Introduction
Cephalopods (Mollusca: Cephalopoda) are exceptionally diverse molluscs with a broad global distribution, and they play a pivotal role in marine ecosystems as both predators and prey (Jereb and Roper, Reference Jereb and Roper2005; Xavier et al., Reference Xavier, Allcock, Cherel, Lipinski, Pierce, Rodhouse, Rosa, Shea, Strugnell, Vidal, Villanueva and Ziegler2015). They inhabit a wide range of environments – from warm tropical tide pools to frigid depths of 5,000 m in the open ocean, including areas beneath polar ice (Hanlon et al., Reference Hanlon, Vecchione and Allcock2018). Cephalopods have adapted to life in various habitats, including the water column and seabed, in both coastal and pelagic zones (Nixon and Young, Reference Nixon and Young2003; Hanlon and Messenger, Reference Hanlon and Messenger2018). Their remarkable ecological versatility is reflected in a wide array of morphological specialisations.
Most cuttlefish species are nekto-benthic in their adult stage. They begin life attached to the substrate, using their fourth (ventral-most) pair of arms (arms IV) and the ventral mantle, which together function like suckers to anchor the hatchling to the surface (Bather, Reference Bather1895; Boletzky and Roeleveld, Reference Boletzky and Roeleveld2000). As they grow, they typically transition to a nekton-benthic lifestyle (Boletzky and Roeleveld, Reference Boletzky and Roeleveld2000) (Supplementary Figure S1A).
In contrast, a few small cuttlefish species – such as Ascarosepion tullbergi (Appellöf, 1886), Ascarosepion pfefferi (Hoyle, 1885), and Ascarosepion bandense (Adam, 1939) – remain benthic throughout their life cycle. These species continue to use arms IV and the ventral mantle for substrate attachment as adults. Instead of swimming, they exhibit a slow, ambling gait using arms IV and ventral flaps (ambulatory flaps) (Adam and Rees, Reference Adam and Rees1966; Roper and Hochberg, Reference Roper and Hochberg1988; Hanlon and McManus, Reference Hanlon and McManus2020; Norman and Reid, Reference Norman and Reid2000; Omura et al., Reference Omura, Takano, Oka and Takei2022, Reference Omura, Takano, Tomita, Yamaguchi and Oka2023) (Supplementary Figure S1B).
As mentioned above, some Ascarosepion species have acquired a unique lifestyle compared to any other cuttlefish. To understand the diversity of cuttlefish, developmental studies play a crucial role. Because many morphological traits are established during embryogenesis, documenting how development progresses in different species can provide insights into evolutionary trajectories and ecological adaptations. However, developmental information of cuttlefish is still limited for many taxa.
To date, numerous developmental studies have focused on the common nekto-benthic species Sepia officinalis Linnaeus, 1758 (Cole and Hall, Reference Cole and Hall2009; Andouche and Bassaglia, Reference Andouche and Bassaglia2016; Arias-Montecino et al., Reference Arias-Montecino, Sykes, Álvarez-Hernán, de Mera-rodríguez, Calle-Guisado, Martín-Partido, Rodríguez-León and Francisco-Morcillo2024). Naef (Reference Naef and Chun1928) and Lemaire (Reference Lemaire1970) established basic developmental criteria, describing stages from post-oviposition to hatching as the yolk is consumed. Boletzky (Reference Boletzky1989), Boletzky (Reference Boletzky2003) and Boletzky et al. (Reference Boletzky, Erlwein and Hofmann2006) expanded this framework by comparing S. officinalis development with that of other cephalopods. Boletzky et al. (Reference Boletzky, Andouche and Aud-Ponticelli2016) later proposed a comprehensive table of 30 stages, divided into five phases: cleavage, gastrulation, and organogenesis (plane, extension, and growth phases). Building on these studies, Lee et al. (Reference Lee, Lin, Chiao and Lu2016) defined 30 stages for Acanthosepion pharaonis Ehrenberg, 1831; Oka and Tezuka (Reference Oka and Tezuka2022) proposed 26 stages for Ascarosepion latimanus Quoy & Gaimard, 1832; and Yamamoto (Reference Yamamoto1982) described 40 stages for Sepiella japonica Sasaki, 1929. Development of Acanthosepion esculentum Hoyle, 1885 and Acanthosepion lycidas Gray, 1849 has also been examined (Yamamoto, Reference Yamamoto1942; Hirose, Reference Hirose1956), with hatching recorded at 40 and 42 days post-fertilisation, respectively.
