Ageing octopods from stylets: development of a technique for permanent preparations

Previous attempts at ageing octopods from stylets have relied on preparations that deteriorate with time. Some techniques require an immediate photographic record, others allow real-time enumeration but do not provide a permanent archive. A technique is described that produces permanent and archivable preparations of octopod stylets. Stylets were dehydrated in ethanol and infiltrated with a low-viscosity resin. Subsequent polymerization of the resin allowed the embedded stylet to be ground and polished to reveal the stylet microstructure. This comprised increments that are probably suitable for age estimation. The technique was developed using stylets of Octopus vulgaris and Eledone cirrhosa. Increments were composed of light and dark bands and were clearly defined at x400 and at x625 magnifications. The number of increments ranged from 189 to 399. The stylets of a deep-sea species (Bathypolypus sponsalis) and an Antarctic species (Megaleledone setebos) were also examined. Each appeared to have growth increments, despite the perception that the environments they inhabited may not provide daily cues. Using the technique developed, the pre-hatch nucleus was seldom well defined, as reported for O. pallidus, stylets of which were prepared using a non-permanent method. Reasons for this are discussed. The microstructure clarity revealed is probably associated with the ultra-low viscosity of the resin used.