Botryllid Tunicates: Culture Techniques and Experimental Procedures

Home , Botrylloides, Botrylloides violaceus, Botryllus, Botryllus schlosseri

Special issue “Proceedings of the 2nd International Invasive Sea Squirt Conference” (October 2-4, 2007, Prince Edward Island, Canada) Andrea Locke and Mary Carman (Guest Editors) Research article

Botryllid tunicates: Culture techniques and experimental procedures

Anya Epelbaum1*, Thomas W. Therriault1, Amber Paulson2 and Christopher M. Pearce1,2 1Fisheries and Oceans Canada, Pacific Biological Station, 3190 Hammond Bay Road, Nanaimo, British Columbia, V9T 6N7 Canada 2Vancouver Island University, Department of Fisheries&Aquaculture, 900 Fifth Street, Nanaimo, British Columbia, V9R 5S5 Canada * Corresponding author E-mail: [email protected]

Received 14 January 2008; accepted for special issue 19 April 2008; accepted in revised form 15 December 2008; published online 16 January 2009

Abstract

Botryllid tunicates have become increasingly important as model experimental organisms in a variety of biological and ecological fields, including invasion ecology. This paper summarizes existing botryllid culture methods and offers new ones for culturing Botryllus schlosseri (golden star tunicate) and Botrylloides violaceus (violet tunicate) under laboratory conditions. Techniques for maintaining adult colonies, obtaining larvae, and establishing juvenile cultures are presented. Considerations for designing laboratory experiments using botryllid tunicates are discussed.

Key words: Botrylloides, Botryllus, culture techniques, golden star tunicate, violet tunicate

Introduction seasonal variations in colony availability, growth rate, and sexual reproduction, and makes it Botryllid tunicates have become increasingly possible to conduct long-term experiments year important as model experimental organisms in a round. Laboratory cultures also make it possible variety of biological fields such as immunology, to directly and accurately assess the effects of physiology, ecology, genetics, developmental various physical, chemical, and biological biology, evolutionary biology, and aging (e.g., factors on colony survivorship, growth, and Boyd et al. 1986; Rinkevich and Shapira 1998; reproduction. Manni et al. 2007 and references therein). Several previous studies have cultured Golden star tunicate, Botryllus schlosseri Pallas, ascidians under directly-controlled laboratory 1766 and violet tunicate, Botrylloides violaceus conditions (Berrill 1937; Grave 1937), including Oka, 1927 – which most likely originated in the botryllid tunicates (Berrill 1937; Grave 1937; Mediterranean Sea and the northwest Pacific, Milkman 1967; Sabbadin 1971 and earlier respectively (Berrill 1950) – have invaded much studies cited therein; Boyd et al. 1986; of the north Pacific, north Atlantic, and the Rinkevich and Weissman 1987; Grosberg 1988; Mediterranean and are becoming a growing Rinkevich and Shapira 1998). However, most of concern in areas of expanding aquaculture these studies focused on specific experimental (reviewed in Lambert 2001, 2005). The ability aspects and described culture methods only of these species to spread rapidly to new briefly. Boyd et al. (1986) provided the most environments and become major components of detailed description of laboratory methods and fouling communities impose a need for more conditions for culturing colonies of B. schlosseri, knowledge of their environmental tolerances and but knowledge gaps still exist. The primary interactions with native species, as well as of the objective of this paper is to summarize existing potential for controlling their spread. For each methods and offer new techniques for culturing of these types of studies, maintenance of healthy botryllid tunicates under controlled laboratory laboratory cultures allows circumventing conditions for various experimental purposes.

