Zhang 1
Understanding the Ecological Impacts of Invasive Tunicates and Their Response to
Climate Change
1
Yingqi Zhang, 2Linda Deegan, and 3Mary Carman
1
Colgate University
Hamilton, NY 13346
2
3
Woods Hole Research Center
Woods Hole, MA 02543
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.
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.
Zhang 7
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.
Zhang 8
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.
Zhang 9
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
Zhang 10
Valigra, L. (2005). Sea squirt threatens native marine life. The Gulf of Maine Times,
retrieved from http://www.gulfofmaine.org/times/summer2005/squirt.html.
Zhang 11
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.
Zhang 12
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.
Zhang 13
Net mussel filtration rate (ug/l/h/g)
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
0
10
20
30
40
50
Percent coverage by epifauna (%)
60
70
80
Figure 4. Net filtration rate of mussels that were covered by tunicates and bryozoans to
different degrees.
Epifauna filtration rate (μg/l/h/g)
7
6
5
4
3
2
1
0
0
2
4
6
8
10
12
Mussel meat filtration rate (μg/l/h/g)
Figure 5. Comparison between epifauna filtration rate and mussel meat filtration rate of
individual blue mussels.
Zhang 14
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.
0
-10
Percent change (%)
-20
-30
-40
-50
Ambient
-60
25 °C
-70
30 °C
-80
-90
-100
A. glabrum (N)
D. vexillum (I)
Species
B. violaceus (I)
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.
Zhang 15
80.00
60.00
Percent change (%)
40.00
20.00
0.00
Ambient
-20.00
25 °C
-40.00
30 °C
-60.00
-80.00
-100.00
A. glabrum
D. vexillum
B. violaceus
Species
Figure 8. Percent change in size of Aplidium glabrum, Didemnum vexillum, and Botrylloides
violaceus after the first round of incubation under different treatments.
0.00
-10.00
Percent change (%)
-20.00
-30.00
-40.00
Ambient
-50.00
25 °C
-60.00
30 °C
-70.00
pH 7.7
-80.00
-90.00
-100.00
D. vexillum (I)
B. violaceus (I)
Species
Figure 9. Percent change in wet weight of Didemnum vexillum and Botrylloides violaceus
after the second round of incubation under different treatments.
Zhang 16
40.00
Percent change (%)
20.00
0.00
-20.00
Ambient
-40.00
25 °C
30 °C
-60.00
pH 7.7
-80.00
-100.00
D. vexillum
B. violaceus
Species
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.