Ascidian Larvae Discriminate Nano-Scale Difference in Surface Structures During Substrate Selection for Settlement

Planktonic larvae of sessile metazoans select substrates for settlement based on various factors. <i>Phallusia philippinensis</i> larvae (Ascidiacea: Phlebobranchia: Ascidiidae) showed a negative preference for nano-scale nipple arrays (dense arrays of papillae-like nanostructures approximately 100 nm in height). To clarify whether ascidian larvae discriminate between nano-structure sizes for substrate selection, three different sizes of periodic nano-folds were fabricated using two-beam interference exposure, and substrate selection assays were performed on the three types of nano-folds and flat surfaces made of the same material. The substrate selection assay with 500-2000 freshly hatched larvae was carried out in nine replicates. The ascidian larvae showed a positive preference for flat surfaces and a negative preference for substrates with a height of 120 nm and pitch of 600 nm. Manly's selection indices differed with the size of the periodic nano-folds, supporting the hypothesis that larvae directly or indirectly discriminate between nano-scale differences upon settlement. The present study is the first to show that differences in nanostructure size affect substrate selection during larval settlement of sessile animals. The evolutionary adaptive reasons for larvae to discriminate between nano-scale structures and select substrates for settlement are potentially important to effectively manage ascidian biofouling using non-toxic methods.