Age validation in common octopus Octopus vulgaris using stylet increment analysis

Abstract

Hermosilla, C. A., Rocha, F., Fiorito, G., González, Á. F., and Guerra, Á. 2010. Age validation in common octopus Octopus vulgaris using stylet increment analysis. – ICES Journal of Marine Science, 67: 1458–1463.

Daily periodicity of growth increments in stylets was validated in wild-caught Octopus vulgaris maintained under controlled conditions. It was corroborated by staining the stylets either with oxytetracycline (OTC) or tetracycline (TC), and comparing the number of rings produced with the number of days elapsed. In all, 19 animals (10 males and 9 females; 680–1470 g body weight, BW) were injected with OTC in Vigo (mean 124 mg kg⁻¹), and another six animals (1 male and 5 females; 248–570 g BW) with TC at Naples (mean 120 mg kg⁻¹). The animals were successfully maintained in captivity until sacrificed for up to 6 (one animal), 9 (one animal), 18 (the six animals from Naples), and 21 (17 animals) days. The number of increments counted in transverse stylet sections was 18.9 ± 1.4 and 20.5 ± 1.5 for octopuses maintained for 18 and 21 d, respectively. The mean rate of increment formation was 1.02 increments per day, suggesting a periodicity of 1 increment per day in the stylet. Consequently, the results successfully validate daily increment deposition in O. vulgaris stylets in the size range analysed.

age validation, increments, Octopus vulgaris, oxytetracycline, stylets, tetracycline

Introduction

Information on age is critical for managing fish resources, especially species experiencing heavy fishing pressure. In the absence of accurate age information, our understanding of octopus growth, recruitment, and productivity relies on methods using morphological and catch data. Such methods are considered inaccurate in estimating growth rates and longevities (Jackson, 1994; Boyle and Boletzky, 1996) owing to the high intrinsic variability of cephalopod growth, the mixture of different age microcohorts at the same size, and the absence of a validated technique to estimate age and growth in these species (Doubleday et al., 2006; Leporati et al., 2008a). The common octopus, Octopus vulgaris, is the most important commercially fished octopus species along the coasts of the Atlantic and Mediterranean (Josupiet, 2000); global catches reached more than 37 000 t in 2007 (FAO, 2009). Although there is a large body of knowledge on the biology and ecology of the species (Mangold, 1983; Guerra, 1992; Domain et al., 2000; Otero et al., 2008, 2009), little is known about its age and growth based on internal hard structures such as statoliths, beaks, or stylets (Raya and Hernández-González, 1998; Hernández-López et al., 2001; Sousa-Reis and Fernandes, 2002).

Age and growth of O. vulgaris has been determined at an individual and a population level in the laboratory or in the wild using different methods (Itami et al., 1963; Nixon, 1969; Mangold and Boletzky, 1973; Guerra, 1979; Hatanaka 1979; Pereiro and Bravo de Laguna, 1979; Smale and Buchan, 1981; Villanueva, 1995; Domain et al., 2000). However, those studies did not achieve consensus in the growth models and age estimates (Guerra, 1979; Hatanaka, 1979; Mangold, 1983; Hernández-López et al., 2001). Therefore, an accurate and validated method of age determination for octopus is still required, ideally a method based on internal structures with approaches similar to those applied to fish (otoliths and vertebrae).

More than two decades of work based on the analysis of statoliths to determine age have proven that this approach is an optimal means of estimating the age and growth of wild cephalopods (Villanueva et al., 2003). The mineral structure of statoliths as well as its biomineralization process is relatively well known, and several hypotheses have been formulated to explain the mechanisms of daily deposition of growth increments in them (Bettencourt and Guerra, 2000). Also, studies have correlated the influence of several environmental factors, especially temperature, with growth and the deposition of increments in the statoliths of several species of squid (Villanueva et al., 2003).

