Sperm storage and mating in the deep-sea squid Taningia danae Joubin, 1931 (Oegopsida: Octopoteuthidae)
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Spermatangium implantation is reported in the large oceanic squid Taningia danae, based on ten mated females from the stomachs of sperm whales. Implanted spermatangia were located in the mantle, head and neck (on both sides) or above the nuchal cartilage, under the neck collar and were often associated with incisions. These cuts ranged from 30 to 65 mm in length and were probably made by males, using the beak or arm hooks. This is the first time wounds facilitating spermatangium storage have been observed in the internal muscle layers (rather than external, as observed in some other species of squid). The implications of these observations for the mating behavior of the rarely encountered squid T. danae are discussed.
Some deep-sea squid are rarely encountered, and the biology of many species remains poorly understood. Observations of live specimens are even rarer, and the examination of dead animals, whether washed ashore, caught by fishing gear or retrieved from stomachs of large predators, remains an important source of biological information. The octopoteuthid squid Taningia danae Joubin, 1931, which may reach 124 kg (González et al. 2003) is one such poorly studied deep-sea species. Recently, one live specimen was photographed (Kubodera et al. 2007), but otherwise the species is mainly known from the stomach contents of sperm whales and sharks (Clarke 1996). Descriptions in the literature of trawl-caught, fresh material include only two mature males, two mature females, two maturing females and 20 juveniles (Okutani 1974; Roper and Vecchione 1993; Santos et al. 2001; González et al. 2003; Quetglas et al. 2006). This study presents the first detailed information on sperm transfer and storage in T. danae.
Reproduction in cephalopods is characterized by separation of the sexes and semelparity, i.e., gonad maturation and spawning (although sometimes protracted), occur once at the end of the animal’s life (Nesis 1987). Spermatozoa are packed into complex spermatophores, which are transferred to the female by the hectocotylus (a modified arm) or, in species lacking a hectocotylus, by a long terminal organ (an extension of Needham’s sac, the storage organ for spermatophores) (Nesis 1995). During mating, the spermatophore is transferred and then everts after initiation of the ‘spermatophoric reaction’ (reported in Loligo pealei by Drew (1919), Octopoteuthis sicula by Hoving et al. (2008b) and Todarodes pacificus by Takahama et al. (1991)). This reaction begins when the spermatophore is removed from the Needham’s sac by the hectocotylus; in unhectocotylized species, the distal tip of the long terminal organ triggers the reaction as the spermatophore exits its aperture (observed in Onykia ingens by Hoving and Laptikhovsky (2007)). The spermatangium, which is the sperm mass enveloped in an outer casing (the ‘inner tunic’ sensu Drew 1919), is then expelled from the spermatophore and deposited on the body of the female. The remaining empty sheath of the spermatophore is lost. Spermatangium attachment locations and mechanisms are species and family specific (Nesis 1995; Hoving 2008); spermatangia may be attached at consistent external sites on the female (the buccal membrane or mantle) or in special seminal receptacles. In some species, spermatangia are deposited seemingly non-selectively over the female’s exterior body surfaces (Nesis 1995; Hoving et al. 2004; Hoving 2008) and are often deeply implanted into the female’s tissues, while spermatangia deposited at specific consistent sites are generally superficially attached (Nesis 1995).
In hectocotylized squids, the male probably holds everting spermatophores against the preferred deposition site, and the spermatangia then attach to the female’s tissues. In species lacking a hectocotylus, the terminal organ positions the everting spermatophores. The exact mechanism by which spermatangia attach to and implant into female tissues remains poorly understood. In some species, attachment is aided by extensions on the oral side of the spermatangium, which penetrate the female’s tissues [e.g., Todarodes pacificus (Takahama et al. 1991) and Octopoteuthis sicula (Hoving et al. 2008b)]. In some onychoteuthids, implanted spermatangia are associated with elongated exterior wounds on the female’s mantle (Torchio 1967; Nesis 1970; Clarke 1980; Okutani and Ida 1986; Bolstad 2008; Bolstad and Hoving submitted), which may be created by the male’s beak (Torchio 1967). Although Norman and Lu (1997) speculated that Architeuthis sp. implants spermatangia deeply into female tissues under hydraulic pressure produced by the terminal organ, recent studies have shown that some squid spermatangia implant autonomously (Hoving and Laptikhovsky 2007; Hoving et al. 2009). Such autonomous implantation may be enabled or aided by proteolytic enzymes present in the spermatophore’s cement body (Nesis et al. 1998).
