- Research article
- Open Access
Mantle margin morphogenesis in Nodipecten nodosus (Mollusca: Bivalvia): new insights into the development and the roles of bivalve pallial folds
BMC Developmental Biology volume 15, Article number: 22 (2015)
Despite extensive knowledge on bivalve anatomy and development, the formation and differentiation of the mantle margin and its associated organs remain largely unclear. Bivalves from the family Pectinidae (scallops) are particularly promising to cast some light on these issues, because they exhibit a complex mantle margin and their developmental stages are easily obtained from scallop farms. We investigated the mantle margin of the scallop Nodipecten nodosus (L. 1758) during larval and postmetamorphic development.
A thorough analysis of the mantle margin development in Nodipecten nodosus, from veliger larvae to mature adults, was conducted by means of integrative microscopy techniques, i.e., light, electron, and confocal microscopy.
Initially unfolded, the pallial margin is divided into distal and proximal regions by the periostracum-forming zone. The emergence of the pallial musculature and its neural innervation are crucial steps during bivalve larval development. By the late pediveliger stage, the margin becomes folded, resulting in a bilobed condition (i.e., outer and inner folds), a periostracal groove, and the development of different types of cilia. After metamorphosis, a second outgrowth process is responsible for emergence of the middle mantle fold from the outer surface of the inner fold. Once the three-folded condition is established, the general adult features are rapidly formed.
Our data show that the middle mantle fold forms from the outer surface of the inner fold after metamorphosis and that the initial unfolded mantle margin may represent a common condition among bivalves. The first outgrowth process, which gives rise to the outer and inner folds, and the emergence of the pallial musculature and innervation occur during larval stages, highlighting the importance of the larval period for mantle margin morphogenesis in Bivalvia.
The molluscan mantle margin, which corresponds to the free portion of the mantle, exhibits great diversity of form and function. In bivalves, the mantle is a membranous organ consisting of left and right lobes, united dorsomedially by an isthmus that lines the interior of the shell valves and surrounds the mantle cavity . The mantle margin is the free edge of this organ and bears tissue extensions named mantle (or pallial) folds, which can bear muscular, sensory or secretory structures . The presence of three mantle folds in this region is claimed to be the general condition for conchiferan molluscs; nevertheless, structural diversity in the pallial margin is very pronounced among and within molluscan classes . Although gastropods and scaphopods most often display a simple, swollen projection in the mantle rim, in a few gastropods the mantle margin is molded into distinctive folds [2, 3]. The monoplacophoran Neopilina galathea displays three folds in the mantle margin, and a periostracal groove is placed between the outer and middle folds . Similarly, among living cephalopods, the mantle of Nautilus pompilius also exhibits the three-folded pattern .
In Bivalvia, the mantle margin is divided into three pallial folds, each with a specific function: the secretory outer fold, the sensory middle fold, and the muscular inner fold [6, 7]. The periostracum is formed in a deep groove between the outer and middle folds, while the shell layers are secreted by the outer mantle epithelium [8, 9]. However, exceptions to this three-folded pattern do exist, e.g., four pallial folds have been described for the Veneridae [10–12], duplication in the middle fold may occur in Donacidae , and two pallial folds may be present in some Arcidae, with the outer one being duplicated [14, 15].
The bivalve mantle margin displays several adaptive traits associated with the different bivalve lifestyles, resulting in the huge morphological diversity observed within the class. Different levels of fusion during mantle margin and siphon formation are among the key features associated with the evolutionary radiation of infaunal bivalves [6, 16, 17]. Specialized secretory glands from the mantle margin are widespread in many groups performing a variety of roles, e.g., cleansing, adhesion, lubrication, and boring [18–20]. Pallial tentacular structures are also present in some families, such as Limidae, Pectinidae, and Galeommatidae, performing sensorial, defensive, and secretory functions [21–23]. In addition, photoreceptors and pallial eyes have evolved independently in the mantle margin of several distantly related bivalve taxa [24, 25]. Even though extensive amount of knowledge concerning the morphology of the bivalve mantle margin has been produced, these studies have mainly focused on adult anatomy, important developmental and functional issues remaining largely unclear .
Bivalve organogenesis is poorly understood compared to other molluscan groups such as gastropods, and further investigations applying techniques of microanatomy are vital to evaluate the functional ontogeny in the Bivalvia . The development of organs and structures, such as gills, foot, velum, and, most of all, the shell, has been thoroughly documented for several bivalve representatives (e.g. [28–34]). Apart from the detailed study on mantle anatomy of Ostrea edulis larvae , details on the larval mantle are fragmentary and restricted to descriptions on general larval morphology [36–40]. As a consequence, several questions on bivalve mantle morphogenesis remain unanswered. For instance, a developmental hypothesis assuming the origin of the middle mantle fold from the inner fold has been proposed based on anatomical features of the mantle margin in some adult bivalves . However, further developmental evidence is still necessary to test this hypothesis and understand the changes and mechanisms underlying mantle fold differentiation.
Scallops (bivalves of the family Pectinidae) are particularly promising to cast some light on the origin and differentiation of the bivalve mantle margin. Due to their economical importance, developmental stages are easily obtained from scallop farms. In addition, pectinids bear an especially complex mantle margin, displaying distinct organs and pallial structures, such as elaborate eyes and tentacles. Scallops are a well-studied group and large literature on their behavior, taxonomy, phylogeny, and aquaculture is available (e.g. [41–44]). Their pallial eyes have been extensively studied due to their intricate structure and optical performance (e.g. [45–49]). The inner mantle fold is a muscular curtain responsible for the regulation of the water flow into and out of the mantle cavity, especially during clapping movements and swimming behavior [50–52]. Although there is a plethora of studies on scallop pallial structures, there is no specific information concerning the anatomy and developmental changes of their mantle margin.
The present study aims at analyzing the morphogenesis of the mantle margin in the scallop Nodipecten nodosus (Linnaeus, 1758). We provide a thorough description of this region throughout its development, from veliger larvae to mature adults, by means of light, electron, and confocal microscopy.
Orientation and axis determination
Larval orientation is herein defined in accordance with comparative lophotrochozoan larval anatomy, i.e., with anterior corresponding to the position of the apical tuft, posterior corresponding to the opposite region, and ventral being defined by the position of the foot. In postmetamorphic bivalves, morphological axis designation is commonly such that the mouth position defines anterior and the hinge line dorsal (with the opposite as ventral). These terms are used herein, although we are aware of the semantic issues concerned with larval versus adult axis designations.
In veliger larvae of Nodipecten nodosus (Fig. 1a, b), the mantle comprises two lobes that enclose the body of the animal and define a small pallial cavity where the larval velum is located (Fig. 1c, d). The mantle margin runs alongside the shell edge from each side of the mantle isthmus (posterior region where the hinge is situated). The pallial margin of live larvae cannot be properly visualized by means of conventional light microscopy because of its transparency and location underneath the shell edge, being also partly obliterated by the large, antero-ventrally located ciliated velum (Fig. 1a, b). Histological cross sections from decalcified veligers show the mantle margin as a slender mantle projection connected to the velum by a short membrane (Fig. 1c, d). A schematic illustration of the larval mantle margin is represented in Fig. 2. The margin, which becomes thinner towards the distal region, is unfolded, and no particular structure is present. The periostracum is a fine layer originating in the inner epithelium of the pallial margin, close to the distal region (Fig. 1e, f, 2). The secretion occurs in a well-defined site, the periostracum-forming zone (PFZ), marked by a line parallel to the margin between the mantle epithelium and the newly formed periostracum sheath (Fig. 1f). From this region, the periostracum begins as a wrinkled band, but becomes smooth when covering the rest of the distal mantle margin (Fig. 1g). In the proximal region, immediately before the PFZ, the exposed inner epithelium contains short microvilli on its surface; no cilia are present (Fig. 1f).
