Laboratory experiments showed that cyprids of Balanus balanoides settled readily only when offered surfaces which bore settled barnacles of their own species, or the cemented bases left on surfaces from which these barnacles had been removed. Without such surfaces they proved to be capable of postponing setting for at least 2 weeks, and they chose such surfaces in preference to similar surfaces which bore related species. Cyprids of B. crenatus and Elminius modestus were also capable of making this choice.
Setting usually occurred in the immediate vicinity of barnacles or bases, but some cyprids crawled short distances away during their final reconnaissances. When they encountered barnacles attached to glass, they were stimulated to proceed with setting, but they swam away from this unfavourably smooth surface and settled on bare stones nearby.
Fragments from various parts of the body, placed upon surfaces which had never borne barnacles, made these somewhat more favourable for setting. Since attached bases alone produced a strong reaction, the cyprids probably respond to contact between their groping antennules and some substance in the cuticle. They did not settle readily when prevented from making contact with adults by a barrier of bolting silk, or with bases by a thin film of nitrocellulose; chemosensory perception of a water-soluble substance, emanating from settled barnacles, is therefore unlikely to be involved.
The power of the bases to induce setting was retained after heating to over 200° C, but not after heating to 275° C, when charring occurred. It withstood prolonged washing in water, heating with fat solvents and dilute acids, and treatment in the cold with caustic alkalis, concentrated acids, pepsin, formaldehyde, benzoquinone, sodium sulphide, phenol, urea, and diazonium solution. It was destroyed in the cold by sodium hypochlorite, which was observed to dissolve the bases, and also by hot concentrated acids and alkalis. The chemical properties of the active substance are thus entirely consistent with those of quinone-tanned proteins, which are known to form the epicuticle and attachment cement.
A field experiment showed that similar behaviour occurs under natural conditions. It leads cyprids to suitable habitats and it facilitates breeding.