A larval-settlement assay method for the gregarious serpulid polychaete, Galeolaria caespitosa

Static settlement assays are considered the standard tool for determining the settlement preferences of marine invertebrates. Often used to assess and evaluate the properties of a given substrate or biofilm cues for colonisation, the static assay format is technically simple, rapid, and inexpensive. Galeolaria caespitosa is a sessile, filter-feeding polychaete worm that inhabits mid to low intertidal regions of exposed rocky shores. Mature adults of G. caespitosa are fertile throughout the year, easy to spawn and rear to settlement competency, and their gregarious settlement behaviour enables easy collection making them an ideal test species. Here we report an optimised protocol for larval settlement assays with G. caespitosa. Unlike other serpulid polychaetes, a bacterial biofilm alone was not sufficient to consistently induce settlement. Instead, a conspecific cue was required for reliable settlement under assay conditions. Yet empty tubes and a homogenate of crushed adult worms had no significant impact on settlement compared to a control, and both treatments showed high variance, inducing <5% of larvae to settle. Only the presence of live conspecific worms consistently induced settlement to the extent necessary for useful assays. In this case, after 48h the proportion of larvae to have successfully settled reached 44%. On average, live conspecific worms increased settlement by 87% compared to the presence of a biofilm alone. The static settlement assay protocol developed in this study provides a reproducible tool for assessing invertebrate settlement with G. caespitosa. Guidelines are provided for adapting this approach to other species.