Introduction
Decapod cephalopods (Superorder: Decabrachia), which transfer sperm to females via complex copulation processes, have diverse methods of female sperm storage among taxa (Marian, Reference Marian2015; Sato, Reference Sato2021). During copulation, males transfer their sperm packed in spermatophores. Transferred spermatophores soon evert to form spermatangia, which are able to attach themselves to the tissues of the female. The attachment site of spermatangia is usually near the mouth (buccal mass) of the female in many coastal squids and cuttlefish (Marian, Reference Marian2015). Sperm are gradually released from the opening at the tip of the spermatangium after attachment has been completed (Sato et al., Reference Sato, Kasugai and Munehara2014).
Generally, female squids and cuttlefish have one or more sperm storage organs (seminal receptacles) on the buccal membrane, and sperm released from spermatangia attached around the buccal mass migrate to the seminal receptacles and are stored there until spawning (Sato, Reference Sato2021). However, spermatangia attachment sites may differ among species. In some loliginid squid species, males have alternative reproductive tactics. Small-sized ‘sneaker’ males attach spermatangia near the buccal mass (Marian et al., Reference Marian, Apostólico, Chiao, Hanlon, Hirohashi, Iwata, Mather, Sato and Shaw2019). In contrast, large-sized ‘consort’ males attach spermatangia near the opening of the oviduct inside the mantle of the female (Marian et al., Reference Marian, Apostólico, Chiao, Hanlon, Hirohashi, Iwata, Mather, Sato and Shaw2019). Firefly squid males attach spermatangia to the nuchal (neck) region of the female, which has spermatangium pockets (Burgess, Reference Burgess1998; Sato et al., Reference Sato, Tsuda, Nur, Sasanami, Iwata, Kusama, Inamura, Yoshida and Hirohashi2020). Alternatively, in some species, especially deep-sea squids, females do not have sperm storage organs (neither seminal receptacles nor spermatangium pockets), so males implant the spermatangia deep within female muscle tissue, where they function as sperm storage organs (Hoving et al., Reference Hoving, Perez, Bolstad, Braid, Evans, Fuchs, Judkins, Kelly, Marian, Nakajima, Piatkowski, Reid, Vecchione and Xavier2014). How this diversity of sperm storage modes has evolved in cephalopods is a theme of considerable interest (Marian, Reference Marian2015; Sato, Reference Sato2021).
In bobtail squids (Family Sepiolidae), the females of many species are unusual in that they store the spermatangium itself, unlike other groups of squid which store the sperm released from the spermatangia in specialized seminal receptacles (Hoving et al., Reference Hoving, Laptikhovsky, Piatkowski and Önsoy2008; Sato, Reference Sato2021). There are two types of spermatangia storage in sepiolids. In Subfamily Sepiolinae, storage is in the bursa copulatrix, which is a storage organ constructed by modification of the distal part of the oviduct (Bello, Reference Bello1995, Reference Bello2020). In Subfamily Heteroteuthinae, storage is in a sac called the seminal receptacle, situated at the posterior end of the visceral mass (Hoving et al., Reference Hoving, Laptikhovsky, Piatkowski and Önsoy2008). Although referred to by the term ‘seminal receptacle’, it is used for spermatangium storage in this subfamily, and not for sperm storage as in most coastal squids. Therefore, these spermatangia storage types are classified as ‘SP (Spermatangia pocket) type’ storage. In Subfamily Rossiinae, storage occurs in the implanted spermatangium itself (classified as ‘IMP type’ storage), in which females have no specialized storage organ and so store spermatangia which are implanted shallowly on the body surface or deeply in the mantle and the head tissues (Hoving et al., Reference Hoving, Nauwelaerts, Van Genne, Stamhuis and Zumholz2009). However, variation of storage type has been reported by Hoving et al. (Reference Hoving, Laptikhovsky, Piatkowski and Önsoy2008) within the Rossiinae and Heteroteuthinae (which have both SP and IMP types). Furthermore, even within the IMP types, the location of spermatangia attachment varies according to species, ranging from inside the mantle (e.g. near the oviductal opening) to the outer surface of the mantle (e.g. the dorsal and ventral mantle and head; Hoving et al., Reference Hoving, Laptikhovsky, Piatkowski and Önsoy2008).
