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Fall 2013
Predation as a Vehicle to Aid Tunicate Invasion in the Biofouling Community
Helen Day University of New Hampshire, Durham
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Recommended Citation Day, Helen, "Predation as a Vehicle to Aid Tunicate Invasion in the Biofouling Community" (2013). Master's Theses and Capstones. 802. https://scholars.unh.edu/thesis/802
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BY
HELEN DAY
BS University of Rhode Island, 2006
THESIS
Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of
M aster o f Science
In
Zoology
September 2013 UMI Number: 1524299
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Dr. Larry G. Harris (Professor, Zoology)
Dr. James F. Hanay (Professor, Zoology)
D. Winsor H. Watson (Professor, Zoology)
Date TABLE OF CONTENTS
TITLE PAGE ...... i SIGNATURE PAGE ...... ii ACKNOWLEDGMENTS...... v LIST OF TABLES...... vi LIST OF FIGURES...... vii ABSTRACT...... xiv
I. CHAPTER ONE - GENERAL INTRODUCTION Introduced Species as an Ecosystem Stressor ...... 1 Benthic Communities ...... 2 Biofouling Communities ...... 2 Predation and the Biofouling Community ...... 4 Biofouling Community Development ...... 6 Tunicates in the Biofouling Community ...... 8 Molgula citrina and Molgula manhattensis...... 10 Ciona intestinalis...... 10 Botryllus schlosseri...... 11 Botryloides violaceus ...... 12 Diplosoma listeranium...... 12 Didemnum species ...... 13 Study Objectives ...... 13 II. METHODS Introduction ...... 16 Benthic Panel Community: Data Collection ...... 22 Benthic Panel Community: Sessile Invertebrates ...... 24 Benthic Panel Community: Identification and Quantification of Molguloid Tunicates ...... 28 Benthic Panel Community: Quantification ...... 30 Benthic Panel Community: Statistical Analysis ...... 31 Laboratory Feeding Trial Overview ...... 34 Predator Inclusion Experiments: Invertebrates ...... 34 Predator Inclusion Experiments: Fish ...... 36 Predator Inclusion Experiments: Statistical Analysis ...... 40 Y-Maze Prey Choice Experiment ...... 40 Y-Maze Prey Choice Experiment: Statistical Analysis ...... 41 III. RESULTS Biofouling Community Composition ...... 44 Surface Biofouling Community Composition ...... 45 Benthic Biofouling Community Composition ...... 47 Benthic Panel Community Composition ...... 51 Community Composition and Seasonal Patterns ...... 55 Tunicates in Panel Community Composition ...... 61 Impact of Predation on Tunicate Community Composition. ..63 Impact of Predation on Tunicate Competition ...... 74 Impact of Predation on Colonial and Solitary Tunicates 76 Caged Control Community Composition ...... 78 Overall Laboratory Feeding Trial Results ...... 79 Predators which did not consume M. citrina...... 79 Predators which consumed M. citrina...... 80 Predator Inclusion Results: Tauiogolabrus adspersus...... 80 Predator Inclusion Results: Asterias rubens...... 82 Comparison of Predator Inclusion Results ...... 83 Y-Maze Prey Choice Preference Results ...... 84 Feeding Trial Results Overview ...... 85 IV. DISCUSSION Community Composition and Successional Development 87 Impact of Predation on Community Development ...... 93 Competitive Interactions between Sessile Species ...... 99 Predators as Eco-system Engineers ...... 107 Limitations to this Study ...... 108 Importance of this Study ...... 109 Future Directions ...... 110 LIST OF REFERENCES...... 1 APPENDIX A - FEEDING TRIAL DATA...... 18 APPENDIX B - IACUC PERMISSION...... 24 ACKNOWLEDGEMENTS
I would like to thank my advisor Dr. Larry Harris for all his help and guidance throughout this project. I would also like to thank my committee members Dr. James
Haney and Dr. Win Watson for their help. I would like to thank the University of New
Hampshire for their academic and financial support including the Summer Teaching
Assistant Fellowship and the Lemer-Grey Fellowship; Finally, I would like to thank my friends and family. LIST OF TABLES
Table 1: The distribution of replicates (all predator access. AA; fish access, FA; no predator access, NA) along the pier, the replicate closest to the shore is on the left.
Table 3: Details of Y-Maze testing with the predator A rubens and the prey items M citrina and M. edulis.
Table 4: An ethogram of behaviors anticipated to occur during Y-Maze prey choice preference experiments.
Table 5: Results of Y-Maze choice test with ,4. rubens.
Table 6: Results of Y-Maze choice test with A rubens.
Table 7: Results of sessile invertebrate species between caged and uncaged treatment types.
Table 8: Table of tunicate species found on benthic community panels.
Table 9: Frequency of abundances of tunicate species between caged and uncaged treatment types.
Table 10: The frequency of disturbance events which reduced the abundances of tunicate species.
Table 11: Tunicate species richness on benthic community panels.
Table 12: Feeding trial results with a suite of potential predators that were commonly found at the CML pier field site.
Table 13: Feeding trial results for T. adspersus.
Table 14: Feeding trial results with A. rubens.
Table 15: Results of video analysis of A. rubens foraging and feeding behavior towards M. citrina.
Table 16: Results of initial choice (i.e. first choice) in Y-Maze prey choice preference between M. citrina and M. edulis.
Table 17: Results of Y Maze prey choice preference for A. rubens between M. citrina and M. edulis.
vi Table 18: Feeding trial results for the predators Flabellina verrucosa, Carcinus maenas and Cyclopterus lumpus with the prey item M. citrina.
Table 19: Table 19: Feeding trial results. Medium size class = 0-9.99 cm: Large size class = 10-20 cm.
Table 20: Results of all feeding trials with T. adspersus.
Table 21: The results ofY Maze Prey Choice Preference between M. citrina and no prey item.
Table 22: Results of video analysis of A. rubens foraging and feeding behavior.
Table 23: Results of feeding trials with T. adspersus.
Table 24: Results of Y Maze prey choice preference for A. rubens between M. citrina and M. edulis. LIST OF FIGURES
Figure 1: Arial photograph of the University of New Hampshire Coastal Marine Laboratory pier where the benthic community panels were located. Photo courtesy of The University of New Hampshire (2010).
Figure 2: The location of the Gulf of Maine (GofM) on the eastern seaboard. The line of separation which can serve as a barrier to dispersal for planktonic larvae is seen in the divide between warmer southern yellow/orange waters and colder arctic blue/green waters. Figure courtesy of National Oceanographic and Atmospheric Administration (NOAA).
Figure 3: Image taken on the floating dock at the University of New Hampshire Coastal Marine Laboratory pier. In frame are the fifteen hanging ropes which are affixed to the fifteen panel replicates suspended in the benthic subtidal.
Figure 4: Underwater image illustrating the layout of a no predator access (NA) replicate and its orientation in the water column in relation to the ocean bottom (benthic substrate). A single caged replicate is seen in the foreground, while a second and third replicate are seen in the background. Video image still from a video courtesy of Dr. James Haney (2010).
Figure 5: Example of an uncaged all predator access (AA) replicate. The replicate has been removed from the water in order to photograph the panel for data collection.
Figure 6: Example of a caged no predator access (NA) replicate. The replicate has been removed from the water and the mesh side opposing the panel has been opened in order to photograph the panel for data collection.
Figure 7: Example of the partial siding found in the cage control replicates.
Figure 8: Example of the solitary tunicate M. citrina on a benthic community panel.
Figure 9: Example of the solitary tunicate C. intestinalis on a benthic community panel.
Figure 10: Example of the colonial tunicate B. violaceus on a benthic community panel.
Figure 11: Example of the colonial tunicate B. schlosseri on a benthic community panel.
Figure 12: Example of the colonial tunicate Didemnum spp. on a benthic community panel.
vi n Figure 13: Example of the dominant types of sponges on benthic community panels; left is most likely Halichondria spp. while right is most likely Haliclona spp.
Figure 14: Example of Anomia species (most likely A. simplex) on a benthic community panel.
Figure 15: Example of the barnacle S. balanoides on a benthic community panel; also indicated are remains of removed S. balanoides.
Figure 16: Example of arborescent Bryozoans (left: most likely Bugula spp.) and encrusting Bryozoans (right: most likely M. membranacea or C. pallasiana) on a benthic community panel.
Figure 17: Example of the hydroid Ectopluera larynx on a benthic community panel.
Figure 18: Example of the bivalve Hiatella arctica on a benthic community panel.
Figure 19: Example of M citrina on a benthic community panel. The panel edges have been delineated and M. citrina have been isolated from other biofouling community members for the purpose of calculations of percentage cover.
Figure 20: The average monthly surface (10 m) water temperature in the western Gulf of Maine for the course of the experiment (May 2009 to November 2010). Source Gulf of Maine Ocean Observing System Western Gulf of Maine buoy.
Figure 21: Six aquaria in the re-circulating seawater system in the University of New Hampshire cold room. Aquaria were used for both housing test animals and to for feeding trials.
Figure 22: The tank at the Coastal Marine Laboratory which was used for both housing test animals and for feeding trials. The feeding arena where feeding trials took place is visible including the foraging shelter and feeding panel.
Figure 23: Close-up of the foraging shelter and feeding panel which was placed in the tank during feeding trials at Coastal Marine Laboratory. Space where M. citrina have been removed by T. adspersus during a feeding trial is indicated, as well as remaining M. citrina.
IX Figure 24: The biofouling community established on the Coastal Marine Laboratory pier near the surface. Indicated are the anemone M. senile, the solitary tunicate C. intestinalis, the solitary tunicate M. citrina, and an Asterias species. Areas not indicated by arrows include more examples of these species as well as other sessile organisms (e.g. Haliclona spp.. Halichondria spp., B. violaceus, B. schlosseri. D. vexillum, D. listerianum, E. larynx, B. simplex, B. turrita, C. pallasiana, M. membranacea, A. simplex, A. aculeata, H. arctica, S. balanoides, Caprella spp., and J. marmorata).
Figure 25: The biofouling community established on the Coastal Marine Laboratory pier near the surface. Indicated are colonial tunicates of the genus Didemnum (e.g. D. albidum and D. vexillum). Areas not indicated by arrows include more examples of these species as well as other sessile organisms (e.g. Haliclona spp., Halichondria spp., M. citrina, C. intestinalis, B. violaceus, B. schlosseri, E. larynx, B. simplex, B. turrita, C. pallasiana, M. membranacea, A. simplex, A. aculeata, H. arctica, S. balanoides, M. senile, Caprella spp., and J. marmorata).
Figure 26: The biofouling community established on the Coastal Marine Laboratory pier adjacent to the benthos. Indicated are Didemnum species (e.g. D. albidum and D. vexillum). Areas not indicated by arrows include more examples of these species as well as other sessile organisms (e.g. Haliclona spp., Halichondria spp., B. violaceus, B. schlosseri, E. larynx, B. simplex, B. turrita, C. pallasiana, M. membranacea, A. simplex, A. aculeata, H. arctica, S. balanoides, M. senile, Caprella species, and J. marmorata).
Figure 27: The biofouling community established on the Coastal Marine Laboratory pier adjacent to the benthos. Indicated are examples of sponges of the genus Haliclona , as well as the colonial tunicates B. violaceus, D. cdbidum, and D. vexillum. In fact, the substrate is almost entirely covered by Didemnum species (e.g. D. albidum & D. vexillum)', the solitary tunicates M. citrina and C. intestinalis are not present.
Figure 28: Video still which shows the presence of T. adspersus in close proximity to the benthic panel community. Also indicated is the bucket beneath a fish access (FA) replicate.
Figure 29: Underwater video still showing the presence of C. irroratus and T. adspersus at the field site in proximity to benthic community panels.
Figure 30: The most abundant sessile invertebrates on predator excluded caged community panels.
Figure 31: Low abundance sessile invertebrates on caged predator excluded community panels.
x Figure 32: The most abundant sessile invertebrates on uncaged predator accessed community panels.
Figure 33: Low abundance sessile invertebrates on uncaged predator accessed community panels.
Figure 34: The percentage cover of the arborescent hydroid Ectopluera larynx. and the arborescent bryozoans Bugula turrita and Bugula simplex on caged and uncaged benthic community panels.
Figure 35: The percentage cover of Semibalanus balanoides on caged and uncaged benthic community panels.
Figure 36: The percentage cover of the encrusting bryozoans Membranipora membranacea and Cryptosula pallasiana on caged and uncaged benthic community panels.
Figure 37: The percentage cover oiJassa marmorata on caged and uncaged benthic community panels.
Figure 38: The percentage cover of Anomia aculeata and Anomia simplex on caged and uncaged benthic community panels.
Figure 39: The percentage cover of Porifera species (e.g. Halichondria spp. and Haliclona spp.) on caged and uncaged benthic community panels.
Figure 40: Percentage cover of Molgula citrina in uncaged predator access treatment replicates.
Figure 41: The settlement and growth of tunicate species in caged treatments over nineteen months of panel immersion.
Figure 42: The settlement and growth of tunicate species in uncaged treatments over nineteen months of panel immersion.
Figure 43: The settlement and growth of B. violaceus in caged and uncaged treatments over nineteen months of panel immersion.
Figure 44: The settlement and growth of B. schlosseri in caged and uncaged treatments over nineteen months of panel immersion.
Figure 45: The settlement and growth of Didemnum spp. in caged and uncaged treatments over nineteen months of panel immersion. Figure 46: The settlement and growth of Diplosoma listerianum in caged and uncaged treatments over nineteen months of panel immersion.
Figure 47: The settlement and growth of C. intestinalis in caged and uncaged treatments over nineteen months of panel immersion.
Figure 48: The settlement and growth of M. citrina in caged and uncaged treatments over nineteen months of panel immersion.
Figure 49: The pattern of growth over time of the average percentage cover of all tunicate species between caged (predator exclusion) and uncaged (predator inclusion) treatments.
Figure 51: The percentage cover of Molgula citrina in predator accessed (uncaged) and predator isolated (caged) treatments throughout nineteen months of panel immersion.
Figure 52: The frequency of occurrence of increases in percentage cover observed in tunicate species throughout nineteen months of panel immersion.
Figure 53: The percentage cover of tunicate species in caged and uncaged treatments at one month of panel immersion. M citrina is present in the caged treatments and is nearly (0.5% cover) absent from the uncaged treatments in the initial community.
Figure 54: The percentage cover of tunicate species in caged and uncaged treatments at nineteen months of panel immersion. M. citrina is present in the caged treatments and is completely absent from the uncaged treatments.
Figure 55: Percentage cover of B. violaceus in caged and uncaged treatments throughout nineteen months of panel immersion.
Figure 56: The percentage cover of M. citrina and B. violaceus on benthic community panels throughout nineteen months of panel immersion.
Figure 57: Changes in percentage cover of M. citrina and B. violaceus throughout June, July, and August of 2010.
Figure 58: The frequency of occurrence of increases in abundance (i.e. percentage cover) of colonial tunicate species in predator accessed (i.e. predator included) replicates and predator isolated (i.e. predator excluded) replicates.
Figure 59: The frequency of occurrence of increases in percentage cover of solitary species in communities accessed by predators (predator inclusion) and communities without predator access (predator exclusion).
Xll Figure 60: The final community (Nov. of 2010) on caged control panels after nineteen months of panel immersion.
Figure 61: The number of feeding trials with consumption of M. citrina (26) versus number of trials without consumption (11) which occurred throughout 37 feeding trials.
Figure 62: The average percent consumption of M. citrina from feeding trials conducted in both 2009 and 2010 (44%). Also indicated are the results from the 2009 (40%) and 2010 (67%) feeding trials. Figure 63: The number of feeding trials with consumption of M. citrina (23) versus the number of trials without consumption (26) which occurred throughout 49 feeding trials.
Figure 64: The average percent consumption of M. citrina by A. rubens in predator inclusion experiments.
Figure 65: The average percent of M. citrina consumed in the feeding trials by the predators A. rubens and T. adspersus.
Figure 66: Comparative percent frequency of trials in which M. citrina was consumed by the predators A. rubens and T. adspersus. ABSTRACT
Predation as a Vehicle to Aid Tunicate Invasion in the Biofouling Community
By Helen Day
University of New Hampshire, Sept. 2013
Competition for space can influence community dynamics in the sessile biofouling community. Within recent decades, community dynamics have shifted towards a community dominated by tunicates. This research proposed predation as a mechanism driving this shift.
In the G ulf of Maine, the non-native species Botrylloides violaceous became abundant when predators (i.e. the benthic fish Tautogolabrus adspersus and the sea star Aster ias rubens) removed the cryptogenic (i.e.native) tunicate Molgula citrina.
Moreover, B. violaceus was present in higher amounts in habitats with low
abundances o f M. citrina than it was in areas in which the two tunicate species were
were both abundant. Furthermore, laboratory feeding trials showed that abundant
local predators T. adspersus and A. rubens readily consumed large amounts o f M.
citrina.
xiv CHAPTER I
GENERAL INTRODUCTION
Introduced Species as an Ecosystem Stressor
Introduced species play a part in the increasing stress load for marine ecosystems, including their ability to remain resilient to the impacts of disturbance and further invasion (Walker, 1995: Steneck et. al., 2002: Stachowicz et. al., 2002: Levin et. al.,
2008). Introduced species can reduce native diversity (Elton, 1927: Hutchinson, 1959:
Naeem et. al., 1994: McKinney & Lockwood, 1999), extirpate native species from the local ecosystem (Preston, 1948: Drake et. al., 1989: Ross, 1991: Fritts 1998: Davis, 2003) and alter existing predator-prey dynamics which can reduce biodiversity (Rosenzweig &
Macarthur, 1963: Krebs, 1972: Pimm, 1989). In turn, lowered species diversity has been correlated with drops in ecosystem health and resilience (Ehlrich & Wilson, 1991:
Simberloff & Holle, 1999: Fischer et. al., 2007). Thus, the spread and control of introduced species can become a marker for the potential health and resilience of an ecosystem (Vitousek, 1989: Gordon, 1998: Folke et. al., 2004).
l Benthic Communities
In marine benthic zones, planktonic larvae settle on rocky bottom substrates to form a
benthic community (Pechenik, 1999: Witman & Dayton, 2001, i.e a complex collection
of organisms structured by a foundation of sessile invertebrates and algae (Mare, 1942:
Dayton, 1971: Dayton et. al., 1974: Sousa, 2001). In temperate ocean waters (e.g. the
Gulf of Maine) benthic community development can lead to algal forests (e.g. Saccharina
latissima, Agarum cribrosum, and Chondrus crispus) that are covered with encrusting
organisms (e.g. Membranipora membranacea, Obelia geniculata, and Electra pilosa;
Lubchenco & Menge, 1978: Berman et. al., 1992: Trussell et. al. 2002). Alternatively,
when mobile macroinvertebrate predators (e.g. green sea urchins Strongylocentrotus
droebachiensis) dramatically reduce kelp density an alternate stable state can arise
creating a benthic community which is dominated by crustose coralline algae and the
most abundant organisms are crustaceans, mollusks, and polychaetes (Witman 1985,
1987: Steneck, 1986: Ojeda & Dearborn, 1989).
Biofouling Communities
In the presence of anthropogenic structures (i.e. piers, floats, boats) planktonic larvae colonize man-made substrates forming a biofouling community (Sutherland & Karlson,
1977: Harris & Irons, 1982: Railkin, 2004: Osman & Whitlach, 2007). Anthropogenic structures can span different habitat areas in the ocean; extending from the surface down to the benthic zone (Halpem et. al., 2008). Thus, the potential is high for differences in community composition throughout biofouling communities (Long, 1970: Heideman &
George, 1981: Hart, 1995: Greene & Grizzle, 2007).
2 Near the surface, a community develops which is often dominated by mussels
(e.g. Mytilus edulis), sponges (e.g. Haliclona spp., Halichondria spp., and Leucosolenia spp.), anenomes (e.g. Metridium senile) and tunicates (e.g. colonials: Botrylloides violaceus, Botryllus schlosseri, and Didemnum spp.; solitaries: Ciona intestinalis,
Molgula citrina, and Styela ctava), and other bivalves (e.g. Anomia aculeata and Anomia simplex)-, while hydroids (e.g. Ectopleura spp.) can become seasonally abundant (Greene
& Grizzle, 2007).
In contrast to surface biofouling communities described above, community development on anthropogenic surfaces located near the bottom can lead to a community dominated by anenomes (e.g. Metridium spp.), sponges (e.g. Haliclona spp.,
Halichondria spp., and Leucosolenia spp.), and colonial tunicates (e.g. Botryllus schlosseri and Didemnum spp.), while bivalves (e.g. Mytilus edulis, Anomia aculeata, and Anomia simplex), barnacles (e.g. Semibalanus balanoides), and solitary tunicates
(e.g. Ciona intestinalis, Molgula manhattensis, and Molgula citrina) are consumed by benthic predators (Greene & Grizzle, 2007).
Biofouling community composition can differ from the surface to the bottom benthic community for several reasons: differences in environmental conditions (e.g.
U.V. radiation, temperature, salinity, and water flow) may alter community composition
(Sanders et. al., 1968: Officer et. al., 1982), surface orientation (e.g. horizontal versus vertical) can alter both predation pressure and settlement (Harris & Irons, 1982: Wendt et. al., 1989: Glasby, 2000: Glasby & Connell, 2001) and differential predation pressures may drive differences in community composition (Harris & Irons, 1982).