In contrast to the many developmental studies on nekto-benthic cuttlefish, developmental descriptions of fully benthic cuttlefish remain extremely limited, despite their distinctive morphology and ecology. Among benthic cuttlefish, A. bandense is the only species for which developmental stages have been documented, with 25 stages recognised by Montague et al. (Reference Montague, Rieth and Axel2021). However, the process preceding yolk consumption remains inadequately understood. Montague et al. (Reference Montague, Rieth and Axel2021) defined the 25th stage as the final developmental stage, during which the embryo retains a large amount of external yolk. It remains unclear whether early hatching with substantial yolk reserves occurs because of artificial stimulation or natural phenomena.
Establishing detailed developmental descriptions of fully benthic species is essential for building a foundation to investigate ecological diversity and life history strategies within cephalopods. Comparative developmental data from such species can help identify stage-specific traits and broaden our understanding of the range of developmental patterns that exist across cuttlefish lineages.
Here, to address the lack of developmental data on benthic cuttlefish, we aimed to contribute to the understanding of cuttlefish development by (1) describing the developmental process of the benthic species A. tullbergi, and (2) comparing its development with that of other species, highlighting the distinctive developmental characteristics of A. tullbergi. Although this study does not reveal the mechanisms underlying benthic trait formation, it provides a developmental framework that can serve as a foundation for future comparative and evolutionary studies on cephalopod diversity.
Materials and methods
The reproductive season of A. tullbergi occurs from March to May in Japan (Nomura et al., Reference Nomura, Sakaji and Horikawa1997). A female typically lays approximately 20 eggs at a time, producing a total of 150–500 eggs over several days in one reproductive season (Nomura, 1998). The eggs are demersal, oval-shaped, and measure approximately 12 mm in length (Nomura, 1985).
Observations were conducted from March to May 2022 at the Okinawa Churaumi Aquarium, Japan.
In March 2022, one male and one female A. tullbergi were housed in a glass tank (58 × 48 × 38 cm) maintained at 22.5°C, which is close to the temperature of their natural habitat. The aquarium was located facing the sea, and seawater was directly pumped from the natural environment and supplied as an open system. The animals were fed thawed frozen shrimp three times a day (09:00, 13:00, and 17:00). Illumination was provided by fluorescent lights with a photoperiod of 07:00 light and 18:00 dark. The tank contained coral stones and empty bivalve shells as potential substrates for egg-laying, with a sand-lined bottom. On the first day of spawning, the female laid approximately 30 eggs beneath an empty bivalve shell. Ten eggs were randomly selected from the cluster and transferred to a smaller glass tank (30 × 18 × 18 cm) filled with natural seawater. The water was aerated with an air stone connected to a pump, and the temperature was maintained between 22°C and 25°C, similar to that of the natural habitat.
To minimize exposure to artificial movement or vibration and to maintain natural conditions, the gelatinous egg capsule was left intact, and no antibiotics or antifungal agents were used. Observations of embryonic development were made daily under a stereomicroscope (ECLIPSE Ts2; Nikon, Tokyo, Japan), and all photographs used in the figures were taken with a digital camera (TG-810; Olympus, Tokyo, Japan) under illuminated conditions. For the final developmental stage (stage 30), only the remaining eggs on the spawning bed were observed directly on the spawning bed, as even slight disturbance could trigger hatching.
We referenced the developmental staging criteria established by Boletzky et al. (Reference Boletzky, Andouche and Aud-Ponticelli2016) for S. officinalis and Lee et al. (Reference Lee, Lin, Chiao and Lu2016) for A. pharaonis, both of which define 30 stages of sepiid embryogenesis. In this study, embryos were observed without removing the egg capsule. Although we attempted to examine the early cleavage stages, the capsule partially obscured cellular details such as cell separation, making it difficult to clearly visualise stages 1–11. Therefore, detailed morphological descriptions begin from stage 12, when epiboly became clearly visible through the capsule.