The present study focuses on two species of Japanese bobtail squids: the rossiine species Austrorossia bipapillata (Sasaki, 1920) and the heteroteuthine species Sepiolina nipponensis (Berry, 1911). Although there are several species of bobtail squid in Japanese waters (Sasaki, Reference Sasaki1929; Jereb and Roper, Reference Jereb and Roper2005), there is scant knowledge of deep-sea species, compared with coastal species which are relatively easy to collect. Little is known about these two deeper-dwelling species, including their sperm storage patterns, breeding seasons and reproductive strategies. The present study investigates their seasonal changes in body size and reproductive traits, as well as the type of sperm storage found in each species. Also the relative size of gonadal tissue and reproductive organ mass in males of these two sympatric species are compared to investigate differences in the reproductive strategy.
Materials and methods
Sample collection and measurements
The two sepiolids A. bipapillata and S. nipponensis were taken as a bycatch in trawl fisheries targeting crustaceans and fish in Suruga Bay, Shizuoka, on the Pacific coast of Honshu, Japan, at a depth of around 500 m (Figure 1). In total, 160 male and 162 female A. bipapillata, and 55 male and 49 female S. nipponensis were collected on 30 September, 27 October, 22 December 2020, 4 March, and 12 May (sampling dates comprising for the 2020/2021 season), and 7 October, 7 December 2021, 9 February, and 8 May 2022 (comprising the 2021/2022 season: Table 1). All individuals collected, whether mature or not, were used in this study. Dorsal mantle length (DML) and body weight (BW) were measured to the nearest 0.1 mm and 0.1 g, respectively, and then the individuals were dissected. The reproductive organs (nidamental glands and ovary in females; spermatophoric complex (the spermatophore storage organ [Needham's sac] and the spermatophoric gland) and testis in males) were removed and weighed to the nearest 0.001 g. The number of spermatophores stored in the spermatophore sac was recorded in males. After observation of the sperm storage mechanism (the presence of a bursa copulatrix or the location where spermatangia were implanted), the number of attached spermatangia was counted in females. Many spermatangia of S. nipponensis were attached to the female's body and were difficult to distinguish, so females were fixed in 10% formalin in seawater and then stained with Azure B (Fujifilm, Japan) solution to dye the spermatangia blue for counting (Øresland and Oxby, Reference Øresland and Oxby2021).
Figure 1. Map of the study area in Suruga-bay, Honshu, Japan. Each sampling point is indicated by a red circle.
Table 1. Information on the collected number between two bobtail squid species
For males, the gonadosomatic index (‘GSI’) was calculated as the ratio of the weight of the testis (‘TW’) to BW (i.e. GSI = [TW/BW] × 100). To calculate reproductive system index (‘RSI’), the entire reproductive system weight (‘RSW’; the sum of testis weight and spermatophoric complex weight) was used (i.e. RSI = [RSW/BW] × 100). The RSI was also calculated in females as the ratio between the weights of RSW (ovary, oviduct, oviductal gland, and a pair of nidamental glands) and BW.
Statistical analysis
Generalized linear models (GLMs) were used to analyse whether GSI, RSI, and stored spermatophore numbers were related to DML in each two species collected in two years (2020/2021 or 2021/2022). The gamma distribution with log link function was used for analysing GSI and RSI, and the negative binomial distribution with log link function was used for spermatophore number. The significance of fixed factors on dependent variables was assessed using a Wald test. A Mann–Whitney U test was applied to assess whether the difference in the number of spermatangia attached to females between two squid species was statistically significant. All analyses were performed in R version 4.0.3 (R Core Team, 2020). We used the glm function and the glm.nb function (MASS library) to fit GLMs and the wilcox.exact function (exactRankTests library).
Results
Seasonal change
In A. bipapillata, there was a large range of size in females (DML range 18.5–86.2 mm), while males were mostly small (20.9–46.3 mm, Figure 2A). Two groups of females widely separated in size can be discerned in October 2020 in Figure 2A, suggesting the presence of two groups of different size: large (65.9–86.2 mm) and small (18.5–38.2 mm). DML in the small group appeared to gradually increase from October 2020 to September 2021. In addition, both small (35.6–36.9 mm) and large females (76.5 mm) were collected in December 2021. These seasonal changes appear to indicate that mature females reached the size peak in their life history in autumn and juvenile squids were recruited into the population.
Figure 2. Seasonal change in two bobtail squid species from September 2020 to May 2022. (A) Dorsal mantle length (DML). (B) Total gonadosomatic index (RSI, all reproductive organs) for females. (C) Gonadosomatic index (testis only) for males. (D) RSI (all reproductive organs) for males. (E) The number of spermatophores stored in the Needham Sac for males. Females and males were indicated orange and blue colours in Austrorossia bipapillata (open circles) and Sepiolina nipponensis (filled triangles).