Fertilization occurs externally in squid, since spermatangia are stored outside of the perivisceral coelom and oviducts (Hanlon and Messenger 1996). In loliginids, two spermatangium storage sites exist—the buccal membrane and the distal oviduct—and spawned eggs are passed along both sites for fertilization. The fertilization mechanisms in oceanic squid remain unknown.
Taningia danae is one such species. Mature males, which lack a hectocotylus, presumably use the long terminal organ (Okutani 1974; González et al. 2003) to transfer spermatophores to the female. González et al. (2003) provided the only information available on spermatophores of T. danae, reporting one intact spermatophore of 54 mm length and one everted spermatophore of 166 mm length; the actual morphology of the spermatophores remains undescribed. Although the implantation of spermatangia into the arms, head and mantle has been reported for a variety of oceanic squid species (Nesis 1970, 1995; Clarke 1980; Okutani and Ida 1986; Norman and Lu 1997; Guerra et al. 2004; Jackson and Jackson 2004; Hoving et al. 2004, 2008b; O’Shea et al. 2007; Bolstad 2008) including T. danae (Clarke 1967; Okutani 1974), implanted spermatangia associated with male-inflicted wounds have never been reported for this species.
Materials and methods
Information on mated female Taningia danae from South African sperm whale stomachs
Catalog no. and collection details
Location of implanted spermatangia/tunics and (length of cuts)
SAM-S2589, Sperm whale caught at 31°16′S, 30°53′E on 16-VII-1973
Spermatangia in both sides of nuchal cartilage under neck collar in cut (50–55 mm), with corresponding cut (50 mm) in dorsal inner mantle; additionally on left side in dorsal neck in cut (45 mm), with corresponding cut (45 mm) in dorsal inner mantle
SAM-S2778, Sperm whale caught at 30°43′S, 31°01′E on 16-IX-1970
Tunics in dorsal neck just above nuchal cartilage in cut (50 mm); under neck collar on right side of nuchal cartilage in cut (30 mm); in dorsal surface of head
Spermatangia under neck collar on left side of nuchal cartilage in cut (40 mm); on inside surface of mantle 500 mm from anterior tip of nuchal cartilage; tunics implanted in dorsal surface of head
Spermatangia in inner mantle margin on left side; tunics implanted in dorsal surface of head
Tunics above neck collar in dorsal cut (65 mm); dorsal to right eye in cut (50 mm)
Tunics implanted in dorsal surface of head
SAM-S3450, no locality data
Tunics on left side of nuchal cartilage under neck collar in cut (45 mm); right side of nuchal cartilage under neck collar in two cuts (50 mm)
Five incisions: two transverse cuts in exterior surface of head/collar musculature, one ventrally near funnel (55 mm), one laterally posterior to eye (65 mm); one transverse cut in head musculature under collar on each side of dorsal midline (right cut 32 mm, left cut 35 mm), with associated cut into head of same length beneath; one transverse (slightly diagonal) cut (35 mm) just posterior to collar muscle; additional individual spermatangia loose and implanted throughout body cavity
Three incisions: two transverse cuts (each 40 mm) under collar muscle (one dorsal, just right of dorsal cartilage, with ~15 deeply embedded spermatangia; one lateral, on left-hand side of head, approximately halfway between dorsal cartilage and funnel cartilage, with ~10 implanted spermatangia); one slightly diagonal transverse cut in interior of mantle wall, 33 mm long and 9 mm deep, posterior and slightly lateral to dorsal nuchal cartilage, with ~12 spermatangia. ~30 spermatangia also implanted singly into dorsolateral surface of head, obliquely under skin, within a small, contained area
Squid D3256, ex 50′ sperm whale (M), 03/08/1965, M.R. Clarke Collection (Acc. 2366),
Two incisions: one on either side of midline dorsolaterally, under collar musculature, with associated spermatangia
Mated Taningia danae from stomachs of sperm whales caught in 1970–1973
In one female, two of the three cuts in the dorsal neck region corresponded to similar cuts on the inner dorsal mantle wall. The lengths of these latter cuts matched those of the corresponding, spermatangia-bearing neck cuts; their depth extended up to 10 mm into the dorsal mantle wall, but did not break its inner surface (Fig. 3).
