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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

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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

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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.

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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. During the first trial, four different tile groups (1 native species, 2 invasive species, and blank) were set up vertically in each treatment tank and further incubated for 19 days. During the second trial, five tiles groups (2 replicates for both invasive species and blank) were set up vertically in each treatment tank and further incubated in each treatment tank for 8 days. I recycled 1/3 of the water within each tank once every two days. I mixed 3.5g of frozen algae paste with 450mL water and added 100mL of diluted algae solution to each tank every day. To determine change in biomass, I measured the wet weight of tunicate pieces on individual tiles at the beginning and the end of each trial and calculated the difference. I also photographed the initial and final tiles and used ImageJ to estimate the change in tunicate size throughout the incubation. To determine the change in metabolic rate, I conducted two metabolic tests at the beginning and the end of each trial. The incubator was set to 18 °C for all initial runs, but was adjusted to different temperature levels to match the conditions in different treatment tanks for all final runs. Statistics I used t-test to determine whether the metabolic rate and growth rate of different tunicate species are significantly different. Results The two invasive tunicates species, Didemnum vexillum and Botrylloides violaceus, had higher oxygen consumption rates than the native species, Aplidium glabrum (Figure 2). B. violaceus had the highest oxygen uptake rate of 0.13 mg/L/h/g under ambient conditions,

Zhang 6 which was significantly higher than the oxygen consumption rate of the native A. glabrum. D. + vexillum regenerated more than twice as much NH4 than the other two species (Figure 3). The filtration rates of individual mussels were not correlated with their percent coverage by tunicates and bryozoans (Figure 4). The filtration rates of both blue mussels and their corresponding epifauna were in the range of 1-10 μg/L/h/g (Figure 5). Based on the stable isotope analysis, blue mussels and invasive tunicates had similar δ 13C values that were both between -22 ‰ and -18 ‰, which fell in the typical range for phytoplankton (Figure 6). Blue mussels, however, had slightly higher δ 15N values than tunicates, which might be contributed to the consumption of zooplankton as part of their diet. In comparison, bryozoan had very different isotopic signatures than the other two species. This may have been related to incomplete digestion of the carbonate skeleton. During the first trial, the biomass of B. violaceus decreased the least for both control and 25 °C treatment groups (Figure 7). The size of B. violaceus increased at the end of the incubation for control group, and decreased the least for 25 °C treatment group (Figure 8). All organisms experienced 100% mortality rate in the 30 °C treatment tank (Figures 7 and 8). Contrary to the results of trial 1, the biomass of D. vexillum decreased less at the end of the incubation for both control and pH treatment groups (Figure 9). The size of D. vexillum increased at the end of incubation for control group, and decreased less for 25 °C treatment group (Figure 10). All organisms experienced 100% mortality rate in the 25 °C and 30 °C treatment tanks (Figures 9 and 10). Both D. vexillum and B. violaceus incubated in control and pH groups took up oxygen at faster rates at the end of the second trial compared to their initial levels (Figure 11). However, only the oxygen consumption rate of D. vexillum that was incubated in the pH tank was significantly higher than its original value. Discussion The higher oxygen demands of Didemnum vexillum and Botrylloides violaceus suggest that these two invasive tunicates are growing at faster rates than Aplidium glabrum, which is consistent with the ongoing decline in native tunicate abundance due to the competition from invasive tunicates (Carman et al. 2010). The high ammonium regeneration rate of D. vexillum could be attributed to its leaky digestive pattern. Overall, native and invasive tunicates are metabolically different to the ecosystems, in terms of how fast they take up oxygen and regenerate ammonium. A potential reason why blue mussel filtration rate was not negatively correlated with the percent coverage by tunicates and bryozoans is that epifauna only occupied the broad surface of mussel shells in my experiment. However, if the invasive tunicates overgrow the two edges of the shells and prevent the mussels from gapping open, mussel filtration rate would potentially be inhibited. The similar δ 13C values of invasive tunicates and blue mussels indicate that the two organisms are both supported by the phytoplankton food web. Additionally, the filtration rates of epifauna were in the same magnitude as those of blue mussels (Figure 5), which means that there is a close competition between blue mussel and epifauna to acquire food. Since tunicates are voracious filter feeders (Colarusso et al. 2016), it is most likely that majority of the filter feeding by epifauna came from invasive tunicates.