By the early pediveliger stage, larvae of Nodipecten nodosus grow and develop an extensible foot (Fig. 3a). The swimming habit prevails, although the crawling behavior becomes gradually common upon available hard surfaces. The general morphology of the pallial margin in pediveligers closely resembles that observed in veligers, including an unfolded projection (Fig. 3b). However, a major difference is present in both mantle margins: the emergence of a row of cilia along the entire periostracum-forming zone (Figs. 3c, 4a, b). These cilia exhibit the classical 9 + 2 microtubular arrangement (Fig. 4c, d). From the PFZ, the periostracum covers the rest of the inner distal pallial margin (Fig. 4c, e) and extends to cover the shell. The proximal portion of the mantle margin is characterized by the presence of several secretory cells, particularly on the inner epithelium (Fig. 4c, f), as well as well-developed rough endoplasmic reticulum around the cell nuclei (Fig. 4c, g). The content of those vesicles are granular, electron-lucent and seems to be released on the inner surface (Fig. 4f). Despite the presence of secretory activity in this region, no evidence for mucopolysaccharides was detected applying standard histochemical techniques (Fig. 3d).
Although the mantle margin of the pediveliger is quite similar to that of the veliger, crucial anatomical changes arise during this period, contributing to the morphogenesis of the pallial system. In scallop veligers, no muscles or nerves were detected associated with the mantle margin (data not shown). In contrast, distinct serotonergic fibers are found in the mantle of pediveligers, where a conspicuous nerve runs along the entire edge (Fig. 5a), likely corresponding to the developing circumpallial nerve. While the mantle is dorsally innervated by projections from the apical-cerebral ganglia, the ventral portion receives fibers from the visceral ganglion (Fig. 5b). A distinct pallial musculature is also developed in pediveliger larvae, containing smooth and striated myofibers (Fig. 5c, d). While some of them are parallel to the edge, other bundles are branching retractors, attached to the shell, that run towards the mantle margin (Fig. 5d).
In late pediveliger larvae, when the foot is completely developed and the velum gradually shrinks, another crucial modification takes place in the mantle margin. An outgrowth process gradually occurs where the ciliary row is located, adjacent to the periostracum-forming zone, resulting in a lateral evagination of the proximal region (Fig. 6a–c). This outgrowth process is responsible for the appearance of an inner pallial extension and, at the same time, for positioning the periostracum-forming zone (and respective ciliary row) at the bottom of the newly formed groove, i.e., the periostracal groove (Fig. 6c). The distal region of the mantle margin, permanently covered by the periostracum, corresponds to the outer pallial fold, while the proximal region is modified into a projection, henceforward named inner pallial fold (but see Discussion).
Metamorphosis results in profound changes in larval anatomy and behavior. The settled larva secretes the dissoconch, while internal organs are reorganized and rotated. The larval velum degenerates and the first gill filaments grow rapidly (Fig. 7a, b). While the larval mantle margin is inconspicuous and not visible in live specimens, the mantle margin in postlarvae is evidenced by the presence of a well-developed curtain-like inner fold bearing cilia (Fig. 7b, c). The mantle lobes become much longer, extending the free margin far from the visceral organs, providing space for the numerous developing gills within the pallial cavity (Fig. 7d, e). The pallial musculature is enlarged, bundles grow thicker, but the combined arrangement of margin-parallel bundles and retractor muscles persists (Fig. 7f, g).
The mantle margin in postmetamorphic scallops comprises the outer and inner folds (Fig. 8), as in late pediveligers. The outer fold is usually reduced, covered by the periostracum, while the curtain-like inner fold is pronounced (Fig. 9a, b, c). Two types of cilia arrangement are present on the inner fold. The first type, found along the rim of the inner fold, corresponds to ciliary tufts sparsely scattered over the epithelium and contains few cilia (Fig. 9d). The second type comprises a ciliary band in which long cilia are densely distributed on the inner surface of the inner fold. This latter type, however, is restricted to the anterior portion of the left mantle lobe (Figs. 7b, 9b, c, e). In live animals, intense ciliary beating from this latter type is produced in this region. In the dorsal region of postmetamorphic individuals, the inner folds are fused both anteriorly and posteriorly, the outer fold and periostracal groove remaining intact (Fig. 9f, g). Such fusion occurs near the mantle isthmus where the right and left mantle lobes are formed.
In juvenile individuals of Nodipecten nodosus, most of the adult morphological features are already established (Fig. 10a). A few weeks after metamorphosis the pallial margin comprises three folds, including the newly formed middle fold, which contains numerous eyes and tentacles (Fig. 10b, c). The scallop three-folded condition is represented in Fig. 11. The outer epithelium of the middle fold (i.e., the one facing the periostracal groove) exhibits densely distributed cilia continuous with the cilia of the periostracal groove (Fig. 10d). The inner fold, usually referred to as “velum” for pectinid bivalves, is a muscular curtain-like fold (Fig. 10c). While mucin cells were not detected in the mantle of N. nodosus larvae, they were observed in juvenile individuals. In those specimens, gland cells producing acidic mucopolysaccharides are spread in the outer epithelium of the mantle and outer fold, suggesting intense mucous secretion in the extra-pallial cavity, i.e., the space between the mantle and the shell (Fig. 10e).
In juveniles, the pallial musculature becomes evident in histological sections due to the increase of thickness and number of bundles (Fig. 12a, b). Within the mantle margin, numerous long muscles are radially distributed, with striated and apparently smooth fibers mixed in parallel bundles running from the pallial line to the mantle margin (Fig. 12c, d). Within the inner fold, besides radial muscles, densely organized striated muscle bundles running parallel to the mantle margin are present (Fig. 12b, c). These bundles are more closely arranged in the distal region of this fold (Fig. 12e).
The nervous system of the mantle margin comprises numerous radial nerves that run towards the distal region and exhibit strong immunoreactivity for α-tubulin (Fig. 13a, e). In addition, the serotonergic pallial system includes a reticulate pattern of distribution over the entire mantle margin (Fig. 13b). The very prominent circumpallial nerve is responsible for innervation of the pallial margin (Fig. 13c, d, f). Unlike other nerves, completely formed by fibrous projections combined with glia cells, the circumpallial nerve displays a typical ganglionic structure. The central portion (i.e., the neuropil) is composed exclusively by neuronal fibers, while the periphery (i.e., the cortical region) contains the cell bodies (Fig. 13d).
Adult individuals of Nodipecten nodosus (Fig. 14a) display a very prominent mantle margin, with numerous tentacles, eyes, and a pigmented velum (Fig. 14b). At the auricular region (dorsal ears), the inner fold remains fused (Fig. 14c). The mantle is proportionally larger and thicker than in juveniles, and the outer fold is smaller than the remaining folds. The pallial musculature consists of large bundles of striated and smooth fibers, which form two major muscle groups that run close to the outer and inner epithelium, respectively (Fig. 14d). Small transverse muscles are also present (Fig. 14d). Embedded within the connective tissue, pallial nerves reach the circumpallial nerve at the level of the pallial folds (Fig. 14e). The circumpallial nerve preserves its ganglionic organization, i.e., with neuronal bodies distributed at the periphery and numerous axons merged at the core (Fig. 14e).
Simple, cuboidal cells are found in the inner epidermis of the outer fold, in the middle fold, and in the inner pallial epithelium. Simple, columnar cells are common in the inner fold, in the outer pallial epithelium, as well as in the outer epithelium of the outer fold. The adult inner mantle margin epidermis is covered by sparsely distributed tufts of cilia (Fig. 14f) and few epithelial mucous cells with neutral mucopolysaccharide content (Fig. 14g). The outer mantle margin epithelium differs from the inner one by the presence of short microvilli (Fig. 14h) and numerous secretory cells containing exclusively acidic mucopolysaccharide vesicles (Fig. 14i). This condition remains the same at the outer epidermis of the outer fold (Fig. 15a). However, the inner epithelium of the outer fold possesses two types of secretory cells related to acidic and neutral mucopolysaccharide secretion (Fig. 15b). At the periostracal groove, a pair of glandular folds is responsible for periostracum secretion (Fig. 15c).