3 Predation and the Biofouling Community
Competition for space is the top interaction among sessile species (Dayton 1971; Jackson
1975; Paine 1984). Predators can alter community composition through the preferential removal of specific prey species (Lotka, 1920: Volterra, 1931: Menge, 1978: Bemstien et. al., 1981: Marsh, 1986). With a competing species removed from the system, the remaining species experience reduced interspecific competition for resources (Paine,
1969: Connell, 1983: Levin et. al., 1996: Chase et. al., 2002). Released from a facet of competition, some species flourish, and the community experiences alterations to abundance and diversity (Lubchenco, 1978: Menge, 1979: Sousa, 1979).
As habitat modifying species, predators have been shown to have a significant impact on the structure and ecology of marine coastal habitats (Paine, 1969: Menge et. al., 1994: Jones et. al., 1994: Carlton, 1996). Predators alter the habitat through several mechanisms: they can structure trophic food webs, create ecological niches, reduce habitat heterogeneity through the removal of foundation species, enhance the colonization success of non-native species, or drastically alter community composition
(Paine, 1969: Hutchinson, 1959: Jeffries, 1984: Dayton, 1971: Vance, 1979). The overall result of predation is one in which community dynamics are altered causing shifts in community composition and richness.
Predation pressures can create significant changes in community composition between surface biofouling communities and biofouling communities in the benthos (Greene & Grizzle, 2007). Benthic predators (e.g. the wrasse Tautogolabrus adspersus) may not forage in the surface zone (Olla & Bejeda, 1975: Green et. al., 1984: Auster,
1989), allowing some prey species to flourish and dominate surface biofouling communities. Differences in surface orientation between horizontal ocean bottom communities and vertical suspended anthropogenic surfaces may cause differences in foraging predators which can be attracted to surface orientation (Wendt et. al., 1989:
Connell & Glasby, 2000: Connell, 2000: Glasby & Connell, 2001: Greene & Grizzle,
2007). While suspended surfaces may not physically touch the ocean bottom, therefore preventing foraging benthic predators from accessing the biofouling community on suspended surfaces (Greene & Grizzle, 2007: Van Colen, 2008).
Disturbance can create open space such as when mobile predators remove organisms from the substrate (Vimstein, 1977: Carter et. al., 1985: Wilson, 1990). The impact of disturbance can vary from frequent and wide-spread, to infrequent and minimal with each regime having a different effect on the availability of open substrate space
(Miller, 1982: Malanson, 1984: Hubbell & Forster, 1986).
Predation has been a significant force altering the biodiversity and succession of tunicates in the biofouling community (Day & Harris, 2009). Predation alters tunicate community dynamics through the removal of native species as well as between solitary and colonial tunicates, thus opening space for non-native species to rapidly colonize through clonal reproduction and colony fusion (Rinkevich & Weissman, 1987: Carlton,
1996: Cohen & Carlton, 1998: Rinkevich & Shapira, 1992: Carver et. al., 2006).
5 Many benthic predators are bottom specific and do not forage in the pelagic zone or on suspended surfaces in the surface zone of the ocean, thus creating a predation refuge for sessile invertebrates in the biofouling community. In the benthic habitat,
Molgula species are eaten by a range of benthic predators including Tautogolabrus adspersus and Asterias rubens, thus shifting community dynamics to a system dominated by invasive species such as Botryllus schlosseri and Botrylloides violaceus (Osman &
Whitlach, 2004: Day& Harris, 2009). In contrast, in the biofouling community cryptogenic Molgula species may experience a refuge from predation pressure and can coexist with invasive colonial tunicate species; the resulting community has the potential to evolve with both colonial and solitary tunicates existing in a stable state dominated by competition and alternative forms of disturbance (Dijkstra et. al., 2007: Greene &
Grizzle, 2007: Sorte et. al., 2010).
Biofouling Community Development
In the biofouling community, available substrate space varies over time and is highly influenced by disturbance (e.g. predation) and the life histories of sessile community members (Dayton et. al., 1971: Connell, 1978: Sousa, 1979: Wilson, 1990: Dijkstra et. al., 2007). Space utilization alters as organisms settle, grow, reproduce, and senesce through a series of successional stages (Connell, 1961: Paine, 1969: Dayton, 1971:
Osman, 1977). In turn, the pattern of succession is determined by the life histories of the organisms, seasonal variations in habitat conditions, and disturbance (Stachowicz et. al.,
2002: Parmesan & Yohe, 2003: Parmesan et. al., 2005).
6 Initially, newly available hard substrate will be colonized by a biofilm of microscopic organisms such as bacteria, small phytoplankton, and other single celled organisms (Corpe, 1972: Marszalek & Gerchakov, 1979: Little & Wagner, 1997). The colonization of a microfilm is thought to facilitate the settlement of later settling species, by allowing an environment habitable for larger macro-organisms (Rowley, 1989: Little,
1997: Dobretsov, 2006: Wahl, 2008). Over time, the community experiences an increase in species richness and diversity, as other invertebrate species be.g.in to settle and grow; community members then either remain, are removed by predation and disturbance, or
naturally senesce (Little & Wagner, 1997: Railkin, 2004: Wahl, 2008).
Following colonization by a biofilm, the substrate begins to be settled by hydroid
species (e.g. Ectopleura larynx and Obelia spp.) and bryozoan species (e.g. Bugula simplex and Bugula turrita). Hydroids and bryozoans flourish and grow to form large
arborescent structures which dominate the substrate (Wahl, 2008). Predators of E. larynx
(e.g. Flabellina spp. and Dendronotus frondosus) rapidly reduce much of the cover of
hydroids, while Bugula spp. remain on the substrate as there are not many predators of
Bugula spp. in the Gulf of Maine (Miller, 1961). However, bryozoans will senesce in the
winter as water temperature drops. The biofilm also allows for a layer of sediment and
detritus to build up, creating habitat for tube-dwelling amphipods (e.g. J. marmorata) and
other small sessile detritivores; these small tube-dwelling organisms can then create
extensive aggregations of sedimentary tubes which cover much of the surface particularly
on upper surfaces (Conradi et. al., 1997).
Juvenile barnacles (e.g. Semibalanus balanoides) settle early in community
development and increase to cover much of the substrate (Little & Wagner, 1997: Railkin, 2004: Wahl, 2008). The cover of S. balanoides is then reduced as the barnacles are rapidly consumed by predatory seastars such as Asterias rubens (Barnes et. al., 1951:
Crisp et. al., 1970: Dare, 1982). S. balanoides settles in the late spring (Silliman &
Bertness, 2002). Later in the spring more species of barnacles will settle (e.g. Balanus improvisus, Balanus ebumeus, and Balanus crenatus) making barnacles a common and persistent member of the biofouling community. Early in community development sponges begin to settle and increase in size (e.g. Haliclona spp., Halichondria spp., and
Leucosolenia spp.); and while largely unpalatable to many predators, sponges are consumed by some specialized predators such as the seastar Henricia sanguinolenta
(Lippert & Iken, 2003: Peters et. al., 2009). Due to their unpalatable body structure (e.g. spicules) sponges often remain in the community; making both sponges and barnacles persistent members of the community.
Historically, the climax community is then settled and dominated by larger bivalves such as Mytilus edulis (Lubchenco, 1978: Little & Wagner, 1997: Railkin, 2004:
Wahl, 2008). In the Gulf of Maine the biofouling climax community has been shown to be dominated by the blue mussel Mytilus edulis with the amphipods J. marmorata and
Caprella spp. being present in high numbers as well as the mollusks Hiatella arctica and
Anomia spp. (Berman et. al., 1992: Greene & Grizzle, 2007). Encrusting bryozoans are common in the biofouling community, including Membranipora membranacea, Electra pilosa, and Cryptosula palliasana (et. al., 2007). However, in recent decades the climax community has begun to be dominated by tunicates (Lambert & Lambert, 2003: Lambert,
2005: Dijkstra et. al., 2007: Greene & Grizzle, 2007).
Tunicates in the Biofouling Community Within recent decades, alterations to succession in the biofouling community have created a shift towards a climax community dominated by tunicates (Lambert & Lambert,
2003: Lambert, 2005: Dijkstra et. al., 2007: Greene & Grizzle, 2007: Sorte et. al., 2010).
Many of the most abundant tunicate species in the biofouling community are non-native colonial species which serve to reduce available substrate space for native species
(Osman & Whitlach, 1995: Lambert, 2001: Dijkstra et. al., 2007). Ascidian biology and ecology is highly variable amongst the genera of tunicates, but often serves to determine the amount of substrate space colonized in initial community development, the ability to hold and maintain that space, as well removal from the substrate by predators which may preferentially consume some species and not others (Yamaguchi, 1975: Stachowicz et. al., 2002: Peterson, 2005).
In the early successional stages of community development, colonial organisms can reproduce asexually (e.g. Botrylloides violaceus, Botryllus schlosseri, Didemnum spp., and Diplosoma listerianum), thus allowing them to rapidly acquire available substrate space following initial colonization (Yamaguchi, 1975: Jackson, 1977: Sousa,
1979: Dean & Hurd, 1980: Stachowicz et. al., 2002). In contrast, solitary tunicates (e.g.
Molgula citrina and Ciona intestinalis) must reproduce sexually and produce new larval individuals which will then settle and hold substrate space (Dean & Hurd, 1980: Greene
& Schoener, 1982: Greene et. al., 1983). Furthermore, predation on colonial and solitary tunicates is drastically different (Greene & Schoener, 1982; Quinn, 1982). Predation events on colonial animals are not always fatal, as a colonial species can recover if a portion of the colony has been consumed (Jackson, 1977; Kopachena, 1991). In contrast, predation on a solitary individual is most often fatal; and in solitary species the
9 survivorship of the solitary tunicate population is reliant on the reproduction of remaining individuals (Jackson, 1977: Greene & Schoener, 1982).
Once colonial invasive species have colonized a substrate, they may then be resistant to the common predators associated with the habitat; this refuge from predation can allow invasive species to sustain a hold on the substrate while native species are removed (Reusch, 1998: Crooks, 1999: Keane, 2002). As a result, the biofouling community has experienced a decline in native species and has arisen as a major vector in the spread of invasive species which carry negative economic and environmental consequences (Minchin & Gollasch, 2003: Forrest, 2007: Davidson et. al., 2009: Hewitt et. al., 2009: Locke et. al., 2009).
Tunicates common in the intertidal benthos and biofouling communities in the
Gulf of Maine include both native and invasive species. Solitary species include the cryptogenic Molgula species and the invasive Ciona intestinalis.
Molsula citrina andMoleula manhattensis
Molgula species are small round solitary tunicates, commonly called “sea-grapes”.
Although solitary, they are often found in dense aggregations covering large areas of substrate space. Molgula spp. can also utilize secondary substrate space, growing as epibionts on algae and arborescent bryozoans. Molguloid species in the Gulf of Maine biofouling communities include Molgula citrina and Molgula manhattensis (Lambert,
2003: Arenas et. al., 2006: Lutz-Collins et. al., 2009). Molgula manhattensis is not native to the Gulf of Maine and has been found in the area since 1977 (Linkletter, 1977).
Molgula citrina is classified as a “cryptogenic” species since it is not definitively known
10 to be native, but has been found in the Gulf of Maine since 1878 (Sumner et. al., 1913).
Predators will consume Molgula species, drastically reducing the amount of substrate space occupied by this tunicate. Predators found in the Gulf of Maine which will consume Molgula species include fish (e.g. Tautogolabrus adspersus, Paralichthys dentatus, and Pseudopleuronectes americanus), snails (e.g. Mitrella spp.), seastars (e.g.
Asterias rubens, Asterias forbesi and Henricia sanguinolenta) crabs (e.g. Carcinus maenas and Cancer irroratus) and lobster (e.g. Homarus americanus) (Osman &
Whitlach, 2004: Dijkstra et. al., 2007: Osman & Whitlach, 2009: Day and Harris, 2009).
Ciorta intestinalis
Ciona intestinalis is a solitary species first reported in the Gulf of Maine in 1940 (Gosner,
1971: Lambert, 2003: Lambert, 2006: Therriault & Herburg, 2008: Ramsey, 2009).
Individuals of the species can grow rather large in comparison to other solitary tunicate species (up to 14 cm in length). Furthermore, individuals often associate together, creating large aggregations of Ciona species which can occupy significant areas of space on the substrate (Lambert, 2003: Blum et. al., 2007: Cohen et. al., 2008: Locke, 2009).
However, when aggregations become too heavy they can slough off the substrate
(Stachowicz & Whitlach, 1999: Stachowicz et. al., 2002: Edwards & Leung, 2008).
Crustacean and gastropod predation can also reduce the amount of substrate space occupied by Ciona species (Osman & Whitlach, 2004: Dijkstra et. al., 2007).
Colonial species of tunicates are more diverse than solitary species in the Gulf of
Maine. Colonial species include the invasive species Botryllus sch/osseri, Botrylloides
11 violaceus, Diplosoma listerianum, and Didemnum vexillum while Didemnum albidum is a native colonial species.
Botrvlius schlosseri
BotryUus schlosseri (i.e. the “star-tunicate”) is a colonial species characterized by a flattened encrusting morphology with zooids arranged in a star shaped pattern in the body matrix. It is considered a non-native species and has been present in the Gulf of Maine since the 1800’s (Millar, 1952: Lambert, 2001: Dijkstra et. al., 2007: Ramsey et. al.,
2009). Individual colonies of Botryllus schlosseri do not grow larger than a few centimeters in diameter, making their footprint on the substrate small in size (Lambert,
2005: Carver et. al., 2006). However, Botryllus schlosseri colonies can utilize both primary substrate by growing directly on vertical hard surfaces, as well as secondary substrate as epibionts on algae and mussels. Botryllus schlosseri go through one reproductive cycle with a life span of 82-247 days and then regress; thus limiting their temporal presence on the substrate (Chadwick-Furman & Weissman, 1995: Rinkevich et. al., 1992). Removal of adult colonies by predatory gastropods and crustaceans can also reduce colony size (Osman & Whitlach, 2004: Cohen et. al., 2005).
Botrylloides violaceus
Botrylloides violaceus is a colonial species similar in morphology to Botryllus schlosseri but differentiated by an irregular pattern of zooids in the body matrix. It is considered non-native, and was first reported in the area in the early 1980’s (Berman et. al., 1992:
Lambert, 2001: Ramsey et. al., 2008). Colonies of Botrylloides violaceus can grow to a meter in diameter. Adjoining colonies can fuse, forming large chimeric colonies which
12 take up a large footprint of substrate space (Lambert, 2001: Carver et. al., 2006). As a result, Botrylloides violaceus is the most conspicuous colonial tunicate on marine hard surfaces. However, like Botryllus schlosseri, Botrylloides violaceus also goes through one reproductive cycle followed by senescence; thus limiting its temporal presence in the community. Removal of adult colonies by predatory seastars such as Henricia sanguinolenta can also reduce colony size (Dijkstra et. al., 2007).
Diplosoma listerianum
Diplosoma listerianum is a colonial species characterized by a- loosely organized colony of zooids encased in a gelatinous matrix. It is a non-native species first found at the Isle of Shoals in 1993 (Harris & Tyrrell, 2001: Lambert, 2001: Ramsey et. al., 2008). Due to the loose organization of zooids within the matrix, this tunicate can grow extremely rapidly in size, allowing it to quickly acquire and dominate substrate space. Diplosoma listerianum can also utilize secondary substrate space by overgrowing algae. The blood star Henricia sanguinolenta as well as small marine gastropods such as Mitrella spp. feed on Diplosoma listerianum, reducing the colony size on the substrate (Osman & Whitlach,
2004: Dijkstra et. al., 2007).
Didemnum species
The colonial Didemnum albidum is native to the Gulf of Maine. The invasive Didemnum vexillum (i.e. “the pancake batter tunicate”) is a colonial species first discovered in the
Gulf of Maine in 2000 (Lambert, 2001: Dijkstra et. al., 2007: Bullard et. al., 2007:
Valentine et. al., 2007: Ramsey et. al., 2008). D. albidum colonies often remain small in size (Valentine et. al., 2007), butD. vexillum tunicate colonies grow rapidly, and can
IB cover vast areas of substrate space - on Georges Bank rocky bottom substrate this species has been found to cover areas upwards of kilometers (Valentine et. al., 2007: Bullard et. al., 2007). Like other colonial species, this tunicate appears to periodically regress. In the
Gulf of Maine, colonies appear to be dormant in the winter and will resume growth the following summer. Predatory seastars such as Henricia sanguinolenta as well as small marine gastropod snails can consume Didemnum species, thus reducing colony size on the substrate (Dijkstra et. al., 2007: Osman & Whitlach, 2007).
Study Objectives
Previus researchers have found that predation can alter successional development with the presence or absence of different predators influencing the development of differences in species richness and abundance between communities (Clements, 1916: Paine, 1969:
Connell, 1978: Sousa, 1979: McCook et. al., 1991). Many previous ecological studies focused on naturally occurring intertidal systems (e.g. the rocky intertidal) and predator and prey interactions among organisms which were once commonly abundant (e.g. local species of barnacles and mussels and their seastar and gastropod predators: Paine, 1978:
Menge, 1978) But in recent decades anthropogenically derived systems (i.e. the biofouling community) are increasing in both scope and impact (Vitousek et. al., 1989:
Orth et. al., 2006: Halpem et. al., 2008); and once commonly abundant native species are being supplanted by newly dominant species and their associated predators (Huxel, 1999:
Mooney & Cleland, 2001: Beyers, 2002: Gurevitch & Padilla, 2004). Within recent decades, anthropogenic impacts on marine ecosystems (Larkin, 1996: Castilla, 1999:
Worm et. al., 2006: Halpem et. al., 2008) and the spread of introduced biofouling species have begun to be studied by ecologists (Lambert & Lambert, 2003: Dijkstra et. al., 2007: Greene & Grizzle, 2007: Sorte et. al., 2010). As such, this study was designed to focus on the benthic biofouling community and to assess to what degree predation can alter community succession with particular emphasis on tunicate species. Acrylic (i.e. plexi glass) panels were deployed in the benthic community of coastal Portsmouth Harbor in
Newcastle New Hampshire and the growth of the biofouling community was catalogued and characterized throughout 18 months of panel immersion with emphasis on changes in percentage cover of the tunicate species on the available substrate. For the assessment of predators of Mcitrina, this study focused on two abundant predators, the benthic predatory fish Tautogolabrus adspersus (Cunner) as well as the local seastar species
Asterias rubens.
The objectives of this study were twofold:
1) To study the impact of predation in the benthic biofouling
community on tunicate community dynamics such as succession
and biodiversity. For the assessment of predators of all tunicates,
this study utilized two predator access manipulations: total
exclusion (suspended caged treatments) and reduced access
(suspended uncaged treatments).
2) To study the potential role of two common predators on Molgula
citrina. This study focused on two abundant predators, the benthic
predatory fish T. adspersus (Cunner) as well as the local seastar
species A. rubens.
15 METHODS
Introduction
This study took place from May of 2009 to November of 2010 at the University of New
Hampshire (UNH) in Durham New Hampshire, as well as the UNH Coastal Marine
Laboratory (CML) and the UNH CML pier located at the mouth of the Portsmouth
Harbor in the Gulf of Maine (Figure 1). Nineteen months of panel studies with manipulated predator access were undertaken in order to assess biofouling community dynamics under varying levels of predation with emphasis on the development of the
Ascidian (tunicate) assemblage. Complementary laboratory studies were designed to identify predators (i.e. Tautogolabrus adspersus and Asterias rubens) of Molgula citrina as it was the sessile organism that was most impacted by predator exclusion in field studies.
'igure I: Ariai photograph of the University o f New Hampshire Coastal ’arine Laboratory pier where the enthic community panels were located, hoto courtesy o f The University of Panel Experiment Location ew Hampshire.
The Gulf of Maine is located at the northern end of the New England coast
(Figure 2). This body of water is characterized by high levels of dissolved oxygen from
16 arctic currents as well as nutrient enrichment from coastal rivers (Bigelow, 1927: Garrett
& Keeley, 1978: Brooks et. al., 1989). In turn, bottom features enhance tidal movements thereby increasing water circulation and allowing for greater oxygen and nutrient availability throughout various oceanic zones (Bigelow, 1927: Garrett & Keeley, 1978:
Brooks et. al., 1989). Therefore, organisms which inhabit the Gulf of Maine find a nutrient rich habitat in which to thrive (Brooks et. al., 1989: Berman et. al., 1992:
Grosholz, 2002). However, cold waters and a prevailing self-contained counterclockwise current act can as dispersal barriers and can inhibit invading organisms (Crowell, 1986:
Grosholz, 2002: Freeman, 2006).
Figure 2: The location o f the Gulf of Maine (GoM) on the eastern seaboard. The line o f separation which can serve as a barrier to dispersal for planktonic larvae is seen in the divide between warmer southern yellow/orange waters and colder arctic blue/green waters. Figure courtesy of National Oceanographic and A tmospheric Administration (NOAA).
Experimental Field Manipulation: Benthic Panel Community
In May of 2009, fifteen 10-centimeter square acrylic panels were deployed in the subtidal at University of New Hampshire Coastal Marine Lab pier (Figures 1 & 3).
