Developmental Dynamics of the Larval Muscle System of Bay Scallop (Argopecten irradians)

SUN Xiujun , LIU Zhihong , ZHOU Liqing , WU Biao , YANG Aiguo , ,and TIAN Jiteng

1) Key Laboratory of Sustainable Development of Marine Fisheries of Ministry of Agriculture and Rural Affairs,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China

2) Function Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology, Qingdao 266071, China

Abstract Though the larval development of bivalves has been extensively studied for commercial purposes, the dynamic development of larval muscle system remains largely unknown. In this study, we characterized the larval muscle system at different developmental stages (D-shaped veligers, umbo veligers and spats) in the bay scallop (Argopecten irradians) by phalloidin staining and under a confocal microscopy. The functional muscles are initially established at the early stage of veligers, which have four pairs of velar retractors and one anterior adductor. At the veliger stage, the velum and posterior retractor muscles are functionally important for velar contractility but undergo an irreversible shrink until they disappear at the end of the larval stage. During metamorphosis, three crucial modifications take place in the larval muscle system. The metamorphosis process involves the gradual degeneration of velum retractors, mantle margin development from an unfolded to a three-fold state, and remodeling of the adductor muscle system from dimyarian (two adductors) to monomyarian condition (one adductor) as in juveniles/adults. All retractor muscles are composed of striated muscle, but both anterior and posterior adductors have smooth and striated components. These findings highlight that the morphological changes at different stages are typical features of myogenesis in scallops. The present knowledge on the developmental dynamics of myogenesis in the bay scallop will not only improve our understanding of phenotypic diversity of larval myoanatomy in bivalves, but also provide useful information on the larval culture in hatcheries.

Key words myogenesis; retractor muscle; metamorphosis; adductor muscle

1 Introduction

Morphological investigations can help us to understand the formation and function of animal organs and the phenotypic diversity of early development in invertebrates(Wanninger, 2015). For bivalve mollusks, studies on larval development have focused on species with commercial potential from shallow temperate to tropical waters(Gosling, 2015; Cragg, 2016). The typical stages of development in bivalves mainly include blastula, gastrula, trochophore, veligers (D-shaped veligers, umbo veligers, and pediveligers), and post-metamorphic spats. The early veligers are also called as D-shaped veligers, when the larvae have the straight hinge line and show D-shaped forms.As veligers grow, the straight hinge line is gradually obscured by the developing umbos, which are usually named as umbo veligers (Slater, 2005; Schejter et al., 2010). The final stage of veligers is termed as pediveligers, which have both of velum retractors and developed foot, showing temporary swimming and crawling (Cragg and Nott, 1977;Hodgson and Burke, 1988). Finally, pediveligers metamorphose to benthic spats, which possess a ctenolium and can produce a byssus to attach to a substrate (Waller, 2006).Usually the early stage of veligers is often referred as Dshaped veligers, and the late veligers represent both the umbo veligers and pediveligers.

Larval musculature during development in bivalves plays a key role in larval swimming, feeding, and survival (Cragg,2016). Generally, no functional muscle is formed at the trochophore stage, but the rapid differentiation of larval musculature starts from the early veliger (Cragg, 1985). Bivalve veligers usually display a remarkably complex anatomy, which has shell, velum, musculature, digestive tract,nervous system, and foot (Haszprunar and Wanninger, 2000;Cragg, 2016). Rapid contraction of the velum in veliger larvae is supported by velar retractor muscles during larval swimming and feeding (Cragg, 1985). These retractor muscles degenerate before metamorphosis, and thus represent a transitory character during myogenesis of bivalves(Odintsova et al., 2007; Dyachuk and Odintsova, 2009;Audino et al., 2015a; Sun et al., 2019). However, these retractor muscles are highly variable in types, pairs, and insertion sites among bivalve species (Cragg, 1985; Altnöder and Haszprunar, 2008; Wurzinger-Mayer et al., 2014).Furthermore, although the posterior adductor appears after the formation of the anterior adductor in most cases, the timing of appearance is highly variable among different species at the veliger, pediveliger stage or even after metamorphosis (Cragg, 1985). During the metamorphosis process of bivalves, some irreversible morphogenetic changes are involved in this life stage transition, such as loss of the velum, greater development of foot organ, degeneration of the anterior adductor, and formation of mantle folds (Cragg, 2016). However, some changes of larval musculature during early development remain largely unknown.