Results
Schematics of gastrulation (stages 12–15) in Ascarosepion tullbergi embryo, viewed from the lateral side.
Images of gastrulation (stages 12–15) in Ascarosepion tullbergi embryo, viewed from the lateral side. Scale bar = 1 mm.
Stage 12
Epiboly 10%
Approximate time after egg laying: 5 days
The germinal disc began to cover the yolk mass in a process known as epiboly, resulting in approximately 10% of the yolk mass being covered. The ring-shaped zone of the germinal disc gradually disappeared.
Stage 13
Epiboly 30%
Approximate time after egg laying: 8–9 days
The germinal disc covered approximately 30% of the yolk mass as epiboly continued.
Stage 14
Epiboly 50%
Approximate time after egg laying: 9–10 days
The germinal disc covered approximately 50% of the yolk mass as epiboly continued to advance.
Stage 15
Epiboly 75%
Approximate time after egg laying: 11 days
The germinal disc covered 75% of the yolk mass at this stage. The epiboly process continued.
Organogenesis
Plane phase (stages 16–18) (Figures 3, 4)
Schematics of the organogenesis plane phase (stages 16–18) in Ascarosepion tullbergi embryo, viewed from the animal pole. a1, a2, a3, a4: arms 1 to 4; e: eye; fp: funnel pouch; ft: funnel tube; g: gill; m; mantle; mo: mouth; ss; shell sac; st: statocyst; t: tentacle.
Images of the organogenesis plane phase (stages 16–18) in Ascarosepion tullbergi embryo, viewed from the animal pole. Scale bar = 1 mm.
Stage 16
Approximate time after egg laying: 12 days
The yolk envelope almost completely covered the yolk. The rudiment of the mantle appeared as a pentagonal crest ring at the animal pole. The gill rudiments developed ventral to, and slightly apart from, the mantle. The eye rudiments manifested as a crescent-shaped oval ring. The arm rudiments were present but had not yet fully differentiated into discrete structures surrounding the embryonic body. The rudiments of the funnel pouch and tube also emerged and curved inward around the mantle. On either side of the funnel pouch, the statocysts developed into small circular thickenings.
Stage 17
Approximate time after egg laying: 13 days
An oval, ring-shaped mouth emerged midway between the eye rudiments at the edge of the embryo. The arm crown, consisting of four pairs of prehensile arms and an additional pair of tentacles, surrounded the embryonic body. The arm rudiments for arms II–IV and the tentacle rudiments were symmetrically located in the ventral part of the embryo, whereas arm I was situated near the eye rudiment in the dorsal part of the embryo. The arm rudiments converged at the base, forming a belt-like structure that encircled the embryo. The statocysts remained circular thickenings, which became more prominent. The rudiments of the funnel pouch and tube are developed and raised but not yet connected. The mantle rudiment formed into a rounded pentagonal ring with slightly elevated inner and outer rims, marking the beginning of shell sac formation with a central opening.
Stage 18
Approximate time after egg laying: 14 days
The relief of the embryonic body became more distinct. The mouth rudiment thickened into a more prominent ring. The eye rudiments, now in slanting oval shapes, were located laterally on the head rudiment. The individual arm rudiments became more pronounced as their edges protruded. The dorsal-most arm pair, arm pair I, were located anterior to the eyes, while the other arm pairs were arranged in an almost equidistant manner. Arm pair III was the least robust, whereas arms II, the tentacles, and arms IV were more uniform in size. Slight cheek humps appeared posterior to the eye rudiments. The funnel tube rudiments were linked to the funnel pouch rudiments, which became elevated into a rib-like shape and extended posteriorly and laterally to the statocysts. The ring-shaped statocysts exhibited clearly defined sunken holes. The rudiment of the mantle expanded inward.
Extension phase (stages 19–22) (Figures 5, 6)
Schematics of the organogenesis extension phase (stages 19–22) in Ascarosepion tullbergi embryo. D: dorsal side; L: lateral side; V: ventral side. A1, a2, a3, a4: arms 1 to 4; c: cheek; e: eye; eys: external yolk sac; fi: fin; fp: funnel pouch; ft: funnel tube; g: gill; m: mantle; le: lens; mo: mouth; t: tentacle.
