Morphological Study and 3D Reconstruction of the Larva of the Ascidian Halocynthia roretzi

The swimming larva represents the dispersal phase of ascidians, marine invertebrates belonging to tunicates. Due to its adhesive papillae, the larva searches the substrate, adheres to it, and undergoes metamorphosis, thereby becoming a sessile filter feeding animal. The larva anatomy has been described in detail in a few species, revealing a different degree of adult structure differentiation, called adultation. In the solitary ascidian Halocynthia roretzi, a species reared for commercial purposes, embryogenesis has been described in detail, but information on the larval anatomy is still lacking. Here, we describe it using a comparative approach, utilizing 3D reconstruction, as well as histological/TEM observations, with attention to its papillae. The larva is comparable to those of other solitary ascidians, such as Ciona intestinalis. However, it displays a higher level of adultation for the presence of the atrium, opened outside by means of the atrial siphon, and the peribranchial chambers. It does not reach the level of complexity of the larva of Botryllus schlosseri, a phylogenetically close colonial ascidian. Our study reveals that the papillae of H. roretzi, previously described as simple and conform, exhibit dynamic changes during settlement. This opens up new considerations on papillae morphology and evolution and deserves to be further investigated.

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>

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.

Microbiome Variability across the Native and Invasive Ranges of the Ascidian Clavelina oblonga

ABSTRACT Ascidians are prolific colonizers of new environments and possess a range of well-studied features that contribute to their successful spread, but the role of their symbiotic microbial communities in their long-term establishment is mostly unknown. In this study, we utilized next-generation amplicon sequencing to provide a comprehensive description of the microbiome in the colonial ascidian Clavelina oblonga and examined differences in the composition, diversity, and structure of symbiont communities in the host’s native and invasive ranges. To identify host haplotypes, we sequenced a fragment of the mitochondrial gene cytochrome c oxidase subunit I (COI). C. oblonga harbored a diverse microbiome spanning 42 bacterial and three archaeal phyla. Colonies in the invasive range hosted significantly less diverse symbiont communities and exhibited lower COI haplotype diversity than colonies in the native range. Differences in microbiome structure were also detected across colonies in the native and invasive range, driven largely by novel bacteria representing symbiont lineages with putative roles in nitrogen cycling. Variability in symbiont composition was also observed among sites within each range. Together, these data suggest that C. oblonga hosts a dynamic microbiome resulting from (i) reductions in symbiont diversity due to founder effects in host populations and (ii) environmental selection of symbiont taxa in response to new habitats within a range. Further investigation is required to document the mechanisms behind these changes and to determine how changes in microbiome structure relate to holobiont function and the successful establishment of C. oblonga worldwide. IMPORTANCE Nonnative species destabilize coastal ecosystems and microbial symbionts may facilitate their spread by enhancing host survival and fitness. However, we know little of the microorganisms that live inside invasive species and whether they change as the host spreads to new areas. In this study, we investigated the microbial communities of an introduced ascidian (Clavelina oblonga) and tracked symbiont changes across locations within the host’s native and invasive ranges. Ascidians in the invasive range had less-diverse microbiomes, as well as lower host haplotype diversity, suggesting that specific colonies reach new locations and carry select symbionts from native populations (i.e., founder effects). Further, ascidians in the invasive range hosted a different composition of symbionts, including microbes with the potential to aid in processes related to invasion success (e.g., nutrient cycling). We conclude that the putative functionality and observed flexibility of this introduced ascidian microbiome may represent an underappreciated factor in the successful establishment of nonnative species in new environments.

Insights into the Complement System of Tunicates: C3a/C5aR of the Colonial Ascidian Botryllus schlosseri

As an evolutionary ancient component of the metazoan immune defense toolkit, the complement system can modulate cells and humoral responses of both innate and (in jawed vertebrates) adaptive immunity. All the three known complement-activation pathways converge on the cleavage of C3 to C3a and C3b. The anaphylatoxin C3a behaves as a chemokine in inflammatory responses, whereas C3b exerts an opsonic role and, ultimately, can activate the lytic pathway. C3aR, one of the mammalian receptors for C3a, is a member of the G-protein-coupled receptor family sharing seven transmembrane alpha helixes. C3aR can act as a chemokine and recruit neutrophils, triggering degranulation and respiratory burst, which initiates an inflammatory reaction. Mining the transcriptome of the colonial ascidian Botryllus schlosseri, we identified a transcript showing homology with both mammalian C3aR and C5aR. The gene (bsc3/c5ar) is actively transcribed in morula cells, the circulating immunocyte triggering the inflammatory reactions in response to the recognition of nonself. Its transcription is modulated during the recurrent cycles of asexual reproduction known as blastogenetic cycles. Moreover, the treatment of hemocytes with C3aR agonist, induces a significant increase in the transcription of BsC3, revealing the presence of an autocrine feedback system able to modulate the expression of C3 in order to obtain a rapid clearance of potentially dangerous nonself cells or particles. The obtained results support the previously proposed role of complement as one of the main humoral components of the immune response in tunicates and stress the importance of morula cells in botryllid ascidian innate immunity.