Age validation in early stages of Sepia officinalis from beak microstructure

This is the first study addressing validation of the early growth stages (including the first increment) in the beaks of juvenile cuttlefishes. The age validation in juveniles of Sepia officinalis was performed by comparison of the number of increments observed in the rostrum surface of lower jaws with their true age. A total of 159 individuals were reared at 18 ºC and 21 ºC, with ages up to 31 days from hatching. The number of growth increments in the beak was counted and contrasted with the days of life after hatching, validating the hypothesis of one increment of growth corresponding to one day of life. The mean coefficient of variation between readings (measuring precision) was 2.95 ± 5.98%. The growth of the reading area (rostrum surface) and the periodicity of increment deposition showed no difference between the two culture temperatures and therefore daily deposition was confirmed at these temperatures.


Introduction
Since Young (1960), hard structures have become a routine tool for age estimation of cephalopods (Arkhipkin et al. 2018). However, despite the fact that in 1965 Clarke observed the growth lines in Moroteuthis ingens beaks (Moroteuthopsis longimana, as it was confirmed later by Cherel 2020), statoliths have been the most frequently used hard structure for ageing cephalopods (Jereb et al. 1991;Morris 1988;Rodhouse and Hatfield 1990;Šifner 2008;Arkhipkin and Shcherbich 2012). Perales-Raya and Hernández-González (1998) suggested that beak sections of Octopus vulgaris are suitable for age estimation, and Hernández-López et al. (2001) improved Clarke's method (1965) by counting increments on the inner surface of the lateral wall of the lower jaw, confirming daily periodicity in octopus paralarvae up to 30 days of age. Both methods were later compared and improved (Perales-Raya et al. 2010), although the daily deposition of beak increments across the entire age range of the species in both the lateral wall surfaces (LWS) and sections was not validated until several years later (Perales-Raya et al. 2014b), confirming later the first increment using embryos and new hatchlings (Armelloni et al. 2020). Moreover, Liu et al. (2015) concluded that beaks present greater advantages in age studies than statoliths, due to the relative simplicity of this processing method. After validation in O. vulgaris, the beaks have been used for age estimation in a number of cephalopod species, mainly squids and octopuses (e.g. Fang et al. 2016;Liu et al. 2017;Donlon et al. 2019;Jin et al. 2019;Schwarz et al. 2019;Batista et al. 2021).
Daily growth increments in other structures such as stylets or statoliths have been validated for several benthic cephalopod species, including Octopus vulgaris (Hermosilla et al. 2010); O. pallidus (Leporati et al. 2008 (Rodríguez-Domínguez et al. 2013). With regard to neritic/pelagic cephalopods as loliginids (Jackson 1990;Lipinski et al. 1998;Cordella de Aguiar et al. 2012;Hoving and Robison 2017) and Oegopsida squids (Hurley et al. 1985;Nakamura and Sakurai 1991), statoliths were the validated structures, although gladius and beaks were cross-verified by comparing their counts with those from statoliths (Arkhipkin and Bizikov 1991;Hu et al. 2016, among others). The common cuttlefish, Sepia officinalis (Linnaeus 1758), is a target species in many industrial and artisanal fisheries in the Eastern Atlantic and the Mediterranean Sea (Boletzky 1983;Denis and Robin 2001), and frequently reported as a by-catch of bottom trawling (Rathjen and Voss 1987;Jereb et al. 2015). Age determination is essential for cuttlefish population dynamic modelling, and therefore for the assessment and management of its fisheries. However, according to Jackson and Moltschaniwskyj (1999), age estimation methods must be accurate and precise and not laborious or very time-consuming. Most of Sepia officinalis age estimation studies have been based on polymodal decomposition of length frequencies (Boucaud-Camou and Boismery 1991;Mion et al. 2014), the analysis of statolith microstructure (Perales-Raya et al. 1994;Bettencourt and Guerra 2001) or the cuttlebone (Nabhitabhata et al. 2022) although limited estimations have been obtained.
The beaks in sepiid species were tested in adults of Sepia apama (Hall et al. 2007), and more recently in S. officinalis by Lishchenko et al. (unpublished results), but validation is still necessary. The aim of the present study is to estimate, for the first time, the ages of the juveniles in sepiid species by counting the growth increments observed in the beaks, to validate daily deposition and the age of the first increment in known-age cuttlefishes reared in captivity.

