Initiating Laboratory Culturing of the Invasive Ascidian Didemnum Vexillum

Research Article

Initiating laboratory culturing of the invasive ascidian Didemnum vexillum

Baruch Rinkevich1* and Andrew E. Fidler2,3 1National institute of Oceanography, Tel Shikmona, PO Box 8030, Haifa 31080, Israel 2Cawthron Institute, 98 Halifax Street East, Private Bag 2 Nelson 7010, New Zealand 3Institute of Marine Science, University of Auckland, PO Box 349, Warkworth 0941, New Zealand E-mail: [email protected] (BR), [email protected] (AEF) *Corresponding author

Received: 13 November 2013 / Accepted: 13 February 2014 / Published online: 13 February 2014

Handling editor: Katherine Dafforn

Abstract

Over the past few decades, the invasive colonial ascidian Didemnum vexillum Kott, 2002 has established in many temperate regions, sometimes bringing detrimental perturbations to native marine ecosystems and local economies. A reliable supply of healthy, genetically defined D. vexillum stocks/strains would greatly assist research into the traits of this species relevant to its invasive success. To this end, here we describe basic methodologies for the growth, maintenance, formation of chimeric colonies and their follow-up observations in ex situ cultures of D. vexillum. Didemnum colonies were collected during the austral late winter/early spring from a marina (Nelson, New Zealand) and were maintained in the laboratory using re-circulated, unfiltered seawater. Ramets (1 – 10 cm2; >70 fragments) from a variety of colony locations (peripheral/central, flat/lobe/tendril-shaped); were isolated by razor blade and secured to glass slides using cotton thread. The ramets attached to the glass slides within 24 hours, displayed 100% survivorship and could be successfully further sub-cloned for up to 10 weeks, when the study was terminated. Two distinct processes for tunic–substrate attachment were observed: (i) the secretion of growing marginal fronts made of tunic matrix containing spicules but without zooids and (ii) the secretion of thin tunic extensions with clusters of tunic cells at their tips. Allogeneic paired colony fusion assays, using naturally growing edges of colony fragments (i.e. mimicking natural fusion scenarios) resulted in fusions and chimeric colony formation. Zooids from both partners within a chimera appeared to intermingle and the resulting chimeric entities grew vigorously. This brief report provides an outline of simple, low-tech laboratory culture conditions for the maintenance, asexual propagation and chimeric colony formation of D. vexillum. The techniques described present a foundation for the development of genetically defined D. vexillum strains which will facilitate research into the traits of this species that are relevant to its invasive success.

Key words: allorecognition, culture, chimeras, management, ramet, tunic cells, tunicate

Introduction communities. Because of their ability to thrive in eutrophic environments and being strong Marine bioinvasions involving a wide range of competitors, some species of ascidians have marine species introduced into regions beyond easily invaded new localities, spread rapidly, and their natural or historic ranges, and the associated integrate with local biofouling communities perturbations such invasive species impose on (Lambert 2001, 2007; Dijkstra et al. 2007; Carman marine ecosystems and human welfare, are et al. 2010; Adams et al. 2011). Introductions of recognized as a major, and growing, environmental non-indigenous ascidians into new harbors, concern (Mooney and Cleland 2001; Occhipinti- aquaculture facilities, infrastructure constructions Ambrogi 2007; Molnar et al. 2008; Simkanin et and natural environments worldwide, in tropical, al. 2012). One of the more prominent invasive subtropical, temperate and subarctic waters alike, marine taxonomic groups is class Ascidiacea are increasing in frequency, and the damage they (subphylum Tunicata, phylum Chordata; Lambert may inflict on existing ecosystems, and the 2001) whose members include both solitary and associated human economies, is well documented colonial forms that inhabit many benthic marine (Molnar et al. 2008; Simkanin et al. 2012).

