A Developmental Ontology for the Colonial Architecture of Salps.

AbstractColonial animals are composed of clonal individuals that remain physically connected and physiologically integrated. Salps are tunicates with a dual life cycle, including an asexual solitary stage that buds sexual colonies composed of jet-propelling zooids that efficiently swim together as a single unit by multijet propulsion. Colonies from different species develop distinct architectures characterized by their zooid arrangement patterns, but this diversity has received little attention. Thus, these architectures have never been formally defined using a framework of variables and axes that would allow comparative analyses. We set out to define an ontology of the salp colony architecture morphospace and describe the developmental pathways that build the different architectures. To inform these definitions, we collected and photographed live specimens of adult and developing colonies through offshore scuba diving. Since all salp colonies begin their development as a transversal double chain, we characterized each adult colonial architecture as a series of developmental transitions, such as rotations and translations of zooids, relative to their orientation at this early shared stage. We hypothesize that all adult architectures are either final or intermediate stages within three developmental pathways toward bipinnate, cluster, or helical forms. This framework will enable comparative studies on the biomechanical implications, ecological functions, evolutionary history, and engineering applications of the diversity of salp colony architectures.

Artificial seawater based long-term culture of colonial ascidians.

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

Neuromesodermal Lineage Contribution to CNS Development in Invertebrate and Vertebrate Chordates.

Ascidians are invertebrate chordates and the closest living relative to vertebrates. In ascidian embryos a large part of the central nervous system arises from cells associated with mesoderm rather than ectoderm lineages. This seems at odds with the traditional view of vertebrate nervous system development which was thought to be induced from ectoderm cells, initially with anterior character and later transformed by posteriorizing signals, to generate the entire anterior-posterior axis of the central nervous system. Recent advances in vertebrate developmental biology, however, show that much of the posterior central nervous system, or spinal cord, in fact arises from cells that share a common origin with mesoderm. This indicates a conserved role for bi-potential neuromesoderm precursors in chordate CNS formation. However, the boundary between neural tissue arising from these distinct neural lineages does not appear to be fixed, which leads to the notion that anterior-posterior patterning and neural fate formation can evolve independently.

MiR-92 Family Members Form a Cluster Required for Notochord Tubulogenesis in Urochordate Ciona savignyi.

MicroRNAs are frequently clustered in the genome and polycistronically transcribed, regulating targeted genes in diverse signaling pathways. The miR-17-92 cluster is a typical miRNA cluster, playing crucial roles in the organogenesis and homeostasis of physiological processes in vertebrates. Here, we identified three miRNAs (csa-miR-92a, csa-miR-92b, and csa-miR-92c) that belonged to the miR-92 family and formed a miRNA cluster in the genome of a urochordate marine ascidian Ciona savignyi. Except for miR-92a and miR-92b, other homologs of the vertebrate miR-17-92 cluster members could not be identified in the Ciona genome. We further found that the mature sequences of urochordate miR-92 family members were highly conserved compared with the vertebrate species. The expression pattern revealed that three miR-92 family members had consistent expression levels in adult tissues and were predominantly expressed in heart and muscle tissue. We further showed that, at the embryonic and larval stages, csa-miR-92c was expressed in the notochord of embryos during 18-31 h post fertilization (hpf) by in situ hybridization. Knockout of csa-miR-92c resulted in the disorganization of notochord cells and the block of lumen coalescence in the notochord. Fibroblast growth factor (FGF), mitogen-activated protein kinase (MAPK), and wingless/integrated (Wnt)/planar cell polarity (PCP) signaling pathways might be involved in the regulatory processes, since a large number of core genes of these pathways were the predicted target genes of the miR-92 family. Taken together, we identified a miR-92 cluster in urochordate Ciona and revealed the expression patterns and the regulatory roles of its members in organogenesis. Our results provide expression and phylogenetic data on the understanding of the miR-92 miRNA cluster's function during evolution.

Contemporary climate change hinders hybrid performance of ecologically dominant marine invertebrates.

