The Genome of the “Sea Vomit” Didemnum vexillum

Tunicates are the sister group of vertebrates and thus occupy a key position for investigations into vertebrate innovations as well as into the consequences of the vertebrate-specific genome duplications. Nevertheless, tunicate genomes have not been studied extensively in the past, and comparative studies of tunicate genomes have remained scarce. The carpet sea squirt Didemnum vexillum, commonly known as “sea vomit”, is a colonial tunicate considered an invasive species with substantial ecological and economical risk. We report the assembly of the D. vexillum genome using a hybrid approach that combines 28.5 Gb Illumina and 12.35 Gb of PacBio data. The new hybrid scaffolded assembly has a total size of 517.55 Mb that increases contig length about eightfold compared to previous, Illumina-only assembly. As a consequence of an unusually high genetic diversity of the colonies and the moderate length of the PacBio reads, presumably caused by the unusually acidic milieu of the tunic, the assembly is highly fragmented (L50 = 25,284, N50 = 6539). It is sufficient, however, for comprehensive annotations of both protein-coding genes and non-coding RNAs. Despite its shortcomings, the draft assembly of the “sea vomit” genome provides a valuable resource for comparative tunicate genomics and for the study of the specific properties of colonial ascidians.

Ecology of Biofouling and Impacts on Mussel Aquaculture: A Case Study with Didemnum Vexillum

<p>Over the past decade, several species of non-indigenous ascidian have had adverse effects in the marine environment and on associated industries. The colonial ascidian Didemnum vexillum is a recent successful invader in temperate marine communities worldwide, proving problematic to mussel aquaculture in New Zealand. At the inception of this thesis, control strategies to manage the threat from Didemnum to mussel aquaculture were implemented in the absence of information on the biological processes underpinning the species’ invasion success. Background information on Didemnum presented in Chapter 2 recognises this paucity of information on several key biological attributes as well as negative impacts of this species. The ability to obtain larvae and culture colonies in the laboratory was a crucial first step. Thus, Chapter 3 presents laboratory experiments that describe the first successful methods to induce spawning in adult Didemnum colonies, as well as techniques for the successful settlement and metamorphosis of the larvae produced, and for laboratory culture of juveniles. Chapters 4 to 6 address key aspects of the biological characteristics of Didemnum that relate to its invasiveness and spread. The recruitment and reproductive development of Didemnum were assessed over a 20-month period at two locations in central New Zealand. Results indicated that the reproductive season for Didemnum in New Zealand (at least 9 months) is considerably longer than previously believed, with recruitment patterns strongly correlated with seasonal water temperature fluctuations at each location. Secondly, the natural dispersal ability of Didemnum was assessed using a weight-of-evidence approach that combined laboratory and field studies. Larval competency trials revealed that > 70 % of larvae were able to settle and undergo metamorphosis following an artificial settlement delay of 2 hours. Larval viability decreased with increasing delay duration; however 10 % of larvae remained viable following a 36 hour delay. These findings were supported by a field-based study documenting larval recruitment at distances up to 250 m from source populations. Exponential decay models indicated that, given favourable conditions, larval dispersal distances greater than 1 km were theoretically possible, which is a much greater distance than previously assumed. Lastly, the impacts of Didemnum on cultured New Zealand green-lipped mussels (Perna canaliculus) were investigated. At the level of invasiveness experienced in a field experiment, only small mussel size classes were vulnerable to direct Didemnum impacts, with negative effects restricted to fouling-related displacement of mussels as opposed to reduced size or condition. However, at the greater levels of invasiveness evident at other places and times, Didemnum impacts have the potential to be considerably larger. As such, the ability to predict invasiveness, and hence impacts, is critical for stakeholders. However, for reasons discussed in the thesis, making reliable specific predictions of invasiveness is difficult. Despite such limitations, it is clear that an understanding of a species’ basic biological attributes can still greatly assist with management decisions, as highlighted throughout the chapters in this thesis. My research findings have led to a better awareness of commercial impacts and potential spread of this species. Simultaneously, my research also highlights the limitations inherent in inferring invasiveness from other situations (e.g. places, times, and related species); there is a need to specifically evaluate a species’ biological attributes and invasive behaviour when introduced into a novel environment.</p>

Ecology of Biofouling and Impacts on Mussel Aquaculture: A Case Study with Didemnum Vexillum