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Culture methods collect fragments of flat colonies as they generally have better re-attachment capabilities I. General description of tunicates studied than those forming thicker clumps or lobes (Milkman 1967; A. Epelbaum pers. obs.). Our study focused on two non-indigenous Fragments can be glued to the substratum with botryllid species (B. schlosseri and B. violaceus) plastic surgery glue (Dijkstra et al. 2007) or left now common in the Strait of Georgia, British to re-attach naturally, either by securing Columbia, Canada. Both are sessile colonial (Grosholz 2001; McCarthy et al. 2007) or simply tunicates that grow as thin, flat, encrusting mats placing (Bullard et al. 2007, our study) them on or as irregular lobes, depending on the sub- the substratum. We found that securing colonies stratum and colony age. Colonies are composed with rubber bands often damaged zooids, while of morphologically identical individuals (zooids) placing them on the substratum (glass slides) and that are embedded in a common tunic and share a adjusting the water flow – so that they remained circulatory system. Both species exist in a vari- in place and were not dislodged – allowed most ety of colour morphs. Colonies of B. violaceus fragments to re-attach within 24 hours, after are a solid colour (usually purple, pink, yellow, which time they began spreading along the or orange), while B. schlosseri often has two- substratum naturally (Figure 1). toned zooids (predominant colours being orange/brown and black/green). Colonies grow by synchronous asexual multiplication (budding of the zooids) and propagate through sexual reproduction and occasionally through colony fragmentation. Like other colonial botryllids, both species are hermaphrodites and brood embryos inside the parent colony. Their tadpole larvae settle within a day and metamorphose into the first zooid (oozooid) of a new colony. Oozooids produce a set of asexual buds that differentiate into new zooids (blastozooids), which in their turn bud synchronously, providing colony growth (Grosberg 1988). Botryllus schlosseri and B. violaceus differ in gonad arrangement, embryo/larval incubation, and some morphological aspects related to the Figure 1. Fragment of B. violaceus colony growing on a digestive system (Brunetti 1979). Species of the glass slide (six days following re-attachment). genera Botryllus and Botrylloides exhibit similar growth patterns and some have been shown to have similar environmental tolerances (Brunetti III. Larval and juvenile cultures et al. 1980). Therefore, culture techniques for B. Obtaining larvae and establishing juvenile schlosseri and B. violaceus are similar, specific colonies: The duration of the breeding season in differences being noted below. botryllid tunicates varies with temperature. Millar (1971) summarized breeding records for II. Establishment and maintenance of adult B. schlosseri from Naples, Italy (January – (broodstock) cultures December); Venice, Italy (April – November); Brittany, France (June – October); and Millport, Botryllid tunicates are often abundant on Scotland (June – September). Along the east artificial structures in harbours (e.g., wharves, coast of Vancouver Island, Canada, B. schlosseri marinas, docks, pilings, lines, buoys, boat hulls) and B. violaceus generally produce larvae from and at aquaculture sites (e.g., cages, trays, floats, early June to late September (A. Epelbaum pers. lines, anchors). Fragments of colonies can be obs.). gently scraped from the substratum and Colonial ascidians are known to release larvae transported to the laboratory in coolers with in response to light stimulation (Cloney 1987). ambient seawater. When experimental design The time of release can be controlled by keeping requires re-attachment of fragments to certain the colonies in the dark for several hours before substrata (e.g., slides or tiles) it is better to the larvae are needed (Watanabe and Lambert

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1973). To stimulate release of larvae, brooding metamorphosis. Larvae that have settled in such colonies can also be gently torn into small a way can be gently collected by pipette, placed fragments (~8 cm2) (Marshall et al. 2006). individually in small drops of water in the Botryllus schlosseri and B. violaceus that we desired location on the target substratum, and collected along the coast of Vancouver Island in arranged so that their papillae (ampullae) are June and kept in the laboratory on a 16h light/8h facing downwards. In the current study, using dark cycle (daytime light intensity 1000 lux, this method, 536 out of 540 larvae of B. ‘dawn’ at 5:30 AM) released larvae in batches violaceus immediately attached to the desired every morning for approximately one week, substratum (glass slides), successfully meta- mostly between 10:00 and 11:00 AM. morphosed into oozooids, and started developing Free-swimming tadpole larvae usually remain blastozooids (Figure 2). in the water column for 2–12 hours (Grave 1937) and do not feed. Offspring size can be measured as the head length of the larva or as the settler’s branchial basket area (Marshall and Keough 2003, Marshall et al. 2006). To make larvae settle on the desired substrata, the latter can be arranged in the culture tank facing parental colonies. Newly-settled oozooids that have attached to non-target substrata may be carefully removed by hand and transferred to the desired location (Berrill 1937; Boyd et al. 1986; Rinkevich and Weissman 1987). A different method of ensuring larvae settle on the desired substratum was described by Karande and Nakauchi (1981) for Aplidium multiplicatum. Tadpole larvae were placed into tap or distilled water for 20 seconds, where they became motionless, and then were immediately released close to the surface of glass slides submerged in normal seawater. Larvae adhered to the glass surface immediately if the freshwater ‘shock’ Figure 2. Oozooid of B. violaceus (24 hours following was adequately timed; seven out of every 10 attachment). tadpoles were reported to successfully settle when this technique was applied (Karande and Nakauchi 1981). This method allows for minimal handling of We attempted to develop a technique that oozooids, which favours their proper develop- would make juvenile colonies grow in the ment. However, it is less effective for obtaining precise location desired, while ensuring minimal young colonies of B. schlosseri as oozooids of handling of the animals. In preliminary trials, this species are almost transparent and much we arranged 60 glass slides horizontally in trays smaller than those of B. violaceus (branchial with seawater (2 cm deep), put a small ring of basket length ~0.3 mm, as opposed to ~1 mm), PVC pipe over the centre of each slide so that making their proper positioning on the desired the top of the ring was above the water surface, substratum more difficult. We found that larvae and placed a single tadpole larva inside each ring of B. schlosseri could be easily collected by (i.e., 60 larvae in total). This method was not suspending glass slides vertically, parallel to efficient, however, as 32 larvae (>50%) settled each other, just below the water surface in the on the sides of the PVC rings rather than the larval containers. In 24 hours, slides could be desired substratum. In subsequent trials, we retrieved and inspected for the presence of developed another method of ensuring that oozooids, with all unwanted oozooids being larvae settle on the desired substratum. Larvae of removed. both species are positively phototactic (Grave Feeding juvenile colonies: After larval settle- 1937) and thus, when held in a dark-sided tank ment and metamorphosis, the oozooid acquires illuminated from the top, most will settle, atrial and branchial siphons and begins feeding inverted, at the water surface and begin within one to two days (Takeuchi 1980). Several