55 B. Rinkevich and A.E. Fidler

Clearly there is a need for research tools to assist colony fragments were placed with their natural in scientific analysis of ascidians’ environmental growing edges juxtaposed. All slides were placed tolerances, biological traits (e.g., growth rates, vertically in the slots of glass staining racks interspecific competitive proficiencies), diseases and (following Rinkevich and Shapira 1998) in interactions with native species. A central unfiltered, recirculated seawater. Such vertical requirement is a constant supply of healthy, positioning of cultured colonies reduces debris, genetically defined strains/lines suitable for food and fecal accumulation around the colonies. laboratory conditions, thereby circumventing The colonies were grown in 16 litre flow- seasonal and unpredictable variations in colony through glass tanks (~1 litre/min; unfiltered, availability along with the impact of unknown recirculated sea water), under a 12:12 hour light- underlying genetic variability. Such strains would dark regimen. Salinity (34.5±0.1), pH (8.07±0.01) allow the initiation of long-term and controlled and temperature (17±1°C; ambient 15 °C) were experiments evaluating biological aspects of monitored and kept constant along with vigorous invasiveness under defined and controlled aeration by air stones. The colonies were fed 4–5 conditions. times / week by adding ~50 ml of an algal mix Over the past few decades, the colonial (~1:1 ratio of Isochrysis galbana Parke [8–9 × ascidian Didemnum vexillum Kott, 2002 has been 106 cells / ml], Pavlova lutheri Green [10–12 ×106 expanding globally impacting aquaculture facilities cells/ml]). This protocol follows the recommen- and natural habitats (Lambert 2009; Smith et al. dations of Rinkevich and Shapira (1998) for 2012; Stefaniak et al. 2012). The rapid expansion botryllid ascidians inland mariculture which of D. vexillum, along with other invasive ascidians, demonstrated that a mixture of diet types is has raised questions about the biological traits superior to a monotype diet. that underlie such impressive adaptive success - Colonies were cleaned during observations, at especially in the context of anticipated changes least once a week, using a soft, small paintbrush in oceanic temperatures and chemistry over to remove debris and fouling organisms. The coming years (McCarthy et al. 2007; Bullard and substrate around the colonies was cleaned using Whitlatch 2009; Lenz et al. 2011; Simkanin et al. a small brush and razor blade. All cleaning 2012; Smith et al. 2012). Addressing such questions protocols were performed under a binocular is currently hampered by experimental and dissecting microscope and the colonies were procedural deficiencies such as there being no submerged in seawater during all procedures. well-established, disease free, genetically-defined The status of the colonies was monitored every strains of D. vexillum available nor have robust 1–2 days and photographs were taken to document procedures for the long-term culturing of D. colonial attachment to the substrate, growth patterns, vexillum in the laboratory been described. To outcomes of isogeneic and allogeneic contacts, help initiate addressing these deficiencies here and any changes in distribution/morphology. we describe basic methodologies for laboratory culturing of D. vexillum colonies and show that such colonies are easily experimentally manipulated. Results To collect colonies having different genotypes Materials and methods (i.e., to avoid colonies that are clonally related), seven D. vexillum colonies, denoted A–G, were Small to medium sized Didemnum vexillum sampled from different pontoons >5 meters apart colonies, growing >5 meters apart, were collected in the Nelson marina. Colonies were peeled off from ropes and other submerged artificial objects by hand, or using single-edge razor blades, from depths of 0.3–1.5 meters in the Nelson together with a very thin substrate layer and marina (Nelson, New Zealand; 41°15.4′S, 173° brought to the laboratory in seawater within an 16.6′E) and transported ~2 km to the Cawthron hour. Upon arrival at the laboratory, each colony Institute (Nelson) in containers filled with was carefully rinsed in seawater, to remove seawater. Each colony was cleaned of fouling sedimentation, and attached macro-epibionts were organisms and cut using a razor blade into removed using forceps. several ramets (n = 2–6; depending on the sizes As D. vexillum colonies fragment easily of colonies). Ramets were secured by cotton (Bullard et al. 2007a,b; Morris and Carman 2012; threads to 5.0×7.5 cm glass slides, one ramet/slide. Reinhardt et al. 2012) we first attempted to secure On some slides, pairs of isogeneic or allogeneic colonial fragments to the glass slides using the

56 ex situ cultures of Didemnum vexillum

Figure 1. Tunic–substrate attachment processes displayed by D. vexillum colonies: production of growing marginal fronts with spicules but without zooids and production of thin tunic extensions with masses of tunic cells at their tips (a) Marginal front attachment of a fragment sampled from the central part of a colony three days after securing the fragment with cotton threats which subsequently became embedded within the tunic matrix; (b) Two days thereafter, formation of a flat, fast growing colony on the substratum with wider growing marginal fronts; (c) A marginal front of a colony showing the embedded cotton threads, and the zooid-free tunic area, with many spicules; (d, e) Development of tunic extensions from growing marginal fronts with the apex of each extension containing tunic cells but no spicules (arrowheads); (f) Newly developed spicule-free tunic extensions, positioned above the substrate, with clear masses of tunic cells (arrowheads). Abbreviations: te = tunic extensions; sc = spicules condensed area.