Materials and methods
A total of 159 spring hatchlings of Sepia officinalis were obtained from fertilized eggs collected in shallow waters off Gran Canaria (Tufia; 27º57'N, 15º22'W). The eggs were attached to a rope at 7 m depth in a sandy bottom. They were collected in a single batch and the water temperature was 20.4 ºC. Eggs and newly hatched juveniles were reared at similar water temperature (21 ºC), and also at 18 ºC (as a small reduction within the "conventional" rearing temperature of 20 ± 2 °C described by Iglesias and Fuentes 2014) to evaluate the effect of small changes in temperature in the pattern of growth increments on beaks. The study was carried out in two phases. Phase I addressed two issues: (a) testing the hypothesis of 1 day = 1 increment, and (b) evaluating the impact of the temperature on the increment deposition rates. After Phase 1, Phase II consisted of replicating Phase I and standardizing the methodology used for the newly hatched Sepia officinalis. In this phase, all cultures were carried out at 21 ºC.
After sacrifice, the dorsal mantle length (DML) of each individual was measured to the nearest 0.1 mm. DML ranged from 5.08 mm to 8.89 mm, and their total weight varied from 0.1098 to 0.2712 g (Table 1). A total of 127 and 32 lower jaws of Sepia officinalis reared in the laboratory at 21 ºC and 18 ºC, respectively, were analysed for age determination following the methodology of Perales-Raya et al. (2018) in octopus paralarvae.

Rearing conditions
The eggs development occurred in the natural environment, collecting the eggs in the organogenesis period (Lemaire 1970). Then, the eggs were incubated in our lab under natural conditions (water temperature around 19-21 ºC, photoperiod of 14 h L / 10 h D, and a salinity of 36 PSU). All juveniles were reared with the aim of sacrificing them randomly to validate the daily deposition in beaks. Newly hatched individuals were reared inside the laboratory in 25 transparent 10-L square glass tanks (20 × 20x25, densities not greater than three individuals per tank), with aeration, recirculating seawater at a natural photoperiod. No artificial shelters were provided to hatchlings, their feeding was performed ad libitum (3 times per day) with live Artemia salina on a daily basis. Thirty-two individuals were reared at 18 ºC, while 127 were kept at 21 ºC. In Phase I, 112 individuals were cultured, of which 32 were reared at 18 ºC and 80 at 21 ºC. There were differences in the number of specimens cultured at each temperature, but the total number of specimens cultured at each temperature condition was enough to check the significance of differences between temperatures. In Phase II, 47 individuals were cultured at 21 ºC. No individuals were cultivated at 18 ºC because in Phase I differences between those cultured at 18 ºC and 21 ºC were no significant. The specimens were euthanized by anaesthetic overdose (clove oil, following Ayala-Soldado 2014) and dissected fresh.
Animal rearing was performed in compliance with Spanish law 53/2013 within the framework of the European Union's adopted directive 2010/63/EU regarding animal welfare for the protection of animals employed for scientific purposes, following the Guidelines for the Care and Welfare of Cephalopods in Research, as proposed by Fiorito et al. (2015). The present study was also approved (register document OEBA-ULPGC 04/2019R1) by the Ethics Committee for Animal Research (Comité Ético de Experimentación Animal of the University of Las Palmas de Gran Canaria, CEEA-ULPGC, Spain).

