Initiating laboratory culturing of the invasive ascidian Didemnum vexillum

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 cm(2); >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.