Statolith increment analysis has been applied systematically in squid (Arkhipkin and Laptikhovsky, 1994; Jackson, 1994; González et al., 1996, 2000; Rocha and Guerra, 1999) and cuttlefish (Raya et al., 1994; Bettencourt and Guerra, 2001; Challier et al. 2002), and it is currently considered a reliable and validated technique. Unfortunately, statoliths have failed to provide useful results for octopus owing to a lack of visible growth rings (Lombarte et al., 2006), and the search for techniques to age octopods based on hard structures has continued.

On the other hand, growth increments in the beaks of O. vulgaris have been considered a reliable indicator of the age of the animal by Raya and Hernández-González (1998). That method was validated for octopus paralarvae for a specific time frame (Hernández-López et al., 2001), but the relationship between age and the number of increments for juvenile and adult O. vulgaris beaks remains unproven. Additionally, the structure of the beak, mainly the rostrum, can be affected by processes such as feeding, yielding inaccurate estimates (Hernández-López et al., 2001). For this reason, new techniques, such as sectioning through the rostrum sagitta and the lateral wall surfaces, have improved the accuracy of readings by enhancing the visibility of growth increments in upper and lower O. vulgaris beaks (Perales-Raya et al., 2009). Consequently, further study of this technique needs to be undertaken to determine the suitability of this method for estimating octopus age.

Stylets have recently been proposed as a useful hard structure to evaluate the age of octopods (Sousa-Reis and Fernandes, 2002; Doubleday et al., 2006; Leporati et al., 2008a). Stylets are the vestigial shell in incirrate octopods, restricted to two very fine, elongate structures in the mantle muscle at the base of the gill (Wells, 1978; Bizikov, 2008). Analysis of trace element composition in O. pallidus stylets suggests that the structure is chitinous, with some inorganic components associated with phosphatic mineralization (Doubleday et al., 2008). Concentric rings have been observed in O. vulgaris stylets, but deposition rhythms have not been validated, although the regular patterns of micro-increments observed during growth suggests constant deposition (Sousa-Reis and Fernandes, 2002; Napoleão et al., 2005). Recently, the daily deposition of growth increments has been validated in Octopus pallidus using animals of known age reared in captivity (Doubleday et al., 2006), and it has subsequently been applied to the study of age, growth, and reproduction in wild populations of O. pallidus (Leporati et al., 2008a, b).

Our aim here was to validate the daily periodicity of growth increments in stylets of adult O. vulgaris maintained in captivity, by staining the structures with oxytetracycline (OTC) or tetracycline (TC) under experimental conditions.

Methods

The 25 O. vulgaris used in the study were in two experimental groups. Group 1 consisted of 19 (10 males and 9 females; 680–1470 g body weight, BW) animals caught during July 2008 in the Ria de Vigo (northwestern Spain, Northeast Atlantic), and Group 2 contained six (1 male and 5 females, 248–570 g BW) animals caught during November 2006 in the Bay of Naples (southwestern Italy, Mediterranean Sea). The two groups of animals were maintained in the laboratory for 30 and 10 d, respectively, to acclimatize them to captivity at ECIMAT (Marine Sciences Station of Toralla, University of Vigo, Spain) and Stazione Zoologica Anton Dohrn (Naples, Italy), respectively. All animals were maintained under the same standardized conditions of 12:12 h light–dark photoperiod and seawater temperature between 17 and 21°C. Their diet in captivity was fish (Micromesistius poutassou and Sardina pilchardus in Vigo) and crabs (Carcinus mediterraneus in Naples), in both cases provided ad libitum.

The Vigo animals were marked with OTC (average dose injected 124 mg kg⁻¹, except one animal that received 250 mg kg⁻¹; Table 1). The Naples animals were stained with TC (average dose injected 120 mg kg⁻¹; Table 1). All were injected subcutaneously at the base of one of the anterior arms, under anaesthesia from MgCl₂ at 7.5% (Messenger et al., 1985). After injection, animals were allowed to recover from anaesthesia, then kept for 18–21 d.

Table 1.

