Perforation with and without vinegar injection as a mitigation strategy against two invasive tunicates , Ciona intestinalis and Styela clava

Ciona intestinalis and Styela clava, two nuisance species for Prince Edward Island’s blue mussel industry, were treated with individual perforations using nails or hypodermic needles. Other treatments using the same species included simultaneous perforations using perforation devices with low, medium and high needle density, either with or without vinegar injections. Mortality levels estimated for all ranges of individual perforations were significantly higher than mortality levels estimated in control groups during treatments conducted at laboratory facilities. Mortality of C. intestinalis reached 100% for 60 individual perforations or injection of 0.05 mL of vinegar. In S. clava, 100 individual perforations resulted in 100% mortality. Two applications of the highdensity perforation device resulted in 80% mortality of C. intestinalis. During field testing, two applications of the same high-density needle device did not significantly decrease C. intestinalis wet weight, regardless of the addition of vinegar. The field applicability of perforation upon tunicates fouling mussel socks was at least in part limited by the uneven surface created by the mussels and the possible inhibition of bacterial growth caused by low water temperatures. Perforation and vinegar injection showed to be successful in laboratory trials and should be further studied with different perforation devices under field conditions.

C. intestinalis has immune system elements that are comparable to those of other invertebrates and even some vertebrates (Di Bella & De Leo 2000) in that they trigger an inflammatorylike response to the injection of alien soluble proteins (Parrinello & Patricolo 1984).The formation of a white circular capsule can be observed in the tunic of these species (De Leo et al. 1996;Parrinello & Patricolo 1984).Healing processes can result in damage to the surrounding tissues (Parrinello & Patricolo 1984), the extent of which is dependent on the dosage and nature of the substance injected (Parrinello et al. 1990).By targeting these inflammatory and healing processes, mortality can be caused,         In the flow-through system at the PAL, mortality was significantly higher among C.  1 for details on treatment groupings).Significant differences between groups are indicated by different letters.

Effect of perforation with or without vinegar application in the field
On average, the wet weight of C.
intestinalis perforated with or without vinegar application was 28 % and 51 % lower than on control socks, respectively (Fig. 5), but this difference was not significant  1983).Leukocytes include phagocytes (Wright & Cooper 1983) which are involved in the healing process of injected substances in C.

Applicability of perforation in the field
The field application of the high-  1981).Normally, the encapsulation is followed by a melanization healing process (De Leo et al. 1996;1997).If an exaggerated reaction from the lysozymes and granulocytes occurs as a result of the alien substance injected in the tunic, severe damage to the tissue is expected (Parrinello et al. 1984).For example, Davies (1991) suggested that fish, Atherina

Differences in
the survival of C. intestinalis and S. clava.The second part evaluated the efficacy of perforation with or without Introduction, Hypotheses and Problems for Management Fouling of aquaculture gear by two solitary tunicates, Ciona intestinalis (L., 1758) and Styela clava (Herdman, 1881), has negative effects on the blue mussel (Mytilus edulis L., 1758) farming industry on Prince Edward Island (PEI), Canada. of individual perforations were significantly higher than mortality levels estimated in control groups during treatments conducted at laboratory facilities.Mortality of C. intestinalis reached 100% for 60 individual perforations or injection of 0.05 mL of vinegar.In S. clava, 100 individual perforations resulted in 100% mortality.Two applications of the highdensity perforation device resulted in 80% mortality of C. intestinalis.During field testing, two applications of the same high-density needle device did not significantly decrease C. intestinalis wet weight, regardless of the addition of vinegar.The field applicability of perforation upon tunicates fouling mussel socks was at least in part limited by the uneven surface created by the mussels and the possible inhibition of bacterial growth caused by low water temperaturescontrast, holding tanks at the PAL were fed by flow-through seawater from the adjacent harbour (13.7-22.4ºC and 27-29 ppt) without additional aeration or feeding.Lighting at both facilities was controlled according to the daylight patterns of the season.Only healthy tunicates were selected for the trials.Tunicates

intestinalis and 4
to 10 for S. clava.Treatments were repeated in replicate trials to increase sample size.Because of limited success in obtaining and sustaining healthy adult S. clava throughout the study, not all replicate trials could be hold the needles in position.Lids were connected with screws at opposing cornersto assess mortality after 7 days.Mortality was defined as a change in tunic colour and decomposition of the tunicate

Figure 3 .
Figure 3. Average (+1 SD) cumulative mortality of C. intestinalis 1 wk after perforation with 20 G needles ranging from 0 (control) to 100 individual perforations (see Table1for details on treatment groupings).Tunicates were held in recirculating artificial seawater at the Atlantic Veterinary College.Significant differences between groups are indicated by different letters.