methodology successful for botryllid ascidians cotton threads were removed, after a colony’s (Rinkevich and Weissman 1987). Peripheral, central complete adherence to the substrate, by cutting and lobe/tendril shaped colony fragments (sizes 1 – with a razor blade the free cotton threads above 10 cm2) were cut from each colony, and transferred and below each colonial fragment and pulling to settlement glass slides, one ramet/slide. The out the embedded section using fine forceps. ramets were allowed to adhere to the slides by Parts of the four colonies denoted A–D were cut placing them in a moisture chamber at ambient into smaller ramets, and distributed onto several temperature for 35–55 minutes before transferring glass slides (3–10 slides / colony). them to still water aquaria. However, most of the In total >70 small ramets and larger fragments, of fragments detached from the slides upon placing sizes ranging 1–25 cm2 were produced and all into the aquaria. We therefore used the alternate (with the exception of a single case where the methodology of securing the fragments to the cotton covered areas degenerated) attached to the slides using thin cotton threads. The cotton threads glass slides, irrespectively of their origin within firmly attached the colonies to the glass slide the colony (i.e., peripheral, central, lobe/tendril- substrate (two loops improved colony attachment) shaped fragments). The attached colonies were and indeed within 24 hours, the cotton threads observed for up to 10 weeks, when this study were embedded within the growing fragments’ was terminated. Under our basic culturing tunic matrices (Figures 1 a–c, 2). Embedded conditions, all attached fragments grew rapidly

57 B. Rinkevich and A.E. Fidler

Figure 2. D. vexillum chimeric colony formation. (a, b) Fusion of tunics between two allogeneic pairs through tunic spreading and secretion of new tunic matrix with spicules but without zooids; (c) Development of chimerism between two allogeneic pairs through thin tunic extensions; (d - f) A condensed chimera, at the center of the glass slide, two days after first tunic to tunic contacts. The zooids area of each of the two partners is still morphologically distinguishable due to colony color differences (d). Five days thereafter (e), the whole intermingled chimeric entity has moved upward and almost half of the chimera is overgrowing the opposite side of the glass slide (f). The deteriorating right tunic area in ‘e’ (marked by a star) corresponds to the low right side of the right partner in the chimera (d, marked by a star). Abbreviations: te = tunic extensions; sc = condensed zooid-free spicules area.

on the slides (forming only flat, encrusting colonial many spicules but with a paucity/absence of forms), and had 100% survivorship. When all zooids, located either all around the fragment’s available space on the slides was covered outlying zones or as a growing front (Figure 1a- (indeed in many cases fragments rapidly grew d). In common with other aplousobranch ascidians, onto the back of the slides; Figure 2 e, f), the Didemnum spp. colonies lack a common vascular colonies were further subcloned and transferred system and their tunics are made of a fibrous, to additional slides, a protocol with the potential semi-transparent gelatinous matrix fringed, and to provide large stocks of ramets of identical supported, by a thin layer of dense fibers covering genotype. the whole tunic matrix (Hirose et al. 1997). Thus, Within 24 hours after transfer to a glass slide, as didemnid spicules are produced intracellularly the D. vexillum colony fragments started an within the zooids’ lateral organs of the thorax active attachment process, apparently initiated (Lafargue and Kniprath 1978; Kniprath and from the periphery, which led to their firm Lafargue 1980) and this method of attachment is connection to their substrate (Figure 1). We rapid, producing, what appears to be new thin observed two distinct processes associated with tunic with many spicules but without zooids, all such attachment used separately or concurrently these conditions point to spreading, not new at each fragment’s edge. The first process tunic secretion. In the second attachment process involved the fast (within 24 hours after transfer) colonies attach to substrates through thin spreading of thin peripheral tunic matrix with extensions originating from the basal tunic area

58 ex situ cultures of Didemnum vexillum

Table 1. Attachment rates to substrate by peripheral and central D. vexillum colony fragments.

Fragment origin Partly or fully attached to the substrate (days) 2 3 4 6 Peripheral 6/6 6/6 6/6 6/6 Central 1/6 4/6 5/6 5/6* *Areas of cotton strings holding a single fragment start to deteriorate before this fragment fully attached to substrate. This ramet was detached from substrate one day thereafter, and when laid on the aquarium bottom, had started developing new attachment fronts.