17 P i s H n p t Figure 3: Image taken on the floating aiThffi (• tbt dock at the University o f New Hampshire Coastal Marine Laboratory pier. In frame are the fifteen hanging ropes which are affixed to the 15 panel replicates suspended in the benthic subtidal.
Replicates were located on the open ocean side of the pier and spaced I meter apart
(Figures 1 & 3). Panels were suspended vertically by ropes which were tied on one end to a cinder block and on the other end to the stationary pier (Figures 3 & 4). Panels were affixed 30 cm above the 20 cm cinder block, giving a combined height of 50 cm from the ocean bottom (Figure 4).
18 Figure 4: Underwater image illustrating the layout of a no predator access (NA) replicate and its orientation in the Kfe [DRSdMrp water column in relation to the ocean bottom (benthic substrate). A single caged replicate is seen in the 0 3 3 ^ ((iKA foreground, while a second and third replicate are seen in the background. Video image still from a video courtesy o f Dr. James Haney.
Hx©'iufr; artiSG M E
Placement of replicates along the pier was folly randomized in order to distribute possible differences in habitat between replicates (Table I).
Illustration of the Distribution of Treatment Replicates across the pier NA FA FA AA NA AA FA AA FANA FA NA AAAANA Table I: The distribution o f replicates (all predator access, AA: fish access. FA; no predator access, NA) along the pier, the replicate closest to the shore is on the left. Benthic Panel Community: Treatments
Three treatment types characterized by level of predator access were deployed with five replicates per treatment and a single panel per replicate (Table 2). Replicate panels were independent in order to reduce the impact of neighboring panels on community growth and to eliminate variations in water flow between interior and edge panels.
The all predator access (AA) treatment was designed to assess the state of the community whilst it was under the influence of predation. Each replicate had a single
19 panel affixed to a wire mesh support thus allowing the panel to remain uncaged and accessible to predators which included fish, sea stars, and crabs (Figure 5).
The fish access (FA) treatment was designed to allow the access of swimming predators (e.g. fish) while other predators (e.g. sea stars, crabs, and lobsters) were prevented access. Each replicate had a single panel affixed to a wire mesh support; beneath the panel was an upside-down bucket which prevented benthic predators from climbing up the rope in order to gain access to the panel (Figure 28). However, it was possible that crabs (e.g. Carcinus maenas) which can swim short distances may have accessed the panels. Thus, when the panels were brought uOp for photography (i.e. every two weeks), predators found on the panels were removed.
The no predator access treatment (NA) was designed to assess the community which developed in the absence of large (<1 cm) predators. Panels were enclosed in complete caging which was constructed from wire mesh of 1 cm in the widest diagonal
(Figure 6). Overall, the no predator access treatment was designed to exclude adults of the fish species Tautogolabrus adspersus, which was a focus animal in this study.
Figure 5: Example o f an uncaged all predator access (AA) replicate. The replicate has been removedfrom the water in order to photograph the panel for data collection.
\ .
beitfck t w witti' reaiS$»4 fVolfiwsi- dittcoffettiM ! phiHo*™pS> (Acrarrtii 2s Maatkly)
20 Figure 6: Example o f a caged no predator access (NA) replicate. The replicate has been removed from the water and the mesh side opposing the panel has been opened in order to photograph the panel for data collection.
Benthic Panel Treatment Types Treatment Title Number o f Treatment Details replicates No predator access (NA) 5 Caged treatment, no predators over 1cm in size can access the community. Fish access (FA) 5 Partially caged treatment, only predators which can swim (e.g. fish) can access the community. All predator access 5 Uncaged treatment, all predators can access the (AA) community. Cage Control 3 Partially caged treatment designed to assess the impact of caging on community development. Table 2: List o f treatment types in the experimental field manipulation involving community panels with various levels o f predator access. Methodological studies on caging experiments have shown that caging can
increase sediment deposition and alter water flow to the animals living on the panel
(Hulberg & Oliver, 1980: Steele, 1996, 1999: Millar & Gaylord, 2007). Thus, in caged
replicates the mesh size was small enough to reduce predator access, but large enough to
minimize impacts to water flow to the panels (Hulberg & Oliver, 1980: Steele, 1996,
1999: Millar & Gaylord, 2007). As a result, several predators were small enough to fit
through the cage mesh and were sometimes found within the cages, these included: small
sized (2-6 inch) juveniles of the fish species T. adspersus and Cyclopterus lumpus, the
nudibranch Flabellina verrucosa, as well as the seastars Asterias rubens and Asterias forbesi (2-6 inch). The crabs Carcinus maenas and Hemigrapsus sanguineus were also
occasionally able to pry open the cage. When panels were brought up for photography
21 (i.e. every two weeks), predators found inside the cages were removed.
Not only can caging impact water flow, but methodological studies have also shown that caging can deter or attract predators (Hulberg & Oliver, 1980: Steele, 1996,
1999: Millar & Gaylord, 2007). Therefore, the field experiment included a cage control which was designed to assess the impact of caging on both water flow and predator behavior. There were three cage control replicates constructed with partial caging; each replicate had a wire mesh top and bottom, one wire mesh side with an affixed community panel, and two strips of wire mesh on the side opposing the panel (Figure 7). The sides of the cage controls remained open in order to allow water flow to the panels and to allow predators to access to the panels. Cage controls were photographed in May, 2009,
November, 2009, May, 2010, and November, 2010.
Figure 7: Example o f the partial siding found in the cage control replicates.
Benthic Panel Community: Data Collection
Underwater video was taken at the Coastal Marine Laboratory pier in order to assess the community of organisms living on the pier pilings and floating docks, as well as to identify some of the predators present at the field site. In order to do so, a video camera inside a water-tight housing was used to pan across the habitats at the field site.
Underwater video occurred in October, 2010, May, 2010, and August, 2010. Video stills were isolated and used to assess the community both at the surface and adjacent to the benthos. Video recording was also used to assess predation on panels. To do so, a video camera inside a water-tight housing was submerged in the subtidal and trained on the benthic panel community for two hours. Underwater video occurred every two weeks from May to August of 2010: one hour of video was taking during daylight hours on eight occasions, giving eight hours of video total.
In order to assess sessile species abundances in the benthic panel community, panel replicates were photographed every two weeks from May to November of 2009, every four weeks from December of 2009 to April of 2010, and every two weeks from
May to November of 2010, resulting in thirty-three sampling points. Prior to photography, 15 replicates affixed to ropes were pulled from the subtidal at the Coastal
Marine Laboratory pier (Figures 3 & 4). Photographs were taken out of water because it was found that, in photographs of submersed panels, smaller sessile organisms were obscured by a canopy of arborescent species (i.e. hydroids and bryozoans). Panels were photographed with the panel in complete image, as well as in sections. Complete panel images were used for quantitative analysis, while detailed subset sections were used for referral of organismal identity. Photographs were taken with a Canon digital SLR (EOS
RebelXT) using direct sunlight as a lighting source.
Uncaged replicates from the all predator access (AA) and fish access (FA) treatments remained affixed to the wire mesh support during photography (Figure 5).
Caged replicates from the no predator access (NA) treatment had a hinged side which could be opened, thus allowing the panel to remain on the wire mesh during photography
(Figure 6). Overall, panels were out of the water for one to two hours for photography.
23 However, disturbance controls were designed to infer possible impacts of removal from the water on experimental panels. As a result, disturbance control panels remained largely undisturbed and were pulled from the water twice for photography (i.e. November of
2009 and November of 2010).
Benthic Panel Community: Sessile Invertebrates
Photographs were used to assess the amount of panel space occupied by sessile invertebrates (i.e. the percentage cover). Tunicates were identified using tunicate guides
(Alder, et. al., 1907: Millar, 1952, 1971: Brunetti et. al., 1988: Bullard et. al., 2007:
Lambert, 2005: Carver et. al., 2006: Daniel & Therriault, 2007: Satoh, 2009: Nishida et. al., 2010). While other sessile organisms were identified using markers from invertebrate identification guides (Gosner, 1971: Pollock, 1998).
In photographs, the solitary tunicate Molgula citrina had a globular body structure which often contained a visible orange oviduct, while the apical surface had two feeding siphons with irregular apertures distinguished by four lobes on the atrial siphon and six on the oral (Figure 8; Lambert, 2005: Satoh, 2009). In contrast, the solitary Ciona intestinalis was a vase-shaped tunicate with yellow markings around the siphon apertures and a clear tunic through which the gut loop was visible (Figure 9; Millar, 1971).
24 Figure 8: Example o f the solitary tunicate M. Figure 9: Example o f the solitary tunicate C. citrina on a benthic community panel. intestinalis on a benthic community panel.
The colonial species B. violaceus and B. schlosseri grew as gelatinous orange, red, brown, or purple sheets (Figures 10 & 11: Carver et. al., 2006). B. violaceus had zooids positioned randomly within the body matrix (Figure 10). While B. schlosseri had zooids which were grouped into star-shaped clusters (Figure 11).
Figure 10: Example of the colonial tunicate Figure 11: Example of the colonial tunicate B. violaceus on a benthic community panel. B. schlosseri on a benthic community panel. The colonial tunicates Didemnum vexillum and Didemnum albidum grew as light-colored, globular, gelatinous sheets (Figure 12; Bullard et. al., 2007: Daniel & Therriault, 2007).
However, Didemnum species were difficult to distinguish and thus were grouped by genus. The colonial species D. listerianum was not commonly found in this study, but was distinguished as flat gray sheets with small, nearly indistinguishable zooids (Brunetti
25 et. al., 1988).
Figure 12: Example o f the colonial tunicate Didemnum spp. on a benthic community panel. Sponges of the genus Haliclona were identifiable as porous encrusting structures found
on both the substrate and other sessile species, while sponges of the genus Halichondria
grew as porous tubular structures (Figure 13: Gosner, 1971).
Figure 13: Example o f the dominant types o f sponges on benthic community panels: left is most likely Halichondria spp. white right is most likely Haliclona spp. Anomia simplex and Anomia aculeata were affixed to the substrate by their ventral valve
and were identifiable by their visible dorsal valve. Under microscopic analysis, the shell
of Anomia simplex had a smooth surface while Anomia aculeata had a roughened surface
(Figure 14: Gosner, 1971). The arthropod Semibalanus balanoides was identifiable by its
light-colored pyramidal-shaped shell (Figure 15: Gosner, 1971).
26 Indications o f removed Semibalanus balanoides
Semibalanus balanoides
Figure 14: Example o f Anomia species (most Figure 15: Example o f the barnacle S. balanoides on likely A. simplex) on a benthic community a benthic community panel: also indicated are panel. remains o f removed S. balanoides. The encrusting bryozoans Membranipora membranacea and Cryptosula pallasiana grew as flat, light-colored sheets with a regular pattern of box-shaped zooids; while the arborescent bryozoans Bugula turrita and Bugula simplex grew as upright branching structures (Figure 16: Gosner, 1971).
Figure 16: Example o f arborescent Bryozoans (left: most likely Bugula spp.) and encrusting Bryozoans (right: most likely M. membranacea or C. pallasiana) on a benthic community panel. The hydroid Ectopleura larynx grew as upright branching structures with pink-colored polyps at the ends of the stolons (Figure 17: Gosner, 1971). The bivalve Mytilus edulis was not found on experimental panels but was identified as blue-colored and oblong while the bivalve Hiatella arctica was light-colored and oval (Figure 18: Gosner, 1971).
27 Figure 17: Example o f the hydroid Ectopluera larynx on Figure 18: Example of the bivalve a benthic community panel. Hiatella arctica on a benthic community panel. The tube-dwelling amphipod Jassa marmorata was a small segmented organism identified in microscopic analysis by extensive tubing structures constructed from mud and detritus. In microscopic analysis, segmented amphipods of the genus Caprella were observed clinging to other sessile species. The anemone Metridium senile was not found on community panels but was identifiable by its flexible tubular body crowned by tentacles on the oral surface.
Benthic Panel Community:
Identification and Quantification of Molguloid Tunicates
Dissection and microscopic analysis of the panel community were used to differentiate between Molgula citrina and Molgula manhattensis, as well as to quantify the number of
M. citrina found on benthic community panels.
Specimens of Molguloid tunicates were dissected in order to identify the species present at the field site. In 2010, specimens were collected and dissected once a month during months of peak Molgula citrina abundance (i.e. July, August, and September) with three dissections total. Since specimen removal would have disturbed the panel
28 community, samples were not collected from the panels. Instead, Molguloid samples were collected from the wire mesh cages and the ropes attached to the cages. Specimens sized 2-4 centimeters were collected and immersed in a cooler of seawater for transportation to the University of New Hampshire for fixation and dissection.
In order to prevent obscuration of anatomical features by body contraction during fixation, tunicates were relaxed with menthol crystals prior to fixation. Relaxation was performed by placing M. citrina in a plastic bag containing seawater and several dissolved menthol crystals for approximately 2 hours. When siphons were not responsive to touch (i.e. did not contract) the animals were immediately removed and fixed with a
95% ethanol and 5% seawater solution.
Molguloid tunicates were identified as M. citrina due to the following anatomical features: siphons were separated by one siphon width, the oral siphon had six lobes, the atrial siphon had four lobes, the brachial basket had seven folds, there was the presence of spiral stigmata, and there was the absence of the renal sack in M. citrina while M. manhattensis had a renal sack on the right side below the gonad (Lambert, 2005: Satoh,
2009). Tunicates were identified as M. citrina while M manhattensis did not appear to be present in area of this study.
Microscopic analysis was used to identify the species of Molguloid tunicates found on community panels as well as quantify the number of individuals. In 2010, the analysis took place during the months of peak M citrina cover which had been observed in 2009 (i.e. June, July, and August). In 2010, caged and uncaged treatments were pulled from the water at the Coastal Marine Laboratory pier and fully immersed in a cooler of seawater. The cooler was then transported to the adjoining Coastal Marine Laboratory.
29 Panels were removed from the wire mesh support and/or cage and assessed under the microscope while immersed in seawater. After being examined under the microscope the panels were placed on their mesh supports or cages and re-immersed at the field site.
Both M. citrina and M. manhattensis were similar in outer morphology. However,
M. citrina and M. manhattensis were differentiated by anatomical features. Firstly, M. citrina had siphons which were separated by a space the width of one siphon, while M manhattensis had no gap between the siphons (Lambert, 2005: Satoh, 2009). Secondly,
M. citrina had a visible oviduct, while M. manhattensis had no oviduct (Lambert, 2005:
Satoh, 2009). In this study, M. manhattensis were not observed on panels. This finding corroborated with historical records of species habitat range, as M. citrina has been found in the Gulf of Maine where the field site was located, while M. manhattensis has commonly been found south of Cape Cod (Alder et. al., 1907: Lambert et. al., 2010).
During microscopic analysis of the panels, tunicates which were unreactive to stimuli were not included in quantification, as siphon retraction and tunic contraction were indicative of a living organism. The ability to correctly identify Molguloid species and to quantify the number of individuals was sometimes obscured by a covering of epibionts
(e.g. colonial tunicates). In these cases, Molguloid species were discounted. In contrast, microscopic epibionts were common but did not obscure the identifying features of M. citrina, or prevent the quantification of individuals. Thus, several small epibionts (e.g.
Membranipora membranacea, Crvptosula pallasiana, and Obelia geniculata) were commonly found on the surface of M. citrina but were not cause for M. citrina to be eliminated.
30 Benthic Panel Community: Quantification
The image analysis program ImageJ was used to quantify the percentage cover occupied by sessile species (Rasaband, 2009). In order to assess the percentage cover, the area of the panel was divided by the area occupied by all individuals of a genera and species. The area of the panel was calculated by delineating the outer panel edges with the pentagon selection ’ tool and using the ‘analyze measure ’ feature to obtain the number of pixels located within the selected area (Rasaband, 2009). To calculate the area occupied by all individuals of a genus and species (e.g. M. citrina), the boundaries of all of the M. citrina on the panel were selected using the ‘wand selection ’ tool followed by the ‘analyze measure’ tool to obtain the number of pixels located within the selected area (Figure 19:
Rasaband, 2009). The area of M. citrina was then compared to the area of the panel as a ratio (i.e. area M. citrina divided by area panel); this proportion was then converted into a percentage cover by multiplying the given value by 100 percent.
Objects too small to identify could not be entered into calculations due to the lack of identifying features available in photographs. Photographs were analyzed using the area of the image and the area of the M. citrina on an Advanced Configuration and Power
Interface (ACPI) x86-based PC computer, with a screen size of 39.1 cm, and screen pixel dimension of 1280x800.
31 Figure 19: Example of M. citrina on a benthic community panel. The panel edges have been delineated and M. citrina have been isolated from other biofouling community members for the purpose of calculations o f percentage cover.
Benthic Panel Community: Statistical Analysis
Photographs of sessile invertebrates on fifteen benthic community panels were used to assess the percentage cover of sessile invertebrate species. Resultant percentage cover calculations were primarily used to analyze the differential impact of predation between three treatment types (i.e. all predator access, fish access, and no predator access).
Throughout 19 months of panel immersion, photographs were taken every 2 weeks from May to November and every month from November to May, giving a total of thirty-three data points. The percentage cover of each tunicate species (i.e. M. citrina, C. intestinalis, B. schlosseri, B. violaceus, or D. listerianum) from bimonthly time points
(i.e. every 2 weeks) were averaged amongst replicates for each treatment type.
Didemnum vexillum and Didemnum albidum were grouped together by genus (i.e.
Didemnum spp.) and treated identically. The percentage cover of non-tunicate sessile invertebrates (i.e. Ectopleura larynx, Bugula simplex, Bugula turrita, Cryptosu/a pallasiana, Membranipora membranacea, Anomia simplex, Anomia aculeata, Hiatella arctica, Haliclona spp. or Halichondria spp.) from quarterly time points (i.e. every three months) were averaged amongst replicates for each treatment type. Sessile organisms
32 were grouped together based on high commonality of ecological niche. Resultant groupings included: the arborescent hydroid E. larynx with the arborescent bryozoans B. simplex and B. turrita, the encrusting bryozoan C. pallasiana with the encrusting bryozoan M. membranacea, the bivalve H. arctica with bivalves of the genus Anomia
(i.e. A. simplex and A. aculeata), and sponges of the genera Haliclona and Halichondria grouped together. The average percentage cover was then used to compare treatments in order to determine the impact of predation on sessile species.
There were no significant differences the percentage cover of sessile species between all predator access (AA) and fish access (FA) treatments (T-Test p>0.05: Table
3). Moreover, as non-fish predators (i.e. seastars and crabs) had been found on fish access panels, the fish access (FA) treatment did not completely isolate fish predation. Thus, the
FA treatment was combined with the all access (AA) treatment into an “uncaged” treatment. The variation amongst replicates for caged and uncaged treatments did not fit a normal distribution, so non-parametric statistics were used with a Kruskal-Wallis ranked group test which is a form of an analysis of variance (ANOVA).
Calculations of percentage cover were also used to analyze the community in variety of ways which included: a quantification of the impact of disturbance events on the tunicate M. citrina, a comparison of the patterns of growth of tunicate species between treatment types, a quantification of the frequency of occurrences of positive increases in percentage cover in tunicate species, a comparison of tunicate abundances in the initial community (i.e. one month of panel immersion) as well as the final community
(i.e. nineteen months of panel immersion), and a comparison of the impact of predation
33 on the abundance of either colonial or solitary tunicates.
Disturbance events occurred throughout the panel immersion; the timing of these events was recorded and compared to the subsequent loss of abundance of M. citrina.
Patterns of growth of tunicate species were compared between caged and uncaged treatment types. In order to do so, the percentage cover of all tunicates in either the caged or uncaged treatment were averaged and then compared between caged and uncaged treatments. The correlation of this pattern of growth was then calculated using a regression analysis to obtain an R-value.
How frequently tunicate species increased in percentage cover was calculated by quantifying the number of times in which the percentage cover had increased in relation to the previous time point (i.e. two weeks prior). This was then utilized to calculate the frequency of occurrence of positive increases, and compared between species.
Comparisons in the abundance of tunicate species were drawn at one month of panel immersion in order to assess the initial tunicate community, as well as at nineteen months of panel immersion in order to assess the final tunicate community.
Comparisons between tunicate abundances were drawn by comparing the impact of predation on either solitary or colonial species. To do so, occurrences of increases in percentage cover were quantified for either solitary or colonial species in either caged or uncaged treatment types. Then, the frequency of positive increases were combined for either solitary or colonial species and compared between caged and uncaged treatment types.
Photographs of benthic community panels were also used to assess differences in tunicate richness between treatment types and to compare species abundances of sessile
34 invertebrates in ihe final community (i.e. at nineteen months of panel immersion). Thus, tunicate richness was calculated by quantifying the number of species of tunicates and comparing between treatment types; while comparisons in the abundance of sessile invertebrate species were drawn at nineteen months of panel immersion in order to assess the final community of sessile invertebrates.
Laboratory Feeding Trial Overview
Field results from the panel experiment showed that Molgula citrina were abundant in caged treatments and settled but were removed from uncaged treatments (Figures 41,42,
& 40). While the panel experiment assessed the impact of predator exclusion on the community, the experiment did not identify the predators which removed M. citrina from the uncaged panels. Thus, laboratory predator inclusion experiments and filmed feeding trials were utilized to identify predators at the field site which would consume M. citrina.