Current knowledge of larval anatomy and ontogeny has contributed substantially to our understanding of larval development, but the previous data on bivalve myogenesis is handicapped by the insufficient technologies available in previous studies (Monk, 1928; Elston, 1980; Cragg,1985; Cragg, 1989). In contrast, substantial new information in this area has been gained with the assistance of advanced techniques, such as phalloidin staining and confocal microscopy (Wanninger, 2007; Wurzinger-Mayer et al.,2014; Audino et al., 2015a; Wanninger, 2015; Merkel et al.,2015). Recently, the developmental dynamics of myogenesis in Yesso scallop Patinopecten yessoensis and Pacific oyster Crassostrea gigas have been revealed by phalloidin staining and confocal microscopy (Li et al., 2019;Sun et al., 2019). However, the development process of the larval muscle system varies considerably from species to species among bivalves. Knowledge on the muscular ground patterns and the evolution of larval muscular features is particularly scarce for the second-largest molluscan class, the Bivalvia.

In the present study, we characterized the larval muscle system at different developmental stages in the bay scallop Argopecten irradians by phalloidin staining and confocal microscopy. The morphological changes of the larval musculature during early development were uncovered for this commercially important species, which demonstrated the typical features of myogenesis in scallops. The findings will greatly improve our understanding on dynamic development of the larval muscle system in scallops, and provide useful information to guide improvements in larval culture.

2 Material and Methods

2.1 Experimental Animals

Samples from different development stages (D-shaped veligers, umbo veligers and spats) of the bay scallop, Argopecten irradians, were collected from Haiyi Hatchery,Yantai, China. In the hatchery, larvae were cultured in sand-filtered sea water at 22℃ ± 2℃. During the culturing days, larvae were fed with a mixture of Isochrysis galbana and Platymonas helgolandica at a density of 3000–150000 cells mL-1. Two thirds of the culture water were changed every day. On days 3, 8 and 16 after fertilization(dpf), healthy specimens of D-shaped veligers, umbo veligers, and settled spats were visually checked under a light microscope, and then collected with a 48-μm mesh.Larvae were anesthetized by gradual addition of 7.5%MgCl2in 1.5 mL collection tubes prior to fixation.

2.2 Phalloidin Staining and Confocal Microscopy Observation

Larval and spat samples were subsequently fixed in 4%paraformaldehyde in 1×PBS at 4℃ for 2 h after the anesthetization. The samples were further rinsed three times with PBS and then stored in the PBS (including 0.1%NaN3) at 4℃. First, decalcification of D-shaped veligers,umbo veligers, and settled spats was performed using 0.05 mol L-1EGTA for 1 h at room temperature. Second,the samples were subsequently treated with PBST (PBS with 2% Triton-X 100) for at least 12 h. To investigate the dynamic development of muscle fibers, we selected phalloidin as a marker of filamentous cell to stain the muscle fibers at different development stages. The working solution of the phalloidin staining reagent was prepared using Alexa Fluor 488 phalloidin (Molecular Probes) with 1000×dilution in PBST. The resulting samples from the previous steps were immersed in the working solution and kept in dark for 24 h at room temperature. The samples were subsequently rinsed three times in PBS, and the remaining solution was removed by pipetting. The samples were mounted using 50 μL Fluoromount G (Thermo Fisher Scientific) on glass slides. The samples were gently covered with cover slips after short-time air drying to remove most of the residual liquid. All the samples were stored in the dark until they were used in microscopic observation and photography. The fluorescent images were obtained from a Leica TCS SP8 confocal laser scanning microscope. Light and confocal microscopic images of D-shaped veligers, umbo veligers and spats of the bay scallop were all collected in the present study. The image stacks were recorded with 0.7 μm step size along the z-axis. The confocal pictures were selected and analyzed by Image-Pro Plus 6.0. The schematic drawings for the umbo veligers and spats were performed using Microsoft Visio 2010.

