A novel geometry for a laboratory-based larval settlement assay

Abstract

A novel configuration, consisting of two apposing surfaces bounding a vertical water column, is presented and evaluated for settlement assays using cyprids of Balanus amphitrite. Assays were conducted on planar surfaces, ranging from hydrophobic polystyrene to hydrophilic glass and including CH₃- and NH₃ ⁺-terminated self-assembled monolayers (SAMs). Identical apposing surfaces generated settlement rates comparable to those obtained in prior studies, while a choice assay yielded consistent results, with individual replicates each indicating the preferred surface for settlement. As gravity favours contact with the lower apposing surface, cyprids trapped at the air/water interface settled on or around the perimeter where the water column meets the lower substratum. These cyprids are capable of selecting a settlement location and are thus not lost to the assay. The assay geometry lends itself to assessing cyprid exploration and settlement on planar surfaces with chemical patterning, including relief microstructures, without using a confining material or requiring the coating of a three-dimensional well.

Keywords:

• settlement assay
• planar surface
• apposing surfaces
• cyprids
• Amphibalanus amphitrite
• wetting confinement
• settlement patterns

Acknowledgement

The authors acknowledge support from the Innovative Marine Antifouling Solutions for High Value Applications (IMAS) Programme, funded by the Science and Engineering Research Council of A^∗STAR.