Beak extraction, preparation and analysis
The beaks were extracted, cleaned, labelled and stored in distilled water at 5 ºC, as recommended by Perales-Raya et al. (2014b for Octopus vulgaris paralarvae. Previously, it had been observed that the conservation of the beaks in 70% ethanol resulted in a low visibility of the growth increments. Upper and lower jaws were tested for age estimation. Each jaw was placed whole, without cutting, in the ventral position (convex side up) on a drop of water. It was then covered with a coverslip left tilted until the jaw was positioned correctly. Once its position was secured, some pressure was exerted on the coverslip to ensure fixation of the jaw and obtain standardized images (see Armelloni et al. 2020) (Fig. 1a). Image acquisition was carried out according to Perales-Raya et al. (2014b and Armelloni et al. (2020).
After the correct placement, two areas of the jaws-the LWS and the rostrum surface-were explored to select the one that showed higher viability for age estimation. The increments were finally counted on the surface of the pigmented area (rostrum) of the lower jaw under transmitted light with a Nikon Microscope Multizoom AZ100 (400 × magnification). The system is equipped with a differential interference contrast attachment (DIC-Nomarski) that creates a 3-dimensional image of the rostrum surface, where the sequence of micro-increments is revealed. The regions of the jaw were identified, as well as the first increment (hatching; Fig. 1b), to standardize the reading methodology.
The increments were counted twice by the same trained reader. The coefficient of variation (CV) was calculated for each specimen to determine the precision (reproducibility) of the counts: where R1 and R2 were the mean number of increments from the first and the second reading, respectively; R was the mean number of increments for both readings . The sample was classified into 7 age groups (Table 1): Group 0: 0-2 days; Group 1: 3-7 days; Group 2: 8-12 days; Group 3: 13-17 days; Group 4: 18-22 days; Group 5: 23-27 days; Group 6: 28-31 days. To avoid any bias by age groups, the CVs were averaged for each age group, and CVs < 7.6% were taken as valid (Campana et al. 2001). The mean CV was then calculated for each temperature group to assess and compare the precision of their readings. The number of increments was compared with the true age. The width of the reading area (WRA) was measured (μm) in the widest region of the reading area, where the increments were counted, in accord with Perales-Raya et al. (2018) (Table 1).

Statistical analysis
All statistical analyses were carried out using R v-4.1.1 (R Core Team 2021). A general linear model (GLM) was used to test the effect of the temperature, the age, and the interaction of age with temperature on increment numbers following Perales-Raya et al. (2018). ANOVA of the GLM fits was carried out to analyse the possible significant differences in relation to the variables described (Blanca- Mena et al. 2017;Foster 2021). In the same way, ANOVA was used to observe differences in the growth of the WRA between both cultures. The function "glm" was used under the package 'stats v.4.0.3' (R Core Team 2021).

Results
The lower jaw rostrum shows clear and continuous deposition of the growth increments on its surface, in a way similar to that described in Perales-Raya et al. (2014b; and Arkhipkin et al. (2018) for the upper-jaw rostrum of Octopus vulgaris paralarvae. The DIC-Nomarski technology facilitated image analysis thanks to differential interference contrast, highlighting the deposited daily increments. Our examination of both jaws in Sepia officinalis did not reveal any teeth but the rostral area showed a strong pigmentation, which apparently allows a better reading of the growth marks. Regarding the upper jaws, the observed increments did not present a clear sequence of deposition in the rostrum. For this reason, and because the increment deposition was observed more clearly in the lower jaws, they were selected for ageing Sepia officinalis. Our exploration of LWS showed unsatisfactory results with no clear pattern of increments for age estimation. Moreover, the observed increments could often be confused with the roughness of the structure due to their poor definition in some areas and the low pigmentation of the lateral walls. The first increment (hatching) in the rostrum surface of the lower jaw was located in the anterior border of the reading area, and the day of death is the latest increment (posterior border) of the reading area (Fig. 1b). We observe a translucent region that extends to the anterior border of the jaw. This area was identified as a developing shoulder (Fig. 1b), as described by Perales-Raya et al. (2018) for the Octopus vulgaris paralarvae. Increments in the rostrum of lower jaws were clear (Fig. 2). Nevertheless, from the culture at 21 ºC, only 121 of the 127 collected beaks (95.28%) were suitable for age estimation. In the case of cuttlefishes reared at 18 ºC, 29 beaks of the 32 collected (90.63%) were readable.
Mean reading precision (CV) of readings in the rostral surface of S. officinalis was 2.95 ± 5.98% for all individuals. Mean values of CV for each age group were lower than the usual adopted value of 7.6% for annual and daily structures (Campana 2001; Table 1), and therefore, no additional individuals were discarded based on the CV. The CV in individuals cultured at 21 ºC (n = 120) was 3.26 ± 6.75%, and 2.01 ± 2.47% for those cultured at 18 ºC (n = 27). The age assigned to each individual was the average of the two readings.
The relationship between the number of increments and the true age (Fig. 3) in newly hatched cuttlefish at both temperatures showed a linear trend ( Table 2). The regressions between age and number of increments shown in Table 2 confirm daily deposition of increments in juveniles of S. officinalis at 18ºC and 21ºC. In Phase I, ANOVA of the GLM fit showed no significant differences in the increment deposition determined by the temperature alone or the factor Age x Temperature (Table 2 b). The same results were obtained for WRA growth, with no significant differences between cultures at different temperatures (ANOVA, p > 0.05). Phase II results are shown in Table 3.

