Isolation and characterization of twelve polymorphic microsatellite markers for the invasive ascidian Styela clava (Tunicata)

The invasive solitary ascidian Styela clava has spread extensively along European coasts since its first occurrence in the early 1950s. In order to characterize spatial and temporal patterns of genetic change during its establishment and subsequent spread, we developed 12 species‐specific loci from an enriched microsatellite library. Polymorphism was explored in one native and two introduced populations (N = 31 ± 1). Number of alleles per locus varied from two to 13 (mean = 7). The average expected heterozygosity within populations ranged from 0.539 to 0.580.     

Reproduction and early life history of an invasive ascidian Styela clava Herdman in Prince Edward Island, Canada

In the past decade, many aquatic invasive species have been introduced into Prince Edward Island, Canada, coastal waters, and they have had adverse effects on the aquaculture and fisheries industry. For instance, the mussel aquaculture industry has been overwhelmed by extremely high abundances of the invasive ascidian Styela clava in some areas. To understand and minimize the risk of spread of S. clava to other shellfish aquaculture sites, basic information on the reproductive biology of the ascidian is needed. This study investigated Styela gonad development, larval abundance and recruitment. Gonad development, using histological methods, indicated ovaries were ripe from late June through late October, 2004. Weekly water samples indicated larvae were present from 24 June to 29 October, with a distinct abundance peak in mid-August. Recruitment occurred from 24 June to 21 October. Timing of larval sampling was judged to be a crucial part of evaluating recruitment potential for monitoring purposes and a field experiment was carried out to document spatio-temporal larval concentrations. Data were gathered over 30 h sampling periods on 4 August and 14 September 2004. Water samples were taken simultaneously on an hourly basis at three depths: surface, 2 m and 4 m below the surface. Results indicated peaks in larval abundance were, in most cases, limited to a 3 h time interval in the early afternoon. Larval concentrations increased with water depth.     

Identification and population genetic comparison of three ascidian species based on mtDNA sequences

Ascidians are sessile marine chordate invertebrates found along seashores worldwide and are typically regarded as invasive organisms. Knowledge concerning their global genetic structure and subsequent invasive potential is limited. Here, we identified three ascidians—Ciona robusta, Ciona savignyi, and Styela clava from the northeast region of China using morphological characteristics and mitochondrial cytochrome c oxidase subunit I (cox1) as genetic marker. We additionally used phylogenetics to aid in the identification of these three species. The results of a population genetic analysis showed that among the three species, the level of haplotype diversity was particularly high within C. savignyi, and nucleotide diversity varied moderately. We divided the three species separately into native and invasive populations using 170 cox1 sequences from global resources to explore population genetic structure and invasive potential. Although in the network analysis Ciona spp. formed haplogroups of native and invasive populations, some haplotypes were still shared. We found that the haplotypes did not cluster within the network of S. clava. Our AMOVA results also showed that Ciona spp. had a weak genetic structure, and less genetic differentiation was present in S. clava. These data suggest that there are extensive incursions of these three ascidians into different geographical regions. Global comparisons of ascidian populations will help in the understanding of their population genetic structure and invasive potential, hence providing important insights regarding conservation as well as management.     

Pathways of spread of the introduced ascidian Styela clava (Tunicata) in Northern Europe, as revealed by microsatellite markers

Styela clava, a solitary ascidian native to the NW Pacific, has become a conspicuous member of fouling communities in NW European waters. As its natural dispersal appears to be limited, the wide distribution of S. clava along coasts within its introduced range may be attributed to secondary spread assisted by human activities. Here, we used six microsatellite loci to examine the genetic diversity and extent of gene flow among S. clava populations in its European introduced range. Samples were collected from 21 populations within Europe (N = 808), 4 populations within the USA and two populations within the native range (Japan). Large variation in genetic diversity was observed among the European populations but were not explained either by the geographic distance from the first introduction area (i.e. Plymouth, UK) nor by the time elapsed since the introduction. No founder effect was observed in the introduced populations, except possibly in Puget Sound (USA). At least two different introductions occurred in Europe, identified as distinct genetic clusters: northern Danish populations (resembling one Japanese population), and the rest of Europe; a sample from Shoreham (England) possibly represents a third introduction. In North America, the population from the Atlantic was genetically similar to the majority of European populations, suggesting a European origin for populations on this seaboard, while populations from the Pacific coast were genetically similar to the same Japanese population as the Danish populations.     