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When tunicates start to actively grow and reproduce during summer, blue mussels could potentially suffer from shortage of food supply and experience reduced growth. Based on the results from aquarium incubation, invasive tunicates might be more resilient to changes in temperature and pH conditions (Figures 7-10). However, this cannot be a solid conclusion, because no native tunicate was available for pH trial and very little tunicates survived after the exposure to very high temperatures. The solution is to conduct this study again in the summer, when tunicates exist in high abundance and are thus easily obtained. More importantly, the summer average water temperature is already around 25 °C. The transition to higher temperature would be less abrupt, thus reducing artificial temperature shock. It is also advisable to gradually increase the temperature of the tank to allow adaptation. There was also a difference in resilience between the two invasive species. Botrylloides violaceus was more robust in the first trial as indicated by its overall more positive change in biomass and size (Figures 7 and 8), whereas Didemnum vexillum appeared to be the more resilient species in the second trial (Figures 9 and 10). I speculate that the possible cause might be that B. violaceus has slower recruitment rate. Based on my observations of the initial and final tile, majority of the parental tunicate tissues gradually degenerated throughout the incubation. What remained at the end were new colonies that were asexually reproduced by the original piece. The reason why B. violaceus had higher mortality rate than D. vexillum in the second trial could be that 8 days was not enough time for B. violaceus to reestablish new colonies. However, when B. violaceus does reestablish, it will recover pretty rapidly, which explains why it had lower mortality rate at the end of the longer trial (trial 1). The results from the post-incubation metabolic tests indicate that temperature is a stronger driver to metabolism than pH change. There might be synergic effect of warming and acidification on oxygen consumption rate and pH factors according to the significant difference between initial and pH treatment for Didemnum vexillum. However, this hypothesis was not tested. Although my study has provided some insight on the unique ecology of invasive tunicates, it would be very helpful for future studies to further examine the difference in filtration rate between native and invasive tunicates. Additional aquaria that are treated with both high temperature and low pH should be established to explore the synergic effect of ocean warming and acidification, which is most likely to happen in the future.

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Acknowledgement This independent project would not have been possible without the help of many wonderful individuals. I would like to thank my advisors Linda Deegan and Mary Carman for their incredible wisdom, guidance and support. Special thanks to Rich McHorney for tackling major technical difficulties and making sure the experiments ran smoothly; to the fantastic TAs, Helena McMonagle, Madeline Gorchels, and Leena Vilonen, for their patience and assistance; to Michelle Woods and Olivia Bispott for good company and help in the field; to Anne Giblin for providing the oxygen probes and her thumb drive; to Marshall Otter for running my stable isotope samples; to Dave Remsen, Janice Simmons, and Katie Dever for providing me with the frozen algae paste; to Dave Grunden for driving me and Mary to his dock on Martha’s Vineyard and helping me collect blue mussels; and to all SES faculty and students for their valuable input and support throughout this project and the entire semester.

