Thomas K. Pool, School of Aquatic and Fishery Sciences, University of Washington, Seattle, Washington 98195 Sean Luis, School of Aquatic and Fishery Sciences, University of Washington, Seattle, Washington 98195 and Julian D. Olden1, School of Aquatic and Fishery Sciences, University of Washington, Seattle, Washington 98195

Assessing Lethal Dissolved Oxygen Tolerance for Invasive Tunicate Ciona savignyi in Puget Sound

Abstract Ciona savignyi is a solitary tunicate (Phylum Chordata, Class Ascidiacea) native to Japan that has invaded coastal habitats in the north-east Pacific and New Zealand. In the Puget Sound of Washington, USA, we examined the ability of C. savignyi to survive in artificially created hypoxic environments to determine if reduced dissolved oxygen (DO) treatments could be a viable control method. In laboratory bioassays, treatment groups that were immersed in DO concentrations ranging from completely hypoxic (1 mg/L) to low DO (5 mg/L) had zero survivorship of individually isolated tunicates after 14 to 22 days of exposure, respectively. Additionally, hypoxic conditions (approximately 1.5 mg/L) were created in the field using polyethylene tarp wraps applied around dock surfaces fouled with C. savignyi in a Puget Sound marina. To estimate mortality rates underneath the tarp wraps, dock units with clusters of C. savignyi remained wrapped for 10, 14 and 18 days and displayed decreasing survivorship with increased wrap time (76%, 51% and 33%, respectively). Our laboratory and field experiments indicate that wrapping docks fouled by C. savignyi with polyethylene tarps may be an effective management option to locally-control and reduce the spread of this tunicate species from marina habitats, which serve as hubs for non-native species transport via hull fouling. These results inform the development of a rapid response plan for C. savignyi in the state of Washington and may be a viable control method for other high priority non-native tunicates pending further work on species-specific tolerances to low DO. Keywords: ascidian, marina, hull fouling, ballast water

Introduction sion of numerous species of solitary and colonial forms (Lambert 2007). Tunicates can occur in Human-assisted introduction of non-native marine such massive numbers they pose a significant species is widespread across the world (Ruiz et al. economic threat to aquaculture production. Despite 2000, Carlton 2001, Molnar et al. 2008). Although the growing need for science that can assist in the some species introductions may have fairly benign effective management of tunicate invasions, the or even positive effects on an invaded ecosystem literature remains relatively limited in this regard. (Schlaepfer et al. 2011), numerous other non-native species have been implicated as a leading cause Managing against the proliferation of non-native of native biodiversity declines (Bax et al. 2003, tunicates has been a challenging task to date. The Thomsen et al. 2011). One striking example in establishment and spread of seven non-native coastal marine ecosystems is the introduction of tunicates along the outer coastline and Puget non-native ascidians and their negative impacts Sound of Washington State, USA, highlights on food web interactions, nutrient cycling, and this management challenge (Cohen et al. 1998, energy flow associated with invertebrate communi- Cohen et al. 2001, Lambert 2006, LeClair et al. ties (McKindsey et al. 2007). Globally, invasive 2009). Control of non-native tunicates after they ascidians (hereafter designated as tunicates) are are established has had limited success, in part, a growing problem, with continuing range expan- because our understanding of the species biology and physiology is typically insufficient (Coutts 1Author to whom correspondence should be addressed. and Forrest 2007, Forrest et al. 2007, Edwards Email: [email protected] and Leung 2008); a theme broadly shared in man-