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Table 1. Diets used in botryllid tunicate culture.

Food Concentration Tunicate species Outcome of Study Source Natural seawater, coarsely N/A Tunicates in N/A Berrill 1937 filtered general Cyclotella nana 5 to 25x107 cells/L B. schlosseri Good growth, up to three Milkman 1967 buds per zooid Mixture of Phaeodactylum Not specified B. schlosseri Juvenile and young adult Brunetti et al. 1980 tricornutum, Dunaliella sp., and B. leachi colonies exhibited positive Chlorella sp. growth at a number of temperature and salinity combinations Liquifry Marine (Interpret Ltd., 3.8 to 60 µL/L B. schlosseri Juvenile colonies exhibited Boyd et al. 1986 Dorking, UK) positive growth at a number of temperature and salinity combinations 1:1 mixture of Dunaliella 2.5 to 5x107 B. schlosseri High mortality and poor Boyd et al. 1986 tertiolecta and Phaeodactylum cells/L each growth tricornutum Marine Invertebrate Diet 0.1 to 0.3 ml per B. schlosseri Juvenile colonies exhibited Grosberg 1988 (Aquarium Products Inc., 1000 zooids positive growth at 15–25ºC Houston, USA) Invertfood (Waterlife Research Not specified B. schlosseri The use of all diets alone Rinkevich and Ltd., Longford, UK) and in combinations Shapira 1998 Hatchfry Encapsulation 50-150 7.5 g/L resulted in positive growth µm (Argent Chemical Lab, of juvenile colonies Redmond, USA) 7.5 g/L Artificial Plankton (Argent Chemical Lab, Redmond, 7.5 g/L USA) Dried Algae Algal 161 (Celsys, 7.5x1010 g/L total Cambridge, UK) Mixture of Nannochloropsis sp., 7.5 g/L Dunaliella salina, and Isochrysis galbana Freeze-dried rotifers Brachionus plicatilis Liquifry Marine (Interpret Ltd., 30 µL/L B. schlosseri Diets were alternated. Our study Dorking, UK) B. violaceus Juvenile colonies exhibited MicroVert (Kent Marine Inc., 30 µL/L positive growth at 10–25ºC. Acworth, USA) B. schlosseri released the 1:1 mixture of Chaetoceros 5x107 cells/L each first batch of larvae in 45 to muelleri and Isochrysis sp. 50 days after settlement at (Tahitian strain, T-iso) 20ºC