The adult middle fold epithelium is intensely ciliated on its outer surface (Fig. 15d). The middle fold tentacles contain mucous and pigment cells (Fig. 15e, f), while the pallial eyes display the typical scallop ocular components (Fig. 15g). The inner fold is the largest structure of the adult pallial margin, bearing small tentacles at its most distal region (Fig. 14b), and sparse tufts of cilia on its surface (Fig. 15h). The inner fold musculature becomes more prominent and regionalized in adults (Fig. 16a–e). Radial muscles emerge from the pallial musculature and extend through the entire fold (Fig. 16b), running from the base of the velum to the marginal end (Fig. 16b–e). The margin-parallel muscles seen in juveniles are now organized in bundles that become more numerous and larger as they approach the middle and distal portions (Fig. 16d, e). During this gradual transition, the radial musculature is laterally compressed against the base of the epithelium, providing space for the margin-parallel fibers (Fig. 16e).
Larval mantle folds and periostracum-forming zone
The larval mantle margin in Nodipecten nodosus is unfolded, comprising a single projection divided into distal and proximal regions by the periostracum-forming zone. Nevertheless, many authors have adopted the term “fold” to designate such division in early larval stages, which may have led to possible misinterpretations of larval morphology, particularly concerning mantle margin development. Two folds were reported at the mantle rim in larvae of Pecten maximus: the “outer fold” being covered by the periostracum, and the epithelium of the “inner fold” being exposed and covered by short microvilli . However, the schematic representation of the mantle margin in P. maximus  exhibits no folds, but proximal and distal regions, quite similar to the larval pallial organization of N. nodosus described herein. Other similar examples include the mantle margin of Crassostrea virginica , Cardium edule , Nucula delphinodonta , Lasaea adansonii , and Ostrea edulis , where the figures (illustrations or photographs based on light or electron microscopy) provided by the authors all clearly show an unfolded larval margin. Considering the results obtained in the present study combined with the above-mentioned information, it seems more suitable to use the terms “distal” and “proximal” regions rather than “fold” to designate specific areas of the larval mantle margin.
Although it is assumed that the presence of two pallial folds corresponds to the typical condition of bivalve larvae , most of the available information for the larval mantle margin is restricted to observations of a single larval stage, generally with no data on previous stages (e.g. ). In a developmental perspective, this could suggest that early mantle traits are unknown for some species. Therefore, it is reasonable to consider that, at least for some bivalves such as scallops, the larval mantle margin may exhibit an unfolded step in early development, with subsequent division into two mantle folds. However, the early mantle margin in other bivalve species undoubtedly does show a folded condition, thus highlighting the importance of more comparative and detailed studies to clarify such issues.
In Nodipecten nodosus, the larval periostracum begins as a wrinkled band before covering the distal margin and shell, a condition also detected for Ostrea edulis , and referred as “convoluted periostracum” . This organic sheath is comprised by three layers formed within a groove between the outer and inner folds of bivalve larvae . However, periostracum secretion commences before the mantle folds are developed, i.e., before a groove is present. In this case, the secretory activity occurs on a line at the distal region of the inner mantle margin epithelium [26, 56], as confirmed by the TEM observations presented herein for N. nodosus. The term “periostracum-forming zone” was proposed to designate the site where the organic sheath is produced in the unfolded mantle margin of Lasaea adansonii larvae , and we agree with the appropriateness of this term.
Mantle margin development
General development of the mantle margin
A hypothesis for the development of the pectinid mantle margin is summarized in Fig. 17 and is proposed based on the data obtained herein for Nodipecten nodosus combined with revisited information from the literature (e.g. [26, 28, 35, 37, 53, 54, 56, 57]). Even though the developmental sequence includes some unique characters from scallops, such as the pallial eyes from the middle fold, the morphogenesis is represented in order to provide general insights into mantle margin development in the Pteriomorphia.
The bivalve mantle is formed shortly after the appearance of the shell field on the dorsal surface of late trochophore larvae [31, 38]. The epithelium of the shell gland then extends, giving rise to the mantle which will permanently underlie the valves [36, 58]. In scallops, the veliger exhibits an unfolded mantle margin, i.e., a single projection beneath the shell margin with no distinct folds. The periostracum formation zone (PFZ) marks the division between the distal region of the mantle margin, which is permanently covered by the periostracum, and the proximal region. During larval development, the transition to the pediveliger stage is marked by the emergence of a row of cilia in the proximal region, adjacent to the PFZ. In addition, the pallial musculature arises, as does the mantle innervation. The unfolded condition is conserved until slightly before metamorphosis, when the first of two major outgrowth processes takes place. An evagination of the proximal region near the row of cilia produces the inner mantle fold, the distal region now corresponding to the outer fold. Consequently, the PFZ is confined within a groove, and the row of cilia, previously present on the surface of the proximal mantle region, comes to lie on the outer surface of the inner mantle fold. Metamorphosis produces a variety of anatomical changes, the inner fold becoming a prominent pallial curtain-like fold. Also, at this stage the second outgrowth process occurs, forming the middle fold through an evagination at the base of the outer surface of the inner fold. Consequently, the ciliated epithelium of the periostracal groove is now located on the outer surface of the middle fold. Once the three-folded condition is achieved, further development includes the growth of sensorial organs (middle fold tentacles and eyes first appearing at the juvenile stage) and further growth of the pallial curtain.
Alternatively, the presence of cilia on the outer epithelium of the middle fold in juveniles and adults could be interpreted as a condition that originates before metamorphosis and is preserved during subsequent development. In this case, the middle mantle fold would form earlier, while the inner fold would emerge later as a projection from its inner surface, after metamorphosis. However, some lines of evidence support the first hypothesis (earlier formation of the inner fold; Fig. 17): 1) the first outgrowth process generates a curtain-like fold, which is morphologically and functionally similar to the inner fold of later stages; 2) pallial tentacular structures emerge only after metamorphosis, which is in accordance with a late formation of the middle mantle fold (that bears these structures); 3) the dorsal fusion of pallial folds is already present shortly after metamorphosis, when a third fold is not yet present. Such fusion corresponds to the merging of both inner folds at the auricular zone of juvenile and adult scallops, thus also supporting the early emergence of this pallial fold.
Implications for hypotheses concerning mantle fold development
The present investigation on the development of the mantle margin in Nodipecten nodosus casts new questions on old issues concerning the anatomy and development of bivalves. For instance, an unfolded mantle margin is proposed as a possible condition, at least for some bivalves. Whereas two folds are achieved in late pediveliger larvae of N. nodosus, apparently such a condition may occur early in other bivalve species (e.g. [28, 35]). In addition, the groups of cilia scattered on the larval inner mantle fold of some bivalves (e.g. [35, 37]) only arise in scallops after metamorphosis. So, despite common pallial features in those bivalves, the timing of the emergence of cilia and mantle folds seems to vary. This could be explained by distinct rates of differentiation during ontogeny [59, 60], leading to heterochronic alterations across the Bivalvia. Heterochrony has been suggested as the reason for modifications in developmental time of bivalve larvae and for significant anatomical and evolutionary changes [61, 62].
In adult Arcoida, the outer mantle fold is usually subdivided into two distinct folds, while the remaining folds are more variable. While species of Arca and Glycymeris exhibit no middle fold , the inner fold in Philobrya, Bathyarca, Barbatia, and Trisidos is duplicated, showing variation in size and shape . Based on epithelial differences observed in the single inner fold of Arca noae, it was suggested that both middle and inner folds are present in ark clams, although they are combined into one single fold . In addition, the two-folded mantle margin of adults of some arks [14, 63] and the presence of two folds in oyster pediveligers  contributed to suppose that the outer surface of the inner mantle fold would be the putative middle fold, not ontogenetically differentiated into a distinctive structure . The present investigation with Nodipecten nodosus has provided the first developmental evidence to support the origin of the middle fold from the outer surface of the inner fold, reinforcing this previous hypothesis .