(Figure 1 d, e) or extensions emerging from were essentially absent. In faster growing colonies, upper parts of the tunic (Figure 1f), but directing an area of tunic devoid of zooids usually towards the substrate. In contrast to the whole developed in older parts of the growing area. In front attachment process described above, the some cases this older tunic area rapidly degenerated, tips of these small extensions which contain many whereas in other cases it remained intact through cells but no spicules, formed distinct aggregates as the full observational period. As the D. vexillum more opaque areas at specific zones (Figure 1 d– colonies were collected during austral late-winter/ f, arrows). Some of these tunic cells (at least four early-spring (August-October) no larvae were different tunic cell types were recently identified seen in the tunic matrix (Fletcher and Forrest 2011). in D. vexillum: denoted phagocytic, morula, Some areas, varying in locations and numbers filopodial and bladder; Sellers et al. 2013) are between observations, containing faecal pellets possibly the source of the newly secreted tunic were observed indicating the zooids were feeding. matrix material at the growing edges of D. vexillum We also performed both iso- and allogeneic colonies. While these attachment processes were paired-colony fusion assays between the naturally seemingly developed irrespective of the existence of growing edges of colony fragments. This work zooids in peripheral zones, the whole course of was undertaken to establish protocols to follow attachment appears to be a highly coordinated the fusion outcomes when D. vexillum colonies activity at the ramet level. of differing genotypes come into tunic–to–tunic Interestingly, while all the isolated fragments contact. All six pair-wise combinations of colonies used in this study eventually attached to their A – D, along with the four isogeneic combinations substrates, fragments taken from colony peripheral used as positive controls, were tested for fusion. zones attached faster than those taken from Paired colony fragments adjacent to each other but central regions (Table 1). Twelve fragments (ca. without direct tunic-to-tunic contact were 1.0 cm2) were isolated from colonies E – G (four attached to glass sides. Growth of the paired samples/colony, two peripheral, two central). colony fragments toward each other was rapid Two days following their securement to the glass and, interestingly, it appeared that the tunic front slides, 6/6 peripheral fragments had attached to of one of the pair generally advanced more rapidly their substrates (some with very distinct growing than the other. Following tunic-to-tunic contact, fronts) as compared to 1/6 central region fragments the outer cortical layers of the tunics dissolved, being attached (p<0.05; G test). However within resulting in fusion and the formation of chimeric three days following securement, 4/6 of the colonies (example shown in Figure 2a, b). Fusion central colony derived fragments attached while also occurred following the formation of tunic and all fragments attached within a week (Table 1). extensions (Figure 2c). The fusion process, from The differential growth patterns of fragments first tunic-to-tunic contact to complete amal- on the glass slide substrates were so rapid that it gamation, took 1–6 days and was observed in all was possible to document daily changes (Figure four isogeneic and six allogeneic pairings. Many 2 d–f), including examples of directional growth, of the chimeric colonies were morphologically growth with no clear trajectory, and colonies highly active and zooids mixed within the new growing in more than one direction. Two of the entity so that within 1–2 days their colony of characteristic features of fast-growing colonies origin was hardly obvious. Like their progenitors were the less compact distribution of zooids the chimeric colonies grew directionally and within the tunic matrix and the appearance of wider after a few days some even grew onto the back peripheral expansion zones from which zooids surfaces of the glass slides (Figure 2 d–f).