types of diets have been previously used in three diets resulted in exponen-tial growth of botryllid culture, with different levels of success juvenile colonies of both species; colonies of B. (Table 1). In these studies, tunicates were schlosseri attained sexual maturity and released cultured for different purposes, and thus life- their first batch of larvae within 45 to 50 days at history parameters and the ways they were 20ºC. registered differed as well. This makes direct Physical setup and culture conditions for comparison of diet performances among the juvenile colonies: Artificial substrata with newly studies rather difficult. attached oozooids need to be kept horizontally in Rinkevich and Shapira (1998) demonstrated standing seawater for about two days in order to that a mixture of several diet types was superior let oozooids attach firmly. After this time, the to any monotype diet. In our culture experiments substrata can be transferred to experimental/ we fed animals every other day with three culture containers where they may be arranged alternating diets: Liquifry Marine, MicroVert, aslant, with the young colonies facing down and a 1:1 (by cell number) mixture of two (Milkman 1967), or vertically (e.g., in the slots species of microalgae (see Table 1). Use of these of glass staining racks as done by Boyd et al.

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(1986)). Such arrangement ensures that faecal pellets, debris, and fouling algae are less likely to accumulate on or around the colonies. However, regular cleaning of colonies might be required (Boyd et al. 1986; Rinkevich and Shapira 1998), especially when natural seawater is used. When algal growth on or around the colonies became considerable, we observed colonies growing away from algae-covered areas Figure 3. Colonies of (a) B. violaceus and (b) B. schlosseri by forming buds towards the unfouled space in growing away from fouled areas on glass culture slides. Arrows show directions of colony growth. each asexual growth cycle (see Figure 3). Cleaning needs to be done gently with something like a soft, small paintbrush to ensure zooids do not get damaged. In our studies, we suspended glass slides with juvenile colonies vertically from the top of the culture containers using rods and clips (e.g., all- plastic cloth pegs or badge clips) (Figure 4). This technique proved to be quite useful as it allowed unrestricted water movement, minimized the surface area to be cleaned, and simplified handling and transfer of slides to new containers. Aeration, if employed, requires careful implementation as faecal pellets and detritus suspended in the water column might cause reflex cessation of pumping, which negatively affects feeding efficiency (Milkman 1967). In all of our experiments we used containers with static seawater and no aeration. When colonies are cultured in static seawater, evaporation needs to be controlled or compensated for. To control evaporation, Milkman (1967) suggested covering culture vessels with polyethylene covers through which oxygen and carbon dioxide can pass and water vapour can not. However, to the best of Figure 4. Sample culture container with vertically- our knowledge, this option has never been suspended colonies of B. violaceus attached to glass slides. experimentally tested in tunicate culture. Both species possess broad temperature and salinity tolerances. In our experiments, young between 5 and 25ºC and salinities between 20 colonies of B. schlosseri survived environmental and 38‰. Best growth of both species was conditions between 10 and 25ºC and 14 and observed at 20ºC and 32‰. These results are 38‰, while B. violaceus tolerated temperatures similar to those reported by Brunetti et al. (1980) between 5 and 25ºC and salinities between 20 for B. schlosseri from the Venetian Lagoon, and 38‰. Best growth of both species was Italy. observed at 20ºC and 32‰. These results are The frequency of water exchange largely culture vessels with polyethylene covers through depends on the size of the culture container, which oxygen and carbon dioxide can pass and colony size, temperature, and type of food used. water vapour can not. However, to the best of When juvenile colonies (1–40 zooids) were our knowledge, this option has never been cultured in 1-L containers, with four juvenile experimentally tested in tunicate culture. colonies per container at 20ºC and 32‰ and fed Both species possess broad temperature and every other day with three alternating feeds as salinity tolerances. In our experiments, young described above, changing water every 48 hours colonies of B. schlosseri survived environmental kept ammonia levels below 0.03 mg/L (Table 2), conditions between 10 and 25ºC and 14 and within the safe limit for marine invertebrates 38‰, while B. violaceus tolerated temperatures (Boardman et al. 2004).

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Table 2. Ammonia concentration (NH3-N in mg/L) in Yamaguchi (1975), Grosberg (1988), and our tunicate culture over time. Young colonies (1-20 zooids) study suggest that growth rates of botryllid were cultured in 1-L containers at 20ºC, four colonies per container. For food concentrations used see Table 1. colonies (in terms of changes in number of Ammonia levels were measured using a DR 2800 portable zooids per unit time) are exponential until the spectrophotometer (Hach, Loveland, USA) with a TNT 830 onset of sexual maturation (Figure 5). We ultra low range reagent (sensitivity: 0.015 - 2 mg/L NH3-N). believe that a modification of Grosberg’s