Human activities alter patterns of biodiversity, particularly through species extinctions and range shifts. Two of these activities are human mediated transfer of species and contemporary climate change, and both allow previously isolated genotypes to come into contact and hybridize, potentially altering speciation rates. Hybrids have been shown to survive environmental conditions not tolerated by either parent, suggesting that, under some circumstances, hybrids may be able to expand their ranges and perform well under rapidly changing conditions. However, studies assessing how hybridization influences contemporary range shifts are scarce. We performed crosses on Pyura herdmani and Pyura stolonifera (Chordata, Tunicata), two closely related marine invertebrate species that are ecologically dominant and can hybridize. These sister species live in sympatry along the coasts of southern Africa, but one has a disjunct distribution that includes northern hemisphere sites. We experimentally assessed the performance of hybrid and parental crosses using different temperature regimes, including temperatures predicted under future climate change scenarios. We found that hybrids showed lower performance than parental crosses at the experimental temperatures, suggesting that hybrids are unlikely to expand their ranges to new environments. In turn, we found that the more widespread species performed better at a wide array of temperatures, indicating that this parental species may cope better with future conditions. This study illustrates how offspring fitness may provide key insights to predict range expansions and how contemporary climate change may mediate both the ability of hybrids to expand their ranges and the occurrence of speciation as a result of hybridization.

The eventful history of nonembryonic development in tunicates.

Tunicates encompass a large group of marine filter-feeding animals and more than half of them are able to reproduce asexually by a particular form of nonembryonic development (NED) generally called budding. The phylogeny of tunicates suggests that asexual reproduction is an evolutionarily plastic trait, a view that is further reinforced by the fact that budding mechanisms differ from one species to another, involving nonhomologous tissues and cells. In this review, we explore more than 150 years of literature to provide an overview of NED diversity and we present a comparative picture of budding tissues across tunicates. Based on the phylogenetic relationships between budding and nonbudding species, we hypothesize that NED diversity is the result of seven independent acquisitions and subsequent diversifications in the course of tunicate evolution. While this scenario represents the state-of-the-art of our current knowledge, we point out gray areas that need to be further explored to refine our understanding of tunicate phylogeny and NED. Tunicates, with their plastic evolution and diversity of budding, represent an ideal playground for evolutionary developmental biologists to unravel the genetic and molecular mechanisms regulating nonembryonic development, as well as to better understand how such a profound innovation in life-history has evolved in numerous metazoans.

MeDAS: a Metazoan Developmental Alternative Splicing database.

Alternative splicing is widespread throughout eukaryotic genomes and greatly increases transcriptomic diversity. Many alternative isoforms have functional roles in developmental processes and are precisely temporally regulated. To facilitate the study of alternative splicing in a developmental context, we created MeDAS, a Metazoan Developmental Alternative Splicing database. MeDAS is an added-value resource that re-analyses publicly archived RNA-seq libraries to provide quantitative data on alternative splicing events as they vary across the time course of development. It has broad temporal and taxonomic scope and is intended to assist the user in identifying trends in alternative splicing throughout development. To create MeDAS, we re-analysed a curated set of 2232 Illumina polyA+ RNA-seq libraries that chart detailed time courses of embryonic and post-natal development across 18 species with a taxonomic range spanning the major metazoan lineages from Caenorhabditis elegans to human. MeDAS is freely available at https://das.chenlulab.com both as raw data tables and as an interactive browser allowing searches by species, tissue, or genomic feature (gene, transcript or exon ID and sequence). Results will provide details on alternative splicing events identified for the queried feature and can be visualised at the gene-, transcript- and exon-level as time courses of expression and inclusion levels, respectively.

The ontology of the anatomy and development of the solitary ascidian Ciona: the swimming larva and its metamorphosis.

Ciona robusta (Ciona intestinalis type A), a model organism for biological studies, belongs to ascidians, the main class of tunicates, which are the closest relatives of vertebrates. In Ciona, a project on the ontology of both development and anatomy is ongoing for several years. Its goal is to standardize a resource relating each anatomical structure to developmental stages. Today, the ontology is codified until the hatching larva stage. Here, we present its extension throughout the swimming larva stages, the metamorphosis, until the juvenile stages. For standardizing the developmental ontology, we acquired different time-lapse movies, confocal microscope images and histological serial section images for each developmental event from the hatching larva stage (17.5 h post fertilization) to the juvenile stage (7 days post fertilization). Combining these data, we defined 12 new distinct developmental stages (from Stage 26 to Stage 37), in addition to the previously defined 26 stages, referred to embryonic development. The new stages were grouped into four Periods named: Adhesion, Tail Absorption, Body Axis Rotation, and Juvenile. To build the anatomical ontology, 203 anatomical entities were identified, defined according to the literature, and annotated, taking advantage from the high resolution and the complementary information obtained from confocal microscopy and histology. The ontology describes the anatomical entities in hierarchical levels, from the cell level (cell lineage) to the tissue/organ level. Comparing the number of entities during development, we found two rounds on entity increase: in addition to the one occurring after fertilization, there is a second one during the Body Axis Rotation Period, when juvenile structures appear. Vice versa, one-third of anatomical entities associated with the embryo/larval life were significantly reduced at the beginning of metamorphosis. Data was finally integrated within the web-based resource "TunicAnatO", which includes a number of anatomical images and a dictionary with synonyms. This ontology will allow the standardization of data underpinning an accurate annotation of gene expression and the comprehension of mechanisms of differentiation. It will help in understanding the emergence of elaborated structures during both embryogenesis and metamorphosis, shedding light on tissue degeneration and differentiation occurring at metamorphosis.

