Cercopagis pengoi from the Great Lakes basin; and Pacific oyster Crassostrea gigas and eastern oyster Crassostrea virginica from the northeast Pacific coast. Individual (δ13C and δ15N) and population (Bayesian ellipses of δ13C and δ15N) level comparisons of isotopic niche revealed, in most cases, greater niche breadth in the more successful species of tunicate but the less successful species of waterflea and oyster. Comparison with the literature suggested that a broad dietary niche is less crucial for widespread distribution of aquatic invasive invertebrates than it is for vertebrates (i.e., fishes). Inconsistency in the association between isotopic niche breadth and invasion success could be due to a greater influence of habitat suitability on variation in invertebrate diets. These findings challenge the common assumption that a broader niche promotes invasion success, and thus, have implications for invasive species risk assessment, management, and our understanding of species spread and distribution.

Manipulating propagule pressure to test the invasibility of subtidal marine habitats

Global patterns show that estuaries are more invaded than open coasts and artificial habitats are more invaded than natural ones. The contention that artificial habitats in estuaries are more invasible than other habitats may result from variation in propagule supply, however, as artificial habitats are closely linked to vectors of non-native propagules, such as ships and boats. True comparisons of habitat invasibility require manipulations of propagule pressure, which has been historically difficult in marine systems. Using in-situ larval dosing, we delivered propagules of the widespread invasive ascidian Botrylloides violaceus into field mesocosms and assessed how habitat type (floating dock vs. benthic rock), resource availability (occupied vs. unoccupied plates), and propagule number (5, 25 and 50 larvae 225 cm−2) affected settlement (survival after 24 h) and recruitment (survival after 56 days) success. In-situ larval dosing was successful, and after eight weeks there were significant differences in recruitment due to initial dose-size, habitat type, and space availability. At the habitat scale, despite equal propagule delivery, PVC plates in natural benthic rock were not equally invasible and few propagules survived to recruitment. At the organism scale, increased habitat complexity, through facilitation by established fouling species, rather than freedom from space competitors, appears to be more important for B. violaceus to establish. Our results offer greater mechanistic understanding of broader invasion patterns between artificial and natural habitats. This work extends the possibilities for further research to counteract the confounding issue of unknown and unknowable propagule delivery when attempting to explain variation in invasion success.