formula (Grosberg 1988) for calculating a pre- Food 1 h 24 h 48 h 72 h reproductive growth rate reflects growth most Liquifry Marine ≤0.015 0.019 0.026 0.026 accurately, as it incorporates both of its (Interpret Ltd.) components (i.e., number of zooids and number MicroVert (Kent Marine ≤0.015 ≤0.015 0.019 0.021 of growth cycles): Inc.) 1:1 mixture of log g = log (zn-1) / (n-1) Chaetoceros muelleri and ≤0.015 ≤0.015 0.023 0.023 where g = pre-reproductive growth rate, z = Isochrysis sp. n (Tahitian strain, number of zooids in a colony at asexual cycle n, T-iso) and n = cumulative number of asexual cycles at sexual maturity. During our preliminary experiments some young colonies of B. violaceus were attacked by ciliate protozoans that penetrated colonies, multiplied rapidly, and consumed colony tissues. Ciliates were able to completely consume healthy juvenile colonies of B. violaceus within 24 hours, but interestingly were never observed attacking B. schlosseri cultured under the same conditions at the same time. To control ciliates in our subsequent cultures, we cartridge-filtered all seawater to 1 µm and treated with UV light (Coralife Turbo-Twist 12x, Energy Savers Unlimited Inc., Carson, USA). As a result, in subsequent experiments ciliates were found in only two out of 536 B. violaceus colonies, thereby enhancing culture success. Monitoring colony condition and growth: About every three to ten days, depending on the water temperature, botryllid colonies pass through an asexual growth cycle, or blasto- genesis. During each cycle, zooids bud bilaterally, shrink, and get replaced by the new Figure 5. Mean colony size of B. schlosseri and B. buds. Some buds can be re-absorbed together violaceus at 20ºC and 32‰. On Day 1 colonies were 10-12 d with their parent, thus the total number of zooids old. Black arrow shows when the first batch of B. schlosseri larvae was released. n = 6 and error bars = SE. in a colony can increase, remain constant, or decrease. The replacement of zooids during each cycle in Botryllus spp. was described in detail by Colony condition, or general health, may be Mukai and Watanabe (1976) and Grosberg monitored using the following parameters: (1988). This synchronous, successive change of morphology of the zooids and ampullae (well- generations complicates colony growth measure- formed vs. shrunken; absence vs. presence of ments. Several approaches have been developed, dense accumulations of pigment cells), integrity including measuring colony area (Millar 1952), of the circulatory system, and integrity and counting the number of zooids (Boyd et al. transparency of the tunic. Overall colony 1986), and recording the number of primary buds condition may be assessed by assigning points to per zooid in each cycle and the duration and each parameter and calculating a mean value number of cycles (Grosberg 1988). Data from (e.g., Brunetti et al. 1980).