Mated Taningia danae from stomachs of sperm whales caught in 1963–1965
Two partial specimens bearing evidence of mating were observed, each comprised of arms, head and anterior ~30% of mantle with associated ragged viscera (but could not be sexed); the arm lengths of each measured ~20 cm. Incisions (up to five in one specimen) and associated spermatangia (Fig. 4d) were observed as above, both under the collar musculature and in the head and inner mantle musculature (Fig. 4a, b; Table 1). Individually implanted spermatangia, not associated with incisions, were also observed in the dorsal head (Fig. 4c).
The trawled T. danae specimen was a maturing female, ML 650 mm. Small oocytes were present in the ovary, and the developing nidamental glands were 245 mm long. One incision, 65 mm in length, was observed underneath the collar musculature, almost directly below the right-hand funnel component of the locking apparatus (Fig. 3c), resembling the incisions found in the specimens from the whale stomachs. Although the specimen’s head was in poor condition, the cut appeared clean and traversed the depth of the musculature, creating a “window” through to the digestive gland. No implanted spermatangia were found inside the cut or elsewhere in/on this specimen.
The presence of consistent wounds in several large female T. danae specimens is suggestive. Since the trawled specimen bore an incision, but did not have any implanted spermatangia, we cannot conclude that its wound was necessarily mating related. However, the wounds in the other female T. danae were clearly the result of mating or attempted mating, since they were all associated with implanted spermatangia. Large wounds and associated implanted spermatangia have been observed in several other squid species (of the genus Onychoteuthis, see Nesis 1970; Clarke 1980; Okutani and Ida 1986; Bolstad 2008; Bolstad and Hoving submitted). However, in these cases, the wounds occurred in the outer mantle surface, while in T. danae, the cuts are found underneath two layers of intact tissue. The position of the incisions in T. danae indicates that the anterior mantle edge and the underlying collar must be somehow held aside while the incisions are created, perhaps by the arm hooks or the beak. On basis of the mandible dimensions, Torchio (1967) argued that the wounds in the outer mantle surface of ‘Chaunoteuthis’ mollis were made by the beak. In our T. danae specimens, the size of the wounds (30–65 mm) could also correspond to the male’s beak.
We assume that the long, muscular terminal organ (which can extend well out of the mantle cavity of the male (Okutani 1974; González et al. 2003) is used to deposit the spermatophores into the wounds. The spermatophoric reaction may be triggered by the tip of the muscular terminal organ, as observed in Onykia ingens (Hoving and Laptikhovsky 2007). The spermatangia would then discharge from the spermatophores and implant into the head retractor muscle and viscera in T. danae, or in other locations such as the head and mantle; the exact mechanism of implantation is restricted to speculation. Not all implanted spermatangia were associated with cuts, indicating that cuts are not required for implantation. Spermatophores of T. danae may be capable of implanting autonomously into body tissue, as observed in the deep-sea squid Onykia ingens (Hoving and Laptikhovsky 2007), possibly aided by the oral filament (Fig. 2b). This filament may either penetrate the tissue and/or distribute the possibly caustic cement expelled during the spermatophoric reaction. This implantation scenario has also been proposed for Octopoteuthis sicula (Hoving et al. 2008b), a confamilial species with similar spermatangium morphology to T. danae.
