Zhang 1 Understanding the Ecological Impacts of Invasive Tunicates and Their Response to Climate Change 1Yingqi Zhang, 2Linda Deegan, and 3Mary Carman 1Colgate University Hamilton, NY 13346 2Woods Hole Research Center Woods Hole, MA 02543 3 Woods Hole Oceanographic Institution Woods Hole, MA 02543 Fall 2016 SES Independent Project Zhang 2 Abstract Invasive colonial tunicates have become widely distributed in estuaries on Cape Cod over the past years. My study aims to understand how invasive tunicates interact with other organisms in the ecosystem, and to explore the response of tunicates to future climate regime. I collected two species of invasive tunicate (Didemnum vexillum and Botrylloides violaceus) as well as one species of native tunicate (Aplidium glabrum), and evaluated their metabolic rates. I also collected sixteen blue mussels (Mytilus edulis), and investigated on the interaction between tunicates and mussels. Finally, I tested the response of tunicates to the changing climate using experimental manipulations of increased temperature and decreased pH. I found that D. vexillum and B. violaceus consumed oxygen at slightly faster rates than Aplidium glabrum. Both tunicates and blue mussels were feeding on phytoplankton as their major food source. Fouling tunicates were strongly competing with mussels to filter feed, but were not inhibiting mussel’s filtration rate. This was in part because the tunicates had not overgrown the shell lip, thus the mussels were still able to gap open to feed. Invasive tunicates might be more resilient to ocean warming and acidification, although this finding needs to be verified by further studies. Key words: Invasive tunicates, metabolic rate, blue mussels, filtration rate, climate change Zhang 3 Introduction Facilitated by global trades and long-distance travels, invasive species has become a worldwide problem in the past few decades (Keller and Perrings 2011). Shallow coastal waters are especially susceptible to invasions by exotic species, as they are heavily influenced by human activities, including ballast water transfer, aquaculture, and aquarium trade (Carlton and Geller 1993). Apart from species assemblage, the physical environment of global ecosystems is also shaped by anthropogenic influences. Oceans are expected to become warmer and more acidic in the future with the increase of atmospheric CO2 concentration (IPCC 2007). Multiple studies suggested that climate change might provide invasive species with competitive advantage to colonize the new habitat and gradually replace endemic species (Anthony et al. 2009; Rahel and Olden 2008; and Hellmann et al. 2008). To test this theory, I examined the potential influence of invasive tunicates in coastal ecosystems. Tunicates, commonly known as “sea squirts”, are marine biofouling organisms that primarily spread themselves by attaching to underwater surfaces of vessels (McKenzie et al. 2016). Once transported to new locations, they are able to quickly colonize local natural or artificial substrates, reproduce, and establish populations. Invasive tunicates are believed to have been introduced into the New England waters in the 1970s and 1980s (Valigra 2005). Little research exists on how they interact with other species in the food web (Dijkstra et al. 2007). Invasive tunicates can be found on a variety of substrates ranging from rocks and moorings to eelgrass and shellfish (Colarusso et al. 2016). The fast range expansion of invasive tunicates over the past few years has raised considerable concerns for the aquaculture industry due to their potentially negative impacts on shellfish community, including increased maintenance cost and reduced shellfish growth (Colarusso et al. 2016 and Carman et al. 2010). It is very likely that the hard surface of cultured shell fish and aquaculture gear suspended in the water column provides ideal platform for tunicates to foul (Carman et al. 2010). The focus of this study is to understand the ecology of invasive tunicates in comparison to native tunicate and blue mussels, as well as understand how invasive tunicates will respond to climate change. My three research questions are: a) Are there any fundamental difference in the metabolic rates of invasive and native tunicates? b) Do tunicates and bryozoans utilize similar food sources as blue mussels and will the presence of these fouling organisms inhibit the ability of shellfish to filter-feed? c) Which species is most resilient to a warmer and more acidic environment and will the change in abiotic conditions alter tunicate’s metabolism? To answer these questions, I collected three colonial species of tunicates and blue mussels (Mytilus edulis) that had various levels of coverage by tunicates. Aplidium glabrum is a native species, while Didemnum vexillum and Botrylloides violaceus are invasive species that originated from East Asian and Europe. Given the understudied nature of invasive tunicate studies, my project will provide valuable insight for invasive species and shellfish