59 B. Rinkevich and A.E. Fidler

Discussion of this species (unknown under either field or relaxed ex situ conditions). We successfully attached Didemnum vexillum Kott, 2002, is one of the well- colonial fragments to substrates, observed the way known marine invasive species that has invaded colonies acquire new substrates, documented temperate water regions across the globe (Lambert allorecognition assays with ramets of various 2009; Stefaniak et al. 2009; http://woodshole.er. genets, and monitored the outcomes. usgs.gov/project-pages/stellwagen/didemnum/ index.htm): This study shows that different Didemnum Atlantic Europe (Gittenberger 2007) including vexillum genotypes, upon tunic-to-tunic contacts, the UK (Minchin 2007; Griffith et al. 2009), New can form stable (at least on the morphological Zealand (Kott 2002; Smith et al. 2012), both coasts level) chimeras that displayed fast and differential of North America (Kott 2004; Bullard et al. 2007b; growth patterns on the available substrates. It has Cohen et al. 2011) and, more recently, the Medi- long been considered that establishing intraspecific terranean (Tagliapietra et al. 2012). Population chimerism in sessile, sedentary colonial marine genetic studies strongly indicate the north-west organisms may confer various ecological advan- Pacific Ocean as being the probable native region tages, culminating in the formation of a ‘novel of D. vexillum (Smith et al. 2012; Stefaniak et al. entity’ with a greater store of genetic variability 2012). Outside its native range, D. vexillum covers and hence with a wider range of physiological submerged manmade substrates as well as natural qualities, characterized by reduced onset of habitats from the very low intertidal down to 80 reproduction, increased competitive capabilities, meters of depth (Bullard et al. 2007b; Valentine et enhanced growth rates, reduced whole-colony al. 2007; Lambert 2009; http://woodshole.er.usgs.gov/ mortality rates, synergistic complementation, and project-pages/stellwagen/didemnum/index.htm). assurance of mate location when needed (Buss Colonies grow as flat, encrusting mats or produce 1981; Grosberg 1988; Bishop and Sommerfeldt irregular lobes, depending on the type of available 1999; Rinkevich 2002; Rinkevich and Yankelevich substratum and colony size/age. Aside from 2004). This state of chimerism may therefore larval dispersal which is very limited due to the present adjustable genotypic combinations of short-lived nature of the tadpoles, both natural organismal traits to alterable and capricious natural and anthropogenic processes that fragment D. selection operations (Pineda-Krch and Lehtila vexillum colonies are probably a major accelerator 2004; Rinkevich 2004; Rinkevich and of the spread of this species (Bullard et al. 2007a,b; Yankelevich 2004), thus better withstanding harsh Morris and Carman 2012; Reinhardt et al. 2012). and/or fluctuating environmental conditions Indeed reattachment success and fragment viability, (Rinkevich 2004). It seems likely that chimerism as reported in this and earlier (Morris and is an important trait in the life history of D. Carman 2012; Reinhardt et al. 2012) studies, are vexillum, allowing this species to spread considered two important ecological traits worldwide into different environmental regimen responsible for D. vexillum’s invasive success. and be successful under various biological/physical This study provides the tools to follow colony conditions (McCarthy et al. 2007; Lenz et al. survivorship, growth and patterns of substrate 2011; Smith et al. 2012). Chimerism is the norm acquisition under controlled laboratory conditions, for all aplousobranchs, especially didemnids using various ramets of the same genotype and (Bishop and Sommerfeldt 1999). Allorejection differing genotypes from a given population to may or may not ensue depending on relatedness reveal genetic components for key biological of the fused colonies or fragments (Ishii et al. attributes. We documented that whole colonies, 2008). or fragments of larger colonies, can be gently In order to improve our understanding of the and easily peeled off the substratum and transported traits controlling invasiveness of D. vexillum to the laboratory, where they are cultured and (Bullard and Whitlatch 2009), and to help predict propagated in line with the research needs. its future invasive potentialities, there is a need The colonies of Didemnum vexillum cultured to develop culture methodologies amenable for ex situ with the methodology described here detailed and controlled ex situ studies. The were very suitable for a variety of scientific ability to obtain D. vexillum ramets/genets on investigations and almost unlimited number of demand is crucial for elucidating relevant life ramets can be established from any single history parameters and the natural variability of genotype. While we used a short culturing period associated biological traits. In this context this (up to 10 weeks), it is perceived that such study describes simple, low-tech inland culture cultures can be extended to the maximal lifespan conditions to which the animals responded well

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(they survived, attached to substrates and expended/ Carman MR, Morris JA, Karney RC, Grunden DW (2010) An grew on artificial substrates) and presents the first initial assessment of native and invasive tunicates in shellfish aquaculture of the North American east coast. Journal of steps in the development of D. vexillum genetically Applied Ichthyology 26: 8–11, http://dx.doi.org/10.1111/j.14 39- defined lines amenable for various research 0426.2010.01495.x needs. It should be noted that colonies collected Cohen SC, McCann L, Davis T, Shaw L, Ruiz G (2011) Discovery and significance of the colonial tunicate in the wild for division into fragments for Didemnum vexillum in Alaska. Aquatic Invasions 6: 263– laboratory cultures may be chimeras, made up of 271, http://dx.doi.org/10.3391/ai.2011.6.3.03 genetically diverse zooids. Thus, it may be also Dijkstra J, Sherman H, Harris LG (2007) The role of colonial practical to use sexually produced zooids. Thus, ascidians in altering biodiversity in marine fouling communities. Journal of Experimental Marine Biology and Ecology combined with recently described methods for 342: 169–191, http://dx.doi.org/10.1016/j.jembe.2006.10.035 collecting and culturing D. vexillum larvae Fletcher LM, Forrest BM (2011) Induced spawning and culture (Fletcher and Forrest 2011), we are now well techniques for the invasive ascidian Didemnum vexillum placed to develop mass cultures of genetically (Kott 2002). Aquatic Invasion 6: 457–464, http://dx.doi.org/ 10.3391/ai.2011.6.4.11 defined lines of D. vexillum thereby opening up Gittenberger A (2007) Recent population expansions of non- exciting new experimental possibilities. This will native ascidians in the Netherlands. 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