The incisions made by male T. danae may ensure that everting spermatophores have sufficient contact with the female’s tissue to ensure successful implantation. The nuchal region seems to be the preferred implantation site for T. danae, since most spermatangia are found there (in the dorsal head and neck region, in the outer neck collar, and under the outer neck collar, and in the underlying head retractor muscle). Deposition of spermatangia in the region of the nuchal cartilage is also known for smaller mesopelagic squid of the families Lycoteuthidae and Enoploteuthidae (Burgess 1998; Hoving et al. 2007). However, these squid have special areas or pockets (seminal receptacles) in the nuchal region for the reception and storage of spermatangia, and the males do not make wounds. As with most other deep-sea squid, the method by which females of T. danae access the sperm when they are ready to spawn is not known. Sperm may become mobile after exposure to chemical cues from the female (e.g., a nidamental gland secretion), as has been suggested for Illex illecebrosus (Durward et al. 1980). The spermatozoa appear to exit the spermatangium through the aboral opening of the free trailing end, which opens in the nuchal area. After coating the spawned eggs with oviducal and nidamental gland secretion, female T. danae could draw their eggs from the mantle cavity along both sides of the neck, where the spermatangia would open, releasing spermatozoa to fertilize the eggs. A similar spawning and fertilization process has been described for enoploteuthid squids (Nesis 1995).
Copulatory struggles between the sexes have been previously reported in other oceanic squids. In one female Bathothauma lyromma, two long arms were found attached to the inner mantle wall, these may have been bitten off during a copulation attempt (Young 1978, cited by Voss 1985 and again in Nesis 1995). Voss (1985) also observed scars and encysted suckers on the inner dorsal and ventral mantle wall surfaces, as well as on the exposed internal organs of the cranchiid squid Teuthowenia pellucida. All mature males of T. pellucida examined in the same study showed recent loss of body parts, although Arms I, and usually Arms II (the two pairs of modified arms), were always intact, suggesting that they may have been inside the mantle of the female during mating, when the injuries were sustained. An examination of the stomach contents revealed that the stomachs of two females were distended with recently consumed flesh, and pieces of gladii and spermatophores identifiable to T. pellucida. Voss (1985) suggested this as an artifact due to trauma in the trawl and also indicated that cannibalism may occur during mating.
Similar reports exist for three additional families. Both female and male specimens of Heteroteuthis dispar have been observed with large sucker scars on their heads, probably as a result of copulation attempts (Orsi Relini 1995; Hoving et al. 2008a). Mating male and female specimens of Onykia ingens, with interlocking beaks, were described by Jackson and Jackson (2004) and Bolstad (2006). And finally, O’Shea et al. (2007) reported that implanted spermatangia in specimens of Pholidoteuthis massyae were associated with dermal abrasion and removal of the dermal papillae consistent with male beak rasping, and less severe papillar abrasion consistent with male arm sucker rings.
It appears that successful mating in T. danae may similarly entail injury for at least one of the individuals involved. Although the empty incision observed in the trawl-caught specimen, and the non-incision-associated bundles of spermatangia in two of the ex-whale specimens, are far from conclusive, it is not difficult to envision an unsuccessful mating attempt in which such incisions or incisionless implantations might arise. The sexual size dimorphism in T. danae is female biased, which may make wounding the female during copulation a risky strategy for the male, and cannibalism (by females) is known for several squid species (e.g., Jackson et al. 1998; Bolstad and O’Shea 2004; Deagle et al. 2005). The nuchal region underneath the outer collar is a small, specific and not immediately accessible target; and the implantations in the exterior of the head and neck could result failed or foreshortened attempts to access the ‘usual’ site.
This study was financially supported by the Schure-Beijerinck-Popping funds (Royal Dutch Academy of Sciences) and the Nicolaas Mulerius Funds (University of Groningen). We also thank José Lamain for her help during dissection of the animals; Deniz Haydar and Prof. Dr. R. E. Young for critically reading the manuscript and providing supportive criticism; Mr. Summerfield for collecting the trawled specimen; and Serena Donati for translating Torchio (1967). This study is dedicated to Martina. A. C. Roeleveld, who sadly passed away during preparation of this work.
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