management. Zhang 4 Methods Field sampling Three tunicate species, Aplidium glabrum, Didemnum vexillum, and Botrylloides violaceus, were collected from the MBL docks at Eel Pond for the first trial during early November (Figure 1). Only two invasive tunicate species, Didemnum vexillum, and Botrylloides violaceus, were found and collected from the intertidal zone at the Cape Cod Canal for the second trial (Figure 1). Sixteen blue mussels were collected from the shellfish dock in Lagoon Pond on Martha’s Vineyard (Figure 1). Preparation of experimental tunicate tiles I divided the colonial tunicates into pieces of similar size (approximately 1 g and 4 cm2), and stabilized the tunicates onto 4.8 by 4.8 cm white ceramic tile with a rough surface by wrapping rubber bands around individual tiles. Tiles with tunicates were held in flowing water for about 48 hours to allow the tunicates to attach to the tiles. When the tunicates were attached, they were used in my experimental tests. Tiles without attached tunicates were also incubated in all trials and used as blanks to account for colonization and metabolism by microbes. Metabolic tests To evaluate metabolism, each species was held in a sealed respiration chamber (473 mL) and oxygen content was measured over time. Each chamber contained 6 tunicate tiles of one species or blanks and was filled water from the treatment tank. The chambers were held in an 18 °C incubator, gently stirred, and oxygen concentration measured with an O2 probe every 5 minutes until oxygen level dropped down to around 5 mg/L. Net O2 consumption rate by tunicates was determined by subtracting the O2 uptake rate of the blank group from the total metabolism. Metabolism was then divided by the total wet weight of each tile group to get O2 consumption rate per biomass (mg/L/h/g) + To assess nitrogen regeneration, I measured NH4 concentration from the chambers at the + beginning and the end of each trial. Net NH4 regeneration rate by tunicates was determined + by subtracting the NH4 regeneration rate of the blank group from the total, which was + further divided by the total wet weight of each tile group to get NH4 regeneration rate per biomass (μM/h/g). Algal filtration rate of mussels and tunicates To assess the relationship between tunicates and mussels, I measured metabolic rates of mussels with varying coverage of tunicates and other fouling organisms. I estimated percent coverage of tunicates and bryozoans on mussels by photographing the mussels and measuring total area using an image-processing software ImageJ. Filtration rates were determined first on live whole mussels with attached tunicates and bryozoans and then the same mussel shells with epifauna only. Sixteen blue mussel/ pairs of mussel shells were individually placed into sixteen 473 mL jars, with each filled with 350 mL of water and 5mL of diluted algae solution. Jars were then transferred to shaker tables to keep algal cells suspended and maintain oxygen levels. Chlorophyll a readings were taken appoximately every two hours. Filtration rate was determined by the change in Chlorophyll a concentration over the linear portion of the uptake curve. After examination of the data, this was standardized to be the first two hours. Net Zhang 5 filtration rate of mussels was calculated as the difference between the filtration rate of whole mussel and that of its shells (μg/L/h/g). Mussels were dissected after the filtration tests, with their total wet biomass weighed and their adductor muscle tissue dissected out. Mussel filtration rate per biomass was calculated as the net filtration rate of each mussel divided by its total wet biomass. I selected the muscle tissues of several mussels along with tissues of tunicates and bryozoans, and sent them to Marshall’s lab for stable isotope analysis. Growth and survival response to temperature and pH experiments I set up four 38-liter aquariums with different treatment in the sea water room, including one control tank, two temperature tanks, and one pH tank. The control tank was maintained under ambient room temperature (around 18 °C) and normal seawater pH (7.9). Under the influence of global warming, sea surface temperature is projected to increase by 0.03 °C per year (Pershing et al. 2015) and ocean pH is projected to decrease by 0.02 per decade (IPCC 2007). To mimic water conditions in 100 years, two temperature tanks were maintained under normal pH, but were heated up by +5 °C (~ 25 °C) and +10 °C (~ 30 °C) above ambient temperature by aquarium heaters. The pH tank was maintained under ambient room temperature, but received extra CO2 from a CO2 source tank and had a steady pH of 7.7 controlled by a pH regulator. All aquariums were equipped with air bubblers to ensure adequate water circulation. The incubation process was divided into two trials. The first trial lasted for 19 days. The pH treatment was not implemented; thus the pH tank was used as a second control ambient temperature tank. The second trial lasted for 8 days and had all the treatment tanks.