Water transport in the ascidians Styela clava Herdman and Ascidiella aspersa (Müller)

Experiments have been carried out on water transport in the ascidians Styela clava Herdman and Ascidiella aspersa (Müller), using a direct and an indirect method in both running and static sea water at various temperatures. Weight-specific rates, in running water at 10°C, ranged from 78 to 175 ml/min/g dry tissue weight for Styela clava and from 53 to 130 ml/min/g for Ascidiella aspersa.Experiments in a closed circulation showed that transport rates were sensitive to the conditions of water flow, falling to a low level within a few minutes or hours of the circulation being stopped. Rates of water transport of both species in static water, as determined by both methods, were lower than those from running water using the direct method.Results reported in the literature suggest that feeding behaviour in bivalve molluscs and some ascidians shows a similar sensitivity to some environmental conditions.     

Variation in the ability of Didemnum sp. to invade established communities

Over the past 30 years southern New England, USA has been invaded by several species of ascidians, including Botrylloides violaceus, Diplosoma listerianum, Styela clava, and Ascidiella aspersa. These species have become dominate in coastal embayments and marinas but are usually absent from more open water coastal areas. A colonial ascidian, Didemnum sp. has invaded southern New England during the past 10 years and we first observed this species in eastern Long Island Sound in 2000. It has become the dominant at several field sites while remaining in low abundance at others. We conducted an experiment at two places, a protected marina and an open coast site, to examine its ability to compete with the established fouling community. Small colonies of Didemnum were transplanted onto panels with communities that varied in age from one to four weeks old and these treatments along with controls with only Didemnum were exposed at both sites. In most treatments Didemnum became a dominant species in the communities at both sites but it reached higher abundances at the open coast site. Potential causes of the observed differences are predation on other species of ascidians at the open coast site reducing recruitment of these species and competition, lower tolerance for elevated temperatures at the marina site, or other environmental parameters that might affect growth rates.     

An investigation of dispersal of the introduced brown alga Undaria pinnatifida (Harvey) Suringar and its competition with some species on the man-made structures of Torquay Marina (Devon, UK)

As part of a study into the ecology of the recently introduced macroalga Undaria pinnatifida in the British Isles, the competitive ability of this adventive kelp with native species was investigated in Torquay Marina (Devon, UK). Sites of introduction were surveyed for the presence of native species filling a similar ecological niche and long-term competition experiments set up over 6 years. Experiments involving clearance, removal and monitoring showed that Undaria has a low competitive ability in the infralittoral and sublittoral of a rocky breakwater. Undaria did not manage to colonise the nearby (200 m) shoreline for at least 7 years from the date of its original introduction to the floating pontoons of Torquay Marina. Undaria did out-compete native species in a marina environment: two kelp species, Laminaria digitata and Laminaria saccharina, and a sea squirt, Styela clava.     

Biofouling ascidians on aquaculture gear as potential vectors of harmful algal introductions