Laboratory feeding trials included predators routinely found at the Coastal Marine
Laboratory pier. The benthic fish Tautogolabrus adspersus was often collected in minnow traps at CML pier, and underwater filming showed the presence of T. adspersus in close proximity to the benthic panels (Figure 29). While Asterias rubens, Carcinus maenas. Cancer irroratus, Cyclopterus lumpus, and Flabellina verrucosa were often found on uncaged community panels and in caged replicates. Thus, laboratory predator inclusion experiments included the invertebrates Flabellina verrucosa, Cancer irroratus,
Carcinus maenas, and Asterias rubens; as well as the fish, Cyclopterus lumpus and
Tautogolabrus adspersus.
Predator Inclusion Experiments; Invertebrates
35 Predator inclusion experiments with the invertebrates Flabellina verrucosa, Cancer irroratus, Carcinus maenas or Asterias rubens and the prey item Molgula citrina were run using a mesocosm style design. As such, a predator and a prey item were encased together in a container (i.e. a mesocosm) for a period of time in order to assess if that predator would consume that prey item.
Mesocosm replicates were kept in two round open-topped tanks which were 91 cm in diameter and 40 cm deep. Seawater in the tanks was continuously replenished with ocean water from area surrounding the Coastal Marine Laboratory pier. Due to the nature of this flow-through seawater system, water conditions were similar to existing surface water conditions at the CML pier in Portsmouth Harbor, NH from May to November of
2010: salinity was unknown, chlorophyll ranged from 9880-10100 (pg/L), and temperatures ranged from 8-18°C (Gulf of Maine Ocean Observing System Western Gulf of Maine buoy; Figure 20).
Figure 20: The average monthly surface (10m) water temperature in the western Water Temperatures Gulf of Maine fo r the course o f the experiment (May 2009 to November 20 2010). Source Gulf of Maine Ocean Observing System Western Gulf of Maine 15 huov. v 10
5
0 July Sept Nov Jan Mar May July Sept Nov
A. rubens, C. maenas, and F. verrucosa used in feeding trials were captured by manual removal from the outside of caged predator exclusion treatments at the CML pier. C. irroratus were captured by hand or by minnow trap in the subtidal. Each predator was run in a single feeding trial in order to reduce possible habituation towards consumption ofM citrina. M. citrina were collected immediately prior to testing and
36 collected from the outside of caged predator treatments at CML or the surface of the
CML pier.
F. verrucosa, C. irroratus, C. maenas, or A. rubens were starved for forty-eight hours prior to testing. At the initiation of testing, solitary M. citrina of size class 2 (1 to
9.99 mm) were affixed with superglue to the bottom of the mesocosm container. M citrina were attached to the mesocosm container to better simulate the removal of attached sessile invertebrates from a hard substrate. A single F. verrucosa, C. irroratus,
C. maenas or A. rubens was enclosed in each mesocosm and the containers were placed
in the tank. Control containers which contained affixed M. citrina but no predators were
also placed in the tank.
Predator inclusion trials with F. verrucosa, C. irroratus, and C. maenas lasted for
4 weeks with four trials per predator. A. rubens trials took place for either one week or
two weeks with 41 trials total. At the end of the trial duration, the remaining M. citrina
were photographed and the absence of M. citrina was taken to indicate consumption.
While M. citrina remaining in control containers was taken to indicate that predator
removal explained the absence of M. citrina in experimental mesocosms rather than other
variables.
Predator Inclusion Experiments: Fish
Predator inclusion experiments with the fish Tautogolabrus adspersus or Cyclopterus
lumpus and the prey item Molgula citrina were designed to assess if the predators would
consume M. citrina. One Cyclopterus lumpus was removed from a caged panel and tested
in the summer of 2010. Wild T. adspersus were captured with minnow traps deployed in the rocky habitat located below the Coastal Marine Laboratory pier. Fifteen T. adspersus
37 were captured and tested in the summer of 2009, while three T. adspersus were captured and tested in 2010, giving a total of eighteen fish which were used in thirty-four trials.
In order eliminate possible behavioral artifacts exhibited by wild animals during laboratory testing, a series of experimental safeguards were designed into predator inclusion experiments. In order to eliminate possible distress caused by movement from a housing tank to a feeding trial tank, feeding trials took place in the aquaria which housed the fish. In the wild, T. adspersus have been found to exhibit foraging behaviors in the presence of other con-specifics; thus, feeding trials were run with groups of fish rather than a solitary individual (Olla et. al., 1975). While foraging in the wild, T. adspersus have been found to remain in close proximity to habitat refuge; thus, prey items were placed near refuge structures in feeding trials (Olla et. al., 1975). In order to reduce the impact of intraspecific aggression on foraging behaviors during feeding trials, fish of a similar size class were grouped together in a tank. In the 10 gallon tanks at the University of New Hampshire, “Large” fish of 10 cm to 20 cm were housed with another large fish, while “Medium” fish of 1 cm to 9.99 cm were housed with two or three medium fish. In the 91 cm by 40 cm tank at the Coastal Marine Laboratory, a “large” fish was grouped with two “large” fish. Fish were measured from the tip of the snout to the fork of the caudal tail.
In 2009, predator inclusion experiments with T. adspersus and M. citrina were run in the recirculating seawater system in the cold room at the University New Hampshire
(Figure 21). Groups (1-4 fish) of T. adspersus were housed in six, ten gallon glass aquarium tanks at a room temperature of 10°C. Salinity was maintained at approximately
22-32 ppt. The salt water used in the recirculating tank system was ocean water collected
38 at the Coastal Marine Laboratory. Prior to testing, fish were starved for forty-eight hours.
At the initiation of testing, five solitary M. citrina of size class 1 (0 to 1.99 mm) were introduced into the tank. M. citrina were placed on a large flat rock located near a refuge structure. Feeding trials lasted for twenty-four hours. At one hour, two hours, and twelve hours the presence or absence of M. citrina was noted. At twenty-four hours the remaining M. citrina was collected, and the absence M. citrina was deemed to indicate consumption.
Figure 21: Six aquaria in the re-circulating seawater system in the University o f New Hampshire cold room. Aquaria were usedfor both housing test animals and to for feeding trials.
Predator inclusion experiments were duplicated in 2010 in two 91 cm by 40 cm tanks in the flow-through seawater system at the University of New Hampshire Coastal
Marine Laboratory (Figures 22 & 23). Water conditions were the same as existing surface water conditions in the mouth of Portsmouth Harbor, New Hampshire in 2010. Water temperature approximated coastal surface seawater temperatures from May to September of 2010 (12-18° Celcius; Figure 20). T. adspersus were fed once daily with fish pellets
(Omega One Tropical Fish Pellets) and once weekly with crushed Mytilus edulis tissue.
39 T. adspersus were kept in groups of two or three because they are reported to have social feeding behaviors, and will forage more confidently in the presence of other con-specifics
(Olla et. al., 1975). Feeding trials were run in the tank which housed T. adspersus throughout the duration of the experiment (Figure 22). Prior to testing, F. adspersus were starved for forty-eight hours. At the initiation of testing, ten solitary M. citrina of size class 2 (1 to 9.99 mm) were affixed with superglue to acrylic panels with dimensions 13 cm2 (Figure 23). After twenty-four hours, panels were photographed and the absence of previously affixed M. citrina indicated consumption. In total, thirty-four feeding trials were mn with T. adspersus and M. citrina.
acrylic feedini
Figure 22: The lank at the Coastal Marine Figure 23: Close-up o f the foraging shelter and Laboratory which was usedfor both housing feeding panel which was placed in the tank test animals and for feeding trials. The feeding during feeding trials at Coastal Marine arena where feeding trials took place is visible Laboratory. Space where M. citrina have been including the foraging shelter andfeeding removed by T. adspersus during a feeding trial is panel. indicated, as well as remaining M. citrina.
Fecal matter analysis was used to corroborate if the absence of M. citrina in feeding trial results was a true indication of consumption. As such, one F. adspersus was
40 placed in a container with ten M. citrina. After twenty-four hours the water in the bucket was strained for solid matter. It was found that T. adspersus eliminated waste products which contained damaged M. citrina; namely, an empty tunic with no visceral mass. This finding is in line with T. adspersus anatomy, as the fish has a pharyngeal plate which can crush or grind prey items (Olla et. al., 1975: Greene et. al, 1984). Thus, the visceral mass of M. citrina can be consumed while the tunic is excreted in feces.
In 2010, predator inclusion testing with the fish C. lumpus took place in the 91 cm by 40 cm flow-through seawater tank at CML (Figures 22 & 23). Ten solitary M. citrina were affixed with superglue to an acrylic panel. There was one trial total, and after four weeks the remaining M. citrina were counted and photographed.
Predator Inclusion Experiments: Statistical Analysis
Predators (i.e. F. verrucosa, C. irroratus, C. maenas, and C. lumpus) which had no instances of consumption in any of the trials were considered not to consume M. citrina within the context of this study.
A. rubens and T. adspersus were found to consume M. citrina. As a result, A. rubens and T. adspersus were assessed as to whether or not the consumption of M. citrina was a rare or a frequent event. As such, a quantifiable measure was determined using the frequency with which A. rubens and T. adspersus consumed M. citrina by calculating the number of trials in which M. citrina was consumed divided by the total number of trials.
It was determined which predator (i.e. A. rubens or T. adspersus) most frequently consumed M. citrina by comparing the frequency of trials with consumption. Due to differences in the numbers of trials between predators, the percent of trials with consumption was used rather than the direct frequency.
41 How much M. citrina was consumed by each predator (i.e. A. rubens or T. adspersus) was calculated using the average portion of M citrina consumed in feeding trials; this was calculated by dividing the number of consumed M citrina by the number of offered M. citrina and averaging amongst trials for each predator. The average proportion consumed was then compared between predators in order to assess differences in consumption. The number of occurrences of 100% consumption (i.e. when the predator consumed all of the available M. citrina) and 50% consumption (i.e. when the predator consumed half of the available M. citrina) were also assessed and compared between A. rubens and T. adspersus.
Y-Maze Prey Choice Experiment
Y-Maze prey choice experiments were run in order to ascertain the prey preference for
Asterias rubens between Molgula citrina and Mytilus edulis. Specimens of A. rubens, M. citrina and M. edulis were collected from the Coastal Marine Laboratory pier. Prior to testing, A. rubens were housed in a 91 cm by 40 cm flow-through seawater tank at the
Coastal Marine Laboratory. They were fed a diet of M. edulis and M. citrina and were not starved before testing. Trials were run using either one or three A. rubens (Table 3).
Testing took place in an opaque plastic container in seawater collected from the flow through system at CML. Wire mesh was used to separate the two arms of the Y-Maze.
Solitary (size class 0-5mm) or aggregated M. citrina were affixed to an acrylic 5 cm2 panel using superglue. A single acrylic panel was then attached to either side of the wire mesh dividing the testing container. A glue control was used to test attraction or aversion to superglue. Trials were videotaped using a Sony Handycam DCR-SR47.
T rial# # oA. f rubens # of prey items Aggregated or per arm solitary
42 1 1 4 Solitary
2 1 4 Solitary
3 1 4 Solitary
4 1 4 Aggregated
5 3 4 Aggregated
6 3 4 Aggregated
7 3 4 Solitary
Table 3: Details o f Y-Maze testing with the predator A. rubens and the prey items M. citrina and M. edulis.
Y-Maze Prey Choice Experiment: Statistical Analysis
Feeding behaviors observed during testing were categorized and assessed using an ethogram detailing possible behaviors which may occur during testing (Table 4).
Category of Name of Description of behavior behavior behavior Handling Handling Time spent by A. rubens crawling Time onto affixed food item and wrapping arms around food item.
Handling Handling Number of times A. rubens crawled Event onto affixed food item and wrapped arms around food item.
Prey Choice First Choice The y-maze arm which was entered Preference first. This indicates a preference for the prey item found within the y- maze arm.
Feeding Detach Removing food item from the substrate. Feeding Reject Touching and then moving away from food item. Feeding No Tunic Removing tunic from M. citrina. Feeding Siphon Stomach eversion into siphon of M. Access citrina tunic. Feeding Shell Access Stomach eversion into M. edulis. Table 4: An ethogram of behaviors, anticipated to occur during Y-Maze prey choice preference
43 experiments.
Behaviors which were inherently short in duration (often occurring in less than 30 seconds) were cataloged as discrete events without duration. Behaviors which were longer than thirty seconds were recorded with the duration of the behavior. Several behaviors which had been observed in mesocosm experiments did not occur in Y-Maze experiments, these were: detaching the prey item, removing the tunic, and everting the stomach into either the siphon of M. citrina or the shell of M. edulis.
Initial prey choice preference was determined by identifying the maze arm that the predator entered when it was first introduced to the testing tank (i.e. within one minute of trial initiation). The choice of maze arm was taken to indicate a preference for the prey item contained within that maze arm. Initial (i.e. first) prey choice preferences were tallied and compared between prey items using a T-Test in order to determine if the predator had an initial preference for a particular prey item (Table 5).
Overall preference was assessed by calculating the number of times A. rubens entered a maze arm containing a particular prey item; this “overall choice” was tallied and compared between prey items (Table 5). A t-test was used to determine if significant differences occurred between the number of times each prey item was chosen. The first choice data was not combined with overall choice..
The number of times A. rubens handled M. citrina was quantified (Table 5). A handling event was considered to be A. rubens touching and then wrapping its arms around a prey item as according to the ethogram (Table 4). Longer handling events also included duration (Table 5).
Trial # # Times handled M. Duration (m:s)
44 citrina
Glue control 3 n/a
1 1 n/a
2 0 n/a
3 0 n/a
4 7 n/a
5 1 5:33
6 2 n/a
7 2 n/a
Total 16 5:33
Table 5: Results o f Y-Maze choice test with A. rubens. RESULTS
Biofouling Community Composition
Habitat areas (i.e. pier pilings, floating docks, and benthic panels) at the University of
New Hampshire (UNH) Coastal Marine Laboratory ( CML) pier were colonized by sessile invertebrates which included: colonial tunicates (e.g. Botryllus schlosseri,
Botrylloides violaceus, Didemnum albidum, and Didemnum vexillum), solitary tunicates
(e.g. Molgiila citrina and Ciona intestinalis), arborescent hydroids (e.g. Ectopleura larynx), arborescent bryozoans (e.g. Bugula simplex and Bugula turrita), encrusting bryozoans (e.g. Cryptosula pallasiana and Membranipora membranacea), bivalves (e.g.
Anomia simplex, Anomia aculeata, Hiatella arctica, and Mytilus edulis), sponges (e.g.
Haliclona spp. and Halichondria spp.), barnacles (e.g. Semibalanus balanoides), anemone species (e.g. Metridium senile), amphipods (e.g. Caprella spp.), and tube- dwelling amphipods (e.g. Jassa marmorata). These sessile invertebrates exhibited seasonal patterns illustrated by changes in community composition and relative species abundances. Seasonal patterns were observed when organisms increased in abundance in the summer and then decreased in the winter, or increased in abundance in the winter and then decreased in the summer (Figures 34 & 35: 36, 37, & 38).
46 Surface Biofouling Community Composition
A community of sessile invertebrates was established at the ocean surface on the docks
and pier pilings of CML pier (Figures 24 &25). This community included B. schlosseri,
B. violaceus, D. albidum, D. vexillum, M. citrina, C. intestinalis, E. larynx, B. simplex, B. turrita, C. pallasiana, M. membranacea, A. simplex, A. aculeata, M. edulis, H. arctica,
Haliclona spp., Halichondria spp., S. balanoides, M. senile, Caprella spp., and J. marmorata.
Ciona intestinalis
4 7 Figure 24: The biofouling community established on the Coastal Marine Laboratory pier near the surface. Indicated are the anemone M. senile, the solitary tunicate C. intestinalis, the solitary tunicate M. citrina, and an Asterias species. Areas not indicated by arrows include more examples oj these species as well as other sessile organisms (e.g. Haliclona spp., Halichondria spp., B. violaceus, B. schlosseri, D. vexillum, D. listerianum, E. larynx, B. simplex, B. turrita, C. pallasiana, M. membranacea, A. simplex, A. aculeata, H. arctica, S. balanoides, Caprella spp., andJ. marmorata).
4 8 Tunicate species were common in the community and colonial species were capable of occupying much of the substrate (Figure 25).
Figure 25: The hiofouling community established on the Coastal Marine Laboratory pier near the surface. Indicated are colonial tunicates o f the genus Didemnum (e.g. D. albidum and D. vexillum). Areas not indicated by arrows include more examples o f these species as welt as other sessile organisms (e.g. Haliclona spp., Halichondria spp., M. citrina, C. intestinalis, B, violaceus. B. schlosseri. E. larynx, B. simplex, B. turrita, C. pallasiana, M. membranacea, A. simplex, A. aculeata, H. arctica, S. balanoides, M. senile, Caprella spp.. and J. marmoraia).
4 9 Benthic Biofouling Community Composition
A community of sessile invertebrates was established on CML pier pilings in the area adjacent to the benthos. This community included B. schlosseri, B. violaceus, D. vexillum, D. albidum, E. larynx, B. simplex, B. turrita, C. pallasiana, M. membranacea,
A. simplex, A. aculeata, M. edulis, H. arctica, Haliclona spp., Halichondria spp., S. halanoides, M. senile, Caprella spp., and J. marmorata (Figures 26 & 27). The solitary tunicates M. citrina and C. intestinalis were not found in this community.
Figure 26: The biofouling community established on the Coastal Marine Laboratory pier adjacent to the benthos. Indicated are Didemnum species (e.g. D. albidum and D. vexillum). Areas not indicated by arrows include more examples o f these species as well as other sessile organisms (e.g. Haliclona spp., Halichondria spp.. B. violaceus, B. schlosseri. E. larynx, B. simplex, B. turrita, C. pallasiana, M. membranacea. A. simplex, A. aculeata. IT. arctica, S. halanoides, M. senile, Caprella species, andJ. marmorata).
50 Colonial tunicates dominated the substrate on the pier pilings, with B. violaceus, D. albidum, and D. vexillum occupying much of the available space (Figure 27). Sponges of the genus Haliclona were also common. The solitary tunicates M. citrina and C. intestinalis were not found in this community.
Figure 27: The biofouling community established on the Coastal Marine Laboratory pier adjacent to the benthos. Indicated are examples of sponges o f the genus Haliclona, as well as the colonial tunicates B. violaceus. D. albidum. and D. vexillum. In fact, the substrate is almost entirely covered by Didemnum species (e.g. D. albidum & D. vexillum): the solitary tunicates M. citrina and C. intestinalis are not present.
51 At the CML field site, both the benthic biofouling and experimental benthic panel community where located proximal to one another in the habitat area adjacent to the benthos. Thus, both communities were accessible to similar benthic predators which
included fish (e.g. Tautogolabrus adspersus: Figures 28 & 29), crabs (e.g. Cancer
irroratus, Cancer borealis, and Carcinus maenas: Figure 29), and seastars (e.g. Asterias rubens and Asterias forbesi).
Bucket beneath a fish access (TAt reDlicate
Tautogolabrus adsversus
Figure 28: Video still which shows the presence ofT. adspersus in close proximity to the benthic panel community. Also indicated is the bucket beneath aJish access (FA) replicate. Tautogolabrus adspersus, Cancer borealis, and Cancer irroratus were observed in close
proximity to the benthic panel community (Figure 29). In fact, thirteen T. adspersus were
identified in a single ten minute video taken near the benthic panel community. In
contrast, no T. adspersus were observed in video of the community on the floating dock
near the surface.
Figure 29: Underwater video still showing the presence oj'C. irroratus and T. adspersus at the field site in proximity to benthic community panels.
S3 Benthic Panel Community Composition
Predator exclusion in the caged replicates of the benthic panel community led to a community which was dominated by tunicates (Figure 30). Tunicates species included the colonial tunicates B. schlosseri, B. violaceus, and Didemnum spp., as well as the solitary tunicates C. intestinalis and M. citrina. Tunicates occupied up to 15 percent of the substrate while the hydroid E. lanmx and arborescent bryozoans of the genus Bugula
(e.g. B. turrita and B. simplex) occupied up to one percent of the substrate (Figure 30).
The remaining substrate was covered with species too small to identify by macroscopic investigation and/or bare substrate space.
Figure 30: The most abundant sessile 14 invertebrates on -oo i; predator excluded no caged community 10 panels at nineteen O months o f panel immersion.
t I V////////////////A ■ Bugula spp. v Eaoplewa spp. • Tunicates
54 The substrate on caged replicates was also occupied by the encrusting bryozoan C. pallasiana, bivalves of the genus Anomia (e.g. A. simplex and A. aculeata), and the
barnacle 5. halanoides (Figure 31). These species were present in abundances lower than one percentage cover (Figure 31).