<p>Over the past decade, several species of non-indigenous ascidian have had adverse effects in the marine environment and on associated industries. The colonial ascidian Didemnum vexillum is a recent successful invader in temperate marine communities worldwide, proving problematic to mussel aquaculture in New Zealand. At the inception of this thesis, control strategies to manage the threat from Didemnum to mussel aquaculture were implemented in the absence of information on the biological processes underpinning the species’ invasion success. Background information on Didemnum presented in Chapter 2 recognises this paucity of information on several key biological attributes as well as negative impacts of this species. The ability to obtain larvae and culture colonies in the laboratory was a crucial first step. Thus, Chapter 3 presents laboratory experiments that describe the first successful methods to induce spawning in adult Didemnum colonies, as well as techniques for the successful settlement and metamorphosis of the larvae produced, and for laboratory culture of juveniles. Chapters 4 to 6 address key aspects of the biological characteristics of Didemnum that relate to its invasiveness and spread. The recruitment and reproductive development of Didemnum were assessed over a 20-month period at two locations in central New Zealand. Results indicated that the reproductive season for Didemnum in New Zealand (at least 9 months) is considerably longer than previously believed, with recruitment patterns strongly correlated with seasonal water temperature fluctuations at each location. Secondly, the natural dispersal ability of Didemnum was assessed using a weight-of-evidence approach that combined laboratory and field studies. Larval competency trials revealed that > 70 % of larvae were able to settle and undergo metamorphosis following an artificial settlement delay of 2 hours. Larval viability decreased with increasing delay duration; however 10 % of larvae remained viable following a 36 hour delay. These findings were supported by a field-based study documenting larval recruitment at distances up to 250 m from source populations. Exponential decay models indicated that, given favourable conditions, larval dispersal distances greater than 1 km were theoretically possible, which is a much greater distance than previously assumed. Lastly, the impacts of Didemnum on cultured New Zealand green-lipped mussels (Perna canaliculus) were investigated. At the level of invasiveness experienced in a field experiment, only small mussel size classes were vulnerable to direct Didemnum impacts, with negative effects restricted to fouling-related displacement of mussels as opposed to reduced size or condition. However, at the greater levels of invasiveness evident at other places and times, Didemnum impacts have the potential to be considerably larger. As such, the ability to predict invasiveness, and hence impacts, is critical for stakeholders. However, for reasons discussed in the thesis, making reliable specific predictions of invasiveness is difficult. Despite such limitations, it is clear that an understanding of a species’ basic biological attributes can still greatly assist with management decisions, as highlighted throughout the chapters in this thesis. My research findings have led to a better awareness of commercial impacts and potential spread of this species. Simultaneously, my research also highlights the limitations inherent in inferring invasiveness from other situations (e.g. places, times, and related species); there is a need to specifically evaluate a species’ biological attributes and invasive behaviour when introduced into a novel environment.</p>

Environmental DNA Based Surveillance for the Highly Invasive Carpet Sea Squirt Didemnum vexillum: A Targeted Single-Species Approach

The presence and diversity of marine non-native species, the number of new invasions, and the impact on native communities and habitats are important metrics used to assess the health of marine ecosystems. Monitoring for marine non-native species, using traditional approaches such as rapid assessment surveys (RASs), requires taxonomic expertise and may still fail to detect rare or inconspicuous species. This study reports a validation process for a quantitative PCR (qPCR) assay based on the cytochrome oxidase 1 gene, designed to detect highly invasive tunicate Didemnum vexillum by targeting environmental DNA (eDNA) present in water samples. The D. vexillum qPCR assay showed high sensitivity, with the threshold limit of detection (LOD) and modeled LOD3 (based on triplicate qPCR reactions) estimated as 9.187 and 1.117 copies reaction–1, respectively and the limit of quantification (LOQ) was calculated as 18 copies reaction–1. Analyses of water samples collected from selected Pacific oyster farms and recreational marinas in Scotland showed 100% concordance between the historical data on presence of D. vexillum from RASs and detection of D. vexillum eDNA. Consistency of detection of D. vexillum eDNA among different sampling points within each infected sampling site varied, ranging between 100% positive throughout the site to some sampling points testing “negative” or only as “suspected” for D. vexillum. Sites with lower within-site detection consistency included sites with a low density of D. vexillum as reported by RASs or were sites undergoing D. vexillum management. The present pilot monitoring program demonstrates the potential to generate important data on presence of D. vexillum. This program will be scaled up across large geographic regions and used in the first instance to focus and target the traditional RASs to D. vexillum eDNA-positive sites in a cost-effective way, with an aim to verify the species presence by visual observation and direct Sanger sequencing of positive qPCR products.

