Coldwater reattachment of colonial tunicate Didemnum vexillum fragments to natural ( eelgrass ) and artificial ( plastic ) substrates in New England

The colonial tunicate Didemnum vexillum Kott, 2002, was introduced to New England in the 1980s and by 2000 it was widespread. This highly invasive species spreads by larval release and fragmentation. We tested the ability of D. vexillum fragments to reattach to natural (eelgrass Zostera marina (Linnaeus, 1753)) and artificial (plastic container) substrates during late fall and early winter. On average, 77% of D. vexillum fragments reattached to eelgrass and plastic in water temperatures between 6 and 10°C. Eelgrass appeared to facilitate D. vexillum reattachment success in early winter but this tendency should be further investigated.


Introduction
The non-native colonial tunicate (Ascidiacea) Didemnum vexillum Kott, 2002 is commonly found in New England on natural and artificial substrates in coastal marine and offshore habitats (Carman and Roscoe 2003;Pederson 2005;Bullard et al. 2007a;Dijkstra et al. 2007;Valentine et al. 2007a, b).Introduced to New England in the 1980s D. vexillum is a rapid global invader (Lambert 2007), probably native to Japan (Stefaniak et al. 2009;Lambert 2009;Stefaniak et al. 2012), that has been documented in New Zealand, along the east and west coasts of the US, the west coast of Canada and in Alaska and Europe (Coutts and Sinner 2004;Bullard et al. 2007a;Minchin and Sides 2006;Gittenberger 2007;Cohen et al. 2011;Tagliapietra et al. 2012) and it will likely continue to spread via multiple vectors to other locations (Herborg et al. 2009).The impact of this invasive species can be significant as it fouls boat hulls, docks, mooring lines; smothers bivalves and other sessile marine invertebrates; and causes economic hardship for shellfish aquaculture (Coutts and Sinner 2004;Valentine et al. 2007b;Carman et al. 2010;Adams et al 2011;Switzer et al. 2011;Rolheiser et al. 2012).
D. vexillum spreads by release of larvae and by fragmentation.Fragmentation is a dispersal mechanism that occurs when pieces of colonies are removed by the colony naturally pinching-off tendrils, by human disturbance and by the effects of storm events (Lacerda et al. 2002;Boyle et al. 2006;Clarke Murray et al. 2012;Reinhardt et al. 2012).Tunicate colonies with brooded larvae (such as D. vexillum) that are attached to broken eelgrass can raft for miles and thus be dispersed into new habitats (Worcester 1994).
If a D. vexillum fragment settles on a suitable substrate, it can reattach.In several earlier experiments, fragments of D. vexillum demonstrated the ability to reattach to hard artificial substrate (PVC settling plates or plastic flow-through containers) and grow by asexual reproduction in southern New England during mid-summer (Stoner 1989;McCarthy et al. 2007;Bullard et al. 2007b;Valentine et al. 2007a;Reinhardt et al. 2012) and early fall (Morris and Carman 2012).Small fragments of D. vexillum tolerated suspension in the water column for weeks without reattaching, suggesting that it can be transported great distances by tidal and storm currents (Morris and Carman 2012).
Eelgrass Zostera marina (Linnaeus, 1753) is a valuable coastal species that often co-occurs with D. vexillum.Eelgrass is a protected species in New England and elsewhere that serves many important functions, including providing habitat for shellfish and fish.D. vexillum and other tunicates including Botryllus schlosseri (Pallas, 1776), Botrylloides violaceus Okra, 1927, Diplosoma listerianum (Milne-Edwards, 1841) and Molgula manhattensis (Dekay, 1843) were recently documented attached to live and detached pieces of eelgrass in Lake Tashmoo on Martha's Vineyard (Carman and Grunden 2010).Larvae of B. violaceus settle on eelgrass (Worcester 1994) and larvae of other tunicates, including D. vexillum, probably also settle on eelgrass.It is unknown however if fragments of D. vexillum will reattach to eelgrass especially in late fall and early winter when D. vexillum colonies begin to show signs of slow growth and regression (Valentine et al. 2007a).We also wondered if the presence of eelgrass would facilitate D. vexillum fragment reattachment to solid surfaces.The water temperature range for D. vexillum fragment reattachment has been 16 to 22°C (Morris and Carman 2012).The purpose of this study was to determine experimentally whether D. vexillum can reattach to eelgrass and plastic substrates in water temperatures lower than 16°C.