Octopus vulgaris parameters and results obtained during age-validation experiments.

Origin	Specimen	Sex	Maturity stage	Initial weight (g)	OTC (mg kg−1 octopus)	TC (mg kg−1 octopus)	Days elapsed after staining	Mean number of increments	s.e.	Number of counts by stylet	Mean number of increments per day
Group 1: Vigo	1	Male	Mature	680	144.9		21	22.1	1.7	8	1.1
	2	Female	Maturing	1 030	121.7		21	19.2	2.2	5	0.9
	3	Female	Maturing	1 340	118.5		21	21.5	4.2	6	1.0
	4	Female	Maturing	1 330	122.1		21	19.0	1.6	7	0.9
	5	Male	Mature	1 260	112.9		21	19.2	4.4	6	0.9
	6	Female	Maturing	770	139.5		21	22.2	1.5	6	1.1
	7	Male	Mature	830	116.3		21	23.3	2.9	6	1.1
	8	Male	Mature	1 470	108.8		21	19.7	0.6	3	0.9
	9	Female	Maturing	1 380	117.6		21	23.3	2.7	6	1.1
	10	Female	Maturing	1 220	125.0		21	20.8	1.9	9	1.0
	11	Male	Mature	920	126.3		21	23.3	1.2	3	1.1
	12	Female	Maturing	880	141.2		21	17.7	3.2	3	0.8
	13	Female	Maturing	1 020	132.1		21	22.2	2.6	6	1.1
	14	Male	Mature	1 050	120.7		21	20.0	1.0	3	1.0
	15	Male	Mature	1 140	116.7		21	16.7	0.6	3	0.8
	16	Male	Mature	960	117.6		9	9.0	1.1	8	1.0
	17	Male	Mature	1 060	123.9		21	20.9	3.7	8	1.0
	18	Female	Maturing	1 140	124.0		21	21.5	3.1	6	1.0
	19	Male	Mature	860	250.0		6	9.0	1.0	3	1.5
Group 2: Naples	33	Female	Maturing	442		120.0	18	19.0	1.0	3	1.1
	34	Female	Maturing	570		120.0	18	21.3	2.3	3	1.2
	35	Male	Maturing	352		120.0	18	20.5	1.7	4	1.1
	36	Female	Maturing	248		120.0	18	16.3	3.5	7	0.9
	37	Female	Maturing	386		120.0	18	16.0	2.9	7	0.9
	38	Female	Maturing	400		120.0	18	19.0	1.0	3	1.1
Average							19.2	19.3	2.1	5.3	1.0

The individual OTC and TC concentrations used for each animal are indicated.

At the end of the experimental period, the animals were sacrificed using terminal anaesthesia (ethanol at 5% in seawater; Moltschaniwskyj et al., 2007). All were weighed (g), and the stylets were extracted and preserved in 4% formaldehyde in the dark. Transverse stylet sections of the post-rostral zone (∼2 mm) were embedded in thermoplastic resin (Crystalbond), then polished by hand with 1200 grade sandpaper (3M Imperial MicroFinishing Film) and lapping film (12, 9, and 5 µm; 3M Imperial Lapping Film) until a thin, translucent section was achieved. As OTC and TC marks were not evident in all stylet segments, several sections of each stylet were prepared and photographed under a microscope (Nikon fluorescence microscope 90i) at ×400 magnification, using an ultraviolet light system to show the markings. Digital photographs were made using NIS-Elements software developed by Nikon, then stored in .jpg format. In each case, at least two photographs of the same section were taken, one using UV light and one using normal light. Digital photos were taken immediately, because the microstructure disintegrated (sections would crack, shrink, and darken) soon thereafter, typically 15–30 min after preparation.

Increments of each stylet were counted using the digital photographs examined using Photoshop software. In all, three to nine different sections of the same stylet were counted by two independent readers; in other words, the rings of each stylet were counted at least three times by the same person (a minimum of six counts by the two readers) using different sections of the same stylet. Only sections that showed a continuous series of increments were used, to preclude any extrapolation in the increment count. Any counts that differed more than 10% from the mean (outliers) were excluded using a Tukey (1977) method. For each stylet, the mean and the standard error (s.e.) of increment counts were estimated (Zar, 1999).

