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).