Methods
During late fall 2012, healthy-looking colonies of D. vexillum were collected from cultured blue mussels and aquaculture gear in Lagoon Pond at Oak Bluffs on Martha's Vineyard, Massachusetts.Colonies were cut into pieces (about 4 cm 2 ) and placed in 7 labeled, flow-through plastic containers.The containers were made from covered Petri dishes, 9cm in diameter and 1.5cm deep, with 5 holes about 0.5cm in diameter in the lid and bottom of the container so that there were 10 holes per container.Blades of green and brown eelgrass were collected nearby from drift on the shore and 2 blades were placed in half of the containers (4 of the 7 sample containers for the first trial) containing D. vexillum.Controls included 5 flow through plastic containers (covered containers were 9cm in diameter and 5cm deep with 5 holes about 0.5cm in diameter in the lid and bottom of the container so that there were 10 holes per container) with a single adult mussel that had previously attached D. vexillum.In addition there were 2 identical containers with just 2 blades of eelgrass inside.Mussels with previously attached D. vexillum were used as controls because they served as a source for D. vexillum fragments for the experiment.
All containers were randomly placed on a horizontal line secured to the Martha's Vineyard Shellfish Group (MVSG) dock on Lagoon Pond (41.45°N, 70.6°W) and maintained at between 1.0m and 1.5m water depth.Water temperature and salinity were measured at the beginning and end of the each of the 3 trials.Tidal range in the area was less than 1m.
At the end of November after 15 days in the water, during which water temperature cooled 2-3°C, containers were retrieved from the dock and examined in the laboratory at MVSG.Reattachment success was evaluated by using a plastic pipette to gently squirt the presumably reattached fragment with ambient seawater followed by submerging the fragment in a tub of seawater.If the fragment moved or floated, it was considered not attached.If a fragment was deemed attached, the substrate type was noted (container, eelgrass, or container and eelgrass) and any new growth was measured using a transparent cm-grid.This process was repeated twice, using freshly harvested D. vexillum fragments and with larger numbers of samples in November (n=39) and December (n=24), so that there were 3 sets of samples.The number of fragments used in the 3 trials varied because we had initially expected fragments to not reattach but when they did, we continued the experiment and collected new fragments for a second and third trial at colder water temperatures.The number of days that sample trials were in the water varied because of weather conditions.After inspection of the first 2 sets of samples, some reattached fragments were returned to the water and allowed to continue to grow.

Results
Water temperatures ranged between 3 and 10°C and salinity remained at 31 throughout the experiment period (Nov 15 to Jan 8).
After the first set of samples were in the water for 15 days, 100% of the D. vexillum fragments were reattached in sample containers with and without eelgrass (Figures 1, 2).During this period water temperatures ranged from 8 to 10°C and new growth occurred in all of the samples.The average amount of new growth was 1.5cm 2 and the greatest amount of new growth was 4cm 2 .Four of the reattached fragments were returned to the water and allowed to continue growing.
After the second set of samples were in the water for 20 days, 77% of the D. vexillum fragments were reattached (Figure 1).In sample containers without eelgrass 79% of the fragments were reattached to the container.In sample containers with eelgrass 75% of the fragments were reattached: 40% to only the container, 10% to only eelgrass and 25% to container and eelgrass (Figure 2).During this period water temperatures   ranged from 6 to 8°C and new growth occurred in 64% of the samples.The average amount of new growth was 0.9cm 2 and the greatest amount of new growth was 3cm 2 .Fragments reattached to eelgrass and the inside of the containers (Figure 3) and in a few samples, fragments reattached to the inside and grew through the holes to the outside of the container (Figure 4).Eight reattached fragments were returned to the water dock and allowed to continue to grow.
After the third set of samples were in the water for 19 days, 71% of the D. vexillum fragments were reattached (Figure 1).In sample containers without eelgrass 58% of the fragments were reattached to the container.In sample containers with eelgrass 83% of the fragments were reattached: 25% to only the container, 25% to only eelgrass and 33% to container and eelgrass (Figure 2).During this period water temperatures ranged from 3 to 6°C and new growth occurred in 29% of the samples.The average amount of new growth was 0.1cm 2 and the greatest amount of new growth was 0.25cm 2 .Fragments were often not as robustly reattached as they were for the first 2 trials and were often only reattached by a small portion of the fragment.Fragments reattached and grew on green eelgrass (Figure 3) and on brown eelgrass (Figures 4, 5).
All but one of the samples of reattached D. vexillum fragments that were allowed to continue growing from the first (n = 4) and second (n = 8) trials remained attached to the same substrate.The exception was a fragment originally reattached to both container and eelgrass but on January 8 was only attached to eelgrass.The greatest amount of new growth in continuing-growth fragments was 1cm 2 in water temperatures that ranged from 3 to 8°C.
D. vexillum colonies attached to mussels in control containers remained attached and healthy in all 5 of the control samples for each of the 3 trials (n=15 total); no new growth laterally or volumetrically was observed.During the experiment, no D. vexillum was observed in the 2 control samples (eelgrass only in plastic containers) for each of the 3 trials (n=6 total).