•< IS Figure 31: Low abundance sessile ■I 16 invertebrates on -3 0 u caged predator o n-t excluded o 0 12 community panels "1 O o at nineteen months < O.uS o f panel <>.06 immersion. i» u4
o 02 !|
v. Anontrn spp. ■ Semibaiamis baianoides •Ciyptositla pattasicma
55 In uncaged predator accessed replicates, the community was dominated by tunicates
(Figure 32). However, the solitary tunicates M. citrina and C. intestinalis were rarely present (Figures 40 & 41). Instead, the substrate was occupied by the colonial tunicates
B. schlosseri, B. violaceus, D. albidum, and D. vexillum. Colonial tunicate species occupied up to eleven percent of the substrate (Figure 32). The hydroid E. larynx and arborescent bryozoans of the genus Bugula (e.g. B. turrita and B. simplex) were also present on the substrate, but occupied less than one percentage cover (Figure 32). The remaining substrate was covered with species too small to identify by macroscopic investigation and/or bare substrate space.
Figure 32: The most abundant sessile invertebrates on
n uncaged predator 3(13 accessed community n panels at nineteen O months o f panel (13 immersion.
m Bunina spp. '✓ E a o p ie u ra >pp. • T u m m ie s
5 6 Other sessile organisms were present in uncaged replicates, but occupied less than one percent of the substrate (Figure 33). These species included sponges of the genera
Halichondria and Haliclona (i.e. Porifera), the encrusting bryozoan C. pallasiana, bivalves of the genus Anomia (e.g. A. simplex and A. aculeata), the barnacle S. halanoides, and the bivalve H. arctica (Figure 33).
Fib Figure 33: Low
* * It? abundance sessile '> 11 invertebrates on O uncaged predator 3O > i: u.l accessed community n panels at nineteen o I os months o f panel 06 immersion. t 04
• i >2 o m r /////A
Porifera v. Anomia %pp. i Seinibaiatius baianouies <
t nptoMtta radasiana : Hicueila tircnca
57 Community Composition and Seasonal Patterns
Community composition was not static and sessile invertebrate species experienced seasonal patterns which manifested as oscillations in the amount of percentage cover they occupied. For example, the arborescent hydroid E. larynx and arborescent bryozoans of the genus Bugula (e.g. B. turrita and B. simplex) followed an oscillating pattern of abundance in which they were most abundant in the summer and least abundant in the winter (Figure 34). Specifically, these species settled in the fall of 2009 and then declined in abundance, reaching a low point in the winter of 2009/2010 (Figure 34). They then increased in abundance throughout the spring, peaking in abundance in the summer of
2010 (Figure 34). The following winter they reached another low point in abundance
(Figure 34). Predation appeared to have a positive impact on E. larynx, B. turrita and B. simplex, as they were more abundant in predator accessed uncaged treatments (Figure 34: time point Aug.).
Figure 34: The percentage Percent Cover of Arborescent cover o f the arborescent hydroid Ectopluera larynx, Hydroids and Bryozoans and the arborescent bryozoans Bugula turrita bO and Bugula simplex on caged and uncaged benthic 50 • • community panels. • • • • 40 • •
30
10
nov feb 2009 2010
58 The barnacle S. halanoides was largely absent in caged treatments, but in uncaged treatments it exhibited seasonal patterns of growth with a peak in abundance in the spring of 2010 and a low point in abundance in the late summer of 2010 (Figure 35). Predation appeared to positively impact S. halanoides as it was more abundant in predator accessed uncaged treatments (Figure 35: time point May).
Figure 35: The percentage Percent Cover of cover o f Semibalanus halanoides on caged and Semibalanus halanoides uncaged benthic community panels. 60
50 I p 40 t 30 • • • uncaged
I 20 ■“ ■•caaed JJ
~ 10
0 L " " aua n&v feb mav aua nov 2009 2010
59 In late summer, the encrusting bryozoans M membranacea and C. pallasiana settled in the benthic panel community (Figure 36). These species increased in abundance in the fall and winter, and then later declined in the summer (Figure 36). The predators excluded by caging in this study (i.e. larger than 1 cm) appeared to have a little impact on the abundance of M. membranacea and C. pallasiana, as there were no differences in abundance between caged and uncaged treatments (Figure 36).
Figure 36: The percentage cover o f the Percent Cover of Encrusting encrusting bryozoans Bryozoans Membranipor a 12 membranacea 10 and Ciyptosula
• _ 8 pallasiana on caged and •caged 2 s uncaged ■uncaged benthic * 4 community panels. 2
2009 2010 aug nov febmay aug nov
60 The community included Jaw a marmorata, a tube-dwelling amphipod which constructed networks of tubes on the substrate from detritus (Figure 37). J. marmorata experienced a peak in abundance in the winter months, followed by a drop in the summer months.
Isolation from predation appeared to positively impact J. marmorata, as it was more abundant in caged replicates (Figure 37).
Figure 3 7: The percentage Percent Cover of cover o f Jassa marmorata on caged and uncaged benthic Jassa marmorata community panels. 9 S T
6
uncaaed = 4 • • • • caaed
aug nov feb may aug uov 2010
61 Bivalves of the genus Anomia (e.g. A. aculeata and A. simplex) settled in the summer of
2009 and remained in low abundances until the fall (Figure 38: time point Nov.). In
November of 2009, Anomia species began to increase in abundance before peaking in abundance the following winter (Figure 38: time point Feb.). The low abundances seen in the summer of 2009 where duplicated in the summer of 2010 (Figure 38: time point Aug.
2010). The predators excluded by caging in this study (i.e. larger than 1 cm) appeared to have a little impact on the abundance of A. aculeata and A. simplex, as there were no differences in abundance between caged and uncaged treatments (Figure 38).
Figure 58: The percentage Percent Cover of cover o f Anomia aculeata and Anomia simplex on Anomia species caged and uncaged benthic community panels.
4
"•u n c a g e d • cased
0 nov tVb au a nov
2009 2010
62 In the benthic panel community, sponges (i.e. Porifera) of the genera Halichondria and
Haliclona primarily increased in abundance over time without discernible seasonal patterns (Figure 39). Predator isolation appeared to have some negative impact on the abundance of Halichondria and Haliclona species. While high variation meant that there were no statistically significant differences , the abundance of Porifera was frequently higher in uncaged treatment than in the caged treatment (Figure 39).
, ; Figure 39: The percentage Percent Cover of cover o f Porifera species (e.g. Halichondria spp. and Porifera species ! Haliclona spp.) on caged | ; and uncaged-henthic ■■ 25 ' community panels. :o
10 •“■■"•uncaged j • • • • cased i
0 nov
2009 2010
In summary, it was observed that seasonal patterns occurred when species abundances changed over time. In the spring, the barnacle S. balanoides was most abundant (Figure 35). In the summer, the arborescent hydroid E. larynx and arborescent bryozoans of the genus Bugula (e.g. B. turrita and B. simplex) were most abundant
(Figure 34). In the fall, the encrusting bryozoans M. membranacea and C. pallasiana were most abundant !Figure 36). In the winter, the tube-dwelling amphipod J. marmorata, bivalves of the genus Anomia (e.g. A. aculeata and A. simplex), and the bivalve H. arctica were most abundant (Figures 37 & 38). Sponges of the genera
Halichondria and Haliclona did not exhibit patterns of seasonal abundances and instead increased in abundance throughout nineteen months of panel immersion (Figure 39).
63 Isolation from predation appeared to positively impact the abundance of the tube-
dwelling amphipod J. marmorata as it was more abundant in caged replicates (Figure 37:
Table 6). In contrast, it appeared that predation, rather than isolation from predation, had
a positive impact on the abundances of the hydroid E. larynx, arborescent bryozoans of
the genus Bugula (e.g. B. turrita and B. simplex), and the barnacle S. balanoides; thus, E.
larynx, B. turrita, B. simplex, and S. balanoides were most abundant in uncaged
treatments (Figures 34 & 35: Table 6).
Species T reatment Significance Result
Arborescent hydroids (e.g. E. Caged vs. uncaged p>0.05 overall larynx) and bryozoans (e.g. B. turrita & B. simplex) p<0.05 in August 2010
Semibalanus balanoides Caged vs. uncaged p>0.05 overall
p<0.05 in May 2010
Encrusting bryozoans (e.g. M. Caged vs. uncaged p>0.05 membranacea & C. pallasiana)
Jassa marmorata Caged vs. uncaged p>0.05
Anomia simplex and Anomia Caged vs. uncaged p>0.05 aculeata
Porifera (e.g. Halichondria spp. Caged vs. uncaged p>0.05 and Haliclona spp.)
Table 6: Overall results o f sessile invertebrate species between caged and uncaged treatment types.
Tunicates in Panel Community Composition
Several species of tunicates were present in both the Gulf of Maine and the Coastal
Marine Laboratory pier where the benthic panel community was located. Tunicate
species present in the Gulf of Maine included B. schlosseri, B. violaceus, D. vexillum, D. albidum, D. listerianum, C. intestinalis, M. citrina, M. manhattensis, and M. provisionalis. Throughout nineteen months of panel immersion, B. schlosseri, B.
violaceus, D. vexillum, D. albidum, D. listerianum, and C. intestinalis were commonly
present on benthic community panels (Table 7). Tunicate dissections and microscopic
64 analysis indicated that the Molguloid tunicate present in caged replicates was M. citrina rather than M. manhattensis or M. provisionalis.
Genus, species Frequency of occurrence on community panels at CML Botrvllus schlosseri 57% of the time Ciona intestinalis 64% of the time M. citrina, M. 82% of the time for M. citrina manhattensis, and M. provisionalis Botrylloides violaceus 88% of the time
Didemnum vexillum and 12% of the time Didemnum albidum Diplosoma listerianum 0.3% of the time Table 7: Table o f tunicate species found on benthic community panels.
In this study, field experiments included levels of predator exclusion between treatment types in the benthic panel community (Table 2). It was found that predation impacted the solitary tunicate M. citrina, which was significantly different in abundance
(i.e. percentage cover) between treatment types (Table 8). However, there were no significant differences between levels of predator exclusion (i.e. All Access and Fish
Access). As a result, data was grouped into an “uncaged” treatment (i.e. Fish Access with
All Access) and a “caged” treatment (i.e. No Access).
Treatment Types Compared in t-test Significance Results (p value)
No Access (NA) and Fish Access (FA) p>0.05
No Access (NA) and All Access (AA) p>0.05
All Access (AA) and Fish Access (FA) p<0.05
“Uncaged” (FA + AA) and “Caged” (NA) p>0.05 Table 8: T Test results between treatment types.
65 M. citrina settled on both uncaged and caged replicates (Figures 40 & 41). However, it
was commonly removed from uncaged replicates (i.e. within two weeks), and it continued to remain largely absent in uncaged replicates throughout nineteen months of panel immersion (Figure 40).
Figure 40: Percentage [mpact of Predator Access cover o f Molgula citrina in uncaged Treatments on the Percent Cover of predator access Molgula citrina treatment replicates.
0.1
u 0 0S \ Z " °5 •All Predator Access i
H 0.04 Fish Access Onlv '
“ 0.02 0 — .Oil Sep N ov .tan Mar M ay Jul Sep N ov
Impact of Predation on Tunicate Community Succession
In caged predator excluded replicates, the panel substrate was colonized by the colonial
tunicates Botry/loides violaceus, Botryllus schlosseri, Diplosoma listerianum, Didemnum
albidum, and Didemnum vexillum; as well as the solitary tunicates Ciona intestinalis and
Molgula citrina (Figure 41).
6 6 Figure 41: The Tunicates: Caged Treatments settlement and growth o f tunicate species in caged treatments over 19 months o f panel immersion.
* * dum tlohies t lutttceoics * • Bo tin in s ic h io w n - Didemnnin spec les
Diplosoma spp • •Cioita imeumaits • M otzuia c nn>?a
In uncaged, predator accessed replicates, the panel substrate was colonized by the colonial tunicates Botrylloides violaceus, Botryllus schlosseri, Didemnum albidum,
Didemnum vexillum, and Diplosoma listerianum (Figure 42). The solitary tunicate Ciona intestinalis was totally absent from uncaged treatments (Figures 42 & 47). The solitary tunicate Molgula citrina was largely absent from uncaged treatments (<1% at four time points and 0.1% at one time point respectively; Figure 40).
Tunicates: Uncaged Treatments Figure 42. The settlement and growth of tunicate species in uncaged treatments over nineteen months o f panel immersion. Overall, the colonial tunicates B. violaceus and B. schlosseri showed oscillating patterns of growth with increases and decreases in abundance (Figures 43 & 44). Predation appeared to positively impact both B. violaceus and B. schlosseri as they appeared to be
more abundant more frequently in uncaged than in caged treatments (Figures 43 & 44:
Table 9).
Figure 43: The Botrylloides violaceus settlement and growth o f B. violaceus in caged 100 and uncaged treatments over nineteen months of so panel immersion.
60
•u n caged 40 ca sed ■>1:o
0
-:o
Figure 44: The Botryllus schlosseri settlement and growth o f B. schlosseri in 14 caged and uncaged treatments over 10 nineteen months of . panel immersion. 6 4 ■ un cased
c a sed JJ
■o
6 8 Colonial tunicates of the genera Didemnum (e.g. D. albidum and D. vexillum) as well as the colonial tunicate D. listerianum remained in low abundances throughout most of the nineteen months of panel immersion (Figures 45 & 46).Predation appeared to have little impact on D. albidum, as it showed equal occurances of higher abundances in either appeared treatment type (Figures 45: Table 9). D. vexillum, and D. listerianum appeared to be positively impacted by predation, as it appeared more frequently in higher abundances in caged treatments (Figure 46: Table 9).
Figure 45: The Didemnum spp. settlement and growth o f Didemnum spp. in caged and uncaged 30 treatments over nineteen months of :o panel immersion.
■uncaged - 10 ca aed
-10
Figure 46: The Diplosoma listerianum settlement and growth of Diplosoma ;o listerianum in caged » x and uncaged treatments 30 over nineteen months of i e panel immersion. :o i5 uncaaed ■10 ca aed
-10
69 Over time, the solitary tunicates C. intestinalis and M. citrina showed oscillating patterns of growth with increases and decreases in abundance (Figures 47 & 48). Isolation from predation had a positive impact on M. citrina as it was significantly higher in caged treatments (Figure 48: Table 9, repeated measures ANOVA p<0.001). Isolation from predation also appeared to have a positive impact on C. intestinalis as it was more frequently found to be in higher abundance in caged treatments (Figures 47 & 48: Table
9).
Figure 47: The Ciona intestinalis settlement and growth o f C. intestinalis in caged and uncaged :o treatments over 15 nineteen months of panel immersion. ^u 10 > ^ •;T ■ uncaged ^ o — * ca aed I a* ^ fTtTi <>t As
~ -10 2010
-20
Figure 48: The Molgula citrina settlement and growth of M. citrina in caged ■>u and uncaged treatments over nineteen months of panel immersion.
■ ca aed
.un cased
n ,------— .- j - . . ) . . .
- 1 0 ^ ^ O'
70 71 Tunicate Species Number of Times Number of Times Preferred Treatment Tunicate Species was Tunicate Species was Type for this Tunicate more Abundant in more Abundant in Species Caged Treatments Uncaged Treatments
Botrylloides violaceous 4 15 Uncaged
Botrvllus schlosseri 4 9 Uncaged
Ciona intestinalis 14 0 Caged
Didemnum species 4 4 No preference •> Diplosoma listeranium J 0 Caged
Molgula citrina 15 0 Caged
Table 9: Overall results o f tunicate species between caged and uncaged treatment types.
It was observed that all tunicate species (e.g. B. schlosseri, B. violaceus, C. intestinalis,
Didemnum spp., and D. listerianum) showed similar oscillations in abundance between treatment types (Figure 49). Similarities between caged and uncaged treatments indicated that caging did not impact tunicate growth patterns. M. citrina was absent from uncaged treatments and therefore had no uncaged pattern for comparison with the caged pattern
(Figure 40). Lower abundances in uncaged treatments were likely due to the absence of
M. citrina and C. intestinalis in predator accessed replicates (Figure 49).
Successional Tunicate Growth : iu o v o : o j «-1 ■too =3 ->0.0 n UKl ^ ' ti
.10
r '
pteudlor HiitlusK^n piefMlof m< hisiun
Figure 49: The pattern o f growth over time o f the average percentage cover o f all tunicate species between caged (predator exclusion) and uncaged (predator inclusion) treatments.
72 The solitary tunicate M. citrina was most commonly the most abundant tunicate in caged replicates (Figure 41). M. citrina was highly abundant, covering an average of 80% of the substrate in the summer of 2009 (Figure 51). Settlement could be quite dense on caged replicates; in fact, quantification of individuals yielded an average of 117 in July and 57 in August of 2010. Individuals of M. citrina were not found on uncaged replicates.
Molgula species, uncaged treatments Figure 51: The Molgiila species, caged treatments percentage cover o f 120 Molgula citrina in predator accessed (uncaged) and predator 100 Summer 2009 isolated (caged) treatments throughout nineteen months of panel immersion. u 40
‘J
-40 100 200 400 500
Tim e (day)
Fall 2009 Fall 2010
73 However, major decreases in abundance occurred (Figure 51). Events which may have decreased the abundance of M. citrina included abiotic factors such as disturbance events
(e.g. storms) and changes to water parameters (e.g. temperature and salinity: Table 10).