Results

All except two octopuses were successfully maintained in captivity, the exception being two animals (860 and 960 g BW) that were sacrificed or died after 6 and 9 d. The first was sacrificed 6 d after injection to determine whether stylet staining had been successful, and the second stopped eating after injection and died 9 d later, probably through stress. The BW of all other animals increased at a rate of 7.55 ± 4.38 (mean ± s.e.) g per day during the experiment.

Examination of the stylet photographs clearly showed the regular deposition of stylet growth increments (Figure 1). All sections showed concentric rings distributed regularly and, in some cases, rings strongly marked (probably stress marks). A possible embryonic nucleus was observed in the centre of stylet sections (Figure 1a). Several photographs revealed cracks and discontinuities in the sections, likely caused by stylet rupture during preparation. When these cracks affected the area near the OTC or TC mark, the photographs were not used for counting.

Figure 1.

Photographs showing growth markings in two sections of the same stylet of one O. vulgaris (female, 1290 g BW, 141 mm mantle length). The regular deposition pattern of growth increments in the stylet is clear. (a) Central section of the stylet showing the nucleus. (b) The border section of the stylet.

There were clear marks in the stylets of all animals, caused by OTC or TC injection (Figure 2). These marks were not evident in all sections of the same stylet as a consequence of the problems related to cracking or damage during preparation. In some (e.g. the female of 1140 g shown in Figure 2), the OTC mark observed with UV light coincided with a stress mark observed using transmitted light, the latter probably caused by the OTC injection event. The number of increments counted was correlated (p > 0.005; n = 25) with the number of days animals were maintained after OTC or TC injection (Figure 3), and the slope did not differ significantly from unity (t =− 0.398, v = 4). Animals of the Vigo group 1 maintained 21 d after OTC injection showed 20.5 ± 1.5 (mean ± s.e.) increments, and those of the Naples group maintained 18 d after TC injection showed 18.9 ± 1.4 increments. From our data, the mean rate of increment formation was estimated to be 1.02 ± 0.14 increments per day (Table 1).

Figure 2.

(a) Section of a stylet showing the fluorescent OTC mark in a female of 1140 g (injected 124 mg kg⁻¹). A second mark close to the border is also clear; this mark is an artefact caused by heating during the preparation procedure. (b) The same section in transmitted light. Arrows indicate the location of OTC marks. (c) Detail of the increments counted indicated by arrows.

Figure 3.

The relationship between number of days elapsed after OTC and TC staining and the number of increment counts between the mark showing injection with OTC/TC and the stylet border.

Discussion

The results suggest that growth increments are laid down in the stylets of O. vulgaris at a rate of one ring per day, as found in O. pallidus (Doubleday et al., 2006). The accuracy of our counts was supported and validated by the close match between increment number and days elapsed after staining with OTC or TC. Consequently, we believe that we have successfully validated daily increment deposition in the stylets of O. vulgaris over the size range of animals analysed (248–1470 g BW). Further experiments are needed, however, to validate the technique across the whole size spectrum for this species, including juveniles and larger animals, providing more-rigorous evidence of daily increment formation in O. vulgaris stylets.

In some cases, the number of increments counted in the stylets deviated from the expected number, but such deviation could be due to the reader error, produced by an artefact of the preparation or the method of counting (Doubleday et al., 2006). In most cases, there was a high level of accord between counts, replicates, and days after injection, with deviations well below the 10% commonly considered acceptable for other cephalopod age studies (Jackson and Lu, 1994; González et al., 1996, 2000; Rocha and Guerra, 1999; Pecl, 2004; Doubleday et al., 2006).