Discussion
This is the first time that the beak microstructure of Sepia officinalis has been shown in its early stages, where the visualization and the count of growth increments in the lower jaw's rostrum was relatively easy. In contrast to the hatchlings of Octopus vulgaris, newly hatched Sepia officinalis showed no teeth as well as higher and wider pigmentation in the rostrum of lower jaws. This pigmentation in the reading area of the lower jaws probably allowed a better estimation of the true age, while for O. vulgaris the visibility of increments is much better in the upper jaws (Perales-Raya et al. 2014b; Armelloni et al. 2020). The lower jaws of O. vulgaris have a narrower and less pigmented rostrum, with deeper teeth and fewer increments visible compared with the upper jaws. It should also be noted that the upper jaws of S. officinalis juveniles are harder than those of O. vulgaris paralarvae, which complicates their manipulation and increase the risk of damaging them.
Regarding the LWS of S. officinalis beaks, they showed unclear growth marks that could be confused with roughness of the structure. Additionally, and according to Armelloni et al. (2020) for O. vulgaris paralarvae, the LWS in cuttlefish did not show highly developed pigmentation during these early stages and, consequently, some of the observed marks could be false increments. On the other hand, the rostral surface, which is already pigmented at hatching, showed a continuous sequence of growth increments in S. officinalis, making it possible to identify the age of the first increment. It is mandatory for correct validation of the methodology (Campana 2001).
The sizable agreement among readings indicates a high precision of readings in the rostral surface of Sepia officinalis. The reading precision (estimated using CV) was high because the readings differed with a maximum of ± 2.95%, although the variability of CV was high too (± 5.98), which is probably related to the complexity of the structure . The juveniles used in the present study were fed with soft diet but feeding may erode the beaks and careful should be taken to avoid age underestimations.
The GLM analysis showed that our results support the validation of this methodology for age estimation using beak microstructure as has been shown in other benthic species such as Octopus vulgaris (Hernández-López et al. 2001;Perales-Raya et al. 2014bArmelloni et al. 2020) andO. maya (Rodriguez-Domínguez et al. 2013;Villegas-Bárcenas et al. 2014). The present study is the first validating the daily deposition of increments in early stages and the age of the first increment in the beaks of Sepia officinalis. Ageing cuttlefish using the beak rostrum seems a suitable method for these species, in comparison with other commonly used methodologies, which are length-frequency analysis and direct ageing methods using statoliths and cuttlebones. Length-frequency distributions have some problems because the age of cohorts are not independently validated, the cephalopods show a high inter-individual variability, and modelling methods ignore variable growth rates which are influenced by environmental conditions (Semmens et al. 2004;Arkhipkin et al. 2021). Jackson et al. (1997) compared this methodology against the use of statoliths, since several authors used length-frequency analysis to analyse growth rate and growth forms (e.g. Jereb and Ragonese 1995; Mohamed 1996 among others). Jackson et al. (1997) concluded that length-frequency analysis should not be used for these age determinations, being the statoliths the promising technique at that time. As for statolith-based age determination, it is relatively laborious involving the extraction, handling, storage, mounting, both-side grinding and polishing, and counting under a microscope (Arkhipkin and Shcherbich 2012). In addition, its application is limited in cuttlefishes due to the complex crystalline structure of their statoliths (Natsukari and Tashiro 1991;Perales-Raya et al. 1994). Using the sequence of increments observed in the lateral dome (Perales-Raya et al. 1994), Bettencourt and Guerra (2001) validated increment deposition till 240 days of age in S. officinalis; but in older specimens, the age was underestimated due to the poor resolution of newer increments. Regarding the internal shell (cuttlebone), it has been used since the 1960s in several sepiid species, but formation of the shell stripes, or lamellae, is determined by physiological and environmental conditions (Yagi 1960;Choe 1963;Richard 1969; Ming-Tsung and Wang 2013, among others). It has also been used in S. officinalis (Richard 1969;Re and Narciso 1994, among others) and Sepia hierredda (Perales-Raya 2001). The absence of a daily deposition in the cuttlebone of S. officinalis was also described by Ré and Narciso (1994) and Le Goff et al. (1998) where they conclude that the cuttlebone lamellae should not be used for age