Frequency of Occurrence of Natural Perturbances Reducing Tunicate Assemblage
Possible Date Initial Final Proportional reduction in Cumulative
Perturbance (Month) percentage percentage %cover Frequency of
Event cover (%) cover (minor loss (%) Storm event Sept. 80+ - 0+- Total (100% loss) 1 2009 Water Nov. 60+- 20+- Major (67% loss) 2 temperature 2009 drop and resultant seasonal senesence Unknown April 20+- 10+- Major (50% loss) 3 2010 Unknown June 2010 60+- 50+- Minor (17% loss) 4 Storm event Sept. 70+- 20+- Major (71% loss) 5 2010 Table 10: The frequency o f disturbance events which reduced the abundances o f tunicate species. 74 As an inverse to decreases in percentage cover, many tunicate species exhibited increases in percentage cover between two week sampling points (Figures 41-48). The frequency of occurrence of these increases in percentage cover could then be quantified in order to compare competitive abilities (i.e. the ability to increase in abundance). It was found that M. citrina was the tunicate which most frequently increased in percentage cover in caged replicates (Figure 52). However, M. citrina infrequently increased in abundance in uncaged replicates (Figure 52). B. violaceus was the second most frequently increasing species in caged replicates, and the most frequently increasing species in uncaged replicates (Figure 52). C. intestinalis, B schlosseri, Didemnum spp., and D. listerianum also increased in abundance (Figure 52). is Figure 52: The 16 frequency o f occurrence I I o f increases in percentage cover i: observed in tunicate species throughout 10 nineteen months of F re q u e n c y pane/ immersion. s n i ■ ■ r n*d CZ c.iged 75 The competitive ability to rapidly occupy newly available substrate space could also be compared through an assessment of the initial tunicate community. This community included M. citrina, B. schlosseri, and B. violaceus (Figure 53). O f these species, M. citrina was the most abundant tunicate in caged replicates, occupying up to 35% cover (Figure 53). In contrast, B. violaceus and B. schlosseri each occupied less than 5% cover (Figure 53). M. citrina was nearly absent from uncaged replicates (Figure 40, < 1% cover). Figure 53: The percentage cover o f tunicate species in caged and uncaged treatments at one month of 3 0 panel immersion. M citrina is present in the caged treatments and is nearly (0.5% cover) 20 absent from the uncaged treatments in the initial community (caged u replicates in dark blue, uncaged in light blue). 0 C ln tn , •III Molgula Botrylloides Botrvllus 76 The ability to maintain an occupation of space could also be compared with an assessment of the final community. At nineteen months of panel immersion, the final community included M. citrina, C. intestinalis, B. schlosseri, Didemnum spp., D. listerianum, and B. violaceus (Figure 54). M. citrina continued to be the most abundant tunicate in caged treatments despite the introduction of new tunicate species and subsequent increases in tunicate species richness and relative abundances (Figure 54). M. citrina was remained absent from uncaged replicates (Figure 40). *0 Figure 54: The percentage cover o f tunicate species in caged and uncaged treatments at nineteen months o f panel immersion. M. citrina is present in the caged treatments and is completely absent from the uncaged treatments (caged replicates in dark blue, 10 uncaged in light blue). J Molgula Ciona Botrvllus Didemnum Diplosoma Botrylloides 7 7 Impact of Predation on Tunicate Species Richness Both the predator isolated (i.e. caged) and predator accessed (i.e. uncaged) communities experienced a positive increase in tunicate species richness over the course of nineteen months; with tunicate species richness being defined as the number of tunicate species present on the panel at the time of assessment (Table 11). However, at nineteen months of panel immersion, both M. citrina and C. intestinalis were absent from the predator accessed community. Thus, predator accessed replicates exhibited lower tunicate species richness than predator isolated replicates (Figures 53 & 54: Table 11). Therefore, it appeared that isolation from predation had a positive impact on tunicate richness, as more species were present in the predator isolated community. Tunicate Species Richness and Predation Treatment Type Initial Community: Tunicate Final Community: Tunicate Richness Richness No predator 3 6 access (caged) All predator 3 4 access (uncaged) Table 11: Tunicate species richness on benthic community panels. Impact of Predation on Tunicate Competition Differences in predation between caged and uncaged communities appeared to alter tunicate community dynamics as isolation from predation led to a community dominated by M. citrina (Figure 54). In turn, predation appeared to impact tunicate competitive interactions. Thus, when Mcitrina and B. violaceus coexisted in the predator isolated (i.e. caged) community, B. violaceus was consistently less abundant than it was in the 78 predator accessed (i.e. uncaged) community (Figure 55). Specifically, B. violaceus was lower in caged treatments at eleven time points with significant differences between caged and uncaged treatments at eight time points (October, ’09, January’09, February’09, July’10, August’ 10, Predatoi inclusion September’ 10, October’ 10, Predatoi exclusion and November’ 10). I H i u 1 L i L I fui "ep Nov Feb Apr Jun Aua Oci Figure 55: Percentage cover o f B. violaceus in caged and uncaged treatments throughout % nineteen months o f panel immersion Cover Time (months) Moreover, these reduced abundances appeared to be mutual and affect both B. violaceus and M. citrina. Thus, these species exhibited an inverse relationship of abundance. As a result, when B. violaceus peaked in abundance M. citrina declined, and when M. citrina peaked in abundance B. violaceus declined (Figure 56: time points May, June, and July of 2010: Figure 57). The inverse relationship appeared to be triggered by increased population size, as it did not occur until B. violaceus reached higher abundances (Figure 56: time point May of 2010). Eventually, this interaction appeared to reach a stable state, with both species present in lower abundances than those that they could potentially occupy (Figure 56: time points Aug., Sept., and Nov. of 2010). 7 9 Figure 56: The Percent Cover of Molgulacitrina percentage cover o f M. citrina and B. violaceus andBotrylloides violaceous in Caged on benthic community panels throughout Treatments nineteen months of panel immersion. ^“ ■■•Moiaula cimna • • • • Botrviloides violaceous : fc o.- x-. .<$> . o' > ° -S' Impact of Predator Exclusion on Mogula and Figure 57: Changes in percentage cover o f M. Botrylloides species citrina and B. violaceus throughout June. July, and August o f 2010. % cover \ Time (months) Impact of Predation on Colonial and Solitary Tunicates Not only did predation allow for greater abundances of invasive colonial species such as B. violaceus, but colonial species more frequently increased in abundance in predator accessed (i.e. uncaged) replicates than in predator isolated (i.e. caged) replicates (Figures 55 & 58). Specifically, when abundances were compared between two week sampling 8 0 points, colonial species often increased in abundance in predator accessed treatments, over a two week period. 32.5 Figure 58: The frequency of occurrence of 32 increases in abundance (i.e. 31.5 percentage cover) of colonial tunicate I 31 +■ predator inclusion species in predator accessed (i.e. predator | 30.5 > predator exclusion included) replicates and predator isolated 30 (i.e. predator ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ excluded) replicates. 29.5 ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ 29 Treatment Type In contrast, solitary species (i.e. Mcitrina and C. intestinalis) were negatively impacted by predation, as solitary species less frequently increased in abundance (i.e. percentage cover) in the uncaged predator accessed community than in the caged predator isolated community (Figure 59). Specifically, when abundances were compared between two week sampling points, solitary species did not often increase in abundance in predator accessed replicates over a two week period. 8 1 30 25 20 tt predator inclusion 15 • predator exclusion 10 5 0 Treatment Type Figure 59: The frequency o f occurrence o f increases in percentage cover o f solitary species in communities accessed by predators (predator inclusion) and communities without predator access (predator exclusion). Cage Control Community Composition Cage controls with partial cage siding were designed to be accessible to benthic predators. As a result, cage controls showed a similar community composition to that which was observed in uncaged predator accessed panels. Thus, cage control replicates were occupied by arborescent bryozoans of the genus Bugula (e.g. B. turrita and B. simplex), the encrusting bryozoan M. membranacea, the tube-dwelling amphipod J. marmorata, bivalves of the genus Anomia (e.g. A. aculeata and A. simplex), and sponges (i.e. Porifera) of the genera Halichondria and Haliclona (Figure 60). There was a total absence of the solitary tunicates M. citrina and C. intestinalis in the initial community (May, 2009), the midway community (Dec., 2009), and the final community (Nov., 2010). Instead, the final community was dominated by the colonial tunicates B. violaceus and D. vexillum (Figure 60). The arborescent hydroid E. larynx was present on cage 82 control panels at other times in the year, but was absent in November of 2010 (Figure 60). 0.1-1 Figure 60: The fm al community (Nov. of 2010) on caged control panels after nineteen - o.OS months o f panel 3 U.06 immersion. 3 o.t)4 0.02 0 Overall Laboratory Feeding Trial Results Feeding trials in the form of predator inclusion experiments assessed predator consumption of Molgula citrina. A selection of predators found at the Coastal Marine Laboratory pier field site were tested to see if they would consume M. citrina. These predators included Cancer irroratus, Carcinus maenas, Flabellina verrucosa, Cyclopterus lumpus, Tautogolabrus adspersus, and Asterias rubens (Table 14). Only T. adspersus and A. rubens were found to consume M. citrina (Table 14). Specifically, T. adspersus consumed an average of 44% of the offered M. citrina while A. rubens consumed an average of 38% of the offered M. citrina (Figure 65). T. adspersus consumed M. citrina in 70% of the feeding trials while A. rubens consumed M. citrina in 47% of the feeding trials (Figure 66). Predators which did not consume M. citrina Predator inclusion experiments with Cancer irroratus, Carcinus maenas, Flabellina verrucosa, and Cyclopterus lumpus as predators and Molgula citrina as prey indicated 8 3 that these predators did not consume M. citrina. After four weeks of testing, it was found that none of the M. citrina was consumed. Thus, due to their lack of consumption of M. citrina, these predators were unlikely to have altered community dynamics in the field experiment with respect to the abundance of M. citrina. Predators which consumed M. citrina Predator inclusion experiments found that T. adspersus and A. rabens consumed M. Predator Prey Consumption? Asterias rubens Molgula citrina Yes Cyclopterus lumpus Molgula citrina No Cancer irroratus Molgula citrina No Carcinus maenas Molgula citrina No Flabellina Molgula citrina No verrucosa Tautogolabrus Molgula citrina Yes adspersus citrina (Table 14). In fact, previous gut content data showed that of forty-two T. adspersus captured at the Coastal Marine Laboratory pier, fifteen had M. citrina in their gut (Day & Harris, 2009). Moreover, M. citrina comprised an average of 38% of the gut content (Day & Harris, 2009). Thus, T. adspersus and A. rubens likely altered community dynamics in the field experiment with respect to the abundance of M. citrina. 84 Table 12: Feeding trial results with a suite ofpotential predators that were commonly found at the CX'tL pier field site. Predator Inclusion Results: Tautogolabrus adspersus T. adspersus consumed Molgula citrina in twenty-six of thirty-seven trials in predator inclusion experiments (Table 13: Figure 61). Thus, it was shown that this behavior occurred 70% of the time and was highly replicable (Figure 61). Moreover, T. adspersus consumed all of the available Molgula citrina in eleven of thirty-seven trials, or 30% of the time (Table 13). However, T. adspersus did not consume Molgula citrina in eleven of trials, or 3()% of the time (Table 13: Figure 61). Predator # # trials with # trials with # trials with trials consumption zero 100% total consumption consumption T. 37 26 11 11 adspersus Table 13: Feeding trial results for T. adspersus. Figure 61: The number T. adspersus Feeding Trials: of feeding trials with consumption o f M. Consumption of M. citrina citrina (26) versus number o f trials without consumption(11) which occurred throughout 37 30 feeding trials. iZ c C on sumption ! -0 *N o Consumption 1 15 2 10 In each trial T. adspersus was offered several (5 to 10) individuals of M. citrina. As a result, T. adspersus consumed an average of 44% of the M. citrina throughout the 85 twenty-six trials with consumption. In 2009, T. adspersus consumed 40% and in 2010 it consumed 67% (Figure 62). Thus, feeding trial results indicated that when T. adspersus was given the opportunity to eat multiple M. citrina it would. In fact, it would consume between 40 to 67% of the available M. citrina. Figure 62: The average T. adspersus Feeding Trials: percent consumption of M. citrina from feeding Consumption of M. citrina trials conducted in both 2009 and 2010 (44%). Also indicated are the results from the 2009 (40%) and 2010 (67%) feeding trials. ^ All Trials ■ 2009 Trials ■ 2010 Trials Fecal matter analysis was used to corroborate if the absence of M. citrina in feeding trial results was a true indication of consumption. As a result, partially digested M. citrina was found in fish fecal matter which was collected after a feeding trial. Predator Inclusion Results: Asterias rubens A. rubens consumed Molgula citrina in mesocosm predator inclusion experiments (Table 14). In fact, A. rubens were found to consume M. citrina in nineteen of the forty-nine trials, or 39% of the time (Table 14). Predator * # trials with # trials with # trials with # trials with Average trials zero consumption >50% 100% consumption total consumption consumption consumption A. 49 30 19 16 3 38% rubens Table 14: Feeding trial results with A. rubens. Thus, it was shown that consumption of M. citrina by A. rubens is replicable and likely to occur 39% of the time (Figure 63). 8 6 "i Figure 63: The number A. rubens Feeding Trials: 1 o f feeding trials with Consumption of M. citrina j consumption o f M. : citrina (23) versus the 1 60 i 1 number of trials without consumption (26) which 50 ! ' occurred throughout 49 i feeding trials. ! i 40 < Consumption j 1 1 30 ! J ■ No Consumption j 20 j i » 10 i i 1 0 In each trial, A. rubens was offered a number of M. citrina (5 to 10). Thus, it was found that A. rubens consumed an average of 38% of the offered M. citrina (Figure 64). Moreover, in sixteen of forty-nine trials (i.e. 33% of the time) A. rubens was found to consume more than 50% of the available M. citrina (Table 15). Figure 64: The average A. rubens Feeding Trials: percent consumption of Consumption of M. citrina M. citrina by A. rubens in predator inclusion _ 45 experiments. 1 40 1 35 -r 30 s -5 2 20 - 15 g 10 I 5 0 Comparison of Predator Inclusion Results Feeding trial results were compared in order to assess differences between T. adspersus and A. rubens in their consumption of M. citrina. These comparisons included the average percent of M citrina consumed, as well as the frequency of trials with or without consumption. 87 The average percent of M. citrina consumed in feeding trials was very similar between T. adspersus and A rubens. However, T. adspersus consumed an average of 44% of the offered M. citrina while A. rubens consumed 38% (Figure 65). Thus, T. adspersus consumed slightly more M. citrina in feeding trials than A. rubens. j Figure 65: The average Comparative Consumption i percent of M. citrina of M. citrina | consumed in the feeding 1 trials by the predators j A. rubens and T. s2 46 = 44 ■ adspersus. 3 S i I 42 s 40 j)t 38 $ 34 i A. rubens < T. adspersus T. adspersus consumed M. citrina in 70% of the feeding trials while A. rubens consumed M. citrina in 47% of the feeding trials. Thus, T. adspersus was much more likely to consume M. citrina in feeding trials than A. rubens (Figure 66). j Figure 66: Comparative Consumption ! Comparative percent of M. citrina i frequency o f trials in i which M. citrina was SO ^0 ! consumed by the | predators A. rubens V* 60 j and T. adspersus. 47 = 40 aA. mbens - T. adspersus Y-Maze Prey Choice Preference Results 8 8 Y-Maze prey choice preference experiments were designed to test possible preferences in prey choice for Asterias rubens between Molgula citrina and Mytilus edulis. As a result, A. rubens was seen to handle Molgula citrina sixteen times during filming (Table 15). While no complete feeding was seen on video, in the time following filming A. rubens consumed the M citrina which had been offered during filmed feeding. Trial: Predator Prey Times Handled 1 A sterias rubens M olgula citrina 3 2 Asterias rubens M olgula citrina 1 3 Asterias rubens M olgula citrina 0 4 A sterias rubens M olgula citrina 0 5 A sterias rubens M olgula citrina 7 Trial: Predator Prey # Choice: M c i t r i n a # Choice: M e d u l i s 1 A s t e r i a s M olgula citrina 0 1 r u b e n s 2 A s t e r i a s M olgula citrina 1 0 r u b e n s 6 A sterias rubens M olgula citrina 1 7 A sterias rubens M olgula citrina 2 8 Asterias rubens M olgula citrina 2 Total 16 Table 15: R esults o f video analysis o f A . rubens foragin g an d feedin g behavior tow ards M . citrina. 89 3 A s t e r i a s M olgula citrina 1 0 r u b e n s 4 A s t e r i a s M olgula citrina 7 6 r u b e n s 5 A s t e r i a s M olgula citrina 2 2 r u b e n s 6 A s t e r i a s M olgula citrina 10 6 r u b e n s 7 A s t e r i a s M olgula citrina 4 3 r u b e n s Total 25 18 Y-Maze prey choice preference results showed no preference between foraging initially (i.e. first choice) in a maze arm with Mcitrina and foraging initially in a maze arm with M. edulis within one minute of trial initiation (Table 16). M olgula citrina M ytilus edulis i t times Is* choice preference 3 3 T able 16: R esults o f initial choice (i.e. first choice) in Y -M aze p rey choice preferen ce betw een M . citrin a an d M . edulis. Overall, there was no difference in choice between foraging in a maze arm withM. citrina and foraging in a maze arm withM. edulis (t-test, p = 0.133: Table 17). Trial: Predator Prey i t Choice: M c i t r i n a i t Choice: M e d u l i s 1 A s t e r i a s M olgula citrina 0 1 r u b e n s 2 A s t e r i a s M olgula citrina 1 0 r u b e n s 3 A s t e r i a s M olgula citrina 1 0 r u b e n s 4 A s t e r i a s M olgula citrina 7 6 r u b e n s 5 A s t e r i a s M olgula citrina 2 2 r u b e n s 6 A s t e r i a s M olgula citrina 10 6 r u b e n s 7 A s t e r i a s M olgula citrina 4 3 r u b e n s Total 25 18 Table 17: R esults o f Y M aze prey choice preference fo r A. rubens betw een M . citrina an d M . edulis. 90 Feeding Trial Results Overview Laboratory feeding trial results gave insight into field results and allowed for a more complete assessment of the results of predation on tunicate community succession. In the field, isolation from predation created a community which was dominated byMolgula citrina while predator access created a community in whichM. citrina was largely absent (Figures 41 & 42). In turn, feeding trial results gave an idea of which predators were most likely to consume M. citrina, how often they would consume M. citrina, and how much M. citrina they would consume. In feeding trials, Tautogolabrus adspersus consumed M. citrina 70% of the time while Asterias rubens consumed M. citrina 41% of the time (Figure 66). In turn, both predators consumed almost half of the availableM. citrina (T. adspersus, 44%; A. rubens, 38%: Figure 65). In the field, these predators likely influenced benthic panel community development through the frequent removal of M. citrina. In turn, isolation from these predators likely resulted in a community dominated byM. citrina. DISCUSSION Community Composition and Successional Development Several structures at the University of New Hampshire (UNH) Coastal Marine Laboratory ( CML) pier acted as substrate for a biofouling community which included: Botryllus schlosseri, Botrylloides violaceus, Didemnum vexillum, Didemnum albidum, Diplosoma listerianum, Molgula citrina, Ciona intestinalis, Ectopleura larynx, Bugula simplex, Bugula turrita, Cryptosula pallasiana, Membranipora membranacea, Anomia simplex, Anomia aculeata, Hiatella arctica, Mytilus edulis, Haliclona spp., Halichondria spp., Semibalanus balanoides, Metridium senile, andJ. marmorata (Figures 24-27: 30- 91 39). These sessile invertebrates were found on the pier and the floating dock at the surface, thus forming a surface biofouling community (Figures 24 & 23); on the pier pilings adjacent to the bottom, thus forming a benthic biofouling community (Figures 26 & 27); and on the experimental panels suspended above the bottom, thus forming a benthic panel community (Figures 5 & 6). Community composition in the benthic panel community was found to change over time in a process of successional development (Figures 34-39,41-48). Successional development describes changing community dynamics as species enter and exit the system (Clement, 1916: Connell, 1961: Paine, 1969: Dayton, 1971: Osman, 1977: Dean & Connell, 1987). Primary succession occurs when random events such as lava flow, glacial retreat, or forest fire create new substrate which is subsequently colonized by organisms (Yarranton & Morrison, 1974: Horn, 1974: Tilman, 1987). While secondary succession takes place when incoming species colonize space previously inhabited by earlier species (Horn, 1974: Tilman, 1987). In this experiment, newly immersed panels acted as new substrate which then experienced primary succession (Figures 34-39 & 53). Secondary succession then took place when seasonal patterns of growth reduced existing species abundances, or disturbance events cleared the substrate, thus allowing other species to colonize the newly available substrate (Figures 34-39,41-49: Table 12). In the experimental benthic panel community, primary succession occurred when species settled on the newly available panel space and community composition changed as species richness increased (Figure 53). Early community composition was dominated by tunicates, and within one month of benthic panel immersion M. citrina, B. violaceus, andB. schlosseri had settled on the substrate (Figures 53; 48,43, & 44). By August of 92 2009, species richness had expanded to include at least seven more species (e.g.M. membranacea, C. pallasiana, J. marmorata, A. simplex, A. aculeata, Haliclona spp., and Halichondria spp.: Figures 35-39). Community composition continued to change as species increased in abundance and new species entered the system. In November of 2009, E. larynx, B. simplex, andB. turrita settled increasing community species richness from ten to thirteen (Figure 34). The tunicates Diplosoma listerianum, Didemnum spp., and C. intestinalis settled later in community development bringing species richness to sixteen (Figures 46,45, & 47). Finally, S. balanoides settled increasing richness to seventeen (Figure 36). The abundances of these species were not static and species showed oscillations in percentage cover with peaks and drops in abundance indicating patterns of growth and senescence (Figures 34-39,41-48). These peaks and drops in abundance allowed for secondary succession to occur as changes in percentage cover created newly available substrate space. In turn, oscillations in abundance may have been a result of seasonal growth patterns. Such patterns of growth can be driven by organismal life history traits such as reproduction, dispersal, and recruitment which are driven by changing abiotic conditions such as water temperature, nutrient availability, and light levels (Little & Wagner, 1997: Railkin, 2004: Wahl, 2008; Peckol & Searls, 1982: Keough, 1983: Underwood & Anderson, 1994). In the spring, abiotic conditions changed as water temperatures, nutrient levels, and light levels increased (Figure 20; Chapman & Lindley, 1980: Valiela, 1984; Orton, 1920: Boreo, 1984). As these parameters continued to increase throughout the summer, M. citrina, B. schlosseri, B. violaceus, E. larynx, B. simplex, andB. turrita increased in 93 size and abundance (Figures 48,44,43, & 34). Increases in species abundances may have been in response to changing abiotic conditions. For example, tunicates such as B. schlosseri andB. violaceus have been shown to increase reproduction and growth as temperature increases (Yamguchi, 1975: Stachowicz et. al., 2002: McCarthy et. al., 2007). While hydroids (e.g. E. larynx) and bryozoans (e.g.B. simplex andB. turrita) have been found to initiate reproduction and increase stolon size when temperatures increase (Orton, 1920: Grave, 1933; Maturo, 1959: Lombardi et. al., 2008). However, different species will have varying responses to changing abiotic conditions. Some species will continue to thrive as temperatures increase, while