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. Figure 1. Photograph showing the fluorescent Oxytetracycline (OCT) stylet mark in a female of 1140 g (stained with 124 mg OTC per Kg). Figure 2. Light photograph showing the stylet increments in the same specimen of Fig.1. Arrows showing the location of OTC mark. Figure 3. Relationship between number of days elapsed after OTC staining and number of increments counts between OTC mark and stylet border. Introduction 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 number of rings produced with the elapsed days. Material and Methods 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 was achieved. Several sections of each stylet were prepared and photographed (Figures 1 and 2). Increments were counted 3-5 times by two different readers. Increments counts for each stylet were analysed and outliers excluded using statistical methods. Results and discussion Results and discussion 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 rate of increments formation was 1.02 increments per day (1 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 deposition in stylets of Octopus vulgaris to the size range analysed. Acknowledgements Acknowledgements This research was supported by ECOSUMMER actions (European Union). C. Hermosilla has an early training ECOSUMMER (Marie Curie Action). F. Rocha is an Isidro Parga Pondal Researcher. Animals were housed and maintained at ECIMAT (University of Vigo, Vigo, Spain) and Stazione Zoologica A. Dohrn (Napoli, Italy). y = 0,8586x+ 2,7868 R 2 = 0,9652 0 5 10 15 20 25 0 5 10 15 20 25 Days afterstaining Increments

Cephalopod International Advisory Council, CIAC’09 Symposium. Vigo (Spain), 3-11 September 2009 Age validation in common octopus, Octopus vulgaris Cuvier

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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.

Figure 1. Photograph showing the fluorescent Oxytetracycline (OCT) stylet mark in a female of 1140 g (stained with 124 mg OTC per Kg).

Figure 2. Light photograph showing the stylet increments in the same specimen of Fig.1. Arrows showing the location of OTC mark.

Figure 3. Relationship between number of days elapsed after OTC staining and number of increments counts between OTC mark and stylet border.

IntroductionIntroduction

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 number of rings produced with the elapsed days.

Material and MethodsMaterial 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 was achieved. Several sections of each stylet were prepared and photographed (Figures 1 and 2). Increments were counted 3-5 times by two different readers. Increments counts for each stylet were analysed and outliers excluded using statistical methods.

Results and discussionResults and discussion

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 rate of increments formation was 1.02 increments per day (1 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 deposition in stylets of Octopus vulgaris to the size range analysed.

AcknowledgementsAcknowledgements

This research was supported by ECOSUMMER actions (European Union). C. Hermosilla has an early training ECOSUMMER (Marie Curie Action). F. Rocha is an Isidro Parga Pondal Researcher. Animals were housed and maintained at ECIMAT (University of Vigo, Vigo, Spain) and Stazione Zoologica A. Dohrn (Napoli, Italy).

y = 0,8586x + 2,7868

R2 = 0,9652

0 5 10 15 20 25

Days after staining

Incre

ments