In S. nipponensis, for both sexes the range of DML among the sampled specimens did not vary as much throughout the year as did that of A. bipapillata (S. nipponensis: 20.4–31.9 mm for males, 23.0–31.1 mm for females; A. bipapillata 20.9–46.3 mm for males, 18.5–86.2 mm for females) but the range in body size for both sexes in S. nipponensis increased from October 2020, reaching a peak in February 2021. Few S. nipponensis were collected in May 2021 (Figure 2A). This occurrence pattern was similar in 2021/2022.
Seasonal changes in female RSI showed a similar pattern to body size with A. bipapillata increasing from December 2020 to October 2021, and in S. nipponensis increasing from May 2021 to February 2022 (Figure 2B). The GSI of A. bipapillata males did not change much throughout the year (0.02–1.56) and was much lower than that of the S. nipponensis (1.06–5.25, Figure 2C). The highest values of GSI for male S. nipponensis were in November in the 2020–2021 season and in September in the 2021–2022 season. In males, especially S. nipponensis, the peaks of RSI and spermatophore number were delayed in comparison with GSI in 2020/2021, but this delay was not observed in 2021/2022 (Figure 2D, E cf. Figure 2C).
Differences in gonadal investment and sperm storage mechanisms
While the RSI values were higher in 2021/2022 than in 2020/2021 (GLM, t = 3.705, p < 0.001), the RSI of females increased with DML in both species (GLM, t = 18.651, p < 0.001), and S. nipponensis had a higher RSI than A. bipapillata (GLM, t = −21.283, p < 0.001; Figure 3A). Male GSI and RSI were higher in S. nipponensis than A. bipapillata (GLM: GSI, t = −10.751, p < 0.001; RSI, t = −23.575, p < 0.001; Figure 3B, C). Although male GSI and RSI did not show a significant relationship with body size (GLM: GSI, t = −1.936, p = 0.054; RSI, t = 1.621, p = 0.11; Figure 3B, C), GSI values were lower and RSI values were higher in 2021/2022 than in 2020/2021 (GLM: GSI, t = −2.029, p < 0.05; RSI, t = 4.699, p < 0.001).
Figure 3. Relationships between dorsal mantle length (DML) and (A) female RSI, (B) male GSI, and (C) male RSI in Austrorossia bipapillata (open circles) and Sepiolina nipponensis (filled triangles). Solid and broken lines represent gamma regressions.
There were fewer spermatophores stored in the Needham's sac in A. bipapillata (mean ± SD = 36.49 ± 29.41) than in S. nipponensis (243.24 ± 219.37) (GLM, z = −4.824, p < 0.001). The number of spermatophores is not significantly correlated with DML and season of collection (GLM: DML, z = −0.542, p = 0.59; season of collection, z = 0.105, p = 0.53), but there was a significant correlation between DML and species (GLM, z = 2.811, p < 0.01: Figure 4A).
Figure 4. Differences in the number of spermatophores stored in males and spermatangia attached to females for the two sepiolid species. (A) Relationship between dorsal mantle length (DML) and the number of spermatophores stored in Needham's sac in Austororossia bipapillata (open circles) and Sepiolina nipponensis (filled triangles). (B) Comparison of the numbers attached in the female body. (C, D) Examples of the spermatangia attachment in A. bipapillata (C) and S. nipponensis (D). Sites of spermatangia attachment are enclosed within broken red ovals. Abbreviations: ovd, oviduct; ng, nidamental gland.
There is no bursa copulatrix in either species. The number of attached spermatangia was higher in S. nipponensis females than in A. bipapillata (Mann-Whitney U test, U = 391, p < 0.001; Figure 4B). While about 6 spermatangia (5.71 ± 6.47, n = 14) were deeply implanted in the oviductal opening in A. bipapillata (Figure 4C), many spermatangia (62.7 ± 61.5, n = 30) were attached by shallow implanting into the left gill and the skin surface above the liver in S. nipponensis (Figure 4D).
Discussion
Although both species lack a specialized sperm storage organ and store sperm by spermatangia implantation, their reproductive strategy, especially regarding sperm competition, seems to differ. Austrorossia bipapillata males have a lower GSI and store fewer spermatophores than S. nipponensis males. It is suggested that GSI is related to the intensity of sperm competition (Rowley et al., Reference Rowley, Daly-Engel and Fitzpatrick2019). While Japanese pygmy squid having higher GSI (3.78%) shows promiscuity, firefly squid having lower GSI (0.4%) shows monogamy in cephalopods (Sato, Reference Sato2021). More spermatangia were attached to female S. nipponensis than female A. bipapillata, which may mean that S. nipponensis females copulated with several males and that there is strong sperm competition. An alternative explanation is that these spermatangia were transferred from a single male in a species where there is markedly strong sperm competition. For either possibility, there may be strong sperm competition in S. nipponensis but not in A. bipapillata.