In our study, stylets were fixed in 4% formaldehyde and not later transferred to ethanol for storage. The procedure yielded good results and the increments could be read with relative ease. In this case, subsequent preservation in ethanol was unnecessary, so the procedure used was reasonably simple. However, this conclusion is based on the stylets of a single species, and it is possible that stylet structure can vary significantly between species. For example, Doubleday et al. (2006) found that the microstructure of formalin-preserved O. pallidus stylets differed slightly from those preserved in ethanol. In contrast, Leporati et al. (2008a) found that there was no significant difference between age estimates derived from formalin- and ethanol-fixed stylets. However, as indicated by Leporati et al. (2008a), the results only corroborated the fact that formalin-treated stylets tended to swell, making them weaker and more susceptible to damage, although no effect could be observed on increment counts.

In accord with Doubleday et al. (2006), it was considered that the best method to count growth increments in the stylets is from digital photographs taken immediately after preparation, because the stylets appear to disintegrate soon thereafter. The reason for disintegration of prepared sections is still unknown. Microstructural disintegration after preparation can be caused by several factors, e.g. excessive heat during preparation or an inadequate medium in which the stylet was preserved (Doubleday et al., 2006). A working hypothesis could be that the organic composition of the stylet, with just 35% of total inorganic content (Doubleday et al., 2008), can influence disintegration, so it is possible that stylet sections can suffer dehydration during preparation causing deterioration of the organic chitin matrix and subsequent collapse.

New protocols have recently been proposed for preparing stylet samples (for O. vulgaris and Eledone cirrhosa, I. M. Barratt and A. L. Allcock, pers. comm.; for Octopus maya, U. Markaida, pers. comm.). In these new methods, resin or gelatinous media are employed to embed stylets to protect the section and preclude structural disintegration. Preliminary results show that these protocols seem to yield good results and also provide the possibility for each section to be recounted at a later date.

The employment of stylets for age determination is still a new technique that requires further evaluation to understand the mechanisms that result in the daily deposition of growth increments. Although some studies on the biochemistry, element composition, and microstructure of stylets in O. vulgaris (Napoleão et al., 2005) and O. pallidus (Doubleday et al., 2008) have been carried out, more information is needed to formulate a better understanding of the mechanism of stylet increment formation. For example, it seems necessary to extend the validation over the whole life cycle of a species because, to date, this has only been done for adults or subadults. Sousa-Reis and Fernandes (2002) stated that stylets of previously frozen material contained sections with cracks, making counting impossible. However, increment counts from stylets obtained from frozen O. vulgaris could be made even when cracks were evident (CAH, unpublished data), implying that stylets can be used for age determination from commercially caught octopuses. However, the effect of freezing on stylets does need to be investigated further, because it is still possible that freezing might prove to be a critical handicap to the use of these structures in age studies of octopuses caught commercially. Notwithstanding, we agree with Doubleday et al. (2006) that growth increments in stylets appear to provide a straightforward and reasonably successful method of assessing the age of octopods. The method could be more useful if other analytical tools were used on stylets together with the increment age analysis, however. For instance, the changes of trace element composition in different sectors of the stylet can be associated with the age to obtain information on its ecology and environmental history (Doubleday et al., 2008).

Octopus age studies are critical for most biological, ecological, and fisheries studies. Owing to their importance as fishery resources, it is vital that a precise and validated technique of determining octopus age, growth, and population characteristics be developed. In this way, the stylet-based age-determination method offers an important advance in the knowledge of the biology, ecology, and management of octopods, a group of cephalopods with many species of commercial interest. Finally, the validation of this age technique for O. vulgaris opens fresh possibilities for studying one of the most important exploited species of octopus.

Acknowledgements

The research was supported by ECOSUMMER (European Union), and CAH received early training ECOSUMMER (Marie Curie Action) support. FR is an Isidro Parga Pondal Researcher. We specially thank Gretta Pecl, ICES JMS editor Andy Payne, and two anonymous referees for providing helpful comments and corrections, and Katherine Murphy for improving the English.