Increase of suckers throughout development
Firstly, to elucidate the increase pattern of suckers in S. esculenta and S. lycidas, the number of suckers on the second arm were counted in embryos, juveniles and adults. No suckers were observed on arms in embryos at early developmental stages (St. 24-25), during which arms start to elongate, but they seemed to form at later stages. Moreover, in sexually matured adults, there were many more suckers than in embryos (Fig. 2A).
The number of suckers (including primordial suckers) on the second arm was counted in S. esculenta. The sucker number (including primordial suckers) per second arm (mean ± S.D.) was 7 ± 1.6 at St. 28-29, 24 ± 0.96 at St. 31-33, 33 ± 2.0 at St. 34-36, 47 ± 2.19 at St. 37-39, 100 ± 9.4 in ML10 juveniles (mantle length: 10 mm), 132 ± 5.1 in ML20 juveniles, and 181 ± 13 in sexually mature adults (Fig. 2B). Similar investigations were also carried out in S. lycidas, showing that the sucker number on the second arm was 45 ± 1.9 at St. 34-36, 62 ± 3.8 at St. 37-39, 110 ± 11 in ML10 juveniles (mantle length: 10 mm), 146 ± 3.4 in ML20 juveniles, and 263 ± 25 in sexually mature adults (mean ± S.D.). Interestingly, S. lycidas possessed more suckers than S. esculenta.
Sucker formation during embryogenesis in S. esculenta
To investigate the pattern of sucker formation based on observations on the external morphologies and internal structures, the nucleus and cytoskeleton (F-actin) were stained and observed using a confocal laser scanning microscope (CLSM). The observation results showed that, in St-25 embryos at the early stage of arm elongation, there were no structures of primordial suckers (Fig. 3A). At St. 26-27, epithelial tissues on the oral side of arm tips were observed to be swollen (Fig. 3B). At St. 28-29, on the oral side of the arm tip, the epithelial tissue formed a ridge along the proximal-distal axis (sucker field ridge). On the more proximal area of the oral side, next to the sucker field ridge, multiple dome-shaped bulges (sucker buds) were located aligned in one or two rows in the proximal-distal direction, and the number of such bulges was increased in the more proximal part of the arm (Fig. 3C). At the middle to late embryonic stages (after St. 32), it was found that the sucker field ridge was located at the tip of the oral side of the arm, and many small sucker buds were aligned on the proximal side of the sucker field ridge (Fig. 3D, E).
From the distal to the proximal part of the arm, the number of suckers per row increased up to four. To examine the pattern of sucker-row formation in detail, observations of optical sections obtained from the basal part of each sucker buds were carried out and revealed that two buds were pinched off from one another, forming a gourd shape (Fig. 3F, G; for other developmental stages see Additional file 2: Fig. S2). Two pairs of gourd-shaped units were arranged alternately, forming 4 rows of suckers. Each unit of sucker buds in the 4 rows was clearly shown by the F-actin localization revealed by staining with phalloidin, and consisted of 2 cell components: a single layer of epithelial cells and the inside cells, which possessed relatively large nuclei (Additional file 3: Fig. S3).
At the stage just before hatching (St. 39), the gradual succession of stages of the process of sucker formation was observed along the distal-proximal axis of the arm; immature sucker buds were at the more distal part while larger and well-developed suckers were at the more proximal part (Fig. 3H). While dome-shaped sucker buds were observed at the distal tip, primordial suckers at more proximal positions showed constricted shapes, but without any detailed structures (Fig. 3H-J). At the most proximal part of an arm, furthermore, the structure of primordial suckers resembled the functional suckers in adults, with an attachment surface, a cup-shaped structure, and a stalk connecting to the arm, that corresponded, respectively, to an infundibulum, an acetabulum and a peduncle (Fig. 3K). In the cup-shaped structure, a ring-shaped accumulation of F-actin was observed along the cup edge (Fig. 3E).
