External sexual dimorphism of Sepia bertheloti was mainly based on the fact that males present median arm suckers with greater diameter than marginal suckers and hectocotylus in the left ventral arm. Apart from sexual structures, females internally show the buccal membrane extending ventrally with two spermathecae [12]. However, minimal attention has been paid to cuttlebone as a potential element to differentiate males from females. This study showed that female cuttlebones are significantly wider probably due to the need for a bigger volume of the paleal cavity to increase the capacity to accommodate the gonads. Jereb and Roper [12] remarked that the mantle is relatively wider in females than in males.
Sepia bertheloti males are larger than females [12], which is also noted in S. latimanus [9, 22], S. koilados, S. rhoda, and S. subplana. However, in most cuttlefish species (i.e., S. acuminata, S. australis, S. bidhaia, S. braggi, S. elegans, S. filibranchia, S. grahami, S. hedley, S. latimanus, S. limata, S. mestus, S. orbignyana, S. plana, S. senta, S. smithi, S. sulcata, Sepiella inermis, and S. weberi) females growth larger than males [12]. Regarding S. latimanus, Dan et al. [22] observed that there were no morphometric differences in the growth pattern of both sexes during the initial life stages, but males become larger than females after 1 year of age. Males and females of S. bertheloti caught more northwards than the distributional range given by Jereb and Roper [12] presented a maximum size of 133 and 100 mm DML, respectively, which is clearly smaller than the maximum sizes reported by Jereb and Roper [12] for the Eastern Atlantic (175 and 134 mm DML, respectively). However, females were usually wider, and this aspect could be associated with the wider morphometric structure of their cuttlebone for a given mantle length.
This differentiation of the cuttlebone between sexes with females with a broader cuttlebone has also been described in other cuttlefish species, such as S. officinalis [4], S. prabahari, S. weberi and Sepiella ornata [12]. Therefore, although S. bertheloti males and females cannot be differentiated macroscopically (except for the presence of a hectocotylus), they show sexual differences in the morphometric structure of the cuttlebone.
The present results are comparable to those obtained by Almonacid-Rioseco et al. [4] for S. officinalis, which also showed a differentiation between sexes for the same measurements of the cuttlebone (FW, CW, ECW). These results reveal that sexes have a relatively different growth pattern. Specifically, males exhibit faster growth in length to reach larger sizes, while females exhibit greater growth in the cuttlebone width in preparation for reproduction (Fig. 3a–c). Vasconcelos et al. [23] also describe this sexual dimorphism for S. officinalis in the Algarve coast (southern Portugal). On the other hand, the gonadosomatic index allowed comparison of gonadal development with the cuttlebone measurements that presented differences between sexes, revealing that the faster growth in width of female cuttlebone is related to an increase in gonadal mass. Accordingly, this relationship can be interpreted as an adaptation of the female body to the gonadal development, which always exhibits increased occupation compared with the male gonad. In this sense, Akyol et al. [24] reported that the total number of oocytes produced by S. officinalis varied from 49 to 828 with an average of 261 ± 27 per female, whereas Laptikhovsky et al. [25] reported a range of large yolk oocytes between 130 and 839. This body adaptation hypothesis was previously proposed by Hewitt and Stait [26] and Sainz [27], who mentioned that these changes in S. officinalis were due to phylogenetic features where females adapted to their reproductive stage by developing a wider cuttlebone than males. The volume of oocytes could justify a larger capacity of the female paleal cavity and therefore the adaptation of female cuttlebone to provide the required support and space.
Due to the lack of immature individuals, we are not able to determine whether the differentiation between sexes based on cuttlebone morphometrics is only possible when analysing ripe individuals prior to spawning (i.e., when the cuttlebone morphology adapts to lodge the voluminous gonad), which would decrease the usefulness of this “tool” for gender identification in juveniles stages.