Biofouling ascidians are ubiquitous in coastal ecosystems and are among the main colonizers of aquaculture gear. Our study tested the hypothesis that the transport, removal, and transfer of fouling ascidian species by aquaculturists provide a mechanism for concentration and distribution of harmful-algal cells to new areas. Wild-caught specimens of common, biofouling ascidian species (Styela clava, Ciona intestinalis, Molgula manhattensis, Botrylloides violaceus, Didemnum vexillum, and Botryllus schlosseri) were exposed individually to cultured strains of co-occurring harmful algae (Prorocentrum minimum, Alexandrium fundyense, Alexandrium monilatum, Karenia brevis, Aureococcus anophagefferens, or Heterosigma akashiwo) at simulated bloom cell densities of each HAB species. After feeding, ascidians were transferred to ultrafiltered seawater. Immediately after exposure, and after 24 and 48 h in ultrafiltered seawater, biodeposits were collected and observed microscopically for the presence of intact, potentially viable cells. Subsamples of biodeposits were transferred into culture tubes with ultrafiltered seawater and monitored for algal growth during 8 weeks. Cells of all HAB species were found to pass intact through the ascidian digestive system, remained viable, and in many cases were capable of re-establishing populations at least 48 h post-ingestion. The results of our study will inform industry and managers of the potential threat and ecological impact of spreading biofouling ascidians, and practices to mitigate adverse impacts. Additionally, these management practices have been formally incorporated into a new cost-share program developed to help shellfish producers prevent the further spread of ascidians and associated HAB species.     

Process of invasiveness among exotic tunicates in Prince Edward Island, Canada

Over the past decade, four exotic tunicates (Styela clava, Ciona intestinalis, Botrylloides violaceus and Botryllus schlosseri) have been reported in the Brudenell estuary in Prince Edward Island (PEI), Canada. Styela clava was the first exotic tunicate to arrive in 1997, rapidly establishing, spreading, invading, and eventually becoming a nuisance in several estuaries of PEI. In the Brudenell estuary, S. clava remained the only exotic nuisance tunicate until 2003. In the fall of 2004, the vase tunicate C. intestinalis, was reported in low abundance, followed by the two colonial species, B. schlosseri and B. violaceus, reported in the spring of 2005. The abundance of C. intestinalis rapidly increased post-introduction, eventually replacing S. clava as the foremost nuisance species on mussel farms in the estuary. To date, C. intestinalis continues to colonize this estuary at epidemic proportions, resulting in the continuing drop of S. clava abundance. The current abundance of C. intestinalis is estimated at 5 cm⁻², which is similar to S. clava abundance at its height in 2003. The 2006 abundance of S. clava is estimated to have fallen to near 0 cm⁻². The dominance of C. intestinalis as a fouling organism on mussel farms is considered a serious threat to this aquaculture industry, mainly due to its unmanageable weight. The process of the detection, establishment, invasiveness, and eventual rise to nuisance level of exotic tunicates in the Brudenell River is presented.     

Ecological implications of invasive tunicates associated with artificial structures in Puget Sound, Washington, USA

The non-native tunicates Didemnum vexillum, Ciona savignyi, and Styela clava are of concern to resource managers of Puget Sound, Washington, USA because they have been shown to threaten native species diversity and shellfish aquaculture in other regions. Invasive tunicates in Puget Sound occur mainly on man-made structures such as floating docks and aquaculture facilities. We conducted studies of the three species of concern and a fourth introduced tunicate, Botrylloides violaceus, that occur on these structures to evaluate their effects on mussels and native invertebrate communities. Because most studies of community effects of tunicates have dealt with sessile fouling organisms, we focused instead on epibenthic organisms such as meiofaunal harpacticoid copepods and macrofaunal polychaetes and amphipods that are known to be important prey for juvenile salmon and other small fish. Similar studies have shown mixed results, with negative, positive, or no effects depending on the species. We also found few community-level effects. Abundances of several species were lower when tunicates were present, but only at some of the sites. Several other species, including a non-native isopod, were significantly more abundant in the presence of tunicates. However, in most cases results were not statistically significant and more intensive, controlled sampling or experiments may be needed to demonstrate any consistent tunicate effects. Although invasive tunicates cause problems for mussel growers elsewhere, we did not find negative effects on mussels at four sites in Puget Sound. Given the large impacts known to accompany tunicate invasions elsewhere and their relatively recent invasions into Puget Sound, monitoring of their populations and effects should continue in the region.     