106 Northwest Science, Vol. 87, No. 2, 2013 © 2013 by the Northwest Scientific Association. All rights reserved. agement of invasive species (Simberloff 2003). and rapidly increase in abundance (LeClair et al. For example, high-pressure water spraying was 2009). Recent work has also shown that a related used to remove non-native golden star tunicates non-native solitary species, Ciona intestinalis, (Botryllus schlosseri) from fouled aquaculture gear can cause detrimental ecological impacts on in- in Prince Edward Sound, Canada. However, the vertebrate communities by decreasing taxonomic tunicate fragments were found to be reproductively diversity and species growth rates (Blum et al. viable, potentially exacerbating the spread of the 2007, Daigle and Herbinger 2009). In late 2006, species (Paetzold and Davidson 2010). Infested Governor Christine Gregoire and the Washington structures and other natural substrata found to State Legislature provided $250,000 in emer- support tunicates can also be cleared by hand gency and supplemental funds to respond to and or by vacuuming (Pannel and Coutts 2007), a prevent the spread of invasive tunicates in the method resulting in variable success for Didemnum Puget Sound (Puget Sound Action Team 2007). vexillum. Other methods include immersion in a A primary agenda item stemming from this report chlorine exposure, and application of anti-fouling was the development of effective containment and agents such as medetomidine to target key stages eradication strategies for C. savignyi in the Puget in the tunicate life cycle (Paetzold and Davidson Sound. We aim to support this management need 2011, Willis and Woods 2011, Paetzold et al. by evaluating the effect of hypoxia on C. savignyi 2012). In Puget Sound, a viable management ap- using a combination of laboratory bioassays and proach incorporating knowledge of the biology of field experiments. non-native tunicates is needed to manage current populations in the region. Methods Efforts have been made by the Washington Laboratory Experimental Design Department of Fish and Wildlife (WDFW) to We collected individuals of C. savignyi for graded examine the efficacy of various non-native tu- bioassays during March, 2011 from an infestation nicate eradication methods within Puget Sound on docks in Elliott Bay Marina at Seattle, WA marinas (Pleus et al. 2008, LeClair et al. 2009); (47° 37’ N, 122° 23’ W; Figure 1A). Although these habitats are known to facilitate the spread of C. savignyi establishes on a variety of substrata, non-native species via hull fouling (Bax et al. 2002, contributing to its extensive range throughout Floerl et al. 2004). One method that successfully the region, individual tunicates for our study dispatched the non-native clubbed tunicate (Styela were collected exclusively from the underside clava) from fouled pilings in New Zealand involved of floating dock units where the highest densities wrapping individual pilings with polyethylene were observed. The species is identifiable by eight tarps creating hypoxic water conditions for the small orange markings around the openings of the epifaunal communities (Coutts and Forrest 2005). siphons with yellow or orange pigment flecks in While this control method was effective, a more the mantle, visible through the translucent tunic. detailed understanding of non-native tunicates’ Adult individuals typically range in size from physiological tolerances to low dissolved oxygen 50-100 mm in total length. (DO) would be useful in developing a method C. savignyi were collected from the marina with for more effective and extensive use with other SCUBA and then individually incubated within invasive tunicate species. 4-L treatment chambers of unfiltered seawater The aim of our study was to provide informa- under dim fluorescent light (Figure 1B). The tion on the lethal DO tolerance of the non-native effects of DO concentration on C. savignyi were solitary tunicate Ciona savignyi, providing relevant investigated in a series of 28-d bioassays. Nitrogen management information to reduce the species gas was immediately bubbled into each treatment secondary spread. The tunicate is currently a chamber after the animals were incubated to reduce priority species for eradication in Puget Sound the DO content. We exposed nine individuals of because it can proliferate quickly into new habitats C. savignyi to each of five DO treatment levels

Lethal Dissolved Oxygen Tolerance for Ciona savigny 107 Figure 1. (A) The current distribution of Ciona savignyi in Puget Sound, WA, as of June 17, 2011 (REEF 2011). (B) Laboratory experiments (pictured are the experimental aquaria) occurred at the University of Washington. (C) Field experiments (pictured is the dock with brown tarp and dissolved oxygen meter) occurred at the Elliott Bay Marina (indicated by star in panel A).

consisting of 11 mg/L (DO level of seawater with to reduce the handling of the tunicates prior to no gas applied), 7 mg/L, 5 mg/L, 3 mg/L and their incubation. All individuals that remained 1 mg/L concentrations. For the duration of the alive at the end of the study were euthanized study, the treatment chambers were sealed and and weighed. Each tunicate was dried for 48 h at submerged in a water table chilled at a constant 50 ˚C (Draughon et al. 2010). The DO level in 7 °C, the ambient marina water temperature at the treatment chambers was also measured using a time the tunicates were collected. With survivor- HACH LDO after the death of each tunicate to ship expected to increase with colder temperatures confirm concentrations remained constant within due to slower individual metabolic rates, incuba- each chamber during the study. The change in tion at 7 °C provides a conservative estimate of DO content of treatment chambers ranged from the tunicates ability to persist at low DO levels. 0 to 0.4 mg/L indicating minimal variation in DO Every chamber was inspected daily to determine if during the laboratory experiment. mortality had occurred. Mortality was determined by the lack of active filtration, general retraction Field Study Design of the oral and atrial siphons, a withered tunic, LeClair et al. (2009) outlined techniques to elimi- and increased yellow pigmentation throughout nate non-native tunicates by wrapping invaded dock the tunicate (Figure 2). areas with polyethylene tarps to create hypoxic The wet and dry tissue weights for C. savignyi conditions under the wraps. In May 2011, clusters were measured immediately after death occurred of C. savignyi were identified on dock units within

108 Pool, Luis, and Olden an airtight PVC piping component (Figure 1C) was installed along with one of the wrapped dock units allowing us to assess the rate of DO decline under the wraps during the experiment. The DO was measured every 24 h for the duration of the field study.