Images of the organogenesis extension phase (stages 19–22) in Ascarosepion tullbergi embryo. D: dorsal side; L: lateral side; V: ventral side. Scale bar = 1 mm.
Stage 19
Approximate time after egg laying: 15 days
Mantle length (ML) = 0.5 ± 0.1 mm.
The embryo began to protrude from the animal pole, forming a cap-like structure over the yolk. The relief of the embryo became more distinct. The tentacles grew more prominent and larger than the other arm rudiments, with arm III remaining the weakest. The eyes projected, and cheek furrows became visible behind them. The funnel tube rudiments became more elevated and wider. The mantle rudiment exhibited a gentle downward dome shape. The fin rudiments transformed into subtly semicircular protrusions. The tips of the gills were observed in the mantle cavity.
Stage 20
Approximate time after egg laying: 16 days
ML = 0.6 ± 0.0 mm.
The embryonic body rose from the external yolk sac. The mantle rudiment exhibited greater width than length. The funnel pouch and tube rudiments shifted beneath the mantle rim, while the funnel tube rudiments developed towards one another, bending inward. The gills still protruded from the mantle cavity. The arm rudiments presented two distinguishable parts: a basal part, which was less elevated and attached to the head; and a distal part, which protruded outwards. The arm rudiments were more closely positioned than in earlier stages, however, arm I remained separate from the others.
Stage 21
Approximate time after egg laying: 18 days
ML = 0.7 ± 0.1 mm.
The embryonic body exhibited a more upright orientation on the external yolk sac. The mantle remained wider than it was long. The edges of the funnel tube rudiments converged but had not yet been fully sealed. Both the funnel pouch and tube rudiments rose toward the mantle cavity, while the gill tips remained protruding from the mantle cavity. The arms began to differentiate, with the tentacles developing into hook-shaped tips. The other arms remained shorter than the tentacles and became distinct from each other and from the head rudiment. Spherical lenses developed precisely at the centre of the eyes. The retina began to show a pale orange colouration accompanied by a thin orange ring at its periphery.
Stage 22
Approximate time after egg laying: 21 days
ML = 1.0 ± 0.1 mm.
The mantle length was approximately equal to the mantle width, resulting in an elongated, dome-like shape. The head remained wider than the mantle. The arms increased in length and began to fuse with each other, with arms III no longer being the smallest. The funnel tubes fused to form a single closed tube, eliminating the seam between them. The gills were covered by the mantle cavity. The ends of the fins extended to half the length of the mantle. The retina in the eye exhibited a darker appearance relative to that of the previous stage.
Growth phase (stages 23–30) (Figures 7–10)
Schematics of the organogenesis growth phase (stages 23–26) in Ascarosepion tullbergi embryo. D: dorsal side; L: lateral side; V: ventral side. A1, a2, a3, a4: arms 1 to 4; af: ambulatory flap; cb: cuttlebone; dr: dorsal ridge; e: eye; eys: external yolk sac; led: lateral edge; fi: fin; f: funnel; is: ink sac; iys: internal yolk sac; m: mantle; pp: papilla; t: tentacle.
Images of the organogenesis growth phase (stages 23–26) in Ascarosepion tullbergi embryo. D: dorsal side; L: lateral side; V: ventral side. Scale bar = 1 mm.
Schematics of the organogenesis growth phase (stages 27–30) in Ascarosepion tullbergi embryo. D: dorsal side; L: lateral side; V: ventral side. A1, a2, a3, a4: arms 1 to 4; af: ambulatory flap; cb: cuttlebone; dr: dorsal ridge; e: eye; eys: external yolk sac; led: lateral edge; fi: fin; f: funnel; is: ink sac; iys: internal yolk sac; m: mantle; pp: papilla; t: tentacle.
Images of the organogenesis growth phase (stages 27–30) in Ascarosepion tullbergi embryo. Scale bar = 1 mm.
Stage 23
Approximate time after egg laying: 24 days
ML = 1.5 ± 0.2 mm.
The mantle increased in size, with its length approximately half that of the external yolk sac. The mantle grew along the anteroposterior axis and transitioned from a dome-like shape to a round cone shape. The ventral mantle began to differentiate, forming a ventral shield, with the lateral edges appearing as white thickenings. The head shape started to change, and the eyestalks became shorter and more robust. The internal Y-shaped yolk sac was visible on the dorsal side. The retina turned orange, making it easier to distinguish the round lens. The arms extended further, tapering at their ends. The base of each arm and the tentacle converged medially, while the base of arm IV expanded into a triangular shape. The tip of the tentacle curved outwards.