The two-folded arrangement has been claimed to be a primitive condition in the Bivalvia [14, 15]. There are, however, few comparative studies on the bivalve mantle margin, particularly in Pteriomorphia, and none of them was analyzed under a phylogenetic perspective, which prevents further inferences on character evolution. We agree that analysis of developmental sequence make no a priori assumptions on the conservation of developmental stages or about how sequence should evolve , so we are not able to ascertain if the two-folded mantle margin is a primitive condition or a derived trait of some species (e.g., in which the middle fold would not be differentiated). Identifying homologous characters in the mantle margin is a challenging issue and represents a central point to the understanding of molluscan mantle evolution. More comparative data are still necessary, as well as developmental evidence for other taxa, to integrate hypotheses of homologies concerning mantle folds and pallial structures across the Bivalvia.
Functional ontogeny and morphology of the mantle margin
Larval mantle margin anatomy
The covering of the inner epithelium of the mantle margin by microvilli seen in Nodipecten nodosus was previously observed in Pecten maximus  and Ostrea edulis [35, 37]. The presence of cilia has been reported for the larval mantle margin of several bivalves, but this information is almost always imprecise. For instance, cilia were described along the pallial margin of veligers of the scallop Argopecten irradians, but no details about type or distribution were provided . In N. nodosus, cilia are not present until the pediveliger stage, when a row of long cilia is formed adjacent to the PFZ. Five ciliary types are present on the mantle margin of P. maximus larvae . Type 1 represents few cilia located in small depressions; type 2 is a row of long cilia in line, close to the distal region of the edge; type 3 includes tufts of numerous cilia scattered on the posterior margin; type 4 is a line of single cilia around the posterior edge; and type 5 is a row of short cilia in a protuberance dorsal to the anus. Even though not all these ciliary types were detected in larvae of N. nodosus, the row of cilia adjacent to the periostracum-forming zone likely matches type 2.
When the mantle margin becomes two-folded in late pediveligers of Nodipecten nodosus, new ciliary groups arise, and after metamorphosis they become more prominent. Within the periostracal groove, dense rows of cilia are distributed on the outer surface of the inner fold. Such a ciliary band was also detected in Ostrea edulis . Epithelial cells containing several electron-lucent vesicles were found in Pecten maximus, close to the ciliated cells at the PFZ . In addition, great abundance of rough endoplasmic reticulum was detected in mantle margin cells of Crassostrea virginica . In general, these ultrastructural characteristics seem in accordance with the present data for N. nodosus. On the edge of the inner fold of postmetamorphic N. nodosus, tufts of cilia are scattered along the margin, exactly like those classified as type b in pediveligers of Argopecten purpuratus , and type 3 in P. maximus . Long cilia, similar to those present in the ciliary band from the inner surface of the inner mantle fold of postmetamorphic N. nodosus, also occur in tufts along the inner larval fold of Mytilus edulis , O. edulis , and Pinna carnea . However, no cilia were detected in the mantle margin of Pandora inaequivalvis larvae prior to or after metamorphosis . Tufts of cilia in the inner fold of oyster pediveligers are related to sensory cells, while the wide ciliary band may display cleansing functions . However, in the case of N. nodosus postlarvae, the wide ciliary band is restricted to the anterior portion of the inner fold, within the inhalant region of the pallial cavity. In live postlarvae, those cilia exhibit intense beating, which may suggest a potential contribution to water flow into the mantle cavity instead of a sensory function.
The larval anatomy of the bivalve pallial musculature and nervous system remains largely unknown. Some muscle fibers were described for the inner fold of some Pinnidae species , but a detailed comparison with the present data is prevented, since no image was provided in that study. Smooth fibers are supposed to be present near the mantle folds of P. maximus based on larval descriptions . In pediveligers of O. edulis, radial muscles were supposed to be present in the larval mantle margin based on mantle contractions of live specimens . The present investigation of Nodipecten nodosus provides clear evidence for the emergence of pallial musculature during the pediveliger stage, including retractor and margin-parallel bundles. The formation of pallial muscles in mid-stage larvae was also observed in Lasaea adansonii, where both muscle types are formed by smooth fibers and spread over the mantle margin . Regarding pallial innervation, the mantle of N. nodosus seems to become assisted by the peripheral nervous system only by the pediveliger stage, when serotonin-like projections extend into the mantle, forming a nerve that runs parallel to the margin. Besides the possible presence of neuronal cells adjacent to the mantle margin , a serotonin-like nerve was detected running along the mantle in Mytilus edulis pediveligers, and catecholaminergic cells in Placopecten magellanicus .
Postmetamorphic mantle margin anatomy
The mantle of adult bivalves has been extensively studied, mainly regarding aspects of shell secretion and siphon anatomy (e.g. [7, 12, 32, 69, 70]). The bivalve mantle epithelium generally comprises cuboidal to columnar cells, with numerous secretory cells spread across both surfaces. The mantle lobes may exhibit free margins partially united in specific regions, as commonly observed in several protobranch and pteriomorphian groups, as well as different degrees of marginal fusion, including a variety of siphons, such as those present in infaunal bivalves [6, 16, 62]. In the auricular region of Nodipecten nodosus, the inner folds from both lobes are dorsally fused, characterizing a very restricted fusion in contrast to the wide free ventral margins. In general, reduced or entire absence of fusion are common features in the mantle margin of epifaunal bivalves from the Pteriomorphia clade .
While the outer mantle fold remains unaltered and covered by the periostracum after metamorphosis, the inner fold of Nodipecten nodosus becomes greatly pronounced into the typical curtain-like fold (“velum”) observed in grown scallops . In this respect, “pallial curtain” is the term applied to the hypertrophied marginal projection of the inner fold that controls the passage of water into and out of the pallial cavity . Pallial curtains are also found in Ostreidae and Pteridae, where the inner mantle fold is enlarged .
As demonstrated herein, the middle fold emerges after metamorphosis, giving rise to tentacles and eyes in Nodipecten nodosus and establishing the three-folded pattern. The typical functions concerning the bivalve mantle fold are present in the Pectinidae, including a secretory outer mantle fold, a sensorial middle fold, and a muscular inner fold [6, 7]. The concentration of chemical, mechanical, and optical receptors in the middle mantle fold have stimulated extensive studies on their functional properties. The pallial eyes, remarkable by their complexity and debatable evolutionary significance, have been deeply studied [45, 47, 49, 73–75], and recent molecular investigations have cast some light on the origin and diversification of such organs [76, 77].
The late larval musculature of the mantle of Nodipecten nodosus seems to be preserved after metamorphosis. Notwithstanding, little is known about the anatomy of the bivalve mantle musculature shortly after metamorphosis. In Mytilus trossulus, the postlarval muscles of the mantle margin comprise margin-parallel bundles that emerge during the pediveliger stage . A rapid development of mantle retractor muscles was also observed after metamorphosis in pinnids , but it is possible that mantle muscles were present in late larvae that were overlooked due to methodological limitations. The emergence of the pallial musculature in N. nodosus pediveligers indicates the possibility of effective retraction of the mantle margin into the mantle cavity, which has already been suggested for Ostrea edulis .
In Nodipecten nodosus, the inner fold is the most muscular region of the mantle margin, being composed mainly of numerous margin-parallel bundles of striated fibers as well as mantle retractors. In contrast, striated muscles are not present in mussels and other bivalves after metamorphosis, resulting in an entire muscular system organized by smooth myofibers .
