First record of vase tunicate , Ciona intestinalis ( Linnaeus , 1767 ) , in coastal Newfoundland waters

Vase tunicate, Ciona intestinalis (Linnaeus, 1767) Type B (sensu Sato et al. 2012), was first recorded in Newfoundland (Canada) in Burin, Placentia Bay, by SCUBA divers conducting rapid visual surveys for invasive species on 19 September 2012. Follow-up surveys of Burin and adjacent communities were conducted in October and November 2012. Ciona intestinalis was present in relatively low abundance in Burin, but a well-established population was located in Little Bay, approximately 15 km north of Burin. Little Bay is located within Mortier Bay, an area of high vessel traffic and the possible introduction source of C. intestinalis. Further surveys are recommended to delineate the present distribution and monitor future dispersal of C. intestinalis in coastal Newfoundland waters. Due to the demonstrated high ecological and economic impacts of this species in other previously invaded regions, options for mitigating population growth and dispersal should be examined to minimize effects of this non-indigenous species.


Introduction
Non-indigenous ascidians continue to be problematic globally as invasive species (Lambert 2002;2007), as they often have wide environmental tolerances, rapid growth rates, early sexual maturity, and are prolific spawners (Lambert 2002).Invasive ascidians may have significant ecological and economic effects, by altering subtidal community structure and diversity (Lambert and Lambert 2003;Blum et al. 2007;Valentine et al. 2007) and by fouling man-made structures, especially bivalve aquaculture sites (LeBlanc et al. 2007;Ramsay et al. 2008;Rocha et al. 2009).
A variety of survey methods using qualitative and/or quantitative sampling techniques have been employed worldwide to detect introduced marine species (Campbell et al. 2007).Visual surveys by SCUBA divers have been demonstrated to be highly effective in detecting artificial Ciona intestinalis decoys (Kanary et al. 2010).Rapid visual surveys (RVS) were carried out opportunistically by SCUBA divers in September 2012 while conducting transect surveys for an ongoing Department of Fisheries and Oceans project: the Wharf and Breakwater Survey Project.These surveys supplemented the Aquatic Invasive Species (AIS) Harbour Survey Monitoring Program of the Department of Fisheries and Oceans and Memorial University.This paper reports the first detection of vase tunicate, Ciona intestinalis (Linnaeus, 1767), in coastal Newfoundland waters.

Methods
The Wharf and Breakwater Survey Project (WB Project) of Canada's Department of Fisheries and Oceans (DFO) investigates changes to habitat, fish, epibenthic macroinvertebrate, and macroalgae species associated with wharf or breakwater construction sites in Newfoundland.Since 2007, 17 sites (11 wharf/breakwater and 6 controls) have been surveyed by SCUBA divers annually in September along the coast of insular Newfoundland (Figure 1A).In 2012, while two divers conducted visual surveys and video recorded habitat along transects, a third diver conducted RVS at all project sites.RVS consisted of brief (15 -20 min) dives to visually examine adjacent wharves, floating docks, breakwater structures, and boat hulls for the presence of aquatic invasive species.1A).In October 2012, divers conducted video transect surveys in Baine Harbour and Spanish Room, and RVS of public wharves and adjacent natural substrates in Burin, Little Bay, Little St. Lawrence, and St. Lawrence (Figure 1B).These RVS were similar to those conducted during the WB Project but were more extensive (30 -45 min).They consisted of visual examination of public wharves, including wharf understructure and pilings, floating docks, adjacent boat hulls, and nearby natural substrates and observations were recorded post-dive.In November 2012, further surveys were completed by the DFO/MUN AIS survey team to delineate the range of C. intestinalis within Little Bay, and Mortier Bay.