Statistical Analysis We analyzed the laboratory bioassay data using a one-way analysis of variance (ANOVA) for between-group differences in mortality rates (days to mortality) of our DO treatment groups. The median effective DO concentration resulting in

death of 50% of individuals (LD50) was calculated according to the Probit method (Finney 1971) for the 5 mg/L, 3 mg/L and 1 mg/L treatments. More simply, we determined if tunicate mortality rates varied significantly between treatment groups by comparing the total number of days before death for every individual in our three most hypoxic groups. A one-way ANOVA was used to test for significant differences (P < 0.05) in wet and dry Figure 2. Ciona savignyi within a treatment chamber (A) alive and (B) after death occurred. weights of four individuals of each of the five treatments. Tunicate mortality in the field experi- ments were calculated based on a comparison of the Elliott Bay Marina using SCUBA surveys. pre- versus post-treatment counts. Analysis was The units consisted of individual 2.5 m by 1.0 m executed in SPSS (SPSS Inc. 2001) and R 2.15.1 floating cement slabs supporting a diverse array (R Development Core Team 2012). of marine invertebrates. Nine dock units (each supporting greater than seven C. savignyi individu- Results als) were identified on the same pier to minimize natural differences in environmental factors that We found decreasing survivorship of C. savignyi might spatially vary within the marina, potentially individuals with decreasing DO levels in the bio- complicating the interpretation of the survivorship assays (Figure 3); groups exposed to the lowest results. The individual dock units were wrapped DO concentration (1 mg/L) exhibited the lowest using the LeClair et al. (2009) method and sealed survival and the 5 mg/L and 7 mg/L treatments (p < using all-weather corrosion protection tape (3M 0.05) displayed the highest survival of C. savignyi. Scotchrap) preventing entry of the surrounding At the end of our 28-day study the control group water inside the wrapped enclosure. Given that had 78% survivorship, the 7 mg/L group had 22% 100% mortality occurred on day 14 for the most survivorship, and the remaining three groups with hypoxic bioassay, field treatment groups were 5, 3, and 1 mg/L exhibited 0% survivorship. The wrapped for 10, 14 and 18 total days to estimate mean day of death (d) for individuals within the mortality rates over time. For each treatment group, three lowest DO groups differed significantly we randomly selected and unwrapped three dock between treatments (F2,24 = 6.852, p < 0.001): units at each time interval. The total number of C. 5 mg/L (mean = 18.1 d), 3 mg/L (16 d) and 1 savignyi remaining alive under each wrap were mg/L (10.6 d). Results from the Probit analysis counted using SCUBA to determine survivorship for treatments with zero survivorship at the end of tunicates in each treatment group. Additionally, of the experimental period produced LD50 values

Lethal Dissolved Oxygen Tolerance for Ciona savigny 109 across all treatment groups. The dissolved oxygen concentration underneath the wrap with the DO observation PVC pipe dropped from an ambient (11.23 mg/L) to highly hypoxic level (1.58 mg/L) over the course of 10 d. The DO remained at this concentration for the remainder of the field study.

Discussion Control of solitary tunicates has focused on remov- al of adults from artificial structures using methods such as plucking by divers, high pressure water blasting of fouled structures, air drying, immersion Figure 3. Laboratory study of survivorship of Ciona savignyi in each dissolved oxygen (DO) treatment group in freshwater, acetic acid or chlorine exposure, and during the 28-d study. Each treatment group had encapsulation (Pannel and Coutts 2007, LeClair et nine tunicates, each in an individual treatment al. 2009, Piola et al. 2010, Paetzold et al. 2012). chamber. In our study, the non-native tunicate C. savignyi of 17, 15 and 10 d for the 5 mg/L, 3 mg/L and 1 was resistant to short-term exposure to hypoxic mg/L DO treatments, respectively. Wet and dry conditions but was effectively eliminated with weights of C. savignyi did not differ significantly sustained exposure to low dissolved oxygen levels. Specifically, our laboratory bioassays (Figure 3) between treatment groups (Wet: F4,16 = 1.520, p and field experiments suggest that by wrapping = 0.24 Dry: F4,16 = 0.738, p = 0.58); therefore size did not appear to be influencing observed docks fouled by C. savignyi with polyethylene tarps mortality rates. for >20 days, the species could be significantly Field experiments largely supported the results reduced and likely eradicated from the treatment from the laboratory bioassays. Increased wrap time areas making it an effective management option corresponded to decreased survivorship (Figure for controlling local abundances and reducing 4). Dead tunicates were significantly withered the spread of this tunicate within marina habitats. and discolored or had completely detached from Interestingly, in a study conducted by the WDFW the substrate. A strong ammonia odor was re- using a similar wrap method during the same time leased when removing the wraps from all three of year (LeClair et al. 2009), 100% mortality of time classes indicating hypoxia had occurred the invasive tunicate D. vexillum occurred in only 14 days, suggesting that tunicate species may respond differentially to hypoxia treatments. In areas with multiple non-native tunicate species (such as Puget Sound), determining the lethal DO tolerance for each species is needed to ensure adequate wrap times are implemented. Surveys of invaded marinas could then dictate the most conservative wrap time required to achieve 100% mortality of the non-native tunicates in that area. Ensuring the wrap method is used for an appro- priate time period is essential because previous studies have shown that epifaunal composition can shift in favor of non-native tunicates when Figure 4. Mean survivorship of Ciona savignyi individuals in communities are exposed to moderately low DO three treatment groups exposed to hypoxic condi- tions underneath experimental tarp wraps. Whiskers (2 mg/L < DO < 4 mg/L) for short (24-hour) represent 1 standard deviation. periods (Jewett et al. 2005).