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Under certain unfavourable conditions, experiment. For each colony, condition and size colonies may undergo reversible colonial (number of zooids) were recorded at the start of regression or irreversible degeneration (Brunetti the experiment and 15 days after the colony et al. 1980). Degenerating colonies go through reached its experimental salinity (Figure 6). distinct stages of deterioration preceding death, Statistical analyses were carried out using described by Rinkevich et al. (1992) and SYSTAT® Version 11 (SYSTAT Software Inc., Chadwick-Furman and Weissman (1995) for B. Richmond, USA). A two-way ANOVA of the schlosseri. First, blood vessels narrow and blood growth data (percent increase in number of flow slows; then, the zooids shrink and become zooids from the beginning of the experiment) densely pigmented; in the third stage, groups of revealed a significant interaction between the zooids become disorganized; in the fourth and factors salinity and acclimation rate (F16,365 = final stage, the tunic gradually disintegrates and 1.98, P = 0.014). Therefore, the effects of all of the colony tissue dies. acclimation rate on colony growth were analysed at each experimental salinity using one-way IV. Experimental designs ANOVAs followed by Tukey’s post-hoc tests. For most salinity levels (15, 21, 27, and 33‰), The following section contains some experimental differences in growth rates among all results and considerations that may be useful when acclimation rate treatments were insignificant designing laboratory experiments using botryllid (P>0.05). However, at 39‰ growth was signifi- tunicates. cantly higher when the salinity acclimation rate Acclimation to experimental conditions: Some was ≥12 hours (Figure 6). For subsequent experimental work requires keeping or growing experiments we used an acclimation rate of 12 colonies under environmental conditions that hours per 2‰ salinity step. differ from those to which the colony would Experimental treatment replicates: In all of normally be exposed to in nature (e.g., our experiments, young colonies of both species, temperature or salinity). In these types of and especially B. schlosseri, demonstrated high experiments it is important to offer an adaptation growth-rate variability within all treatments regime that is compatible with the biological (Figures 5, 7). Previous studies also have shown properties of the test animal. When a stepwise high variability among colonies in several life- acclimation technique is applied, salinity is history aspects, such as survivorship, growth, usually adjusted in 1 to 5‰ steps to allow longevity, and reproduction (Brunetti et al. 1980; gradual attainment of osmotic equilibrium Boyd et al. 1986; Rinkevich and Shapira 1998). (Karpevich 1960). For our experiments, we Therefore, in order to obtain statistically signi- chose a 2‰ salinity acclimation step and ficant results, sufficient numbers of replicates of experimentally explored the effects of varying experimental treatments is necessary. In certain the acclimation rate (i.e., time intervals between cases it might be useful to conduct a preliminary salinity adjustments) on post-acclimation growth study first and run a power analysis of survival and growth of young colonies of B. the results in order to determine a sufficient violaceus. Newly established (7-10 day old) number of replicates. For example, for our study colonies, initially maintained in ambient on the combined effects of temperature (5, 10, seawater (28.5‰), were gradually brought to 15, 20, and 25ºC) and salinity (14, 20, 26, 32, experimental salinities (15, 21, 27, 33, and 39‰) and 38‰) on survival and growth of B. in 2‰ steps. Water of various salinities was schlosseri, a minimum of six replicates per prepared by addition of freshwater or artificial treatment were required (based on the results of marine salt Coralife (Energy Savers Unlimited a power analysis). Inc., Carson, USA) to natural seawater. Use of colour morphs: Botryllid tunicates are Acclimation rates were 24, 12, 6, 2, and 0 hours known for their colour polymorphism. They exist per 2‰ salinity step (an acclimation rate of “0” in a variety of colour morphs, but all zooids hours means that colonies were immediately within a colony are always the same colour or exposed to experimental salinities). The experi- combination of colours. Sabbadin (1959) and ment combined five levels of salinity and five Sabbadin and Graziani (1967) demonstrated that levels of acclimation rate in a totally-crossed the presence/absence of orange, blue, and experimental design (25 treatments in total). reddish pigment cells in colonies of B. schlosseri There were 15 to 18 replicates per treatment, were controlled by three independent Mendelian with a total of 431 colonies used for the loci, while two other loci controlled the distri-

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Figure 6. The effects of salinity acclimation rates on B. violaceus at various salinities: A – survivorship at day 15, B – percent growth over 15 d (i.e., percent increase in number of zooids from the beginning of the experiment). n = 15-18 and error bars = SE. Treatments within salinities denoted by different lower case letters differ significantly (1-way ANOVAs followed by Tukey’s post-hoc tests, P<0.05).

bution of nephrocytes around and between the siphons, giving rise to a variety of colour morphs. There currently is no evidence of colour morph-specific differences in life-history patterns of botryllids; in nature, colonies of various colours often occupy the same substratum, growing right next to each other (A. Epelbaum pers. obs.). This indicates that botryllid colour morphs likely exhibit the same physiological characteristics and environmental tolerances and preferences. The pigmentation pattern can thus be used as an experimental marker that allows easy distinguishing among colonies. Control of environmental parameters: Many types of experiments require precise control of the environmental parameters involved in colony growth, such as temperature, humidity, photoperiod, and light intensity. The use of growth chambers, or incubators, may be considered as it allows consistent reproduction of these Figure 7. Box plot showing mean colony size of parameters throughout the experiment. Growth B. schlosseri (30 d old) at 25ºC and various salinities. n = chambers have been used for culturing a variety 24, boxes = middle 50% of data, horizontal lines in boxes = medians, dots = outliers. of marine organisms, such as larval sea stars and crabs, anemones, and polychaetes (e.g., Watts et al. 1982; Forward 1989; Nii and Muscatine 1997;