Recent studies suggest that the diversification of sperm storage organs may be driven by post-copulatory sexual selection mechanisms, such as sperm competition, in many animals (Peretti and Aisenberg, Reference Peretti and Aisenberg2015; Sloan and Simmons, Reference Sloan and Simmons2019). Hence, the diversification of sperm storage organs in cephalopods may also have been influenced by sperm competition, while in contrast it is considered that the spermatangia implantation seen in deep-sea cephalopod species (having a low opportunity to encounter a mate; Hoving et al., Reference Hoving, Perez, Bolstad, Braid, Evans, Fuchs, Judkins, Kelly, Marian, Nakajima, Piatkowski, Reid, Vecchione and Xavier2014) is likely not associated with sperm competition. However, there are variations in traits related to sperm competition among IMP species. In Subfamily Rossiinae, Semirossia patagonica (with a high GSI: mean 2.75%) stores many spermatophores (ranging between 13 and 229) in Needham's sac (Önsoy, Laptikhovsky and Salman, Reference Önsoy, Laptikhovsky and Salman2008), which is markedly different from A. bipapillata, with its low GSI (mean 0.4 ± 0.25%; present study) and storage of fewer spermatophores (36.49 ± 29.41). Similar variation has been reported also among SP species: Rondeletiola minor has a high GSI (mean 3.17%; Önsoy, Ceylan and Salman, Reference Önsoy, Ceylan and Salman2013) but in Sepiola steenstrupiana it is low (mean 1.32%; Salman and Önsoy, Reference Salman and Önsoy2004). Therefore, it is difficult to understand the evolution of sperm storage mechanisms in cephalopods judging only from the apparent relationships between sperm competition and the mechanism of sperm storage.
Sperm storage by IMP is typical of Rossiinae (Hoving et al., Reference Hoving, Laptikhovsky, Piatkowski and Önsoy2008; Marian, Reference Marian2015), but the location of spermatangia attachment differs among species. While spermatangia are deeply implanted in the head and mantle tissues of some species (Zumholz and Frandsen, Reference Zumholz and Frandsen2006; Hoving et al., Reference Hoving, Nauwelaerts, Van Genne, Stamhuis and Zumholz2009), they are implanted near the oviduct in others (Cuccu et al., Reference Cuccu, Mereu, Cannas, Follesa, Cau and Jereb2007; Önsoy et al., Reference Önsoy, Laptikhovsky and Salman2008; Laptikhovsky et al., Reference Laptikhovsky, Nigmatullin, Hoving, Onsoy, Salman, Zumholz and Shevtsov2008). Reid (Reference Reid1991) reported for some species that the opening of the oviduct is modified as a seminal receptacle, often with spermatophores embedded on the inner edge of the opening to the oviduct. In view of these characteristics described by Reid (Reference Reid1991), Hoving et al. (Reference Hoving, Laptikhovsky, Piatkowski and Önsoy2008) classified these species as SP, rather than IMP. In the present study, it was found that A. bipapillata females did not have a special modification of the oviduct for spermatangia storage, but about six spermatangia were typically found deeply implanted in its inner wall. Given this configuration, it is not easy to determine whether this situation should be classified as SP or IMP. However, the presence of spermatangia implanted at the opening of the oviduct may be a hint concerning the evolution of sperm storage in Sepiolidae. Perhaps ancestral males attached spermatangia to the head or external surface of the mantle, later attempted to transfer their spermatangia inside the mantle and implant them near the oviduct to achieve priority and improve their chances of successful fertilization: that is, vying for first contact with the ova. Recently, it has been reported that the structure of the bursa copulatrix is related to the morphology of the hectocotylus in Sepiolinae. In closed-bursa copulatrix genera, males have enlarged sucker pedicels in the distal part of the hectocotylus, while there are no hectocotylus modifications in the open-bursa copulatrix group, suggesting that the evolution of the bursa copulatrix is related to coevolution between males and females (Bello, Reference Bello2020). Their specialized sperm storage organs may also have evolved to prevent the removal of all spermatangia transferred by males who had previously copulated with the female.