110 Pool, Luis, and Olden Despite increased research focus on non-native of those species into surrounding coastal ecosys- species in coastal systems (Lewis et al. 2003, tems. Additionally, when early detection activities Levin et al. 2006, Harvey et al. 2008), evaluation discover new (and small) invasions in marinas, the of cost-effective and ecologically robust control wrap treatment may be a valuable management methods to manage these species remains limited tool to quickly eradicate the infestation. (Pleus et al. 2008). The wrap method in our study In conclusion, when developing techniques did not require expensive equipment or technical to manage an invasion, a fundamental under- experience and could be replicated in marinas standing of the non-native species physiology throughout the Puget Sound. The WDFW Tuni- and biology is essential to maximize efficiency cate Response Advisory Committee identified and cost-effectiveness of the management effort this method as requiring relatively low human (Simberloff 2003). Our study indicates that control effort and materials costs but that assessment of the non-native tunicate C. savignyi on marina applied only to their relatively small-scale pilot structures with wrap treatments is a viable man- study (LeClair et al. 2009). At a larger scale, a agement option, but sustained periods of low DO similar method was used to wrap 178 wharf piles are required to ensure 100% mortality. Given that to control the spread of D. vexillum in Shakespeare at least six other non-native tunicates have been Bay, New Zealand (Coutts and Forrest 2007). established in the region and propagule pressure Although eradication from the whole bay was remains high from the continued importation not successful, the method was very effective at of species via hull fouling and ballast water killing the non-native tunicate under the wrapped releases, developing this management approach piles. Post-treatment monitoring should also a to eradicate multiple non-native species within component of any treatment effort to determine the same treatment is needed. Importantly, this if the recovery of the epifaunal community is research has demonstrated that treatment wraps native species dominated, potentially providing have the potential to contribute to a larger regional future resistance to subsequent species inva- plan to control non-native invertebrate species in sions. Furthermore, ongoing monitoring can identify if future reintroductions of the target Puget Sound. non-native species occur, precipitating further Acknowledgments management action. In aquatic environments where a non-native spe- The authors graciously thank Greg Jensen, Kristian cies have become well established and widespread, Happa-Aho and Jon Wittouck for their assistance such as C. savignyi within Puget Sound, examples with our laboratory experimental design. We also of successful eradication are limited. However, thank Sarah Hu, Mike Luis and Beth Sosik for control methods to reduce local abundance are their assistance conducting the field component, valuable (see Culver and Kuris 2000). Recent work Lauren Kuehne for providing statistical assistance, has identified recreational boating as a significant Christy Semmens for supplying the distributional vector for hull fouling invertebrates (Fofonoff et data presented in Figure 1A and the staff at Elliott al. 2003, Murray et al. 2011), making marinas Bay Marina for accommodating our study. Funding potential propagule sources for the secondary support was provided by the School of Aquatic transport of non-native species. Although targeting and Fishery Sciences, University of Washington, marinas with rapid and effective control measures including a H. Mason Keeler Endowment Profes- may not eradicate target non-native species from sorship to JDO. Comments from two anonymous the entire region, such actions may successfully reviewers also contributed significantly to improv- stem the local dominance and subsequent spread ing the quality of the final manuscript.

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Received 13 August 2012 Accepted for publication 12 December 2012

Lethal Dissolved Oxygen Tolerance for Ciona savigny 113