Stage 24
Approximate time after egg laying: 25 days
ML = 1.7 ± 0.1 mm.
The mantle experienced further differentiation, exhibiting a slight curvature into a bow-like shape at the midpoint of the dorsal mantle rim. The head covers, which are formed by the fusion and expansion of the arm bases to cover the head region (Boletzky et al., Reference Boletzky, Andouche and Aud-Ponticelli2016), grew and extended to the cheek zone. The dorsal and ventral arm rudiments converged. The internal yolk sac elongated posteriorly, forming thumb-like structures that bent inward and converged at their tips. The first translucent increment of the cuttlebone became visible on the dorsal side. The retina darkened to a deep orange.
Stage 25
Approximate time after egg laying: 28 days
ML = 2.2 ± 0.3 mm.
The dorsal mantle length was approximately equal to the diameter of the external yolk sac and continued to elongate posteriorly. Dorsal ridges became visible, resulting in gentle unevenness on the surface of the dorsal mantle. The lateral edges of the ventral mantle grew stronger, with two white oval spots forming along these edges. The posterior third of the lateral edges of the ventral mantle curved delicately outwards; these curvatures corresponded to ambulatory flaps, representing large skin papillae (Omura et al., Reference Omura, Takano, Tomita, Yamaguchi and Oka2023). The dorsal mantle, head, and arms were pale yellow because of the increasing number of chromatophores. A black ink sac was visible on the ventral side. The tentacles extended to the middle of the external yolk. The rims of arms IV displayed small white spots. The head covers continued to grow, extending to the edge of the eyes on the dorsal side and slightly beyond the eyes on the ventral side. The fin rudiments exhibited a distinctive shape: posteriorly tapered and anteriorly expanded. The first increment of the cuttlebone was calcified and completely opaque white, with two chambers. The eyes exhibited a fleshy red colouration.
Stage 26
Approximate time after egg laying: 31 days
ML = 2.9 ± 0.8 mm.
The mantle had enlarged, with its lower part nearly corresponding to the width of the head. The dorsal ridges and lateral edges of the mantle became more pronounced, and the outward curvature along the lateral edges became more distinct. The dorsal colour of the embryo turned pale pink-brown, intensifying with the emergence of additional chromatophores on the mantle, head, and arms. White spots on the body became more prominent.
The tips of arm IV extended to approximately half of the length of the external yolk sac. Arm IV also developed broader lateral rims, accompanied by the emergence of small white patterns interspersed among the previously established larger ones. Two or three small papillae weakly projected along the rims of arms I–III. The tips of these arms were darker than their bases. The head covers, corresponding to the secondary lids, covered nearly 50% of the eyes. Small projections were observed below the eyes. The eyes displayed a deep red colour. The cuttlebone showed three calcified chambers.
Stage 27
Approximate time after egg laying: 34 days
ML = 3.5 ± 0.4 mm.
The pigmentation on the dorsal side of the mantle, head, and arms progressively intensified. Four white oval patterns appeared along the base of the fin on the dorsal mantle. The raised portion of the dorsal ridge turned white. Additional white patterns emerged on the dorsal side of the head. The eyes turned brown, and the secondary eyelid folds became more prominent, giving the eyes a crescent-shaped appearance. Thin white ridges radiated outwards from the eyes. The tip of the arms IV nearly reached the end of the external yolk sac. The small papillae on the rims of arms I–III grew more robust. The tentacles retracted into tentacle pouches, rendering them invisible externally. The cuttlebone contained four calcified chambers.
Stage 28
Approximate time after egg laying: 36 days
ML = 3.8 ± 0.4 mm.