The innervation of the mantle margin in adult scallops is relatively well-known for some species, the studies covering several anatomical and physiological topics [44, 71, 73, 74, 80]. Nevertheless, little information is available on its development. The circumpallial nerve is the most significant nerve in the scallop pallial margin. It runs parallel alongside its entire extension and is responsible for the innervation of the pallial folds and organs . The anterior dorsal portion of the nerve is formed by fibers from the cerebral-ganglia, while the ventral and posterior regions of the mantle margin are innervated by long fibers from the visceral ganglion [73, 81]. Strong serotonergic immunoreactivity was detected in the circumpallial nerve of juvenile Nodipecten nodosus, as well as in projections towards mantle organs. Considering that these characteristics were also detected in the nervous system of pediveligers, it seems reasonable to conclude that the nerve along the larval mantle margin is, in fact, the circumpallial nerve (formed, therefore, even before marginal folding). The development of this specific nerve along the mantle margin may contribute to the innervation of this region, allowing for sensory functions and muscular control. Serotonin is known to play a vital role in regulation of cilia activity in bivalve and other molluscan larvae [82–84] . Such information is relevant to discuss hypotheses of the early emergence and subsequent development of neuronal activity in the pallial margin. The presence of a neuropil and a cortex in the circumpallial nerve is regarded by some authors as an anatomical evidence for the crucial role of this nerve in the mantle margin, which was even considered a marginal ganglion . Finally, larval development of mantle muscles and innervation demonstrate how anatomical organization anticipates fold establishment during mantle margin morphogenesis. After metamorphosis, further modifications in both systems are deeply associated with specialization of the mantle folds and their associated structures.
Shell secretion is generally regarded to be the main function of the mantle, which exhibits different secretory cells on the outer epithelium adjacent to the outer fold (e.g. [9, 85–87]. Biomineralization has been extensively studied in bivalve molluscs, including mechanisms of calcification and shell microstructure (e.g. [8, 88]). During development, shell formation is initiated at the late trochophore stage, when mantle gland cells are involved in secretion of the larval prodissoconch I and II, and subsequently, the postmetamorphic dissoconch [32, 33]. Apart from shell secretion, functional studies on the larval mantle margin are very scarce, although some efforts have been made to elucidate the roles of chitin secretion in bivalve larvae . PAS-reactive vesicles and acidic mucopolysaccharide-protein complex content were detected in cells from the outer and inner mantle folds of Ostrea edulis pediveligers . Although the histochemical techniques applied to Nodipecten nodosus have provided no evidence for such secretory roles at least until metamorphosis, secretory vesicles were detected in pediveligers by transmission electron microscopy studies. Further work is still necessary to investigate the developmental origin and possible changes in mantle secretory activity during development.
In juvenile and adults of Nodipecten nodosus, secretory cells are present in all three pallial folds, as well as in the remaining inner and outer mantle epithelia. The columnar cells of the outer surface of the outer fold gradually become the cuboidal epithelium observed on the inner surface of the same fold and in the rest of the mantle. Reduction of mantle epithelial thickness throughout the outer fold was reported for other bivalves, such as Mytilus edulis, Cardium edule, Nucula sulcata , and Gomphina veneriformis , and related to possible differences in secretory activities . Similar to the gland cells observed in the outer mantle fold of N. nodosus, positive reaction to PAS and Alcian Blue was found in secretory cells from the outer fold of Pinctada fulcata  and Cerastoderma edule , suggesting intense secretion of neutral and acidic mucopolyssacharides in this region. In the extra-pallial cavity of the outer mantle fold, the calcified shell is deposited in an organic matrix formed by proteins, mucopolyssacharides, glycoproteins, and lipids [9, 85, 92]. The importance of the organic matrix for shell formation may explain the role of the several mucous-secreting cells concentrated in the outer mantle epithelium observed in these bivalves. Additionally, it has also been suggested that the products of mucous cells in this region may be concerned with mantle lubrication [86, 92]. Mantle secretory cells of Pinctada margaritifera exhibit mucous or granular content in all three mantle folds, although the number of cells responding to PAS-Alcian blue decreases in the middle one , a condition also observed in C. edule . In contrast, numerous cells secreting acidic mucopolysaccharides were detected in the middle fold tentacles of N. nodosus. Although PAS-positive and Alcian Blue-positive gland cells are present in both middle and outer folds, we observed some differences within each group when applying hematoxylin to stain nuclei (different purple to bluish tones), suggesting that more than one type of mucous secretion is present.
In several bivalves, including scallops, the inner mantle fold and the inner pallial epithelia commonly display mucous cells, usually associated with ciliated cells [42, 94–96]. Cilia and mucocyte distribution on the inner mantle epidermis of Nodipecten nodosus are in accordance with previous observations on Placopecten magellanicus, where mantle cilia are suspected not to contribute much to the water flow or particle transportation inside the mantle cavity due to their sparse distribution . Furthermore, the high viscosity of the acidic mucopolysaccharides secretions in the scallop mantle epithelium, in addition to the absence of a specialized mantle ciliary transportation, suggest that this mucous secretion might facilitate the passage of water over the mantle surface [42, 97, 98].
Taken together, within an evolutionary context, several issues concerning the development and diversification of bivalve mantle folds remain unclear. The evolution of the bivalve mantle margin might have been associated with the emergence of new functions, new pallial structures, and fold specialization . Nevertheless, general hypotheses concerning both phylogenetic and developmental diversification of the bivalve mantle margin are still challenging. Although our current knowledge of mantle margin diversity is very fragmentary, the plasticity of this region is obvious . Future investigations on the bivalve mantle margin should aim at identifying and testing hypotheses of homologies for mantle folds and pallial structures across bivalve taxa. Our study provides a developmental background for large-scale comparative evodevo analyses into these issues. The mantle margin is a complex system, and its evolution should be regarded as an anatomical and developmental innovation which, under varying selective forces, has led to considerable diversification of form and function.
Our results support a previous hypothesis  concerning the origin of the middle mantle fold from the outer surface of the inner fold, and that an initially unfolded condition of the mantle margin may represent a common feature for at least some bivalve mollusks. In addition, our study highlights the importance of the larval period for mantle margin morphogenesis, during which the first outgrowth process (by which the outer and inner folds are formed) and the emergence of the pallial musculature and the innervation of the mantle occur. Consequently, both larval and postmetamorphic changes are crucial for the establishment of the later three-folded condition seen in juveniles and adult bivalves.
Specimens of Nodipecten nodosus at different developmental stages were obtained from the scallop farm Institute of Eco-Development from Baía da Ilha Grande (IED-BIG), Rio de Janeiro, Brazil. Veligers (about 100 μm length) correspond to larvae 2 weeks after fertilization, while pediveligers (around 150 μm length) were 3 weeks old and settled individuals (about 400 μm length) reached metamorphosis slightly before the fourth week. Juveniles (maximum of 4 mm length), around a few weeks after metamorphosis, and adult mature individuals (about 8 cm length) were obtained as well. The specimens were removed from artificial hatcheries and, in case of larvae, observed under the light microscope to check if they exhibited healthy morphology and behavior. Then, samples were anesthetized by gradual addition of drops of 7.5 % MgCl2 for 2 h prior to fixation.
Specimens were fixed for 3 h at 4 °C in a modified Karnovsky solution (2 % paraformaldehyde + 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4 and 1 Osm adjusted with sucrose ). Larvae, postlarvae and juveniles were decalcified for 12 h at room temperature in 3 % ascorbic acid in distilled water, while adult specimens were dissected after anesthesia to remove fragments from the pallial margin. Then, specimens were dehydrated in a graded ethanol series and embedded in glycol-methacrylate resin (Leica Historesin Kit). Serial sections of 2–3 μm were produced on a Leica RM2255 microtome (Leica, Wetzlar, Germany) and stained with hematoxylin and eosin (HE) or toluidine blue and basic fuchsin (TB). In order to evidence possible secretory cells in the mantle, histochemical methods were applied using periodic acid-Schiff stain (PAS) and Alcian blue (AB) for neutral and acid mucopolysaccharide staining, respectively. Digital images were captured using a Nikon eclipse 80i microscope equipped with a Nikon DS-Ri1 camera (Nikon Instech Co. Ltd, Kawasaki, Japan).