Discussion
This study demonstrated that D. vexillum fragments can reattach to natural and artificial substrates in water temperatures between 6 and 10°C.The water temperature at the cessation of larval release by D. vexillum in fall is between 9 and 11°C at a nearby site (Valentine et al. 2009).Therefore, during our experiment containers were not examined for larval recruits because water temperatures were <10°C.
The presence of eelgrass appeared to facilitate reattachment success in the third trial but not in the first and second trials.At the conclusion of the first 2 trials (on Nov 30 and Dec 20), the percent of reattachment fragments in containers without eelgrass was similar to containers with eelgrass but on January 8 the percent of reattached fragments in containers with eelgrass (83%) was greater than in containers without eelgrass (58%).The presence of eelgrass or other natural substrate may enhance the ability of D. vexillum to reattach in cold water especially in early winter.However, this tendency should be further investigated.
Earlier studies documented the successful reattachment of fragments of D. vexillum on artificial substrates in summer and early fall in water temperatures ranging from 22 to 16°C (McCarthy et al. 2007;Bullard et al. 2007b;Valentine et al. 2007a;Morris and Carman 2012).This study extends the lower temperature range to at least 6°C, perhaps lower.During the third trial, reattachment occurred while water temperatures ranged between 3 and 6°C but we do not know exactly when the reattachment occurred.The ability of D. vexillum to reattach at water temperatures <6°C needs to be tested directly.The D. vexillum fragment reattachment success declined with declining water temperature and suggests that colder temperatures negatively influenced fragment reattachment success.However, reattached fragments that were allowed to grow remained attached as water temperatures dropped.Thus, it may be easier for a fragment to stay attached than it is for a new fragment to reattach in cold water.As well, the growth rate of reattached fragments declined with declining water temperatures.
Fragmentation of D. vexillum may encourage growth along the incised edges of the colonies.In contrast to the observed growth of fragments in our experimental containers, we observed no growth by the D. vexillum colonies attached to mussels in the control samples.Disturbance at the colony edge might be a requirement for colony growth at the low temperatures experienced during this study period.
Rafting plant debris should be considered as a dispersal mechanism for D. vexillum.Fall is a period of natural shedding and rafting of Zostera leaves and a period of storms (end of hurricane season and beginning of nor'easters).The possibility of long distance dispersal of D. vexillum on eelgrass by rafting occurs in fall and would seem to extend well into winter.The ability of this highly invasive species to reattach and grow in cold water and to be dispersed during fall and early winter contributes to its potential spread.

Figure 1 .Figure 2 .
Figure 1.Percent of D. vexillum fragments that were reattached in the first set of samples (Nov 15 to Nov 30), second set of samples (Nov 30 to Dec 20), and third set of samples (Dec 20 to Jan 8), in containers without eelgrass and containers with eelgrass.Blue bars are containers without eelgrass and white bars are containers with eelgrass.

Figure 3 .
Figure 3. Fragment of D. vexillum reattached to and growing on the inside of the plastic flow-through container and on green eelgrass; December 20, 2012; cm grid in background (Photo credit: D. Blackwood).

Figure 4 .
Figure 4. Fragment of D. vexillum reattached to and growing on the inside and outside of the plastic flow-through container (through a hole in the container); December 20, 2012; cm grid in background (Photo credit: D. Blackwood).

Figure 5 .
Figure 5. Fragment of D. vexillum reattached to and growing on brown eelgrass; January 8, 2013; cm grid in background (Photo credit: D. Blackwood).