污损性海鞘的生态特点研究展望

海鞘生长快,繁殖迅速,能产生大量在短时间内附着的幼虫,是海洋污损生物群落中的重要成员,对海上人工设施会产生严重危害。污损性海鞘主要由悉尼海鞘(Ascidia sydneiensis)、史氏菊海鞘(Botryllus schlosseri)、米氏小叶鞘(Diplosoma listerianum)、柄瘤海鞘(Styela clava)、红贺海鞘(Herdmania momus)等9科29属103种组成,其中在太平洋海域64种、印度洋23种、大西洋44种,而北冰洋海域仅3种;另外,其附着污损具有明显的地域性和季节特点,并与深度有关。今后应加强污损性海鞘的生态调查和分类研究,阐明深海及两极海域附着污损特点,揭示幼虫附着变态过程的分子调控机理,完善幼虫采集培养技术,以期更好地掌握海鞘生物学特性与生态特点,丰富和发展海洋生态学内容,并为海洋污损生物的防除奠定基础,促进海洋经济产业的发展。     

Renewal of the gonads in Styela clava (Urochordata: Ascidiacea) as revealed by autoradiography with tritiated thymidine

DNA-synthesizing cells in the gonads of the ascidian Styela clava were labeled with tritiated thymidine and detected with autoradiography. In the testis, spermatogonia and primary spermatocytes are labeled after 1 hr. Labeled spermatozoa occur in the lumen of the testis follicles after 10 days and in the sperm ducts after 20 days. In the ovary, only germ cells (oogonia and pre-leptotene primary oocytes) and follicle cells are labeled after 1 hr. By 60 days, oocytes with basophilic cytoplasm (15–65 μ in diameter) are labeled; test cells embedded in larger eosinophilic oocytes (150 μ in diameter) are also labeled. Germ cells give rise to both oocytes and follicle cells. Through continued cell division, follicle cells give rise to test cells.     

Spatial and temporal effects of pre-seeding plates with invasive ascidians: Growth, recruitment and community composition

Many shallow water subtidal habitats in Massachusetts, USA have recently been invaded by five non-indigenous ascidian species: Ascidiella aspersa, Botrylloides violaceus, Didemnum sp., Diplosoma listerianum and Styela clava. This study examined the effects of seawater temperature, as a proxy for climate change, on B. violaceus and D. listerianum and the impact these ascidians have on native sessile fouling communities. Field experiments were conducted over a four month period at two locations (Lynn and Woods Hole, MA) to examine growth dynamics over regional thermal and geographic ranges. Invasive ascidians occupied as much as 80% of the primary substratum and accounted for the majority of species richness. B. violaceus and D. listerianum growth were similar at both study sites, but initial colony growth of D. listerianum was positively affected by temperature. B. violaceus and D. listerianum exhibited rapid two-week growth rates during the summer months with more rapid growth at the warmer Woods Hole site. Competition for space between B. violaceus and D. listerianum typically resulted in neutral borders between colonies. Overgrowth occurred if the colony of one species was disproportionably larger than the colony of the other species. Recruitment and growth of native species influenced the long-term composition of experimental communities more than the pre-seeding with B. violaceus or D. listerianum colonies. Elevated temperatures, however, increased initial growth of B. violaceus and D. listerianum and may have facilitated the species success to invade the communities during crucial periods of introduction. With projected global climate change, a rise in sea surface temperatures may exacerbate the cumulative impacts of invasions on benthic communities and facilitate the invasion of other non-native ascidian species.     

Evolution of the chordate muscle actin gene

Summary The ascidians Styela plicata, S. clava, and Mogula citrina are urochordates. The larvae of urochordates are considered to morphologically resemble the ancestral vertebrate. We asked whether larval and adult ascidian muscle actin sequences are nonmusclelike as in lower invertebrates, musclelike as in vertebrates, or possess characteristics of both. Nonmuscle and muscle actin cDNA clones from S. plicata were sequenced. Based on 27 diagnostic amino acids, which distinguish vertebrate muscle actin from other actins, we found that the deduced protein sequences of ascidian muscle actins exhibit similarities to both invertebrate and vertebrate muscle actins. A comparison to muscle actins from different vertebrate and invertebrate phylogenetic groups suggested that the urochordate muscle actins represent a transition from a nonmusclelike sequence to a vertebrate musclelike sequence. The ascidian adult muscle actin is more similar to skeletal actin and the larval muscle actin is more similar to cardiac actin, which indicates that the divergence of the skeletal and cardiac isoforms occurred before the emergence of urochordates. The muscle actin gene may be a powerful probe for investigating the chordate lineage. Offprint requests to: C.R. Tomlinson     

