Age validation in early stages of Sepia officinalis from beak microstructure

Abstract

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.

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Marine Biology (2023) 170:24
https://doi.org/10.1007/s00227-022-04165-1
ORIGINAL PAPER
Age validation inearly stages ofSepia officinalis frombeak
microstructure
AiramGuerra‑Marrero1 · CatalinaPerales‑Raya2· FedorLishchenko3,4· AnaEspino‑Ruano1·
DavidJiménez‑Alvarado1· LorenaCouce‑Montero1· JoséJ.Castro1
Received: 28 July 2022 / Accepted: 22 December 2022 / Published online: 14 January 2023
© The Author(s) 2023
Abstract
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.
Keywords Daily increments· Age· Growth· Beak microstructure· Central Eastern Atlantic
Introduction
Since Young (1960), hard structures have become a rou-
tine 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) con-
cluded that beaks present greater advantages in age studies
than statoliths, due to the relative simplicity of this process-
ing 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 ben-
thic cephalopod species, including Octopus vulgaris (Her-
mosilla etal. 2010); O. pallidus (Leporati etal. 2008),
Responsible Editor: Z. Doubleday .
* Airam Guerra-Marrero
airam.guerra@ulpgc.es
1 IU-ECOAQUA, Universidad de Las Palmas de Gran
Canaria, Las Palmas de Gran Canaria, Edf. Ciencias Básicas,
Campus de Tafira, 35017LasPalmas, Spain
2 Centro Oceanográfico de Canarias (IEO, CSIC),
Calle Farola del Mar, 22. Dársena Pesquera,
38180SantaCruzdeTenerife, Spain
3 Vietnam-Russia Tropical Centre Marine Branch, Nguyễn
Thiện Thuật 30, NhaTrang650000, Vietnam
4 A.N. Severtsov Institute ofEcology andEvolution
oftheRAS, Leninsky Prospect 33, Moscow, Russia119071
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Marine Biology (2023) 170:24
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and O. maya (Rodríguez-Domínguez etal. 2013). With
regard to neritic/pelagic cephalopods as loliginids (Jack-
son 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), stato-
liths 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 assess-
ment 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 estima-
tion 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 tempera-
ture 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 repli-
cating 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 determina-
tion following the methodology of Perales-Raya etal. (2018)
in octopus paralarvae.
Table 1 Data summary of
juveniles of Sepia officinalis
analysed for each age group and
temperature (Tª)
n number of individuals, DML dorsal mantle length, WRA width of the reading area, Mean CV (%) mean of
coefficient of variation; mean values of DML and WRA in brackets
Age group
(Days)
Tª (ºC) nDML (mm) WRA (μm) Mean CV (%)
0 18 2 5.70–5.80 [5.75] 40,32–47.85[44.09] 0.000 ± 0.000
21 8 5.08–5.72 [5.43] 35.12–58.57 [43.27] 0.000 ± 0.000
1 18
21 19 5.6–7.01 [6.45] 42.52–141.23 [97.23] 2.717 ± 1.492
2 18 2 6.96–7.01 [6.98] 158.26–186.25 [172.25] 0.000 ± 0.000
21 10 6.27–7.20 [6.87] 140.25–170.24 [153.63] 1.288 ± 0.859
3 18 6 6.98–7.70 [7.36] 180.24–241.23 [219.72] 1.464 ± 0.900
21 27 6.85–7.32 [7.08] 138.55–242.12 [184.18] 2.024 ± 0.571
4 18 13 6.99–7.78 [7.52] 200.02–261.24 [241.27] 1.152 ± 0.501
21 46 6.54–7.99 [7.20] 158.07–281.23 [211.36] 2.018 ± 0.395
5 18
21 8 7.28–8.89 [7.79] 222.35–323.27 [269.92] 1.461 ± 0.553
618 4 7.75–7.89 [7.82] 301.95–321.24 [298.21] 1.336 ± 0.772
21 2 7.60–7.77 [7.69] 344.56–355.25 [349.91] 0.000 ± 0.000
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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 natu-
ral conditions (water temperature around 19–21 ºC, pho-
toperiod of 14h L / 10h D, and a salinity of 36 PSU). All
juveniles were reared with the aim of sacrificing them ran-
domly to validate the daily deposition in beaks.
Newly hatched individuals were reared inside the labora-
tory 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 feed-
ing 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 num-
ber 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 Span-
ish 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 docu-
ment 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, 2018) for Octopus vulgaris paralarvae. Pre-
viously, 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, 2018) 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 trans-
mitted light with a Nikon Microscope Multizoom AZ100
(400 × magnification). The system is equipped with a differ-
ential 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 (hatch-
ing; 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:
Fig. 1 Lower jaw in Sepia officinalis juvenile a. Anterior region of
the lower jaw: Location of the main parts b. R rostrum, H Hood, S
Shoulder, LWS Lateral Wall Surface. The black arrow indicates the
first increment, and the white segment indicates the width of the read-
ing area (scale bar = 100μm)
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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 (Perales-Raya
etal. 2018). 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 tempera-
ture group to assess and compare the precision of their read-
ings. 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 interac-
tion of age with temperature on increment numbers follow-
ing 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 deposi-
tion of the growth increments on its surface, in a way simi-
lar to that described in Perales-Raya etal. (2014b; 2018)