other species will reach a temperature threshold and then decline in abundance. For example, as water temperatures continued to increase in the late summer, the abundanceE. of larynx, B. simplex, andB. turrita began to drop. Hydroids (e.g. E. larynx) and bryozoans (e.g. B. simplex, andB. turrita) have been shown to experience reduced fitness beyond a high temperature threshold (Orton, 1920: Grave, 1933; Maturo, 1959: Lombardi et. al., 2008). In contrast, M. citrina, B. schlosseri, B. violaceus continued to thrive in the late summer, and tunicates (e.g.B. schlosseri andB. violaceus) have been found to be resilient to higher temperatures (Figures 48,44, & 43; Yamguchi, 1975: Stachowicz et. al., 2002: McCarthy et. al., 2007). Changes to successional development and community composition continued into the late fall and winter as other species increased in percentage cover when water temperature declined. Fall and winter can be associated with growth, as some species have greater reproduction at cooler temperatures, or may increase reproduction when temperatures drop after a period of increase (Brunetti et. al., 1988: McCarthy et. al., 2007: 94 Osman & Whitlach, 2007; Gulberg & Pearse, 1995). In this study,M. membranacea, C. pallasiana, Didemnum spp., Diplosoma listerianum, J. marmorata, A. simplex, andA. aculeata increased in late fall (Figures 36,45, 46, 37, & 38). The encrusting bryozoans M. membranacea andC. pallasiana have been shown to increase in the fall (Berman et. al., 1992: Lambert et. al., 1992: Harris & Tyrrell, 2001; Amui-Vedel, 2007). While Didemnum species have been shown to be more successful in cooler temperatures (McCarthy et. al., 2007: Osman & Whitlach, 2007).Diplosoma listerianum shows optimum growth between 10°C and 15°C., which would occur in the fall (Brunetti et. al., 1988). However, fall peaksin A. simplex, A. aculeata, andJ. marmorata may have been a result of detection methods rather than a seasonal peak as these species have previously been shown to increase in growth in the summer (Franz, 1989; Hadfield & Anderson, 1988); due to size, these species were not detected until they reached a certain size threshold which then occurred in the fall rather than the summer. These seasonal patterns of growth created oscillations in cover which may have allowed secondary succession to occur, as one species decreased it opened space for another species to increase and occupy. For example, in the late summer months,M. citrina, B. schlosseri, B. violaceus, E. larynx, B. simplex, andB. turrita were abundant (Figures 48, 44,43, & 34). Then, in the late fall, E. larynx, B. simplex, andB. turrita decreased and the decline of these abundant summer species allowed for a variety of new species to settle and increase. Thus, in the fallM. membranacea, C. pallasiana, J. marmorata, A. simplex, andA. aculeata increased (Figures 36,37, & 38). Three of the abundant summer species(M. citrina, B. schlosseri, andB. violaceus) did not decrease in the fall and remained in high abundances until the winter (Figures 48,44, & 43). In the 95 winter they then decreased whileDidemnum spp. andDiplosoma listerianum increased (Figures 48,44, & 43; 45 & 46). In the final spring of panel immersion S. balanoides peaked in abundance, then decreased whileM. citrina, B. schlosseri, B. violaceus increased (Figures 48,44, & 43). Some species did not exhibit oscillations in growth and appeared to utilize newly available space throughout all the seasons: sponges (e.g. Haliclona spp. andHalichondria spp.) steadily increased in abundance throughout all o f the seasons (Figure 39); C. intestinalis remained in low abundances throughout nineteen months, only beginning to increase in percentage cover in the fall o f 2010 (Figure 47). Secondary succession may also have been influenced by disturbance. Disturbance events took place several times throughout the course of the study (Table 12). These events often cleared much of the panel substrate, which was then followed by secondary succession as species settled on the newly cleared substrate. A storm in September of 2009 cleared 70% of the substrate whenM. citrina was removed; thus potentially creating a large amount of available space for secondary succession (Figure 41: time point Aug.- Sept. 2009). In caged treatments in whichM. citrina had been removed, several species exhibited a jump in percentage cover in the months following the storm. M. citrina resettled and increased to 70% cover (Figure 41). S. violaceus and C. intestinalis increased from <1% to 10% cover (Figure 42). M. membranacea and C.pallasiana jumped from <1 to 5% cover (Figure 36). J. marmorata went from <1 to 4% cover (Figure 37). Anomia simplex andAnomia aculeata increased from <1 to 2% (Figure 38). The uncaged treatments also experienced loss of percentage cover during the September storm with resultant recovery (Figure 42: time point Aug.-Sept 2009). In the months following the storm: B. violaceus increased from 0 to 35% cover (Figure 42); M. 96 membranacea and C.pallasiana jumped from <1 to 10% cover (Figure 36); and Anomia simplex andAnomia aculeata increased from <1 to 2% (Figure 38). In conclusion, successional development appeared to take place in this community. Over time, the community increased in richness and abundance to include other sessile species. However, abundances were not static and seasonal patterns of growth occurred with some species being more abundant in the summer (e.g. M. citrina, B. schlosseri, B. violaceus, E. larynx, B. simplex, andB. turrita: Figures 41-48 & 34) while others were abundant in the winter (e.g. M. membranacea, C. pallasiana, Didemnum spp., andDiplosoma listerianum: Figures 36,45, & 46). Seasonal growth also influenced secondary succession as decreases in percentage cover allowed for other species to utilize newly available substrate. Finally, disturbance allowed secondary succession to occur as species rapidly occupied newly available space. This pattern of changing successional development was similar to patterns of change historically found in a variety of different ecosystems both terrestial and marine (Clement, 1916: Connell, 1961: Paine, 1969: Dayton, 1971: Osman, 1977: Dean & Connell, 1987). However, while similar patterns of successional development can be observed between terrestrial rainforests, the rocky intertidal, and the fouling community observed at this site, the organisms within each community vary between habitats, between sites, and even between studies (Connell, 1961: Paine, 1969). In this study, the initial community exhibited primary succession which was dominated by tunicates (e.g. M. citrina, B. schlosseri, andB. violaceus: Figures 34-39 & 41-48; 30 & 32). This finding was similar to results by other researchers which have found that succesional development in the fouling community at coastal marinas and piers has led to a 97 community dominated by tunicates (Sutherland & Karlson, 1977: Railkin, 2004: Osman & Whitlach, 2007: Greene & Grizzle, 2007). In contrast, other researchers have found that community development can lead to a community structured by a foundation of sessile invertebrates and algae (Mare, 1942: Dayton, 1971: Dayton et. al., 1974: Sousa, 2001). A similar study conducted by Greene and Grizzle found that mussel species were commonly abundant, but they were absent in this study (Figures 24 & 25: Greene and Grizzle, 2007). In habitats similar to the Gulf of Maine, benthic community development can lead to algal forests (e.g.Saccharina latissima, Agarum cribrosum, andChondrus crispus) which are covered with encrusting organisms (e.g. Membranipora membranacea, Obelia geniculata, andElectra pilosa\ Lubchenco 1978: Lubchenco& Menge, 1978: Berman et. al., 1992: Trussell et. al. 2002). In this study, algae were largely absent (Figures 24 & 25). However, the encrusting organism Membranipora membranacea was present both on the panels themselves and growing on other sessile organisms including the tuniate species M. citrina (Figure 16). While some differences were present, this study was overall in line with findings indicating changing community patterns to a community dominated by tunicates (Lambert & Lambert, 2003: Lambert, 2005: Dijkstra et. al., 2007: Greene & Grizzle, 2007). Impact of Predation on Community Development Predation can change community composition through the preferential removal of prey species and alter successional development when prey species are removed from the system thus creating available open space for other species to occupy (Lotka, 1920: Volterra, 1931: Bemstien et. al., 1981: Wootton, 1993). In this study, predation dramatically altered the successional development of sessile invertebrates in the benthic 98 panel community by decreasing the abundances o f the solitary tunicatesMolgula citrina andCiona intestinalis. Refuge from predation also allowed for the continuing persistence of the solitary tunicateM. citrina at the Coastal Marine Laboratory pier and created a patchy distribution of M. citrina between habitat areas at CML pier. Furthermore, predator consumption of M. citrina reflected predator response to changing community dynamics in which the historically abundant speciesMytilus edulis has diminished in abundance whileM. citrina has become abundant. Predation impacted successional development through the removal of sessile invertebrates which created newly available substrate space for other species to occupy. Predation appeared to be a factor in the removal oS. f balanoides, E. larynx, B. simplex, andB. turrita. The predatory fish T. adspersus has been shown to consume barnacles including S. balanoides and is commonly more active in the summer when water temperatures are warmer (Olla et. al, 1975). As a result, juvenilesS. obalanoides f were present in high amounts during the early spring but were dramatically reduced in percentage cover by August (Figure 35). Nudibranch species such asFlabellina spp. have been shown to be more active when water temperatures are warmer and have been found to consume hydroids such as E. larynx (Miller, 1961: Gosner, 1971). In turn, E. larynx, B. simplex, andB. turrita were abundant in the summer months but declined in the fall (Figure 34). It was possible to observe the impact of predation on the process of secondary succession when the physical remnants of sessile species remained on the substrate after predation and those remnants then became overgrown by other sessile species. In S. balanoides the base of the carapace or the empty carapace remained while the visceral 99 body of was absent; the empty carapace was then overgrown by other sessile species such asB. violaceus andC. pallasiana (Figure 15). In E. larynx, B. simplex, andB. turrita the base of the stolon remained while the rest of the organism was absent. In turn, the base of the stolon was often overgrown by C. pallasiana or encrusting sponges (e.g. Haliclona spp. andHalichondria spp.). Predation altered the successional development of tunicate species such that divergent communities developed in either the presence of predation (i.e. uncaged treatments) which were dominated by solitary tunicate species (i.e.M. citrina), or the absence o f predation (i.e. caged treatments) which were dominated by colonial tunicate species (i.e. B. violaceus-, Kruskal-Wallis one-way analysis of ranks with post-hoc Tukey Test, p<0.00). As the influence of predation altered tunicate community dynamics, it was of interest to identify the predators which were influencing the abundance of M. citrina as well as their relative levels of consumption. In order to do so, predators found at the field site were tested for consumption of M. citrina. It was found that both Tautogolabrus adspersus and Asterias rubens consumed M. citrina (Table 14). Historically, the predatory fishT. adspersus has been found to abundant in the Gulf of Maine (Olla et. al., 1979: Bradbury et. al., 1995: Able & Hales, 2005); makingT. adspersus a major predator of M. citrina of the uncaged benthic panel community at the Coastal Marine Laboratory pier. In response, caged treatments which were inaccessible to T. adspersus had an abundance M.of citrina (Figure 41). Moreover, T. adspersus is highly habitat specific, being limited to the bottom near the benthic zone (Olla et. al., 1979: Bradbury et. al., 1995: Able & Hales, 2005. Field observations showed thatT. adspersus was not found at the surface biofouling community were M. citrina was 100 abundant, andT. adspersus was commonly found in the benthic community where M citrina was largely absent (Figures 24, 25,26, & 27). Thus, field observations showed that the distribution of T. adspersus was inversely correlated with the abundanceM. of citrina (Figure 24 & 25). Therefore, it appeared that predation byT. adspersus had the potential to significantly alter tunicate community succession through the removal of M. citrina. In contrast to T. adspersus, specimens of A. rubens were found in the surface and benthic biofouling communities as well as the caged and uncaged treatments in the benthic panel community. Thus, there was the potential for predation byA. rubens on M citrina in all the communities at the CML pier. Moreover, T. adspersus andA. rubens showed differences in consumption which could then translate into differing levels of predation on the biofouling community. Feeding trials showed that both T. adspersus and A. rubens consumed roughly equal amounts of M. citrina (T. adspersus, 44%: A. rubens, 38%: Figure 65). However, T. adspersus more frequently consumed M. citrina (T. adspersus, 70%: A. rubens, 47%: Figure 66). Thus indicating that while both T. adspersus andA. rubens will consume M. citrina, it is T. adspersus which is much more likely to consume the prey item. In the field, this could translate to a greater level of influence of T. adspersus on the successional development of tunicate species through a more frequent removal of M. citrina. Predation altered community succession in the relative abundances of solitary and colonial tunicate species as well as the frequency of colonization events by solitary and colonial tunicates. In predator accessed uncaged treatments solitary species (i.e.M. citrina andC. intestinalis) were largely absent while colonial species (i.e.B. violaceus) 101 occupied up to 80% of the substrate (Figures 40 & 42). Moreover, colonial species more frequently increased in size and abundance (i.e. percentage cover) in uncaged treatments than in caged treatments (Figure 55). Indicating that predation had a positive impact on colonial species. It also appeared that solitary species were negatively impacted by predation, as the frequency of occurrence of successful colonization events was much lower in communities which predators could access (Figure 55). Predation appeared to influence the distribution and abundance ofM. citrina between the communities found at CML pier. M. citrina was nearly absent from uncaged treatments in the benthic panel community and was largely absent from the benthic biofouling community (Figures 40,26, & 27). In contrast, M. citrina was abundant in caged treatments in the benthic panel community and was observed in the surface biofouling community (Figures 42,26, & 27). Moreover, M. citrina has been present in the Gulf of Maine for more than a century and has been found to be a common and abundant member of biofouling communities (Sumner et. al, 1913; Otuska & Dauer, 1982: Carmen et. al., 2009: Carmen et. al., 2010).As a result, this tunicate may persist in Gulf of Maine due to differential predation pressure between habitat areas including areas of refuge from predation. Pockets of refuge with high abundance likely supported the persistence of the population of M. citrina at CML pier. The surface biofouling and caged benthic panel communities were isolated from T. adspersus and showed a high abundance ofM. citrina (Figures 24, 25, & 41). Moreover, M. citrina was highly abundant in the absence of predation, reaching up to 70% cover in caged benthic community panels (Figure 41). In turn, other areas of colonization appeared to be less successful for colonization by M. 102 citrina. The benthic biofouling and uncaged benthic panel communities were accessible by T. adspersus and showed very low abundances ofM. citrina (Figures 26,27,40, & 42). It was observed thatM. citrina settled on uncaged benthic community panels but was rapidly removed (Figure 40). The differential abundances ofM. citrina between communities at CML pier indicate that whileM. citrina is present in the area, it is not homogenous in distribution. Furthermore, some habitat areas were found to be more successful for colonization. Indicating the persistence of M. citrina in the area may be the result of differential predation pressures and the availability of habitat areas which provide refuge from predation. Predator behavior reflected predator response to changing community dynamics which have seen a shift in dominance from the historically abundantMytilus edulis to the recently abundant tunicate speciesM. citrina. Historically,Mytilus edulis was a common member of the benthic and biofouling community (Dayton, 1971: Paine, 1974, 1976: Paine & Levin, 1981: Harris & Irons, 1982: Jackson, 1983). In 1982, Harris and Irons found M. edulis to be a dominant sessile invertebrate at the University of New Hampshire Coastal Marine Laboratory pier (Harris & Irons, 1982). In contrast, throughout the course of this thesis study (May 2009-Nov. 2010) observations taken at UNH CML pier found no M. edulis on the benthic community panels or the benthic habitat. However,M. edulis was not entirely absent from the field site, as underwater video showedM. edulis on the floating dock at the surface. Despite this,M. edulis is becoming less abundant, and tunicates such asM. citrina are becoming a major component of the biofouling community (Sumner et. al, 1913; Otuska & Dauer, 1982: Carmen et. al., 2009: Carmen et. al., 2010). In turn, predators which were able to alter their diet to include the newly 103 abundantM. citrina may have a resultant competitive advantage. Predators suchT. as adspersus andA. rubens were found to consume M. citrina (Table 14). Thus, these predators may have experienced a shift in diet to include the newly abundant tunicate M citrina in response to changing community dynamics. Furthermore, A. rubens prey choice feeding trials found no difference in choice between M. citrina andM. edulis (Table 19; t-test, p = 0.133). Prey choice preference can influence community dynamics in that predators will consume the preferred item when both are available. In the field this could translate to removal ofM. edulis from the community while M. citrina remains. However, A. rubens showed no distinct preference between prey items indicating that the influence of A. rubens may not be a major force driving changing community dynamics. In conclusion, predation had major impacts on community development and succession. It allowed for secondary succession to occur as some species were removed from the system and other species increased in abundance. It altered successional development through changes to the abundances of solitary and colonial tunicate species. It allowed for the continuing persistence of M. citrina in the area and drove the distribution of this tunicate species. Furthermore, it was found that predators will consume the abundantM. citrina indicating a positive response to changing community dynamics which reflect a shift from a community dominated byM. edulis to one dominated by tunicates. Results from this study were similar to studies conducted by other researchers in which predators acted as habitat modifying species, altering the structure and ecology of marine coastal habitats through the preferential removal of prey species (Paine, 1969: Menge et. al., 1994: Jones et. al., 1994: Carlton, 1996). In this study, predators removed 104 the tunicate species M. citrina, thus allowing colonial tunicate species to dominate the habitat. Similar alterations to species abundances and community dominance have been found following the removal of a predator including the removal o f starfish on the mussel population (Paine, 1969). Thus, this study was in line with ridings which indicated that predators can structure trophic food webs and dramatically alter community composition (Paine, 1969: Hutchinson, 1959: Jeffries, 1984: Dayton, 1971: Vance, 1979). Competitive Interactions between Sessile Species Competition for space is the top interaction among sessile species (Dayton 1971: Jackson 1975: Paine 1984). Species compete for primary substrate when they occupy space which was has not been colonized by other species and secondary substrate when they colonize the surface of other species (Luckhurst & Luckhurst, 1978: Aldredge & King, 1984). Competitive interactions for space can take many forms with impacts which can be positive, negative, or neutral (Hairston et. al., 1960: Dayton, 1971: Levine, 1976). In this study, competitive interactions between sessile species occurred which were both negative and positive. Positive interactions have been shown to be beneficial for the community, serving to increase species richness and diversity (Turner et. al., 1966: Connell & Slatyer, 1977: Boucher & Keeler, 1982: Whitman, 1987: Stachowicz, 2001). Positive interactions can include positive instances of epibiosis and facilitation. In positive epibiotic interactions, some species (i.e. basibionts) act as secondary substrate for other species (i.e. epibionts) with resultant increases in the availability of habitable substrate as well as possible increases to fitness (Wahl et. al., 1989: Floerl et. al., 2004: Claar et. al. 2011). In turn, facilitation can occur when species provide nutrients or make it easier for other species to 105 obtain nutrients (Vitousek et. al., 1987). As a mode of facilitation, species can provide physical structure, thereby increasing habitat complexity and providing shelter and refuge for other species (Egler, 1954: Stachowicz, 2001: Bruno & Stachowicz, 2003). The end result of facilitation may be one which allows other species to achieve larger population sizes within a community, thus imparting the beneficial effect of increased genetic diversity (Bertness, 1989: Bruno et. al., 2003: Mooney & Cleland, 2004). In this study, tunicates were the major occupant o f space (Figures 30 & 32). As the dominant occupant of space in a community in which competition for space was paramount, it is of interest to assess the competitive interactions between tunicates and other sessile species. It appeared that positive epibiotic interactions occurred between solitary tunicates and other sessile species through the utilization of M. citrina for secondary substrate space. This interaction was observed to occur betweenM. citrina and several sessile species (e.g.B. simplex, B. turrita, C. pallasiana, M. membranacea, andS. balanoides) including colonial tunicates (e.g. B. schlosseri, B. violaceus, andDidemnum spp.). However, while some species (e.g. B. simplex, B. turrita, C. pallasiana, M. membranacea, andS. balanoides ) utilized M. citrina for substrate it did not result in a greater abundance of these species in the caged community which was dominated byM. citrina (Figures 34, 35, 36, & 38). Nor did it influence species richness, as these species were present in both community types. However, throughout the course of this experiment, primary substrate space remained available throughout panel immersion which likely ameliorated the outcome of competitive interactions for space. In turn, B. simplex, B. turrita, C. pallasiana, M. membranacea, andS. balanoides most often occupied primary substrate which was still readily available. Thus, if more primary 106 substrate space had become occupied, these species may have received quantifiable benefits from their ability to utilize the increased substrate made availableM. by citrina. Facilitation was observed to occur when aggregations of M. citrina were seen to increase habitat complexity and provide refuge for other species.B. simplex, B. turrita, J. marmorata, andH. arctica commonly utilized the structure provided by aggregations o f M. citrina as habitat and refuge.J. marmorata appeared to derive a quantifiable advantage from this interaction, occurring in much greater abundance in theM. citrina dominated community (Figure 37). In fact,J. marmorata were commonly found in close association with M. citrina. Another species which was observed to be using aggregations of M. citrina for habitat refuge wasH. arctica. However, H. arctica was also commonly observed settled between sessile species other than M. citrina, including individual colonies of colonial tunicates. Therefore, facilitative effects from other sessile species may have masked the impact of facilitationM. by citrina, and as resultH. arctica appeared in equal amounts between caged and uncaged treatments (Figure M.38). citrina was commonly found in close proximity to B. simplex andB. turrita and small colonies of these species were found on the surface of M. citrina. In fact, previous studies have found that Bugula species will settle on solitary tunicates (Meyers, 1990); indicating a measure of facilitation between these species. However, in this study, the majority of the populations of B. simplex andB. turrita were observed to be colonizing available primary substrate. In turn, there was more primary substrate available in uncaged