Role of cell interactions in ascidian muscle and pigment cell specification

Summary Muscle and brain pigment cell specification was studied by disrupting cell adhesion, cell dissociation, and reaggregation in embryos of the ascidianStyela clava. Treatment of embryos with Ca²⁺-free sea water between the 2-cell and gastrula stages disrupted blastomere adhesion but did not prevent acetylcholinesterase or muscle actin expression in presumptive muscle cells. Similar treatments initiated between the 2- and 32-cell stages caused more ectoderm cells to express tyrosinase and develop pigment granules than expected from the cell lineage. Whereas 2 pigment cells become the otolith and ocellus sensory organs in normal embryos, up to 33 pigment cells could differentiate in embryos after disruption of cell adhesion. Replacement of Ca²⁺-free sea water with normal sea water restored cell adhesion and usually resulted in development of embryos containing the conventional number of pigment cells. Dissociation of embryos into single cells between the 2- and 64-cell stages and culture of these cells beyond the fate restricted stage had no effect on the accumulation of muscle actin mRNA and muscle actin synthesis, but blocked pigment cell differentiation. Reaggregation of the dissociated cells did not enhance the number of cells that developed muscle features, but rescued pigment cell development. The results indicate that ascidian muscle cell specification occurs by an autonomous mechanism, whereas pigment cell specification occurs by a conditional mechanism involving cell interactions. In addition, the results suggest that negative cell interactions may restrict the potential for pigment cell development in the ectoderm of cleaving ascidian embryos.     

Recent population expansions of non-native ascidians in The Netherlands

Over the last 50 yrs seven non-native ascidians have settled in The Netherlands, concentrated in the two periods 1974-1977 and 1991-2004 (i.e., Styela clava, Aplidium glabrum, Diplosoma listerianum, Didemnum sp., Botrylloides violaceus, Molgula complanata and Perophora japonica). The year of the introduction of B. violaceus remains a matter of dispute because many of the Botrylloides specimens that are recorded in western Europe, have been identified as the closely resembling species B. leachi. Only Didemnum sp. has become a true invasive species and has become a threat to native ecosystems, especially in the province of Zeeland, by its ability to overgrow virtually all hard substrata present. This includes rocks, stones, sand, algae and almost all sessile marine animals. The sudden population expansion of the didemnid from 1996 onward, coincided with the cold winter of 1995-1996, which caused decreased population sizes of many marine animals. The resulting increase in the availability of suitable substrates for settlement and the strong decrease of grazing sea urchins, may have triggered the population expansion. Studying its population dynamics, the optimal growing temperature for Didemnum sp. appears to be 14-18 °C. Virtually all colonies die when the water temperature gets colder than 5 °C. Colonies growing on live marine animals seem to be more resistant to the cold, than those growing on rocks, stones and plants. Two potential predators of the didemnid have also been recorded in Dutch waters: the gastropods Trivia arctica and Lamellaria sp.     

Mutual effects of several fouling organisms of the White Sea (<Emphasis Type="Italic">Mytilus edulis,Styela rustica</Emphasis>, and <Emphasis Type="Italic">Hiatella arctica</Emphasis>) on their growth rate and survival

The mutual effects of several fouling species (the bivalves Mytilus edulis and Hiatella arctica and a solitary ascidian Styela rustica) on their growth rate and mortality were studied through field experiments. The interactions between S. rustica and H. arctica appeared to be the least antagonistic. In contrast, the mussel was the most “aggressive” species with regard to both competitors. It was observed that the ascidians died, because they were intensively braided and gummed up with the byssus threads of the mussels. However, in some cases the intraspecific competition was stronger than the interspecific one.