Results and discussion
On 19 September 2012, four Ciona intestinalis specimens (Figure 2A) were identified during the WB Project RVS in Burin, Newfoundland (Figure 1).This was the first observation of C. intestinalis in Newfoundland waters (Ma et al. 2011;DFO 2012) Newfoundland and Labrador, pers. com.).In turn, M. membranacea were attached to the native kelp, Saccharina longicruris (Bachelot de la Pylaie) Kuntze, 1891, growing on wharf and floating dock structures.Specimens were observed at 2 -5 m depth at 15°C.Several small colonies (1 -4 cm diameter) of the non-native golden star tunicate, Botryllus schlosseri (Pallas, 1766), were also observed on the floating dock, growing on M. membranacea attached to S. longicruris and on a car tire.Botryllus schlosseri has been reported in Placentia Bay since 2006 (Callahan et al. 2010;Ma et al. 2011).Green crab, Carcinus maenas (Linnaeus, 1758), an invasive species observed in Placentia Bay since 2007 (Klassen and Locke 2007), was not observed at the Burin site, but local people reported seeing this species in the area.The Burin site was previously surveyed on 20 September 2007, during DFO/MUN AIS surveys.At that time only indigenous ascidian species were observed, including Aplidium glabrum (Verrill, 1871), Ascidia sp., Boltenia echinata (Linnaeus, 1767), and great numbers of large Halocynthia pyriformis (Rathke, 1806).During the 2012 WB Project RVS, many large H. pyriformis appeared dead or dying, although many small individuals were observed on the outer wharf pilings.One small Molgula sp. was also observed attached to a piece of S. longicruris.Through the remainder of the RVS, the only invasive species observed was M. membranacea, which was detected at all sites, except Renews, and both Terra Nova sites (see Figure 1A), although it was observed at other locations in Terra Nova in 2012 (P.Sargent, per.obs.).Three C. intestinalis specimens collected from Burin were preserved in ethyl alcohol (anhydrous).Two specimens were deposited in The Rooms, Provincial Museum of Natural History Annex (St.John's, Newfoundland and Labrador, Canada) under Catalogue #NFM CH-1.The remaining specimen was dissected to examine gonads and estimate sexual maturity.Presence of red ocelli, 6 on the atrial siphon and 8 on the buccal siphon (Van Name 1945; Figure 2A), lack of white pigment flecks in the body wall (Lambert and Lambert 1998; Figure 2B), and presence of single subterminal refringent bodies in the follicle cells of the eggs (Byrd and Lambert 2000; Figure 2C) confirmed species identification as C. intestinalis.Genomic studies have revealed the presence of two cryptic species of C. intestinalis, presently referred to as Types A and B (Sato et al. 2012).Yellow pigmentation on the distal end of the siphons (Figure 2A) and absence of tubercles on the sides of the siphons (Figure 2B) further identified the Burin specimen as C. intestinalis Type B (Sato et al. 2012).This individual was approximately 70 mm in length.Its vas deferens were white with sperm and its oviducts and ovary were full of eggs (170 m diameter; Figure 2D).It was sexually mature, and capable of reproduction (G.Lambert, University of Washington, Seattle, Washington, USA, pers.com.).
Follow-up DFO/MUN AIS video transect surveys on 17 October 2012 did not detect C. intestinalis at Baine Harbour or Spanish Room (Figure 1B).The Burin public wharf site was re-examined on 18 October 2012 and divers observed that C. intestinalis was widespread at this site, but in low densities, attached to kelps, S. longicruris and Agarum cribosum (Bory de Saint-Vincent, 1826), mussels, Mytilus sp., wharf structure, and sea cucumbers, Cucumaria frondosa (Gunnerus, 1770).Ciona intestinalis was not detected at Little St. Lawrence or St. Lawrence.Only indigenous Ascidia callosa (Stimpson, 1852) and Molgula complanata (Alder and Hancock, 1870) were observed on kelp attached to the public wharf in St. Lawrence.In Little Bay, large and small individuals of C. intestinalis were detected in very high densities with up to 100% coverage on the undersurface wharf structure and pilings, kelp, and mussels.Most individuals were attached to permanent wharf structures (Figure 2E), but they were also attached to the two adjacent floating docks and hull of one vessel at the wharf (Figure 2F).Ciona intestinalis were also observed attached to blades of eelgrass, Zostera marina (Linnaeus, 1753), near the wharf (< 1 km away) individually (Figure 2B) and in clumps of 5 -10 animals and to the stipe of an individual S. longicruris.Given the abundance and variation of size classes of C. intestinalis observed, it is likely to have been established in Newfoundland waters for more than one year.Further surveys conducted 26 -27 November 2012 found C. intestinalis throughout Little Bay, except midharbour where depth increased and substrate changed to sand with little to no vegetation.The public and shipyard wharves, immediately adjacent the WP Project site in Marystown were also surveyed.Small C. intestinalis were observed near the base of pilings on the public wharf and on mussels and ladders of the adjacent private wharf.General abundance in Marystown was much lower than in Little Bay.
The present observations of C. intestinalis in Newfoundland waters are approximately 375 km east of previous reports of this species in Cape Breton, Nova Scotia (DFO 2012; Figure 1).Swimming larvae of C. intestinalis are planktonic for only 1 -6 days before settlement (Svane and Havenhand 1993) which suggests planktonic dispersal is limited.Kanary et al. (2011) predicted planktonic dispersal of C. intestinalis may be up to 6 km per generation.Limited planktonic dispersal and extensive distance between the Burin Peninsula population and other Atlantic Canadian populations of this species suggests vessel traffic was the likely transmission vector.