treatments due to the fact thatB. violaceus occupied 50% cover in uncaged treatments while M. citrina occupied 70% cover in caged treatments (Figure 56). Thus, confounding effects of available primary substrate space may have allowedB. simplex and B. turrita to be more 107 abundant in uncaged treatments (Figure 34). In contrast, negative interactions in a competition for space can take many forms including: negative epibiotic interactions, exploitation competition, interference competition, and apparent competition. Negative epibiotic interactions can occur when an epibiont decreases the fitness of a basibiont or increases the predation pressure on both basibiont and epibiont (Wahl et. al., 1989: Floerl et. al., 2004: Claar et. al. 2011). Exploitation competition can occur through both direct and indirect inhibition. In direct inhibition, species can release noxious chemicals which inhibit the growth of neighboring species (Muller, 1969: Pickett, 1976: Morris & Wood, 1989). Through indirect inhibition, species can co-opt available nutrients through rapid nutrient uptake which inhibits the growth of other species (Steinwascher, 1978: Sousa, 1979: Menge, 1995). Interference competition can occur when species utilize rapid physical growth in order to occupy of much of the available habitat space (Connell, 1977: Sousa, 1979: Wootton, 1993). Interference competition can also occur when species act as the sole occupant of a unit of space and exert “competitive exclusion” thereby preventing other species from utilizing habitat area (Gause, 1932, 1934: Boaden et. al., 1976: Keough, 1984). While apparent competition can occur due to differences in predation; as when competition between species is altered and enhanced by the differential removal of prey species by predators (Paine, 1969: Hutchinson, 1959: Jeffries, 1984: Dayton, 1971: Vance, 1979). Negative interactions through interference competition commonly occurred between tunicates and other sessile species. Tunicates exerted direct interference competition as they rapidly utilized available substrate space and occupied a dominant proportion of the substrate (Figures 30 & 32). For example, M. citrina increased from 108 <1% to 70% cover in one month and B. violaceus increased from <1% to 10% cover (Figures 48 & 43) Furthermore, M. citrina occupied from 40 to 70% of the substrate at four time points, while B. violaceus was capable of occupying from 40 to 80% of the substrate at several time points (Figures 48 & 43). The ability to increase in abundance and population size in order to occupy more substrate is an important aspect in a competition for space. Many of the tunicates showed frequent increases in percentage cover (Figure 52). Moreover, there were differences between tunicates in their relative abilities to increase in percentage cover, with some species being more competitive in the ability to occupy new space. In particular,M. citrina showed the most frequent increases in percentage cover (Figure 52). Increases in the percentage cover of M citrina were highly common, occurring sixteen times throughout nineteen months of panel immersion (Figure 52). While the colonial species B. violaceus also frequently increased, with fourteen increases in percentage cover (Figure 52). As a solitary species,M. citrina is limited in growth by the rate of sexual reproduction, larval development, and juvenile growth rate (Snell et. al., 1986). While colonial species can reproduce rapidly through clonal growth and colony expansion (Lambert & Lambert, 2003: Lambert, 2005: Carver et. al., 2006). However, despite differences in growth rate between solitary and colonial species,M. citrina more frequently increased in percentage cover than any other tunicate species (Figure 52); indicating that tunicates in general andM. citrina in particular can be highly successful through interference competition in the competition for space. Tunicate abilities towards interference competition were enhanced by the fact that other sessile species did not overgrow or settle on colonial tunicates while colonial 109 tunicates where commonly observed to overgrow and colonize other species. Therefore, colonial species were able to both exert competitive exclusion and to negate other species abilities towards competitive exclusion. As colonial tunicates occupied a large proportion of the substrate in uncaged treatments (<50%: Figure 42), one would expect the abundances of sessile invertebrates to be lower in a community dominated by colonial tunicates. However, abundances were most likely confounded by the amount of available substrate as many species settled on primary substrate and were not directly impacted by competitive exclusion with colonial tunicates. As a result, several species were similar in abundance between treatment types (e.g.M. membranacea, C. pallasiana, A. simplex, A. aculeata, Haliclona spp., andHalichondria spp.: Figures 36, 38, & 39). Other species (e.g. E. larynx, B. simplex, andB. turrita) were actually higher in uncaged treatments (Figure 34). While the solitary tunicate Mcitrina commonly acted as secondary substrate for many species,M. citrina exhibited interference competitive through competitive exclusion towards several species. The solitary tunicateM. citrina did not act as secondary substrate for several sessile species (e.g.A. simplex, A. aculeata, Haliclona spp., andHalichondria spp.). Thus, these species experienced the impact of competitive exclusion in a community in which the substrate was dominated byM. citrina. These species were also unable to utilize colonial tunicates as secondary substrate. As a result, these species (e.g. A. simplex, A. aculeata, Haliclona spp., andHalichondria spp.) showed equal abundances between caged and uncaged treatments (Figures 38 & 39); indicating that the relative abundances o f solitary and colonial tunicates did not influence their abundance. 110 Negative competitive interactions occurred between tunicates. Competitive exclusion was widespread as colonial tunicates occupied much of the substrate and did not act as secondary substrate for other tunicates. In contrast, solitary species did not act as the sole occupant of space and colonial species often colonized the surface of the solitary speciesM. citrina. There also appeared to be bilateral negative competitive interactions as population size increased. In the initial community, Mcitrina was highly abundant (70% cover) whileB. violaceus was present in low amounts (<10% cover: Figures 41 & 42). As the community developed and the population of B. violaceus increased there appeared an inverse relationship in growth: whenM. citrina decreased,B. violaceus increased, whenM. citrina increasedB. violaceus would drop again (Figures 56 & 57); indicating that increased population size in one species caused a decrease in population size in the other. This pattern then appeared to stabilize over time with both species present in reduced amounts than those they had occupied earlier (Figure 56). This reduced population size when in competition was evident in B. violaceus which showed reduced abundances at all time points in caged treatments when compared to uncaged treatments (Figure 55); indicating that competitive interactions with a large population of M. citrina could reduce the abundance ofB. violaceus. Apparent competition can occur due to differences in predation. Competitive interactions between sessile invertebrates do not occur in isolation; instead these species are part of an ecosystem which includes predators. As a result competition between species can be altered and enhanced by the differential removal of prey species by predators (Paine, 1969: Hutchinson, 1959: Jeffries, 1984: Dayton, 1971: Vance, 1979). I ll Moreover, in this study, differences in predation appeared to increase the invasion succession of the invasive colonial tunicate B. violaceus. Differences in predation can act as a mechanism for invasion success as native species are consumed and invasive species (i.e.B. violaceus) flourish in a new habitat due to an absence of predators which will readily consume the invasive species (Williams & Meffe, 1998: Crooks et. al., 1999: Zavaleta et. al., 2001: Eiswerth & Johnson, 2002). In this study, the predators T. adspersus andA. rubens were commonly found at CML pier and were found to consume M. citrina (Table 14). Thus, predation may have altered competitive interactions between M. citrina andB. violaceus through the preferential removal of M. citrina. Competitive interactions between B. violaceus and M. citrina appeared to have a detrimental effect on the abundance ofB. violaceus (Figure 55). Thus, predator removal of M. citrina may have enhanced the success B.of violaceus. B. violaceus was an abundant tunicate in both the benthic and surface biofouling community at this site and has been found to be a common invasive species of biofouling communities (Figures 26, 27, & 42: Otuska & Dauer, 1982: Carmen et. al., 2009: Carmen et. al., 2010). However, in this study,B. violaceus reached higher abundances in habitats which had low abundances ofM. citrina (Figures 55, 26, & 27). Thus, the effects o f apparent competition likely enhanced the success of B. violaceus in some habitat areas through predator removal of the competing species M. citrina. In contrast, the high abundance of M. citrina in the surface biofouling community potentially reduced the abundance ofB. violaceus (Figures 23, 24, & 55). In conclusion, competitive interactions for space took many forms in this 112 community with impacts which were positive or negative. Species interactions were complicated by the fact that much of the primary substrate space remained throughout panel immersion. However, epibiosis and facilitation with the solitary tunicate Mcitrina appeared to benefit other sessile species in the community. Interference competition, competitive exclusion, and differences in predation impacts appeared to have negative impacts on the community. Moreover, differences in predation appeared to increase the invasion succession of the invasive colonial tunicateB. violaceus. Other studies have found that the competition for space was a dominant interaction among sessile species (Dayton 1971; Jackson 1975; Paine 1984). In this study, primary substrate space remained throughout the entire course o f the experiment, thus it was difficult to assess if the competition for space was a dominant interaction among sessile species. However, other researchers have found that when a competing species is removed from the system, the remaining species experience reduced interspecific competition for resources (Paine, 1969: Connell, 1983: Levin et. al., 1996: Chase et. al., 2002). In line with these findings, the tunicate species B. violaceus appeared to experience greater fitness when predators removed the possible competitor M citrina in this study (Figures 56 & 57). Predators as Eco-system Engineers Predators have been shown to work as “eco-system engineers”, modifying habitats through significant impacts on the structure and ecology of communities of organisms (Paine, 1969: Menge et. al., 1994: Jones et. al., 1994). In turn, eco-system engineers often increase community diversity. In this study, predators altered the physical environment of the benthic community through the preferential removal of species, thus altering the 113 available substrate space in a community already operating under heavy competition for space. Autogenic engineers modify their habitat through their physical form. In caged treatmentsM. citrina provided substrate and habitat for other organisms. Predator removal of M. citrina removed the habitat modifications provided byM. citrina from the system, thus potentially decreasing community diversity. Allogenic engineers alter the habitat by changing the physical state of community members (e.g. consuming a prey item). In uncaged treatments predators acted as allogenic engineers through the removal of prey species such asM. citrina. In the benthic biofouling community predators altered the physical environment through the preferential removal of species, thus altering the available substrate space in a community already operating under heavy competition for space. When local and abundant predators (i.e. benthic predator fishT. adspersus, seastarsA. forbesi andA. rubens) removed resident species such as M. citrina, the predators acted as an agent to create habitat space which was then capitalized upon by opportunistic species. Predation can also change community competition dynamics when competing species are predated upon and removed from the system (Lotka, 1920: Volterra, 1931: Bernstein et. al., 1981). Limitations to this Study While extremely important research questions can be addressed with ecological field studies, there are limitations to these studies. Field conditions can be variable and some parameters cannot be isolated or controlled. In this study, disturbance events (i.e. storms) cleared the panel substrate thus “resetting” successional development back to the 114 “starting point”. Subsequent species abundances then reflected changing seasonal dispersal patterns of sessile species and their subsequent recruitment levels on benthic panels. The experimental limitations of variable field conditions turned out to benefit to this study when disturbance events served to further strengthen observed results. It was found that the pattern of community development was highly replicable following disturbance events. As a result, similar patterns of abundanceM. of citrina andB. violaceus between caged and uncaged treatments arose at several time points and in several various seasonal conditions (Figures 41 & 42). Caged predator exclusion methods are often used in ecology to assess the impact of predation on the community. However, predator exclusion cannot identify the predators impacting the community under field conditions. Efforts to address this experimental limitation where twofold: firstly, local predators were collected in an attempt to assess the potential suite of predators present at the site; and secondly, feeding trials were used to assess the consumption of the prey items by those potential predators. The predatory fish species T. adspersus, the invertebrate predatorsA. rubens, A.forbesi, andCarcinus maenas were also found commonly found at the field site. Subsequent feeding trials found that T. adspersus andA. rubens consumed M. citrina', while C. maenas were not found to consume M. citrina (Table 14). However, laboratory feeding trials of wild animals have their own limitations; such as the conservation of natural foraging and feeding behavior in a laboratory setting and possible artifacts of laboratory conditions. Therefore it is not definitive if C. maenas will consume M. citrina under other conditions. Neither is it definitive what the rate of consumption of M. citrina by T. adspersus andA. rubens would be under field conditions. 115 Importance of this Study Within recent decades, invasive tunicate species such as Botrylloides violaceus, Botryllus schlosseri, Ciona intestinalis, Didemnum vexillum, and Diplosoma listerianum have grown to cover much of the benthos (Lambert & Lambert, 2003: Lambert, 2005: Dijkstra et. al., 2007: Greene & Grizzle, 2007). In turn, invasive species can exhibit a variety of negative impacts in their new habitat. Often, invasive tunicates are not consumed by local predators, and this reduced predation pressure then allows invasive species greater success than native species (Keogh, 1984: Osman & Whitlach, 2004, 2007). Invasive tunicates can prevent the recruitment of other invertebrate species, and can overgrow and smother native species of economic interest such as mussels, scallops, and clams (Lutz- Collins et. al., 2009). As a result, the biofouling community has arisen as a major vector in the spread of many non-native marine nuisance species which carry negative economic and environmental consequences (Locke et. al., 2007: Osman & Whitlach, 2007). In this study, T. adspersus and A. rubens were found to consume M. citrina and T. adspersus was found to consume M. citrina more frequently than A. rubens (Table 14: Figure 66). Thus, it is likely that T. adspersus predation on the resident tunicate species M. citrina led to a tunicate assemblage which was dominated by the invasive tunicate B. violaceus (Figure 41). Furthermore, B. violaceus was present in lowered amounts when it co-occurred with M citrina (Figure 55). Therefore, T. adspersus predation of M citrina likely enhanced the invasion success of B. violaceus through the removal of a competing species. As a result, the range of T. adspersus could be used as an indicator of habitat areas which have low abundances of M. citrina and may be more susceptible to invasion by B. violaceus. 116 Future Directions Competitive interactions between Molgula citrina and Botrylloides violaceus should be identified and defined, in order to quantify and assess their impact. Possible competitive interactions could include: reduced levels of recruitment and colonization when the species co-occur, reduced abundances due to a direct competition for physical space, as well as reduced abundances or growth rates due to interference competition such as chemical interaction. 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Invertebrate species included the nudibranch Flabellina verrucosa and the crab Carcinus maenas. Vertebrate species was the benthic fish species Cyclopterus lumpus. Predator Prey Trial duration T rial# Results F labellina verrucosa M olgula citrina 4 weeks 1 No feeding F labellina verrucosa M olgula citrina 4 weeks 2 No feeding F labellina verrucosa M olgula citrina 4 weeks 3 No feeding F labellina verrucosa M olgula citrina 4 weeks 4 No feeding C arcinus m aenas M olgula citrina 4 weeks 1 No feeding C arcinus m aenas M olgula citrina 4 weeks 2 No feeding C arcinus m aenas M olgula citrina 4 weeks 3 No feeding Carcinus maenas M olgula citrina 4 weeks 4 No feeding C ancer irroratus M olgula citrina 4 weeks 1 No feeding C ancer irroratus M olgula citrina 4 weeks 2 No feeding C ancer irroratus M olgula citrina 4 weeks 3 No feeding C ancer irroratus M olgula citrina 4 weeks 4 No feeding C yclopterus lum pus M olgula citrina 4 weeks 1 N o feeding Table 18: F eeding trial results fo r the predators F labellina verrucosa, C arcinus m aenas and C yclopterus lum pus w ith the prey item M . citrina. 19 Predator inclusion feeding trials with the predator Tautogolabrus adspersus and the prey item Molgula citrina were used to determine if T. adspersus would consume M. citrina (Table 19). Year T rial# # T. Size class T. # M . c i t r i n a Percentage a d s p e r s u s per a d s p e r s u s per tank eaten tank 2009 1 2 Large 5 0% 2009 2 3 Medium 5 20% 2009 3 3 Medium 5 0% 2009 4 3 Medium 5 0% 2009- ..... 5 . 4- Medium— 5 - 0%— 2009 6 Large 5 0% 2009 7 3 Medium 5 6% 2009 8 3 Medium 5 60% 2009 9 3 Medium 3 0% 2009 10 2 Large 5 20% 2009 11 Medium 5 60% 2009 12 3 Medium 5 60% 2009 13 3 Medium 5 40% 2009 14 3 Medium 5 80% 2009 15 Medium 5 100% 2009 16 3 Medium 10 40% 2009 17 3 Medium 10 100% 2009 18 Medium 10 90% 2009 19 3 Medium 5 20% 2009 20 3 Medium 10 40% 2009 21 3 Medium 8 37.5% 2009 22 3 Medium 10 40% 2009 23 Large 4 100% 2009 24 3 Medium 4 100% 2009 25 3 Medium 4 100% 2009 26 3 Medium 4 100% 2009 27 3 Medium 4 50% 2009 28 Medium 4 100% 2009 29 3 Medium 10 0% 2009 30 3 Medium 10 0% 2009 31 3 Medium 10 100% 2010 32 3 Large 10 100% 2010 33 3 Large 10 100% 2010 34 3 Large 10 100% Table 19: F eeding trial results. M edium size class = 0-9.99 cm : Large size class = 1 0 - 2 0 c m . 20 Predator inclusion feeding trials with the predator^, rubens and the prey item M. citrina showed that A. rubens would consume M. citrina (Table 20). Trial # o f A . # M. % Trial # o fA. UM. % Eaten Trial # o fA. # M % r u b e n s c i t r i n a Eaten r u b e n s c i t r i n a r u b e n s c i t r i n a Eaten 1 1 4 0% 19 1 4 0% 37 1 4 75% 2 1 4 0% 20 1 4 75% 38 1 4 75% 3 1 4 0% 21 1 4 75% 39 1 4 0% 4 1 4 0% 22 1 4 100% 40 1 4 0% 5 1 4 0% 23 1 4 75% 41 1 4 0% 6 1 9 100% 24 1 4 75% 7 1 9 88.9% 25 1 4 75% 8 1 9 66.7% 26 1 4 75% 9 1 4 0% 27 1 4 75.00% 10 1 4 0% 28 1 4 100% 11 1 4 0% 29 1 4 75% 12 1 4 75% 30 1 4 75% 13 1 4 0% 31 1 4 0% 14 1 4 0% 32 1 4 0% 15 1 4 0% 33 1 4 0% 16 1 4 0% 34 4 0% 17 1 4 0% 35 1 4 0% 18 1 4 0% 36 1 4 0% T able 20: R esults o f all feedin g trials w ith T. adspersu s. 21 Video analysis withAsterias rubens in Y-Maze Prey Choice Experiments betweenM. citrina andMytilus edulis were inconclusive. There was no difference in choice between foraging in a maze arm withM. citrina (Choice: M. citrina) and foraging in a maze arm with M. edulis (Choice: M. edulis) within ten minutes of video duration (t-test, p = 0.133: Table 21). T rial# #o f A. # o f Choice: Choice: r u b e n s prey M, M Table 21: The results o f Y M aze P rey C hoice items e d u l i s c i t r i n a P reference betw een M . citrina and no prey item . per arm Video analysis withX rubens in Y- 1 1 4 Yes No Mfl7P Prsv CVinirf* FYnprim pntc hptw ppn 2 1 4 No Yes 3 1 4 No No M. citrina and M. edulis showed that A choice choice n j h f > n s will hnnrllp and 4 1 4 No Yes 5 3 4 No Yes consume M. citrina ( table 22). 6 3 4 Yes No Table 22: R esults o f video analysis o f A . rubens 7 3 4 Yes No Joraging and Jeedm g behavior. Trial # # o f A # o f # times #M Duration r u b e n s M. M. c i t r i n a o f c i t r i n a c i t r i n a consumed feeding handled (min. & sec.) Glue 1 4 3 0 n/a control 1 1 4 1 0 n/a 2 1 4 0 0 n/a 3 1 4 0 0 n/a 4 1 4 7 0 n/a 5 3 4 1 1 5:33 6 3 4 2 0 n/a 7 3 4 2 0 n/a 22 Feeding trial results showed that there was no preference between aggregated and solitary M. citrina or aggregated and solitaryM. edulis (Table 23). Trial #A. Choice 1 Choice 2 Result Table 23: R esults o f feedin g trials w ith T. r u b e n s a d s p e r s u s . Glue 1 Solitary Glue. No The overall number of times A. rubens cont M . citrina. feeding. rol entered a maze arm containing a 1 1 4 solitary 4 No M . citrina. solitary feeding. particular prey item was also tallied and M. e d u l i s , - differences between prey items were 2 1 4 solitary 4 Consumed M . citrina. solitary M . citrina. compared (Table 24). M. e d u l i s . Tria Predator Prey M M 3 1 4 solitary 4 Consumed h c i t r i n a e d u l i s M c i t r i n a solitary M . citrina. 1 A s t e r i a s M o l g u l a 0 1 M r u b e n s c i t r i n a e d u l i s . 2 A s t e r i a s M o l g u l a 1 0 4 1 Aggregate Aggrega Consumed r u b e n s c i t r i n a d M ted M. both M. 3 A s t e r i a s M o l g u l a 1 0 c i t r i n a . e d u l i s . c i t r i n a r u b e n s c i t r i n a and M. 4 A s t e r i a s M o l g u l a 7 6 e d u l i s . r u b e n s c i t r i n a 5 3 Aggregate Aggrega Consumed 5 A s t e r i a s M o l g u l a 2 2 d M ted M both M r u b e n s c i t r i n a c i t r i n a . e d u l i s . c i t r i n a M o l g u l a and M. 6 A s t e r i a s 10 6 e d u l i s . r u b e n s c i t r i n a 6 3 Aggregate Aggrega Consumed 7 A s t e r i a s M o l g u l a 4 3 d M ted M both M. r u b e n s c i t r i n a c i t r i n a . e d u l i s . c i t r i n a Total 25 18 and M. e d u l i s . 7 3 Solitary 4 Consumed Table 24: R esults o f Y M aze prey choice M . c i t r i n a solitary both M. preference fo r A. rubens betw een M . citrina an d M. c i t r i n a M . e d u l i s . e d u l i s . and M. e d u l i s . 23 APPENDIX B - IACUC PERMISSION University of New Hampshire Research Integrity Services, Office of Sponsored Research Service Building, 51 College Road, Durham, NH 03824-3585 Fax: 603-862-3564 06-Apr-2009 Harris, Larry Biological Sciences, Rudman Hail Durham, NH 03824 IACUC # : 090301 Project: The Impact of a Native Predatory Fish Species, Tautogolabrus adsperus (Cunner) on Local and Invasive Tunacates in the Gulf of Maine Category: C Approval Date:25-Mar-2009 The Institutional Animal Care and Use Committee (IACUC) reviewed and approved the protocol submitted for this study under Category C on Page 5 of the Application for Review of Vertebrate Animal Use in Research or Instruction - the research potentially involves minor short-term pain, discomfort or distress which will be treated with appropriate anesthetics/analgesics or other assessments. Approval is granted for a period of three years from the approval date above. Continued approval throughout the three year period is contingent upon completion of annual reports on the use of animals. At the end of the three year approval period you may submit a new application and request for extension to continue this project. Requests for extension must be filed prior to the expiration of the original approval. Please Note: 1. All cage, pen, or other animal identification records must include your IACUC # listed above. 2. Use of animals in research and instruction is approved contingent upon participation in the UNH Occupational Health Program for persons handling animals. Participation is mandatory for all principal investigators and their affiliated personnel, employees of the University and students alike. A Medical History Questionnaire accompanies this approval; please copy and distribute to all listed project staff who have not completed this form already. Completed questionnaires should be sent to Dr. Gladi Porsche, UNH Health Services. If you have any questions, please contact either Dean Elder at 862-4629 or Julie Simpson at 862-2003. For the IACUC, Jessica A Bolker, Ph.D. Chair cc: File Day, Helen 25