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Lindsay et al. 2004), as well as tunicates and B. violaceus growing away from algae- (Grosberg 1988). However, when precise control covered areas of the slides (Figure 3), indicating is important we would recommend that that fouling may inhibit colony growth. More incubators be thoroughly tested for the uni- studies comparing growth of field- and formity of conditions inside the chamber prior to laboratory-raised botryllid colonies would be starting culture experiments. Our tests on low- valuable. At present, growth of laboratory-raised temperature incubators showed that temperature colonies should be interpreted with caution as in was not consistent throughout the chamber, some cases it might not directly reflect growth especially at lower temperature ranges (≤15ºC) rates and survivorship observed in nature, but and when photoperiod control was used. For our rather reveal the physiological capabilities of the culture experiments we used a water-bath system species. Laboratory studies are still a useful with heating/cooling coils that provided means for testing environmental limits of species temperature control of ±0.3ºC (SD). in a replicated controlled environment.

Concluding Remarks Acknowledgements Laboratory culture of botryllid tunicates has several important advantages. It allows We are grateful to John Blackburn, Laurie Keddy (Fisheries and Oceans Canada), and Brian Loos (Vancouver Island maintaining genetically defined populations and University) for their assistance in various aspects of the producing animals or tissues continuously culture experiments. We also thank the editors and two throughout the year for a variety of studies. anonymous reviewers for their helpful comments and Laboratory experiments in ecological research suggestions. Support for the project was provided by funds from the Aquatic Invasive Species program of Fisheries and provide the means by which cause and effect can Oceans Canada. A. Epelbaum was funded through the be established by allowing precise control of Visiting Fellowship program of the Natural Sciences and variables and accurate replication of trials. Engineering Research Council of Canada (NSERC). A. However, life-history traits exhibited by Paulson was funded by an Undergraduate Student Research Award and a Discovery Grant (to C.M. Pearce) provided by laboratory cultures – including patterns of NSERC. growth, reproduction, and longevity – may not always reflect the evolutionary or ecological processes that act upon species in nature References (Chadwick-Furman and Weissman 1995). For Berrill NJ (1937) Culture methods for ascidians. In: Galtsoff example, life-history patterns in field-grown PS, Lutz FE, Welch PS, Needham JG (eds) Culture colonies of B. schlosseri in Monterey Bay, methods for invertebrate animals. Dover Publications, California were found to differ from colonies New York, USA, pp 564-571 raised in the laboratory: field cohorts exhibited Berrill NJ (1950) The Tunicata, with an account of the British species. Ray Society, London, 354 pp rapid growth, short and intense reproduction, Boardman GD, Starbuck SM, Hudgins DB, Li X, Kuhn DD short lifespan, and senescence soon after (2004) Toxicity of ammonia to three marine fish and reaching maturity, while colonies raised in the three marine invertebrates. Environmental Toxicology laboratory exhibited slow growth or shrank over 19: 134-142, doi:10.1002/tox.20006 Boyd HC, Brown SK, Harp JA, Weissman IL (1986) Growth many months, ceased reproduction long before and sexual maturation of laboratory-cultured Monterey death, and lived for more than two years (Boyd Botryllus schlosseri. Biological Bulletin 170: 91-109, et al. 1986, Rinkevich and Weissman 1987). doi:10.2307/1541383 Similar life-history differences were observed Brunetti R (1974) Observations on the life cycle of Botryllus schlosseri (Pallas) (Ascidiacea) in the between field- and laboratory-raised colonies in Venetian lagoon. Bolletino de Zoologia 41: 225-251 Mediterranean populations (Brunetti 1974; Brunetti R (1979) Ascidians of the Venice Lagoon. I. Brunetti and Copello 1978). Annotated inventory of species. Annales de l'Institut Several factors may be responsible for these Oceanographique, Paris 55: 95-109 Brunetti R, Copello M (1978) Growth and senescence in differences. Under laboratory conditions water colonies of Botryllus schlosseri (Pallas) (Ascidiacea). motion is slower and food might be less varied Bolletino de Zoologia 45: 359-364 than in the field (Milkman 1967; Brunetti and Brunetti R, Beghi L, Bressan M, Marin MG (1980) Copello 1978). In addition, the absence of Combined effects of temperature and salinity on colonies of Botryllus schlosseri and Botrylloides leachi natural grazers in the laboratory may lead to the (Ascidiacea) from the Venetian Lagoon. Marine formation of a fouling film (Chadwick-Furman Ecology Progress Series 2: 303-314, doi:10.3354/meps and Weissman 1995, A. Epelbaum pers. obs.). 002303

We observed juvenile colonies of B. schlosseri

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