The mantle underwent considerable enlargement. The bow-shaped curve at the rim of the middle dorsal mantle became more pronounced. The dorsal ridges grew more distinct. The ambulatory flaps continued to develop and assumed a slight ‘W’ shape. The pigmentation on the dorsal mantle, head, and arms intensified. The eyes transitioned to dark brown in colour. Thin white ridges around the eyes progressively formed. The tips of arms IV extended beyond the end of the external yolk sac. The rims, corresponding to the swimming membranes of arms IV, further developed. In addition, the white patterns of the rims enlarged. The projections on the lateral rims of arms I–III became more pronounced. The cuttlebone was no longer externally visible as the mantle became more opaque. The embryo expressed chromatophore patterns (the embryo changed its body colour when it was stimulated).
Stage 29
Approximate time after egg laying: 38 days
ML = 4.0 ± 0.2 mm.
The outer shape of the embryo closely resembled that of a newly hatched animal, except for the presence of an external yolk sac. The mantle cavity exhibited no notable new differentiation, although the existing structures were further enhanced. The body darkened as the chromatophores became more numerous. White patterns on the body became increasingly pronounced. Raised structures, including the dorsal ridges, ambulatory flaps on the ventral mantle, and small papillae on the arms, became more defined. The ambulatory flaps formed a clear W-shape with white patterns at the tips visible from the ventral view.
The eyelids were fully formed. The eyes were dark brown, and the iris folds transformed into the W-shape typical of sepiids. The external yolk sac was nearly consumed and invisible from the dorsal side. Its diameter was approximately one-quarter of the length of arm IV. The dorsal arms extended beyond the external yolk sac. The ink sac turned silvery in colour. At this stage, the embryo could hatch in response to external stimuli.
Stage 30 – hatching
Approximate time after egg laying: 40 days
ML = 4.3 ± 0.5 mm.
The external yolk sac was completely consumed. The length of arm IV was 4.1 ± 0.1 mm. The external overall shape of the embryo closely resembled that of an adult. This stage represented hatching, during which the hatchling emerged from the egg (Figure 11).
An image of Ascarosepion tullbergi hatching at stage 30.
Discussion
This study provides the first description of embryonic development in A. tullbergi. Our observations indicate that A. tullbergi follows a developmental sequence generally consistent with that reported in other cuttlefish species (Table 1). All examined species share a common sequence: following epiboly, embryonic organs differentiate on the yolk surface, the embryo elevates from the yolk sac, adopts adult orientation, and progressively matures into a hatchling. Although the total number of developmental stages varies slightly among species – for example, 26 in A. latimanus (Oka and Tezuka, Reference Oka and Tezuka2022) and 30 in S. officinalis (Boletzky et al., Reference Boletzky, Andouche and Aud-Ponticelli2016) – these differences are attributed to variations in staging criteria rather than in actual developmental processes. This suggests that the staging framework is conserved not only within the genus Ascarosepion but also across species with differing lifestyles and taxonomic affiliations. These findings align with those of Montague et al. (Reference Montague, Rieth and Axel2021), who reported comparable staging patterns among A. bandense, S. officinalis, and A. pharaonis.
Comparison of the embryonic staging series of cuttlefish, from 10% epiboly to hatching, among Ascarosepion tullbergi, Ascarosepion latimanus, with data from Oka and Tezuka (Reference Oka and Tezuka2022), Ascarosepion bandense, with data from Montague et al. (Reference Montague, Rieth and Axel2021), Sepia officinalis, with data from Boletzky et al. (Reference Boletzky, Andouche and Aud-Ponticelli2016) and Cole and Hall (Reference Cole and Hall2009), Sepiella japonica, with data from Yamamoto (Reference Yamamoto1982), Acanthosepion pharaonis, with data from Lee et al. (Reference Lee, Lin, Chiao and Lu2016), Acanthosepion lycidas, with data from Hirose (Reference Hirose1956; incubation temperature 19°C–24°C), and Acanthosepion esculentum with data from Yamamoto (Reference Yamamoto1942; incubation temperature 18°C–24°C)
St.: stage, Dpf: days post fertilisation.