estimation. The number of lamellae does not correspond to the true age and the temporal periodicity can be defined only if the temperature where the animal lives is considered (Bettencourt and Guerra 2001). The internal shell was recently analysed in several loliginid and sepiid species of known age by Nabhitabhata et al. (2022), reevaluating the accuracy in the neritic species living the tropical zone where environmental conditions are more stable. Moreover, some studies proposed to use the concentration of lipofuscin, a pigment that accumulates in tissues, as a proxy of age (Gras et al. 2016), but this method is considered complex and inaccurate (Doubleday and Semmens 2011). The use of these methodologies for age estimation were promising, although biological factors (length-frequency analysis) and morphological factors of the analysed structures (statolith and cuttlebone analysis) may influence the under-or over-estimation of age.
Using the rostrum surface of juveniles to determine age is equivalent to using rostrum sagittal sections in adults, as demonstrated in Octopus vulgaris (Perales-Raya et al. 2018, Lishchenko et al. unpublished results). Their similarities indicate that both structures are equivalent and suitable for S. officinalis. Cuttlefish beaks, once validated in adult stages, could be a promising method to determine the age of cuttlefishes. The water temperature seems to be one of the most powerful factors influencing cuttlefish growth in cultured conditions. Domínguez et al. (2006) compared wild and reared Sepia officinalis observing that temperature and culture space play an important role in the growth of individuals. In the case of Octopus vulgaris, Perales-Raya et al. (2018) showed that the readings in octopuses cultured at 14 ºC and 21 ºC differed in width, whereas octopuses cultured at 21 ºC had a bigger reading area than octopuses reared at 14 ºC. Moreover, octopus paralarvae reared at 14 ºC showed apparent compaction of increments in the beaks and growth slowed or stopped at that temperature, thus affecting increment depositions . However, in our study, cuttlefishes reared at 18 ºC and 21 ºC did not show significant differences in the WRA. This could be due to the experimental temperatures, since they were both within the optimal range for S. officinalis, and this seems irrelevant for the increase in rostrum width during the first month of life. Nevertheless, temperature impact on the structure size was observed in other hard structures of cephalopods, such as statoliths. In particular, Villanueva (2000) reported that the size of the statoliths of Loligo vulgaris is determined by the temperatures at which the animal grows.
In conclusion, our results proved the validity of using the rostrum surface of beaks for age estimation in the early stages of S. officinalis and show that this methodology (using the microstructure of the beak rostrum) can be promising, once validated in adult stages, to determine the age in cuttlefishes since other structures such as statoliths or cuttlebone have limitations for a routine ageing method. The feeding factor should also be evaluated in future studies to estimate how much it might affect eroding the beak rostrum to avoid age underestimation. This study confirms the daily deposition in the first 31 days of life, fulfilling the initial hypotheses of one increment per day, but it is necessary to validate the age estimation of beaks in the full ontogenetic range of the species.
Author contributions All the authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by AGM, CPR and JJC. The first draft of the manuscript was written by AGM and all the authors commented on previous versions of the manuscript. All the authors read and approved the final manuscript.
Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Guerra-Marrero was supported by a PhD-fellowship (PIFULPGC-2017-CIENCIAS-2) from the University of Las Palmas de Gran Canaria.

Data availability
The datasets generated during and/or analysed during the current study are not publicly available due to are being processed for further analysis, but are available from the corresponding author on reasonable request.

Conflict of interest
The authors have no relevant financial or non-financial interests to disclose.
Ethical approval This study was performed in compliance with Spanish law 53/2013 within the framework of the European Union's adopted directive 2010/63/EU regarding animal welfare for the protection of animals employed for scientific purposes, following the Guidelines for the Care and Welfare of Cephalopods in Research. The present study was also approved (register document OEBA-ULPGC 04/2019R1) by the Ethics Committee for Animal Research (Comité Ético de Experimentación Animal of the University of Las Palmas de Gran Canaria, CEEA-ULPGC, Spain).

Consent to publish Not applicable.
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