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References Anthony, A., Atwood, J., August, P., Byron, C., Cobb, S., Foster, C., Fry, C., Gold, A., Hagos, K., Heffner, L., Kellogg, D.Q., Lellis-Dibble, K., Opaluch, J.J., Oviatt, C., Pfeiffer-Herbert, A., Rohr, N., Smith, L., Smythe, T., Swift, J., Vinhateiro, N. (2009). Coastal lagoons and climate change: ecological and social ramifications in U.S. Atlantic and Gulf coast ecosystems. Ecology and Society 14(1): 8. http://www.ecologyandsociety.org/vol14/iss1/art8/ Carlton, J.T. and Geller, J.B. (1993). Ecological roulette: the global transport of non-indigenous marine organisms. Science 261: 78–82. http://dx.doi.org/10.1126/science.261.5117.78 Carman, M. R., Morris, J. A., Karney, R. C., & Grunden, D. W. (2010). An initial assessment of native and invasive tunicates in shellfish aquaculture of the north american east coast. Journal of Applied Ichthyology/Zeitschrift Fur Angewandte Ichthyologie, 26, 8-11. doi:http://exlibris.colgate.edu:2070/10.1111/j.1439-0426.2010.01495.x Colarusso, P., Nelson, E., Ayvazian, S., Carman, M. R., Chintala, M., Grabbert, S., & Grunden, D. (2016). Quantifying the ecological impact of invasive tunicates to shallow coastal water systems. Management Of Biological Invasions, 7(1), 33-42. doi:10.3391/mbi.2016.7.1.05 Dijkstra, J., Sherman, H., Harris, L.G. (2007). The role of colonial ascidians in altering biodiversity in marine fouling communities. Journal of Experimental Marine Biology and Ecology, 342: 169– 171. http://dx.doi.org/10.1016/j.jembe.2006.10.035. Hellmann, J. J., Byers, J. E., Bierwagen, B. G., & Dukes, J. S. (2008). Five potential consequences of climate change for invasive species. Conservation Biology : The Journal of the Society for Conservation Biology, 22(3), 534-543. doi:http://exlibris.colgate.edu:2070/10.1111/j.1523-1739.2008.00951.x. Keller, R. P., & Perrings, C. (2011). International policy options for reducing the environmental impacts of invasive species. Bioscience, 61(12), 1005-1012. Retrieved from http://exlibris.colgate.edu:3933/docview/912504309?accountid=10207. IPCC. (2007). Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change, 2007. Retrieved from https://www.ipcc.ch/publications_and_data/ar4/wg1/en/contents.html Mckenzie, C. H., Matheson, K., Caines, S., & Wells, T. (2016). Surveys for non-indigenous tunicate species in Newfoundland, Canada (2006 – 2014): a first step towards understanding impact and control. Management Of Biological Invasions, 7(1), 21-32. doi:10.3391/mbi.2016.7.1.04 Pershing, A. J., Alexander, M. A., Hernandez, C. M., Kerr, L. A., Bris, A. L., Mills, K. E., . . . Thomas, A. C. (2015). Slow adaptation in the face of rapid warming leads to collapse of the gulf of maine cod fishery. Science, 350(6262), 809-812. doi:http://exlibris.colgate.edu:2070/10.1126/science.aac9819. Rahel, F. J., & Olden, J. D. (2008). Assessing the Effects of Climate Change on Aquatic Invasive Species. Conservation Biology, 22(3), 521-533. doi:10.1111/j.1523-1739.2008.00950.x

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Valigra, L. (2005). Sea squirt threatens native marine life. The Gulf of Maine Times, retrieved from http://www.gulfofmaine.org/times/summer2005/squirt.html.

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Figures

Figure 1. Tunicate and blue mussel samples were collected from Cape Cod Canal (Sandwich, MA), Eel Pond (Woods Hole, MA), and Lagoon Pond (Martha’s Vineyard, MA). All sites were labeled by red pins. Retrieved from https://www.google.com/maps.

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Figure 2. Oxygen uptake rate of Aplidium glabrum (native), Didemnum vexillum (invasive), and Botrylloides violaceus (invasive) measured at the beginning of trial 1. Error bars represent standard errors. Different letters indicate that statistical significance exist between two species.

Figure 3. Ammonium regeneration rate of Aplidium glabrum, Didemnum vexillum, and Botrylloides violaceus measured at the beginning of trial 1.

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Figure 4. Net filtration rate of mussels that were covered by tunicates and bryozoans to different degrees.

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Figure 5. Comparison between epifauna filtration rate and mussel meat filtration rate of individual blue mussels.

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Figure 6. Measured δ 13C and δ 15N of blue mussels, tunicates, and bryozoan. Isotopic values of phytoplankton were retrieved from week 9 food web isotope analysis lab.

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Figure 7. Percent change in wet weight of Aplidium glabrum, Didemnum vexillum, and Botrylloides violaceus after the first round of incubation under different treatments. Positive values on the y axis indicate growth, whereas negative values indicate mortality.

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Figure 9. Percent change in wet weight of Didemnum vexillum and Botrylloides violaceus after the second round of incubation under different treatments.

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Figure 10. Percent change in size of Didemnum vexillum and Botrylloides violaceus after the second round of incubation under different treatments.

Figure 11. Oxygen uptake rate of Aplidium glabrum, Didemnum vexillum, and Botrylloides violaceus measured at the beginning and end of trial 2 under different treatments.