Genetic diversity and relatedness in aquaculture and marina populations of the invasive tunicate Didemnum vexillum in the British Isles

Introductions of invasive, non-native species in the marine environment are increasing as human activity within coastal areas rises. Genetic datasets are useful tools to identify source populations, track routes of invasions, and illuminate the role of genetic variation in the establishment and subsequent spread of novel introductions. Here, a microsatellite dataset is used to estimate the genetic diversity and population structure of 7 introduced Didemnum vexillum populations in Britain and Ireland, 4 of which are associated with aquaculture and 3 with marinas. Genetic differentiation observed between these populations indicates human-mediated transport as the main mechanism underlying the population structure of D. vexillum in Britain and Ireland. In addition to elucidating patterns of population structure we found that aquaculture sites showed significantly higher genetic diversity (measured as allelic richness) in comparison to the marina sites. We discuss these findings in relation to the history of each invasion, the complex life history of D. vexillum, and available evidence of the relative invasiveness of these populations. Our results show numerous interesting patterns which highlight further research avenues to elucidate the complex factors underlying the global spread of this successful invader.

Development of eDNA tools for the detection of marine invasive non-native species to support European flat oyster (Ostrea edulis) restoration projects.

The European flat oyster (Ostrea edulis) is an important keystone species in Scottish coastal waters. However due to anthropogenic pressures, significant reductions to oyster beds have been observed across Europe. In Scotland, several projects are currently aiming to restore European flat oyster habitats through the translocation of juvenile oysters from various sources including hatcheries and aquaculture. However, translocation of shellfish is not risk free and can increase the risk of accidental translocation of invasive non-native species (INNS). If INNS become established outside of their native range they can cause irreversible harm to native organisms and habitats. This study aims to develop molecular tools to detect environmental DNA of INNS which can be potentially associated with the translocation of live shellfish stocks. We have developed a species-specific real-time PCR assay for detection of Pacific oyster (Crassostrea gigas) and tested its sensitivity in a large-scale replicated mesocosm based experiment with varying densities of C.gigas. A secondary objective of the experiment was to assess the detection of another invasive species, the carpet sea squirt Didemnum vexillum which was cohabited with C. gigas. We aim to quantify the detection probability of increasing densities of C. gigas from repeat water samples and qPCR replicates. This project also aims to investigate the feasibility of using portable, real-time sequencing technologies such as the Oxford Nanopore MinION to develop robust tools to support native oyster restoration programmes.

The genome of the "Sea Vomit" Didemnum vexillum

Background: Tunicates are the sister group of vertebrates and thus occupy a key position for investigations into vertebrate innovations as well as into the consequences of the vertebrate-specific genome duplications. Nevertheless, tunicate genomes have not been studied extensively in the past and comparative studies of tunicate genomes have remained scarce. The carpet sea squirt Didemnum vexillum is a colonial tunicate considered an invasive species with substantial ecological and economical risk.Results: We report a newly re-assembled genome of Didemnum vexillum. We used a hybrid approach that combines two genome sequencing technologies and also its first transcriptome. Started from 28.5 Gb Illumina and 12.35 Gb of PacBio data a new hybrid scaffolded assembly was obtained comprised of a total size of 517.55 Mb that increases contig length about 8-fold compared to previous, Illumina-only assembly. While still highly fragmented (L50=25284, N50=6539), the assembly is suffcient for comprehensive annotations of both protein-coding genes and non-coding RNAs.Conclusions: The draft assembly of the "sea vomit" genome provides a valuable resource for comparative tunicate genomics and for the study of the specific properties of colonial ascidians.Availability: Genome and annotation data as well as a link to a UCSC Genome Browser hub are available at http://tunicatadvexillum.bioinf.uni-leipzig.de/.

THE EFFECTS OF INVASIVE EPIBIONTS ON CRAB–MUSSEL COMMUNITIES: A THEORETICAL APPROACH TO UNDERSTAND MUSSEL POPULATION DECLINE

Blue mussels (Mytilus edulis) are important keystone species that have been declining in the Gulf of Maine. This could be attributed to a variety of complex factors such as indirect effects due to invasion by epibionts, which remains unexplored mathematically. Based on classical optimal foraging theory (OFT) and anti-fouling defense mechanisms of mussels, we derive an ODE model for crab–mussel interactions in the presence of an invasive epibiont, Didemnum vexillum. The dynamical analysis leads to results on stability, global boundedness and bifurcations of the model. Next, via optimal control methods, we predict various ecological outcomes. Our results have key implications for preserving mussel populations in the advent of invasion by non-native epibionts. In particular, they help us understand the changing popluation dynamics of local predator–prey communities, due to indirect effects that epibionts confer.