Scanning and transmission electron microscopy
For scanning electron microscopy (SEM), samples were fixed in modified Karnovsky solution. Post-fixation was performed for 30 min in 1 % OsO4 in buffer solution (sodium cacodylate buffer at pH 7.4), followed by 15 min in 1 % tannic acid in buffer solution and additional 15 min in fresh solution of 1 % OsO4 at 4 °C. Then, specimens were decalcified as previously described for histological procedures and dehydrated in a graded ethanol series. Samples were critical point dried using CO2 as intermediate in a Balzers CPD 030 (Electron Microscopy Sciences, EUA), mounted on stubs, coated with gold in a Balzers SCD 050 sputter coater (Electron Microscopy Sciences, EUA), and observed in a Zeiss DSM 940 (Carl Zeiss, Oberkochen, Germany). For transmission electron microscopy (TEM), post-fixation was performed for 1 h in 1 % OsO4 in buffer solution. Larval samples were embedded in Epoxy resin; ultrathin sections (50–70 nm) were cut using a Leica Ultracut UCT microtome (Leica, Illinois, USA), mounted on copper slot-grids, contrasted with uranyl acetate and lead citrate, and analyzed using a Zeiss EM 900 electron microscope (Carl Zeiss, Oberkochen, Germany).
Immunocytochemistry, confocal laser scanning microscopy and 3D reconstruction
Specimens were fixed in 4 % paraformaldehyde in 0.1 M phosphate buffered (PB) for 1 h, followed by four rinses with buffer solution. Until further preparation, all samples were stored in 0.1 M PB containing 0.1 % NaN3 at 4 °C. Prior to staining procedures, larval and postmetamorphic individuals were decalcified in 0.05 M EGTA for 1 h. Juvenile specimens were dissected in order to remove small fragments of the mantle margin for confocal laser scanning microscopy (CLSM).
For F-actin staining, specimens were permeabilized in PB containing 2 % Triton-X 100 (PBT) overnight and then incubated in a 1:40 dilution of Alexa Fluor 488 phalloidin (Molecular Probes) in PBT for 24 h at room temperature in the dark. For neuronal staining, larval specimens were incubated in 6 % normal goat serum in PBT (block-PBT) overnight at room temperature. Subsequently, primary antibodies (e.g., anti-serotonin raised in rabbit and anti-α-tubulin raised in mouse) were applied at a concentration of 1:400 in block-PBT for 24 h. Then, specimens were rinsed several times in block-PBT prior to application of a secondary fluorochrome-conjugated antibody (goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Flour 633, Molecular Probes) in block-PBT at a concentration of 1:200 for 24 h in the dark. Nuclei were stained by adding a 1 μl drop of 4′, 6-diamidino-2-phenylindole (DAPI, Invitrogen) in conjunction with secondary antibody or phalloidin incubation. Then, all samples were washed three times in PBS for about 30 min and mounted in Fluoromount G (Southern-Biotech, Birmingham, EUA) on standard microscope slides which were stored in the freezer prior to analysis. Analysis and image acquisition were performed on a Leica TCS SP5 II confocal laser scanning microscope equipped with the software Leica Application Suite Advanced Fluorescence (LAS AF), Version 2.6.0 (Leica Microsystems, Wetzlar, Germany). Confocal image stacks were recorded with 0.3 μm step size along the z-axis and digitally merged as maximum intensity projections. 3D reconstructions were created from selected confocal stacks using the imaging software Imaris, Version 4.1 (Bitplane, Zürich, Switzerland).
Confocal laser scanning microscopy
Phosphate buffer solution containing Triton-X 100 and 6 % normal goat serum
Hematoxylin and eosin
Periodic acid-Schiff stain
Phosphate buffer solution
Phosphate buffer solution containing Triton-X 100
Scanning electron microscopy
Toluidine blue and basic fuchsin
Transmission electron microscopy
Carter JG, Harries PJ, Malchus N, Sartori AF, Anderson LC, Bieler R, et al. Illustrated Glossry of the Bivalvia. Treatise Online. 2012;48:1–209.
Stasek CR, Mcwilliams WR. The comparative morphology and evolution of the Molluscan Mande Edge. Veliger. 1973;16:1–19.
Steiner G. Observations on the anatomy of the scaphopod mantle, and the description of a new family, the Fustiariidae. Am Malacol Bull. 1991;9:1–20.
Lemche H, Wingstrand K. The anatomy of Neopilina galatheae, 1957 (Molllusca, Tryblidiacea). Galathea Rep. 1959;3:9–71.
Westermann B, Schmidtberg H, Beuerlein K. Functional morphology of the mantle of Nautilus pompilius (Mollusca, Cephalopoda). J Morphol. 2005;264(March):277–85.
Yonge CM. Mantle fusion in the Lamellibranchia. Pubbl Stn Zool di Napoli. 1957;29:151–71.
Yonge CM. Symmetries and the role of the mantle margins in the Bivalve Mollusca. Natl Widl. 1983;16:1–10.
Taylor JD. The structural evolution of the bivalve shell. Paleontology. 1973;16:519–34.
Wilbur K, Saleuddin A. Shell formation. In: Wilbur KM, editor. The Mollusca, v. 4. New York: Academic; 1983. p. 235–87.
Ansell AD. The functional morphology of the British species of Veneracea (Eulamellibranchis). J Mar Biol Assoc United Kingdom. 1961;41:489–515.
Hillman RE, Shuster CNJ. A comment on the origin of the fourth fold in the Mantle of the Quahog, Mercenaria mercenaria. Chesap Sci. 1966;7:112–3.
Sartori AF, Mikkelsen PM: Siphonal Structure in the Veneridae (Bivalvia : Heterodonta) with an assessment of its phylogenetic application and a review of venerids of the gulf of Thailand. Raffles Bull Zool. 2008:103–25.
Passos FD, Domaneschi O. Biologia e anatomia funcional de Donax gemmula Morrison 674 (Bivalvia, Donacidae) do litoral de São Paulo, Brasil. Rev Bras Zool. 2004;21:1017–32.
Waller TR. Scanning electron microscopy of shell and mantle in the order Arcoida (Mollusca: Bivalvia). Smithson Contrib Zool. 1980:1–58.
Morton B, Peharda M. The biology and functional morphology of Arca noae (Bivalvia: Arcidae) from the Adriatic Sea, Croatia, with a discussion on the evolution of the bivalve mantle margin. Acta Zool. 2008;89(January):19–28.
Yonge CM. Formation of Siphons in Lamellibranchia. Nature. 1948;161:198–9.
Yonge CM. Mantle margins with a revision of siphonal types in the Bivalvia. J Molluscan Stud. 1982;48:102–3.
Beedham GE, Owen G. The mantle and shell of Solemya parkinsoni (Protobranchia: Bivalvia). Proc Zool Soc London. 1965;145:405–30.
Morton B, Scott PJB. Morphological and functional specializations of the shell, musculature and pallial glands in the Lithophaginae (Mollusca: Bivalvia). J Zool. 1980;192:179–203.
Morton B. The mantle margin and radial mantle glands of Entodesma saxicola and E. inflata (Bivalvia: Anomalodesmata: Lyonsiidae). J Molluscan Stud. 1987;53:139–51.
Gilmour T. The defensive adaptations of Lima hians (Mollusca, Bivalvia). J Mar Biol Assoc United Kingdom. 1967;47:209–21.
Morton B. Dymantic display in Galeomma polita Deshayes (Bivalvia: Leptonacea). J Conchol. 1973;28:365–9.
