Cephalopod International Advisory Council, CIAC’09 Symposium. Vigo (Spain), 3-11 September 2009 Age validation in common octopus, Octopus vulgaris Cuvier 1797, using stylet increment analysis Hermosilla, C.A.1, Rocha, F.1, González, A.F.2, Guerra, A.2 and Fiorito, G. 3 1. Dpto. Ecología y Biología Animal. Universidad de Vigo. Spain. 2. Instituto de Investigaciones Marinas (CSIC). Eduardo Cabello 6, 36208 Vigo, Spain. 3. Laboratorio di Neurobiologia, Stazione Zoologica A. Dohrn, Villa Comunale, 80121 Napoli, Italy. Introduction The common octopus, Octopus vulgaris Cuvier, 1797, is the most important commercially harvested octopus species. However, although this is a well-known cephalopod species, only a few ageing studies were made. Two hard structures have been used in ageing studies regarding octopods: beaks and stylets. In this sense, stylets seem to be the most promising structure to age studies. We present here the validation of the daily periodicity of growth increments in stylets of laboratory reared Octopus vulgaris specimens. Daily periodicity was corroborated by staining the stylets either with oxytetracycline or tetracycline and comparing the Figure 1. Photograph showing the fluorescent number of rings produced with the elapsed days. Oxytetracycline (OCT) stylet mark in a female of 1140 g (stained with 124 mg OTC per Kg). Material and Methods A total of 19 individuals (10 males and 9 females, 680 to 1470 g body weight) were marked with oxyetracycline (OTC) in Vigo facilities (mean of 124 mg OTC per Kg of octopus) and 6 individuals (1 male and 5 females, 248 to 570 g body weight) were stained with tetracycline (TC) in Naples (mean of 120 mg/Kg). The animals were successfully maintained in captivity until sacrificed for up to 6 (1 specimen), 9 (1 specimen), 18 (6 specimens from Naples) and 21 (17 specimens) days. Stylets were removed from specimens and fixed in 4% formalin. Transverse stylet sections were embedded in thermo-plastic resin and polished until a thin and translucent section Figure 2. Light photograph showing the stylet was achieved. Several sections of each stylet were prepared and photographed increments in the same specimen of Fig.1. Arrows (Figures 1 and 2). Increments were counted 3-5 times by two different readers. showing the location of OTC mark. Increments counts for each stylet were analysed and outliers excluded using statistical methods. 25 Results and discussion 20 The number of increments counted was 18.9±1.4 and 20.5±1.5 days for 18 and 21 days specimens, respectively (Figure 3). Mean .s rate of increments formation was 1.02 increments per day (1 Increments 15 increment = 1 day). Photograph analysis corroborated the regular deposition pattern of stylet growth increments (Figures 1 and 2). Consequently, this study successfully validates daily increments 10 deposition in stylets of Octopus vulgaris to the size range y = 0,8586x + 2,7868 analysed. 2 R = 0,9652 5 0 0 5 10 15 20 25 Acknowledgements Days after staining This research was supported by ECOSUMMER actions (European Union). C. Hermosilla has an early training ECOSUMMER (Marie Curie Action). F. Rocha is Figure 3. Relationship between number of days elapsed after OTC staining and an Isidro Parga Pondal Researcher. Animals were housed and maintained at number of increments counts between OTC mark and stylet border. ECIMAT (University of Vigo, Vigo, Spain) and Stazione Zoologica A. Dohrn (Napoli, Italy).