Little Bay is a small, sheltered embayment of Mortier Bay, 3 km east of Marystown, the largest community on the Burin Peninsula.Mortier Bay is a busy area for vessel traffic and may be the point of introduction for C. intestinalis.Invasive ascidians survive particularly well in sheltered harbours where they may rapidly reproduce and further colonize (Lambert and Lambert 1998).In Little Bay, environmental conditions may have been exceptionally favourable to allow the establishment and colonization of C. intestinalis, whereas nearby Marystown and Spanish Room are more exposed and may be less favourable (Figure 1B).However, as other parts of Mortier Bay remain unsurveyed, undetected populations of C. intestinalis may exist elsewhere.
Atlantic Canadian waters are considered at high risk for establishment of C. intestinalis (Therriault and Herborg 2008), and this species poses a serious threat to the shellfish aquaculture industry (Ramsay et al. 2008).Suspended shellfish culture sites inadvertently provide abundant available substrate and refuge from predators with little interspecific competition (Vercaemer et al. 2011), thus increasing survival of nonnative tunicates (Carver et al. 2003).In mussel aquaculture, tunicates, such as C. intestinalis, may compete with mussels for food and result in decreased mussel size and condition and increased mortality rates (Daigle and Herbinger 2009).In addition, the associated costs of tunicate removal results in lost revenue for mussel growers (Daigle and Herbinger 2009).In Atlantic Canada, C. intestinalis was first reported as a significant biofouling problem for the mussel aquaculture industry in Nova Scotia in 1997 (Cayer et al. 1999).It was later detected in Prince Edward Island in 2004 and by 2007 had colonized its east coast in epidemic proportions, and replaced the clubbed tunicate, Styela clava Herdman, 1881, as the most problematic invasive species to the local mussel aquaculture industry (Ramsay et al. 2008).
Unlike their neighbours in other parts of Atlantic Canada, the Newfoundland mussel aquaculture industry has not had to deal with the problem of invasive ascidians to date.Also, unlike their Atlantic Canadian counterparts, the Newfoundland mussel aquaculture industry must contend with higher transportation costs associated with getting their product from the island to mainland markets.Thus any extra costs associated with the removal of invasive ascidians during harvesting and processing would be especially problematic to the industry (D. Green, Newfoundland Aquaculture Industry Association, St. John's, Newfoundland and Labrador, pers.com.).There were 53 mussel aquaculture sites licensed in Newfoundland in 2012, including 5 sites located in Placentia Bay (DFA 2012) within 90 -100 km of Little Bay.Given the limited dispersal range of C. intestinalis (Kanary et al. 2011), it would take more than 7 years to reach these sites through natural dispersal alone.However, due to the high volume of commercial and recreational vessel traffic moving within Placentia Bay, this time may be significantly shortened unless utmost vigilance is taken on behalf of users of this bay.
A variety of environmental factors influence growth, reproduction and survival in ascidians.Temperature and salinity are factors that influence distribution, persistence, and spread of C. intestinalis (Vercaemer et al. 2011).In general, C. intestinalis has wide environmental tolerances for temperature (-1 to 30ºC) and salinity (12 to 40) (Carver et al. 2006).However, these wide ranges likely overestimate tolerances of specific populations (Vercaemer et al. 2011).For example, C. intestinalis from southern Nova Scotia do not tolerate long term exposure to salinity ≤ 20 and temperature ≥ 24ºC (Vercaemer et al. 2011).Ciona intestinalis is essentially an annual species, living 12 -18 months (Millar 1952).Individuals that settle in May and June may spawn by August of the same year, and mature individuals may spawn continually at water temperatures > 8ºC (Carver et al. 2003).In sheltered waters, two generations per year may be produced (Dybern 1965;Vercaemer et al. 2011).As with spawning, recruitment occurs when mean water temperature exceeded 8ºC (Howes et al. 2007;Ramsay et al. 2009).In Prince Edward Island, C. intestinalis recruitment occurs mid-June to late November (Ramsay et al. 2009) while in Nova Scotia it occurs late June to mid-November (Howes et al. 2007).Howes et al. (2007) also found that C. intestinalis recruitment in southern Nova Scotia is highest in sheltered locations at depths of 4.5 -8.5 m.Environmental conditions in southern Nova Scotia and eastern PEI are especially suitable for invasive ascidians (Carver et al. 2003;Locke et al. 2007;Sephton et al. 2011), but it has yet to be determined whether environmental conditions within coastal Newfoundland waters will allow C. intestinalis to attain invasive status.Temperature and salinity data collected from 2009 -2012 from three nearshore locations at the northern end of Placentia Bay (Arnold's Cove: 47°45'30.65"N,53°59'16.97"W -Ma 2012, Lowen et al. in prep.;North Harbour: 47°50'46.24"N, 54°05'49.12"W -McKenzie, unpublished  Future effects of global warming and increases in vessel transportation will only increase the likelihood of spread of invasive species (Myerson and Mooney 2007;Stachowicz et al. 2002).Until current regulations regarding domestic discharge of ballast water and movement of hull-fouled vessels between ports change, the spread of non-indigenous species will continue to escalate (Lambert and Lambert 1998).It is therefore crucial to have a system in place focused on early detection, prevention, and public awareness to recognize when and where introductions occur.