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 ß-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.

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

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

The Effects of Microplastics on Dolioletta gegenbauri (Tunicata, Thaliacea).

Oceanographic studies revealed the abundance of minute plastic particles in coastal regions. Such particles, called microplastics, are abundant in sizes smaller than 100 µm ESD (Equivalent Spherical Diameter) and can be collected and ingested by planktonic copepods. Those animals are the most abundant metazoans on our planet. Abundantly co-occurring with planktonic copepods in subtropical and temperate neritic waters are doliolids (Tunicata, Thaliacea), which can dominate subtropical shelves because of their high asexual reproductive performance. Our studies were designed to examine the effects of polystyrene beads at low abundance, compared with phytoplankton, on abundantly occurring gonozooids of Dolioletta gegenbauri. Our findings reveal that such abundance of microplastic particles, in the presence of environmental concentrations of phytoplankton, reduces rates of feeding, growth, and oxygen consumption of this tunicate. Feeding rates on phytoplankton in the presence of beads were reduced by up to 58%, growth rates by up to 85%, and oxygen consumption rates by up to 33%. We conclude that such microplastic particles could limit the often in situ encountered pronounced proliferation of this tunicate species (Deibel in: Bone (ed) The biology of pelagic tunicates, Oxford University Press, Oxford, 1998).

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri (Uljanin 1884) for Experimental Studies.

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, and trophic interactions primarily due to methodological challenges, including the ability to culture them. This is particularly true for the doliolid, Dolioletta gegenbauri. D. gegenbauri commonly occurs in productive subtropical continental shelf systems worldwide, often at bloom concentrations capable of consuming a large fraction of daily primary production. In this study, we describe cultivation approaches for collecting, rearing, and maintaining D. gegenbauri for the purpose of conducting laboratory-based studies. D. gegenbauri and other doliolid species can be captured live using obliquely towed conical 202 µm mesh plankton nets from a drifting ship. Cultures are most reliably established when water temperatures are below 21 °C and are started from immature gonozooids, maturing phorozooids, and large nurses. Cultures can be maintained in rounded culture vessels on a slowly rotating plankton wheel and sustained on a diet of cultured algae in natural seawater for many generations. In addition to the ability to establish laboratory cultures of D. gegenbauri, we demonstrate that the collection condition, algae concentration, temperature, and exposure to naturally conditioned seawater are all critical to the culture establishment, growth, survival, and reproduction of D. gegenbauri.

Dynamics of a biofouling community in finfish aquaculture: a case study from the South Adriatic Sea.

Biofouling is one of the challenges that can strongly affect the finfish farm economy. Although several studies on biofouling in aquaculture have been conducted in the Mediterranean Sea, they focused on specific taxa or were limited to a particular period of sampling. The present study investigated for the first time the development, composition and variation in a biofouling community in a finfish farm with immersion time, season and depth. The results indicate that all these factors influence biofouling succession and recruitment. Moreover, the species that had a crucial role in structuring the community and in the farm cleaning activities were the ascidian Styela plicata and the bivalve Mytilus galloprovincialis. Compared with the literature data, the results highlight the heterogeneity in the composition of the biofouling present in the Mediterranean Sea. Moreover, such knowledge of the biofouling community could provide important information about management efforts and the costs that farmers will face when siting new fish farms.

Exotic species dominate marinas between the two most populated regions in the southwestern Atlantic Ocean.