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 pigmenta-
tion, 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
CV
(%)=100x
√
(R1−R)2+(R2−R)
2
R
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 cul-
ture 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 individu-
als. Mean values of CV for each age group were lower than
the usual adopted value of 7.6% for annual and daily struc-
tures (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 tem-
peratures 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 deposi-
tion 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 visu-
alization and the count of growth increments in the lower
jaw’s rostrum was relatively easy. In contrast to the hatch-
lings 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 estima-
tion of the true age, while for O. vulgaris the visibility of
increments is much better in the upper jaws (Perales-Raya
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Marine Biology (2023) 170:24
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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 par-
alarvae, 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 (Perales-Raya etal. 2018). 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. 2014b, 2018; Armelloni etal. 2020) and
O. maya (Rodriguez-Domínguez etal. 2013; Villegas-Bár-
cenas 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.
Fig. 2 Beaks of Sepia officinalis
individuals reared for this study
at A. 0days old (hatching day),
B. 15days old and C. 20days
old (scale bar = 50μm)
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Fig. 3 Growth of cultured newly
hatched Sepia officinalis: a
Relationship between the mean
number of increments counted
in the lower jaw and the true
age; b Dorsal Mantle Length
and the true age; c Lower jaw
growth (width of the reading
area; see Fig.1) with true age.
Data grouped by temperature in
the different phases
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Marine Biology (2023) 170:24
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Ageing cuttlefish using the beak rostrum seems a suitable
method for these species, in comparison with other com-
monly 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 sta-
toliths, since several authors used length-frequency analy-
sis 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 incre-
ments. 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 physiologi-
cal 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 cuttle-
bone 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 estima-
tion. 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 (Betten-
court and Guerra 2001). The internal shell was recently ana-
lysed 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 environmen-
tal conditions are more stable. Moreover, some studies pro-
posed 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 (Dou-
bleday and Semmens 2011). The use of these methodologies
for age estimation were promising, although biological fac-
tors (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 similari-
ties 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.
Table 2 Results of: (a) lineal
regression analysis for the
number of growth increments
(NI), the age (days from
hatching) and width of the
reading area (WRA) by rearing
temperature (21 ºC or 18 ºC)
in Phase I. (b) General lineal
model (GLM) coefficients;
***p < 0.001
(a) Regressions Intercept Slope n r2P
Age–NI 21º −0.092 0.998 73 0.992 < 0.001
Age–NI 18º −0.2341 1.016 29 0.982 < 0.001
NI–WRA 21º 47.481 1.435 68 0.858 < 0.001
NI–WRA 18º 53.217 1.544 29 0.947 < 0.001
Age–WRA 21º 48.783 1.445 68 0.863 < 0.001
Age–WRA 18º 55.733 1.566 29 0.954 < 0.001
(b) GLM coefficients Estimate SE t p ( >|t|)
Intercept 91.49176 108.88807 0.840 0.403
Age 7.36607 5.96327 1.235 < 2 × 10–16 ***
Temperature −2.03376 5.36685 −0.379 0.770
Age x Temperature 0.08407 0.29585 0.284 0.777
Table 3 Results of lineal regression analysis for relationships
between the number of growth increments (NI), age (days in cultured
individuals) and width of the reading area (WRA) at 21 ºC in Phase II
Regressions Intercept Slope n r2p
Age–NI 21º −0.030 0.998 48 0.997 < 0.001
NI–WRA 21º 43.296 1.719 35 0.896 < 0.001
Age–WRA 21º 44.041 1.730 35 0.902 < 0.001
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Marine Biology (2023) 170:24
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24 Page 8 of 10
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 cul-
ture space play an important role in the growth of individ-
uals. 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 cul-
tured 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 (Perales-Raya etal.
2018). 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 sta-
toliths 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 concep-
tion 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.
Declarations
Conflict of interest The authors have no relevant financial or non-fi-
nancial 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 Experi-
mentación Animal of the University of Las Palmas de Gran Canaria,
CEEA-ULPGC, Spain).
Consent to participate No applicable.
Consent to publish Not applicable.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
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otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
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need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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