Figure 4 .
Figure 4. Average (+1 SD) cumulative mortality of C. intestinalis held in a flow -through system (Portable Aquatic Laboratory) 1 wk following perforation with a 20 G hypodermic needle.Perforations were applied with a single needle on individual tunicates, ranging from 0-50 repeated individual perforations (see Table1for details on treatment groupings).Significant differences between groups are indicated by different letters.

Figure 5 .
Figure 5. Wet weight of C. intestinalis on 15 cm mussel sock sections one week after treatment.Five mussel sock sections each were either left untouched (control), or treated twice with a high-density perforation device with or without vinegar application.Box blot depicts sample minimum, 1 st quartile, median, 3 rd quartile and maximum.
density perforation device with or without vinegar on tunicate-fouled mussel socks reduced C. intestinalis biomass but did not result in any significant differences compared to the control group.The low sample size (n = 5) along with the inherent the site (De Leo et al.
needle density was limited by the diameter of the needle base.This connector must either be removed or a different type of needle must be used to increase the needledensity of the device.Alternatively, using three or more applications of the high-density device would increase mortality levels as shown in variability cutting boards used as surfaces in laboratory tests, the surface of a mussel sock is uneven and with multiple crevices between mussels.This factor alone likely limited the number of actual perforations into the C. intestinalis tunic.While the perforations could have still weakened the tunicates and potentially lead to mortality, a trial duration of over a week might have been necessary to detect a slower mortality rate.Second, time of the year with its associated low temperature may also have been a factor in the reduced mortality observed during the field trial. of vinegar that is not introduced into the visceral mass of the tunicate would not have a negative impact on the epifauna.

(
Hepsetia) boyeri Risso, 1810, can perceive the presence of acids in the water and swim away from the source, thus preventing injury.The same applies to mobile epifauna but not necessarily to epifaunal species that attach to the mussel socks and cannot swim away.acetic acid spray or dip for 1 minute followed by rinsing in seawater resulted in 100 % mortality of C. intestinalis (Carver et al. 2003).Immersion of S. clava in 5% acetic acid for 1 et al. 2009; Paetzold et al. 2008).The injection of vinegar directly into solitary tunicates, C. intestinalis and S. clava, reduces the dispersion of acetic acid on the mussel sock.Limiting the mortality of other epifauna is beneficial in that it may control the settlement of invasive species by favouring the settlement of competitive species the needles, for example springs.Such an adaptation would buffer the needles if they hit a hard substrate such as mussel shells but would allow the piercing of soft tissues such as the tunic of C. intestinalis.treatment would eliminate the need of lifting mussel lines from the water, thus reducing substantial labour costs.

Table 1 .
Number Figure 1.Approximate location of the field site (St.Marys Bay) and the laboratories (AVC in Charlottetown and PAL in Georgetown) were the perforation trials were conducted.of individual perforations applied to adult C. intestinalis either above or underwater in re-circulating tanks at the Atlantic Veterinary College (AVC) or in flow-through tanks at the Portable Aquatic Laboratory (PAL).Treatment groupings indicate which perforation frequencies were pooled for statistical purposes, so that the sum of the number of replicate trials is equal to the sample size per treatment grouping (e.g. for AVC 0<x≤30, N=8). a Perforations applied above water; b Perforations applied below water

Table 2 .
Needle density and the approximate number of needles perforating each Ciona intestinalis during the application of multiple simultaneous perforations of C. intestinalis using various devices.Treatments were performed at the Portable Aquatic Laboratory.

Table 3 .
Individual and multiple perforations of C. intestinalis with vinegar.Treatments were performed at the PAL.

Table 4 .
Individual perforations of adult S clava.All perforations were performed out of water.

Table 5 .
Multiple perforations of S. clava with vinegar.Treatments were performed at the PAL.ISSN 1989-8649

Table 6 .
C. intestinalis mortality caused by the application of perforation devices (low, medium and high) once, twice and in conjunction with vinegar.For application of the high-density perforation device with vinegar injection, 0.2 mL was taken in a syringe attached to each needle of the device.The number of tunicates in each trial is indicated by n.