The larger the S. nipponensis males, the fewer spermatophores stored in Needham's sac. This may be because larger males copulate with more females and use up more spermatophores. In contrast, the opposite trend was observed in A. bipapillata males. Given the higher GSI in S. nipponensis males, this species is suspected to have a tendency towards promiscuity compared with A. bipapillata and the females to store more spermatangia transferred by many mates. It will be necessary to identify the paternity of the stored spermatangia to confirm their actual mating system.
In Subfamily Heteroteuthinae, Heteroteuthis dispar shows the SP type of sperm storage (Hoving et al., Reference Hoving, Laptikhovsky, Piatkowski and Önsoy2008), while in Stoloteuthis leucoptera it is the IMP type, involving the attachment of many spermatangia to the external dorsal and ventral surfaces of the head (Villanueva and Sanchez, Reference Villanueva and Sanchez1993). The present study identifies another type of sperm storage in S. nipponensis, an example of a heteroteuthinid strategy in which many spermatangia are attached to the left gill and to the surface of the connective-tissue capsule enclosing the digestive gland. There are variations of sperm storage mechanisms in this subfamily, but unfortunately detailed information on reproduction of the Heteroteuthinae is scarce so further investigation is necessary.
Seasonal changes of DML and GSI in females suggest that the reproductive season occurs from May to October (i.e. during summer and autumn) in A. bipapillata and from December to February (i.e. during winter) in S. nipponensis. In particular, RSI in both sexes in S. nipponensis shows the highest value in February, which strongly suggests that this month is the peak of the reproductive season in this species. Reproductive seasons vary among species in Sepiolidae. The broad range of habitat depth and geographical distribution of Sepiolidae may affect their reproductive season, resulting in the observed variations. In Rossiinae, Neorossia caroli distribution includes the Aegean Sea, where N. caroli GSI peaks in autumn (Salman, Reference Salman2011). However, there are no peaks of female GSI during the year in Semirossia patagonica from the Falkland Islands (Önsoy et al., Reference Önsoy, Laptikhovsky and Salman2008). Among the Sepiolinae whose distribution includes the Aegean Sea, Sepietta oweniana has two peaks of female GSI (spring and winter; Salman, Reference Salman1998), and Rondeletiola minor has one peak (spring; Önsoy et al., Reference Önsoy, Ceylan and Salman2013).
The peak of RSI found in the present study did not coincide with the timing of GSI in males. Sepiolid males presumably invest their energy in testis development for producing sperm during earlier stages of maturation and then at maturation produce and store spermatophores in Needham's sac. A similar pattern of investment of male reproductive traits is found also in other decabrachians species (e.g. as shown for Ommastrephes bartramii by Brunetti et al., Reference Brunetti, Ivanocic, Aubone and Pascual2006; and for Idiosepius paradoxus by Sato, Reference Sato2017).
In summary, it may be challenging to conclude Heteroteuthinae and Rossiinae reproductive seasons because our study includes some sampling months where the numbers sampled were few. However, the results do provide new knowledge on the reproductive biology of the Sepiolidae: in the males of two Japanese deep-sea species S. nipponensis and A. bipapillata, the former and the latter have invested more and less, respectively, in the reproductive organ. However, female sperm storage occurs by spermatangia implantation into the opening of the oviduct in both species. This family, with its various patterns of sperm storage, provides a good example to examine the evolutionary process of each sperm storage type and to investigate if post-copulatory sexual selection has influenced sperm storage diversification. Identification of the conditions influencing development of the bursa copulatrix and spermatangia implanting would bring us closer to understanding the evolutionary process. However, sepiolid species inhabit a wide range of depths and are widely distributed, so there is still insufficient information available about the reproductive ecology of many Japanese species, and further research is needed.
Data
The data that support the findings of this study are available from the corresponding author (NS) upon reasonable request.
Acknowledgements
We thank K. Yamada, M. Ohmura, and the crew of the vessel Hinode-maru for their support with sample collection. We also thank an editor, Dr V. Laptikhovsky and two referees for their helpful comments. This research was financially supported by JSPS KAKENHI [Grant Number 22K05788 to NS].
Author contributions
The study conception was by Noriyosi Sato. Material preparation was performed by Noriyosi Sato, Ryohei Tanabe and Takeru Uezu. Data measurements were conducted by Takeru Uezu, Toshiki Matsuoka and Asuka Nakajima. Analysis and manuscript writing were by Noriyosi Sato. All authors approved the final manuscript.
Competing interests
None.
Ethical standards
Not applicable.