Based on potential impacts posed by C. intestinalis in Newfoundland waters, the following measures are recommended.Immediate and annual AIS surveys are recommended throughout Mortier Bay and adjacent harbours to detect other colonization sites and monitor changes in current distribution and abundance of this and other non-native species.Ciona intestinalis and other non-native species including a green macroalgae, Codium fragile (Suringar) Hariot, 1889, are present in nearby St. Pierre and Miquelon (F.Urtizberea, Department of Agriculture and Forestry, St. Pierre and Miquelon, France, pers.com.; Figure 1B).As there is regular ferry service between St. Pierre Harbour and Fortune, Newfoundland, AIS surveys are recommended to determine if these species have been introduced to Fortune Bay.Early detection is crucial to mitigation measures since invasive ascidians, once established, provide a local source of larvae from which they may perpetuate the invasion regionally (Lambert 2002(Lambert , 2007)).The public should be made aware of potential economic and ecological impacts in order to minimize further anthropogenic transmission of C. intestinalis.Mitigation measures should be prepared immediately to alleviate potential problems caused by this species.The aquaculture industry, in consultation with government departments, should also prepare a contingency plan to prepare for further spread of C. intestinalis.Continued use of RVS with collaborative partners is also recommended.Due to economic and logistic constraints, researchers studying aquatic invasive species cannot possibly survey all locations regularly.Qualitative survey methods such as RVS provide rapid evaluations of the presence of non-native species in area and baseline information that may be improved upon by follow-up AIS surveys (Campbell et al. 2007).Therefore, RVS performed by collaborative partners while conducting their own fieldwork may increase chances of early detection.This in turn may enhance implementation of mitigation tools against population growth and further spread of these species.
Projects like the WB Project represent a useful means to conduct RVS for invasive species to supplement the DFO/MUN AIS survey program.Invasive ascidians thrive on wharf structures, floats and boat hulls in sheltered harbours (Lambert 2007).Increased vessel transportation in recent years has led to increases in supporting infrastructure providing more available space upon which opportunistic ascidian larvae may settle (Lambert and Lambert 1998) with little competition from native species (Lambert 2007).The WB Project focuses on such sites with broad scale coverage of insular Newfoundland.Its continuation for another four years, and the opportunity for further RVS, increases the chances of early detection and effectiveness of monitoring aquatic invasive species in Newfoundland.

Figure 1 .
Figure 1.Ciona intestinalis distribution in eastern Canada from the National Aquatic Invasive Species Database (DFO 2012) (•) and collection site of specimens in Burin, Newfoundland on 19 September 2012 (): A -Inset of insular Newfoundland showing Wharf and Breakwater Survey Project sites (■), and Department of Fisheries and Oceans and Memorial University of Newfoundland Aquatic Invasive Species Survey sites 2011 -2012 (×); B -Inset of southern Burin Peninsula, Newfoundland showing follow-up surveys (▲), collection site in St. Pierre Harbour, St. Pierre and Miquelon, France on 4 September 2012 (F.Urtizberea, pers.com.; ) and site of C. intestinalis observations in Little Bay, Newfoundland on 18 October 2012 (♦).

Figure 2 .
Figure 2. Photographs of Ciona intestinalis discovered in Newfoundland: A -C. intestinalis specimen from the public wharf in Burin showing red ocelli and yellow pigmentation on distal end of siphons; B -C. intestinalis attached to Zostera marina showing absence of tubercles on sides of siphons; C -C. intestinalis egg showing follicle cells with single subterminal refringent bodies; D -C. intestinalis showing oviduct with eggs (arrowhead) and white sperm duct (right); E -C. intestinalis specimens attached to the wharf in Little Bay; F -C. intestinalis specimens attached to a boat hull in Little Bay.A -Photograph by P. Sargent; B, E, F -Photographs by R. O'Donnell; C, D -Photographs by T. Wells.
Warmer summers with little freshwater input, as in 2012, may extend the spawning and recruitment periods of C. intestinalis and further its spread within Placentia Bay.Sheltered areas within Placentia Bay, especially those adjacent to Mortier Bay, or such sites along vessel traffic routes from Mortier Bay, may be at risk of colonization by C. intestinalis.