Despite the above-mentioned overall similarities, we identified two distinguishing features in the development of A. tullbergi:
(1) Early elongation of arms IV: In A. tullbergi, arms IV extend beyond the end of the external yolk sac at stage 28, which is prior to the dorsal arms reaching the end of the yolk sac at stage 29. This represents a prominent distinction from two other cuttlefish species, in which arms IV do not extend beyond the yolk sac until much later, typically during the final stages of development. For example, arms IV in S. officinalis and A. pharaonis extend beyond the yolk sac at stage 30, simultaneously with, or subsequent to, the dorsal arms, in each species, respectively (Boletzky et al., Reference Boletzky, Andouche and Aud-Ponticelli2016; Lee et al., Reference Lee, Lin, Chiao and Lu2016) (Figure 12A, Table 1). (No data are available on the developmental sequence of the dorsal arm and arm IV in other cuttlefish species.) The relative length of arm IV, defined here as the ratio of arm IV length to the dorsal mantle length, was measured to be 66.7% in A. tullbergi hatchlings. This value is 1.2–2 times greater than that of other cuttlefish species hatchlings, which we calculated from the photographs in published papers; i.e., 40.5% in A. latimanus, 55.2% in S. officinalis, 54.5% in A. pharaonis, 48.0% in A. esculentum, 35.7% in A. lycidas, and 37.8% in Sepiella japonica (Yamamoto, Reference Yamamoto1942; Hirose, Reference Hirose1956; Yamamoto, Reference Yamamoto1982; Boletzky et al., Reference Boletzky, Andouche and Aud-Ponticelli2016; Lee et al., Reference Lee, Lin, Chiao and Lu2016; Oka and Tezuka, Reference Oka and Tezuka2022).
(A) Comparative developmental timelines of arm development across cuttlefish species: Ascarosepion tullbergi, Sepia officinalis, and Acanthosepion pharaonis. The white circle marks the stage when arm IV reaches half the length of the external yolk sac. The white triangle indicates when arm IV reaches the end of the external yolk sac. The white square represents when arm IV extends beyond the end of the external yolk sac. The black circle marks the stage when the dorsal arms reach or extend beyond the yolk sac. St.: stage, Dpf: days post fertilisation. The data are referenced as follows: S. officinalis: (Cole and Hall, Reference Cole and Hall2009; Boletzky et al., Reference Boletzky, Andouche and Aud-Ponticelli2016), A. Pharaonis: (Lee et al., Reference Lee, Lin, Chiao and Lu2016). (B) Comparison of the developmental timeline of the lateral edge of the ventral mantle between Ascarosepion tullbergi and Sepia officinalis (Boletzky et al., Reference Boletzky, Andouche and Aud-Ponticelli2016). The black square indicates the stage of ventral edge formation on the ventral mantle, occurring at stage 23 in A. tullbergi and at stage 26 in S. officinalis.
(2) Earlier development of the lateral margin of the ventral mantle: A. tullbergi exhibits earlier development of the lateral edges of the ventral mantle than does S. officinalis (no data are available on lateral edge formation for other cuttlefish species) (Figure 12B, Table 1). Although both species commence organogenesis at approximately stage 23, the stage at which the lateral edge appears differs. In A. tullbergi, the lateral edge becomes visible at stage 23, which is the onset of the organogenesis growth phase. Conversely, in S. officinalis, it emerges later at stage 26, in the middle of organogenesis (Boletzky et al., Reference Boletzky, Andouche and Aud-Ponticelli2016).
We hypothesize that morphological differences in hatchlings are related to their habitat environments. The well-developed arm IV and lateral edges of the ventral mantle in A. tullbergi hatchlings may facilitate substrate attachment in stronger water currents than those inhabited by hatchlings of other cuttlefish species. Unfortunately, detailed information on the environmental conditions of hatchling habitats in A. tullbergi and other cuttlefish is still lacking. To test this hypothesis, further field observations and ecological studies of hatchlings in their natural habitats are required.
We also hypothesize that these earlier developments of arm IV and the ventral mantle edge in A. tullbergi are linked to its strictly benthic lifestyle. In typical sepiid cuttlefish, they shift from benthic to nekto-benthic modes of life. The young individuals spend most of the time on the substrate, likely preventing predation by fishes that feed higher in the water column or are displaced by the strong environmental water current (Boletzky and Roeleveld, Reference Boletzky and Roeleveld2000; Hanlon and Messenger, Reference Hanlon and Messenger2018). In association with this benthic ecology, the hatchlings and early juveniles use the ventral mantle and arms IV as suckers for substrate attachment (Boletzky et al., Reference Boletzky, Andouche and Aud-Ponticelli2016). The ventral mantle features a ventral shield formed by the lateral edges as broad rims, allowing the body to attach to the substrate (Andouche and Bassaglia, Reference Andouche and Bassaglia2016; Boletzky et al., Reference Boletzky, Andouche and Aud-Ponticelli2016; Boletzky and Roeleveld, Reference Boletzky and Roeleveld2000). The swimming membranes along the aboral side of arm IV also enhance the attachment force to the substrate (Andouche and Bassaglia, Reference Andouche and Bassaglia2016; Boletzky et al., Reference Boletzky, Andouche and Aud-Ponticelli2016; Boletzky and Roeleveld, Reference Boletzky and Roeleveld2000).