The inner structures of arm tips were observed histologically, utilizing paraffin sections. At St. 24-25, neither distal sucker field ridge nor dome-shaped sucker buds were observed on the arms. However, the arrangement of epithelial cells was different between the oral and aboral sides (Fig. 3L). On the aboral side, the epithelial cells were arranged in orderly alignment, while the arrangement of epithelial cells on the oral side appeared random and disordered (Fig. 3L). At St. 28-29, the epithelium at the tip of the oral side was single-layered and cells under the epithelium looked randomly arranged without any clear structures (Fig. 3M). The size of sucker buds gradually increased from distal to proximal parts. At St. 37-38, although the distal part with undifferentiated sucker buds was similar to those at the earlier stages (Fig. 3N), in the proximal part of the arm, the base of a primordial suckers was constricted, forming a cup-shaped structure, which was not observed at earlier stages. At St. 39, the distal arm tip was similar to that in the earlier stages; a sucker field ridge with a single epithelial layer, from which sucker buds were differentiated, and multiple sucker buds were observed (Fig. 3O-Q). In the proximal part of the arm, the attachment surface and the cup-shaped structure (i.e., infundibulum and acetabulum) became more obvious, and nerve-like tissues were observed in the stalk of the cup structure, i.e., the peduncle (Fig. 3O, R).
Sucker formation during embryogenesis in S. lycidas
The sucker formation in S. lycidas was also observed to investigate whether the process seen in S. esculenta was shared among species. These observations showed that also in S. lycidas, a sucker field ridge was formed on the oral side of the arm tip along the proximal-distal axis, and a cluster of multiple dome-shaped sucker buds was observed in the more proximal part, adjacent to the sucker field ridge (Fig. 4A-D; St. 30-39). The number of primordial rows was only one at the distal arm tip, while it increased to two to four at more proximal positions. CLSM observations revealed that, as seen in S. esculenta, 2 buds were present in one unit with a gourd shape, and 4-sucker rows were formed by zig-zag alignment of these gourd-shaped units (Fig. 4E, F; for other developmental stages see Additional file 2: Fig. S2). At St. 39 (Fig. 4C, D), the size of primordial suckers was much larger in the proximal part than in the distal part of the arm, and an attachment surface (infundibulum), a cup-shaped structure (acetabulum) and a primordial stalk (peduncle) were clearly differentiated in the proximal part, whereas dome-shaped undifferentiated sucker buds were still observed in the distal part (Fig. 4G-I).
Histological observations in S. lycidas also showed that, at the arm tip, the most distal sucker field ridge with a single epithelial layer was observed on the oral side, from which dome-shaped sucker buds were differentiated from the proximal end (Fig. 4J, K; St. 34-39). At St. 39, in the distal part, dome-shaped sucker buds, in which the inner cell mass was undifferentiated, were similar to those seen at the earlier stages (Fig. 4L, M). In the proximal parts, each primordial sucker consisted of a cup-shaped structure and a stalk, which were presumed to develop into the adult sucker components (infundibulum, acetabulum and peduncle) (Fig. 4N).
Overall, in S. lycidas, both the morphology and the developmental pattern of suckers seemed similar to those in S. esculenta. However, focusing on the late embryonic stages (St. 34-39), the detailed sucker structures looked well-differentiated in S. lycidas, in comparison with those in S. esculenta (Fig. 3D, E, 4B, C). Moreover, the increase rate of sucker numbers was higher in S. lycidas. (Fig. 2B, 3A-E, 4A-C).
Sucker formation during postembryonic development in S. esculenta and S. lycidas
The external morphologies of arms and suckers in juveniles of S. esculenta and S. lycidas, observed by CLSM, showed that small dome-shaped sucker buds were seen only in the distal part of the arm, and larger suckers in the proximal arm part seemed to have functional structures like those seen in adult cuttlefishes (Fig. 5A-F, Additional file 4: Fig. S4). Histological observations showed that the sucker size was larger in the proximal part than that in the distal part. Most suckers, except for sucker buds in the distal tip, had distinct adult sucker structures, such as infundibulum, acetabulum and peduncle (Fig. 5G-L). As in embryos, at the most distal sucker field ridge a single epithelial layer was observed on the oral side of an arm tip, in which no clear tissue structures were seen (Fig. 5G, H, J, K).
Furthermore, SEM observations of the external morphology revealed that the epithelial tissues were expanded from the side of the arm’s tip, covering the undifferentiated area at the distal end of the tip. This sheath-like structure was observed throughout postembryonic development (Fig. 5M, Additional file 5: Fig. S5). The sucker field ridge and dome-shaped sucker buds were completely covered by the epithelial sheath. The epithelial sheath cover was also confirmed by observations on live specimens, to exclude the possibility that the epithelia had shrunk due to fixation for SEM observations (Fig. 5N, Additional file 5: Fig. S5).