Human occupation of coastal areas promotes the establishment of non-native species but information on bioinvasions is usually biased toward the Northern Hemisphere. We assessed non-native species' importance in sessile communities at six marinas along the most urbanized area of the Southwestern Atlantic coastline. We found 67 species, of which 19 are exotic. The most frequent species was the exotic polychaete Branchiomma luctuosum, while the most abundant was the exotic bryozoan Schizoporella errata that monopolized the substrata in three marinas. Along with S. errata, the exotic polychaete Hydroides elegans and ascidian Styela plicata dominated space in the three remaining marinas, while native species were in general rare. We show that communities associated with artificial substrata along this Brazilian urbanized area are dominated by exotic species and that using abundance data along with species identity can improve our understanding of the importance of exotic species for the dynamics of biological communities.

Potential roles of nuclear receptors in mediating neurodevelopmental toxicity of known endocrine-disrupting chemicals in ascidian embryos.

Endocrine Disrupting Chemicals (EDCs) are molecules able to interfere with the vertebrate hormonal system in different ways, a major one being the modification of the activity of nuclear receptors (NRs). Several NRs are expressed in the vertebrate brain during embryonic development and these NRs are suspected to be responsible for the neurodevelopmental defects induced by exposure to EDCs in fishes or amphibians and to participate in several neurodevelopmental disorders observed in humans. Known EDCs exert toxicity not only on vertebrate forms of marine life but also on marine invertebrates. However, because hormonal systems of invertebrates are poorly understood, it is not clear whether the teratogenic effects of known EDCs are because of endocrine disruption. The most conserved actors of endocrine systems are the NRs which are present in all metazoan genomes but their functions in invertebrate organisms are still insufficiently characterized. EDCs like bisphenol A have recently been shown to affect neurodevelopment in marine invertebrate chordates called ascidians. Because such phenotypes can be mediated by NRs expressed in the ascidian embryo, we review all the information available about NRs expression during ascidian embryogenesis and discuss their possible involvement in the neurodevelopmental phenotypes induced by EDCs.

Antero-posterior ectoderm patterning by canonical Wnt signaling during ascidian development.

Wnt/ß-catenin signaling is an ancient pathway in metazoans and controls various developmental processes, in particular the establishment and patterning of the embryonic primary axis. In vertebrates, a graded Wnt activity from posterior to anterior endows cells with positional information in the central nervous system. Recent studies in hemichordates support a conserved role for Wnt/ß-catenin in ectoderm antero-posterior patterning at the base of the deuterostomes. Ascidians are marine invertebrates and the closest relatives of vertebrates. By combining gain- and loss-of-function approaches, we have determined the role of Wnt/ß-catenin in patterning the three ectoderm derivatives of the ascidian Ciona intestinalis, central nervous system, peripheral nervous system and epidermis. Activating Wnt/ß-catenin signaling from gastrulation led to a dramatic transformation of the ectoderm with a loss of anterior identities and a reciprocal anterior extension of posterior identities, consistent with studies in other metazoans. Surprisingly, inhibiting Wnt signaling did not produce a reciprocal anteriorization of the embryo with a loss of more posterior identities like in vertebrates and hemichordate. Epidermis patterning was overall unchanged. Only the identity of two discrete regions of the central nervous system, the anteriormost and the posteriormost regions, were under the control of Wnt. Finally, the caudal peripheral nervous system, while being initially Wnt dependent, formed normally. Our results show that the Ciona embryonic ectoderm responds to Wnt activation in a manner that is compatible with the proposed function for this pathway at the base of the deuterostomes. However, possibly because of its fast and divergent mode of development that includes extensive use of maternal determinants, the overall antero-posterior patterning of the Ciona ectoderm is Wnt independent, and Wnt/ß-catenin signaling controls the formation of some sub-domains. Our results thus indicate that there has likely been a drift in the developmental systems controlling ectoderm patterning in the lineage leading to ascidians.

Expansion of a colonial ascidian following consecutive mass coral bleaching at Lizard Island, Australia.

Mass coral bleaching is challenging today's coral reefs. However, our understanding of dynamics in benthic space holders, following such disturbances, is limited. To address this, we quantified successional dynamics of the ascidian, Didemnum cf. molle using a series of temporally and spatially matched photoquadrats across both the 2016 and 2017 mass coral bleaching events on the Great Barrier Reef. Unlike corals, D. cf. molle appeared to flourish in the warm temperatures and rapidly expanded. Indeed, colony density increased nearly 6-fold over two years with one quadrat experiencing an increase of over 1000 ind. m-2. However, this increase did not simply track the increase in space due to coral mortality, but may have benefitted from reduced predation or increased nutrient availability following mass coral mortality. This study highlights the potential for D. cf molle to expand under bleaching conditions and to become a more prominent component of future reef configurations.