In contrast to these sepiid cuttlefishes, A. tullbergi maintains a benthic lifestyle throughout life (Nomura, Reference Nomura1996; Nomura et al., Reference Nomura, Sakaji and Horikawa1997). Although benthic attachment had generally been regarded as a typical juvenile adaptation (Boletzky, Reference Boletzky1974), adults of some medium- and small-sized, strongly benthic cuttlefish species also attach themselves to hard substrates in the same manner as juveniles (Adam and Rees, Reference Adam and Rees1966; Boletzky and Roeleveld, Reference Boletzky and Roeleveld2000). In A. tullbergi, the lateral edges of the ventral mantle function as locomotor structures – ambulatory flaps – directly involved in interaction with the substrate. Reflecting this strongly benthic ecology, A. tullbergi possesses large adhesive surface areas on the ventral mantle and arms IV even in the adult stage (Omura et al., Reference Omura, Takano, Tomita, Yamaguchi and Oka2023). Additionally, the mantle morphology of the species is highly specialized; the posterior third of the lateral edge is broadened, forming an appendage-like structure called an ambulatory flap, and the animal ‘walks’ on the substrate using this projected flap and arms IV (Roper and Hochberg, Reference Roper and Hochberg1988; Omura et al., Reference Omura, Takano, Oka and Takei2022, Reference Omura, Takano, Tomita, Yamaguchi and Oka2023) (Supplementary Figure S1B).
We can expect that these functional requirements of the lateral edges of the ventral mantle and arm IV for a benthic lifestyle need an early investment in benthic traits, enabling the further development of these characteristics in adults, and this may explain why these structures are developed earlier in A. tullbergi compared to other species. This hypothesis can be further tested by observing the developmental process of other benthic cuttlefish species, which could not be achieved in this study.
Our observations begin from stage 12 because early cleavage stages (stages 1–11) were not clearly visible due to the presence of the gelatinous egg capsule. Additional efforts, including in situ imaging or removal of the egg capsule, will be needed to describe earlier developmental processes such as cell division and blastoderm formation.
This study lays the foundation for future developmental comparisons among benthic cuttlefish and provides insights into how benthic traits emerge in cephalopods. Accumulating such data will be critical for advancing our understanding of cephalopods’ evolution, ecological adaptation, and life history diversity.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0025315426101039.
Acknowledgements
We extend our gratitude to the staff at the Okinawa Churaumi Aquarium for their invaluable assistance with data collection. We also thank K. Hasegawa, H. Senoh, and T. Kon for their help in conducting this research and A. Packard, M. Yoshida, S. Shigeno, T. Kishida, and K. Nomura for sharing important research papers that contributed to this study. We respectfully acknowledge the contributions and insightful guidance of T. Okutani, who sadly passed away during the course of this research.
Author contributions
A.O. designed the study, analysed the data, and wrote the manuscript. S.-i.O. and T.T. provided critical biological specimens and wrote the manuscript. H.T. and D.F. aided in data collection.
Funding
This study was supported by the Japan Society for the Promotion of Science, KAKENHI [grant numbers JP20K15605 and JP24K09068].
Competing interests
There is no conflict of interest.
Ethical standards
All observations in this study were non-invasive and conducted at the Okinawa Churaumi Aquarium under the institutional and facility-specific ethical standards for observing marine organisms in captivity. Animal handling was carried out in strict accordance with the ‘Guidelines for Animal Experiments of the Okinawa Churashima Foundation’, with the same level of care and welfare consideration applied to higher vertebrates.
Data
All data are available in this manuscript.