Moir AJ. Ultrastructural studies on the ciliated receptors of the long tentacles of the giant scallop, Placopecten magellanicus (gmelin). Cell Tissue Res. 1977;184:367–80.
Morton B. The evolution of eyes in the Bivalvia: New Insights*. Am Malacol Bull. 2008;26(July 2007):35–45.
Serb JM, Eernisse DJ. Charting evolution’s trajectory: using molluscan eye diversity to understand parallel and convergent evolution. Evol Educ Outreach. 2008;1:439–47.
Cragg SM. Chapter 2 development, physiology, behaviour and ecology of scallop larvae. Dev Aquac Fish Sci. 2006;35:45–122.
Wanninger A, Koop D, Moshel-Lynch S, Degnan BM. Molluscan evolutionary development. In: Ponder W, Lindberg D, editors. Phylogeny and evolution of the Mollusca. California: University of California Press; 2008. p. 427–46.
Cole H a. The fate of the larval organs in the metamorphosis of Ostrea edulis. J Mar Biol Assoc United Kingdom. 1938;22:469–84.
Ansell AD. The functional morphology of the larva, and the post-larval development of Venus striatula (da Costa). J Mar Biol Assoc United Kingdom. 1962;42:419–43.
Hodgson C, Burke RD. Development and larval morphology of the spiny scallop, Chlamys hastata. Biol Bull (Woods Hole, MA). 1988;174:303–18.
Mouëza M, Gros O, Frenkiel L. Embryonic, larval and post larval development of the tropical clam, Anomalo Cardiabrasiliana (Bivalvia, Veneridae). J Nolluscan Stud. 1999:73–88.
Carriker M. Embryogenesis and organogenesis of veligers and early juveniles. In: Kraeuter J, Castagna M, editors. Biology of the hard clam. Amsterdam: Elsevier Ltd; 2001. p. 77–116.
Weiss IM, Tuross N, Addadi L, Weiner S. Mollusc larval shell formation: Amorphous calcium carbonate is a precursor phase for aragonite. J Exp Zool. 2002;293(May):478–91.
Cannuel R, Beninger PG, McCombie H, Boudry P. Gill Development and its functional and evolutionary implications in the blue mussel Mytilus edulis (Bivalvia: Mytilidae). Biol Bull. 2009;217(October):173–88.
Cranfield HJ. Observations on the morphology of the mantle folds of the pediveliger of Ostrea edulis L. and their function during settlement. Marine Biological Association of the United Kingdom, Journal 54(1):1–12, 2 pls. J Mar Biol Assoc United Kingdom. 1974;54:1–12.
Raven C. Morphogenesis: the analysis of molluscan development. New York: Pergamon Press; 1958.
Waller TR. Functional morphology and development of Veliger Larvae of the European Oyster, Ostrea edulis Linne. Smithson Contrib Zool. 1981;328:1–80.
Moor B. Organogenesis. In: Wilbur K, editor. The Mollusca, v. 3. New York: Academic; 1983. p. 123–77.
Carriker M. Functional significance of the pediveliger in bivalve development. In: Morton B, editor. The Bivalvia: Proceedings of a Memorial Symposium in honour of Sir Charles Maurice Yonge (1899–1986) at the 9th International Malacological Congress, 1986, Edinburgh, Scotland, UK. Hong Kong: Hong Kong University Press; 1990. p. 267–82.
Cragg SM. The phylogenetic significance of some anatomical features of bivalve veliger larvae. In: Taylor JD, editor. Origin and evolutionary radiation of the Mollusca. Oxford: Oxford University Press; 1996. p. 371–80.
Alejandrino A, Puslednik L, Serb JM. Convergent and parallel evolution in life habit of the scallops (Bivalvia: Pectinidae). BMC Evol Biol. 2011;11:164.
Beninger PG, Pennec M Le. Structure and Function in Scallops. In: Shumway SE, Parsons GJ, editors. Scallops: biology, ecology and aquaculture. Amsterdam: Elsevier B.V.; 2006. p. 123–227.
Waller TR. New Phylogenies of the Pectinidae (MoUusca : Bivalvia): reconciling morphological and molecular approaches. In: Scallops: biology, ecology and aquaculture. 2008. p. 1991–1.
Wilkens LA. Neurobiology and behavior of the scallop. In: Shumway SE, Parsons GJ, editors. Scallops: biology, ecology and aquaculture. Elsevier B.V.; 2006. p. 317–56.
Dakin WJ. The Eye of Pecten. Q J Microsc Sci. 1910;55:49–112.
Land MF. Image formation by a concave reflector in the eye of the scallop, Pecten maximus. J Physiol. 1965;179:138–53.
Morton B. The function of pallial eyes within the Pectinidae, with a description of those present in Patinopecten yessoensis. Geol Soc London Spec Publ. 2001;177:247–55.
Malkowsky Y, Jochum A. Three-dimensional reconstructions of pallial eyes in Pectinidae (Mollusca: Bivalvia). Acta Zool. 1871;2014:1–7.
Audino JA, Marian JEAR, Wanninger A, Lopes SGBC: Development of the pallial eye in Nodipecten nodosus (Mollusca : Bivalvia): insights into early visual performance in scallops. Zoomorphology. 2015. doi:10.1007/s00435-015-0265-8.
Buddenbrock W. Untersuchungen über die Schwimmbewegungen und die Statocysten der Gattung Pecten. Sitz Heidelb Akad Wiss. 1911;28:1–24.
Yonge CM. The evolution of the swimming habit in the Lamellibranchia. Mem Mus R Hist Nato Belg. 1936;3:77–100.
Moore JD, Trueman’ ER. Swimming of the Scallop, Chlamys Opercularzs (L.). J Exp Mar Biol Ecol. 1971;6:179–85.
Elston R. Functional anatomy, histology and ultrastructure of the soft tissues of the larval American oyster, Crassostrea virginica. Proc Natl Shellfish Assoc. 1980;70:65–93.
Creek GA. The development of the lamellibranch Cardium edule L. Proc Zool Soc London. 1960;135:243–60.
Drew GA. The life history of Nucula delphinodonta (Mighels). Q J Microsc Sci. 1901;44:313–91.
Altnöder A, Haszprunar G. Larval morphology of the brooding clam Lasaea adansonii (Gmelin, 1791) (Bivalvia, Heterodonta, Galeommatoidea). J Morphol. 2008;269(April):762–74.
Bellolio G, Lohrmman K, Dupré E. Larval morphology of the scallop Argopecten purpuratus as revealed by scanning electron microscopy. Veliger. 1993;36:322–42.
Silberfeld T, Gros O. Embryonic development of the tropical bivalve Tivela mactroides (Born, 1778) (Veneridae: Subfamily Meretricinae): A SEM study. Cah Biol Mar. 2006;47(October 2005):243–51.
Smith KK. Heterochrony revisited: the evolution of developmental sequences. Biol J Linn Soc. 2001;73:169–86.
Mcnamara KJ. Heterochrony: the evolution of development. Evol Educ Outreach. 2012;5:203–18.
Yonge CM. On the primitive significance of the byssus in the Bivalvia and its effects in evolution. J Mar Biol Assoc United Kingdom. 1962;42:113–25.
Stanley SM. Functional morphology and evolution of byssally attached bivalve mollusks. J Paleontol. 1972;46:165–212.
Morton B. Functional morphology of Bathyarca pectunculoides (Bivalvia: Arcacea) from a deep Norwegian fjord with a discussion of the mantle margin in the Arcoida. Sarsia. 1982;67:269–82.
Sastry AN. The development and external post-larval stages of the bay scallop, aequipecten irradians concentricus say, reared in the laboratory. Bull Mar Sci. 1965;15:417–35.
Bayne BL. Some morphological changes that occur at the metamorphosis of the larvae of Mytilus edulis. In: Crisp DJ, editor. Fourth European Marine Biology Symposium. Cambridge: Cambridge University Press; 1971. p. 259–80.