Prevalence of Mutation-Prone Microhomology-Mediated End Joining in a Chordate Lacking the c-NHEJ DNA Repair Pathway.

Classical non-homologous end joining (c-NHEJ), a fundamental pathway that repairs double-strand breaks in DNA, is almost universal in eukaryotes and involves multiple proteins highly conserved from yeast to human [1]. The genes encoding these proteins were not detected in the genome of Oikopleura dioica, a new model system of tunicate larvaceans known for its very compact and highly rearranged genome [2-4]. After showing their absence in the genomes of six other larvacean species, the present study examined how O. dioica oocytes and embryos repair double-strand DNA breaks (DSBs), using two approaches: the injection of linearized plasmids, which resulted in their rapid end joining, and a newly established CRISPR Cas9 technique. In both cases, end joining merged short microhomologous sequences surrounding the break (mainly 4 bp long), thus inducing deletions larger than for the tunicate ascidian Ciona intestinalis and human cells. A relatively high frequency of nucleotide insertions was also observed. Finally, a survey of genomic indels supports the involvement of microhomology-mediated repair in natural conditions. Overall, O. dioica repairs DSBs as other organisms do when their c-NHEJ pathway is experimentally rendered deficient, using another mode of end joining with the same effect as alternative NHEJ (a-NHEJ) or microhomology-mediated end joining (MMEJ) [5-7]. We discuss how the exceptional loss of c-NHEJ and its replacement by a more mutation-prone mechanism may have contributed to reshaping this genome and even been advantageous under pressure for genome compaction.

Living on the edge: Early life history phases as determinants of distribution in Pyura praeputialis (Heller, 1878), a rocky shore ecosystem engineer.

The distribution of intertidal organisms can depend on processes operating early in their life history. The ascidian Pyura praeputialis, a mid- to low-intertidal habitat-forming ecosystem engineer, was strongly associated with specific types of habitat (biogenic vs. bare rock). We examined field patterns and performed laboratory and field experiments to assess the nature of this association. Recruits were frequently found on the tunics of conspecifics and clumps of turfing coralline algae. Larvae preferred these same habitats in a series of laboratory settlement assays. Laboratory-reared juveniles (20- & 50-days-old) survived poorly on bare rock in the laboratory, while those on rugose surfaces - the tunic of adults and turfing corallines - showed high survivorship. Field-collected juveniles (<2 cm) affixed to these rugose habitats also exhibited high survivorship in the field. We conclude that both pre and post-settlement processes determine spatial pattern in this important habitat-forming taxon. The acute sensitivity of juveniles to desiccating conditions was unexpected in an intertidal organism.

Effect of substrate deployment timing and reproductive strategy on patterns in invasiveness of the colonial ascidian Didemnum vexillum.

The colonial ascidian Didemnum vexillum is a high-profile marine invader, with a geographically widespread distribution after introductions to several temperate regions. D. vexillum has been the focus of several eradication and control programmes globally and the need for specific biological knowledge that relates to establishment processes, persistence, impacts and potential for spread remains. The present study describes temporal patterns of D. vexillum percent cover on experimental substrates over 1.5-years in relation to seasonality of substratum availability, in conjunction with key physical (i.e. temperature and sedimentation) and biological factors (i.e. interspecific competition) at two sites in New Zealand. Colonies showed large fluctuations in percent cover between the two study sites and with reference to timing of bare substratum availability. Colonies generally exhibited an initial lag phase, with peak levels of D. vexillum cover reached during the second summer or autumn post-deployment. The long-term competitive dominance of colonies founded from the reattachment of fragments, as opposed to ambient larval recruitment alone, was also investigated. Increases in colony size as a result of ambient recruitment alone were initially much slower. However, after 12-months colony cover exceeded that of plates inoculated with fragments, suggesting the benefit of the apparent competitive dominance conferred by fragment inoculation is restricted to the early establishment phase. This information will provide increased understanding of the population dynamics of this species, as well as assist in the implementation of effective management strategies through knowledge of environmental drivers of prolific infestations.