Allen JA. On the functional morphology of Pinna and Atrina larvae (Bivalvia: Pinnidae) from the Atlantic. J Mar Biol Assoc United Kingdom. 2010;91:1–7.
Allen JA. The development of Pandora inaequivalvis (Linne). J Embryol Exp Morphol. 1961;9(June):252–68.
Croll RP, Jackson DL, Voronezhskaya EE. Catecholamine-containing cells in larval and postlarval bivalve molluscs. Biol Bull. 1997;193(January):116–24.
Kellogg JL. A contribution to our knowledge of the morphology of Lamellibranchiate Mollusks. Washington: GPO; 1892.
Vitonis JEVV, Zaniratto CP, Machado FM, Passos FD. Comparative studies on the histology and ultrastructure of the siphons of two species of Tellinidae (Mollusca: Bivalvia) from Brazil. Zool. 2012;29:219–26.
Drew GA. The habits, anatomy and embryology of the giant scallop (Pecten Tenuicostatus Mighels). Maine: University of Maine Studies; 1906.
Nelson TC. The feeding mechanism of the oyster. I. On the pallium and the branchial chambers of Ostrea virginica, O. edulis and O. angulata, with comparisons with other species of the genus. J Morphol. 1938;63:1–61.
Dakin WJ. The Anatomy and Phylogeny of Spondylus, with a Particular Reference to the Lamellibranch Nervous System. Proc R Soc London Ser B. 1928;103:337–54.
Ciocco NF. Anatomia de la “vieira tehuelche”, Aequipecten tehuelchus (d’Orbigny, 1846) (=Chlamys tehuelcha). IV. Sistema nervoso y estructuras sensoriales (Bivalvia, Pectinidae). Rev Biol Mar y Oceonografía. 1998;33:25–42.
Speiser DI, Johnsen S. Comparative Morphology of the Concave Mirror Eyes of Scallops (Pectinoidea)*. Am Malacol Bull. 2008;26(July 2007):27–33.
Pairett AN, Serb JM. De Novo Assembly and Characterization of Two Transcriptomes Reveal Multiple Light-Mediated Functions in the Scallop Eye (Bivalvia: Pectinidae). PLoS One. 2013;8.
Serb JM, Porath-Krause AJ, Pairett AN. Uncovering a gene duplication of the photoreceptive protein, opsin, in scallops (bivalvia: Pectinidae). Integr Comp Biol. 2013;53:68–77.
Dyachuk V, Odintsova N. Development of the larval muscle system in the mussel Mytilus trossulus (Mollusca, Bivalvia): Original Article. Dev Growth Differ. 2009;51:69–79.
Dyachuk V, Wanninger A, Voronezhskaya EE. Innervation of bivalve larval catch muscles by serotonergic and FMRFamidergic neurons. Acta Biol Hung. 2012;63:221–9.
Spagnolia T, Wilkens LA. Neurobiology of the scallop. II. Structure of the parietovisceral ganglion lateral lobes in relation to afferent projections from the mantle eyes. Mar Behav Physiol. 1983;10:23–55.
Drew GA. The Circulatory and Nervous Systems of the Giant Scallop (Pecten Tenuicostatus, Mighels), with Remarks on the Possible Ancestry of the Lamellibranchiata, and on a Method for Making Series of Anatomical Drawings. Biol Bull. 1907;12:225–58.
Gosselin RE. The cilioexcitatory activity of serotonin. J Cell Physiol. 1961;58:17–25.
Beiras R, Widdows J. Effect of the neurotransmitters dopamine, serotonin and norepinephrine on the ciliary activity of mussel (Mytilus edulis) larvae. Mar Biol. 1995;122:597–603.
Kuang S, Goldberg JI. Laser ablation reveals regulation of ciliary activity by serotonergic neurons in molluscan embryos. J Neurobiol. 2001;47:1–15.
Wada K. Studies on the mineralization of the calcified tissue in molluscs. VII. Histological and histochemical studies of organic matrices in shells. Bull Natl Pearl Res Lab. 1964;9:1078–86.
Timmermans LPM. Studies on shell formation in molluscs. Neth J Zool. 1969;19:417–523.
Furuhashi T, Schwarzinger C, Miksik I, Smrz M, Beran A. Molluscan shell evolution with review of shell calcification hypothesis. Comp Biochem Physiol B Biochem Mol Biol. 2009;154:351–71.
Addadi L, Joester D, Nudelman F, Weiner S. Mollusk shell formation: a source of new concepts for understanding biomineralization processes. Chem Euro J. 2006;12:980–7.
Weiss IM, Schönitzer V. The distribution of chitin in larval shells of the bivalve mollusk Mytilus galloprovincialis. J Struct Biol. 2006;153:264–77.
Bubel A. An electron-microscope investigation of the cells lining the outer surface of the mantle in some marine molluscs. Mar Biol. 1973;21:245–55.
Lee JS, Joo JY, Park JJ. Histology and Ultrastructure of the Mantle Epidermis of the Equilateral Venus, Gomphina Veneriformis (Bivalvia: Veneridae). J Shellfish Res. 2007;26:413–21.
Beedham GE. Observations on the mantle of the Lamellibranchia. Q J Microsc Sci. 1958;99(June):181–97.
Fang Z, Feng Q, Chi Y, Xie L, Zhang R. Investigation of cell proliferation and differentiation in the mantle of Pinctada fucata (Bivalve, Mollusca). Mar Biol. 2008;153:745–54.
Richardson CA, Runham NW, Crisp DJ. A histological and ultrastructural study of the cells of the mantle edge of a marine bivalve, Cerastoderma edule. Tissue Cell. 1981;13:715–30.
Jabbour-Zahab R, Chagot D. Mantle histology, histochemistry and ultrastructure of the pearl oyster Pinctada margaritifera (L.). Aquat Living Resour. 1992;5:287–98.
Beninger PG, Veniot A, Poussart Y. Principles of pseudofeces rejection on the bivalve mantle: integration in particle processing. Mar Ecol Prog Ser. 1999;178:259–69.
Beninger PG, Lynn JW, Dietz TH, Silverman H. Mucociliary transport in living tissue : the two-layer in the Mussel Mytilus edulis L. Biol Bull. 1997;193:4–7.
Beninger PG, St-Jean SD. The role of mucus in particle processing by suspension-feeding marine bivalves: Unifying principles. Mar Biol. 1997;129:389–97.
Marian JEAR. Spermatophoric reaction reappraised: Novel insights into the functioning of the loliginid spermatophore based on Doryteuthis plei (Mollusca: Cephalopoda). J Morphol. 2012;273:248–78.
The authors acknowledge funding provided by FAPESP (graduate fellowship and research funding; 2012/11708-1). This study is part of the first author’s Master’s dissertation through the Graduate Program in Zoology of the “Departamento de Zoologia−IB-USP”. The authors thank also the following laboratories and institutions, which provided the necessary facilities for the development of this study: Centro de Biologia Marinha da USP (logistic support for animal collection and maintenance), Laboratório de Biologia Celular, IBUSP (electron microscopy facilities). Dr Alvaro E. Migotto provided assistance during in vivo studies, and Dr. André C. Morandini provided support during image acquisition from histological data. Dr. Alen Kristof, Dr. Martin Fritsch, Dr. Thomas Schwaha, and M. Sc. Emanuel Redl provided invaluable assistance in confocal microscopy and 3D reconstructions. The authors also thank two anonymous reviewers for valuable comments that helped to improve the manuscript.
The authors declare that they have no competing interests.
JA generated all data, analyzed the results, and drafted the manuscript. JM participated in light and electron microscopy procedures and analysis. AW contributed to confocal microscopy analysis and supervised research in Vienna. SL supervised research and contributed to data interpretation. All authors read and approved the final version of the manuscript.