3 Results and Discussion

3.1 Musculature in D-Shaped Veligers

Larval dispersal and feeding are assisted by rapid muscle contraction and shell closing (Cragg, 1985). A rapid differentiation of the larval musculature in bivalve larvae usually coincides with the secretion of the prodissoconch at the D-shaped veliger stage (Cragg, 2016). Larvae start active feeding and swimming with the help of the velum retractors and the adductor muscles. In this study, we also prove that the functional muscles are initially established at the D-shaped veliger stage, having four pairs of velar retractor muscles (vrm), and one anterior adductor muscle(aa; Fig.1). All the velar retractor muscles are exclusively composed of striated muscle fibers, whereas the anterior adductor muscle has both smooth and striated components. In contrast, the anterior adductor muscle is mainly related to shell opening and closure during swimming.During larval development, the branching of the retractor muscles becomes more and more profuse, but the arrangement of velar retractor muscles remains the same order as the D-shaped veligers until they are lost at metamorphosis.

Fig.1 Light and confocal microscopic images for major muscles in D-shaped veligers of the bay scallop, Argopecten irradians. A, light microscopic image of the D-shaped veligers; B, fluorescent image produced by phalloidin staining of the same specimens showing the anterior adductor (aa) and four velum retractor muscles (v1–v4). Scale bar = 25 μm.

The striated retractor muscles are responsible for rapid retraction and protraction of the velum into or out of the pallial cavity (Cragg, 1985). Knowledge on previous and present studies in bivalves emphasizes the functional roles of retractor muscles during larval swimming and feeding(Cragg, 1985; Audino et al., 2015a). In addition, this evidence suggests that initial diets for larval feeding during early ontogeny in bivalves should potentially meet a dietary requirement for muscle development (Milke et al.,2006).

3.2 Musculature in Umbo Veligers

The image of an umbo veliger illustrates its anterior,posterior, ventral and dorsal sides (Fig.2). Based on the phalloidin staining and confocal imaging, larval musculature in the umbo veliger displays a more developed muscle system compared to the D-shaped veligers. The new muscle system mainly includes velum retractor muscles(v1–v4), posterior retractor muscles (prm), anterior (aa) and posterior adductor muscles (pa), digestive smooth muscles(dmm), and mantle margin-parallel muscles (mmp).

The appearance of retractor muscles, including velum and posterior retractor muscles, is the most prominent feature in veligers (Fig.2B). The four velum retractor muscles attach to the hinge region of the shell and profoundly branch into the ventral portion of the velum. They form a pattern that is symmetrical with regard to the plane between the shell valves. The retractor muscle v1 attaches to the hinge region and extends into the anterior-ventral portion of the velum ((Fig.2B, Fig.3). In contrast, the retractor muscle v2 has extensively branched into the ventral position, which runs across the larval body to the ventral portion of the velum and overlaps with the retractor muscles v3 and v4 (Fig.3). The retractor muscle v3 is the major branch reaching to the anterior portion of the velum.The abundant fibers are also found in the retractor muscle v4, which spread all over the ventral-posterior velum (Fig.3).

Fig.2 Light and confocal microscopic images of the umbo veliger of the bay scallop, A. irradians. A, light microscopic image of the umbo veliger, with ventral on the top (vent), dorsal on the bottom (dors), anterior on the left (ant), and posterior on the right (post). B, larval musculature demonstrated by phalloidin staining for the same specimen. v1–v4, velar retractor muscles; prm, posterior retractor muscles; dmm, digestive smooth muscles; aa, anterior adductor muscle; pa, posterior adductor muscle; ft, foot; mmp, mantle margin-parallel muscle. Scale bar: 25 μm.

Fig.3 The schematic drawing for the umbo veliger of A.irradians. The cross stripes in blue background represent the striated muscles, while black dots in green background indicate the smooth muscles. vent, ventral; dors, dorsal;ant, anterior, post, posterior; v1–v4, velar retractor muscles;prm, posterior retractor muscles; dmm, digestive smooth muscles; aa, anterior adductor muscle; pa, posterior adductor muscle; ft, foot; mmp, mantle margin-parallel muscle.

Besides the four velum retractor muscles, two posterior retractor muscles run across the middle of larval body overlapping with v2 and v4 (Fig.2B). The schematic drawing for the umbo veliger of A. irradians is displayed in Fig.3.The posterior retractor muscles attach to the posterior body wall, lying dorsal and posterior to the velum. All the retractor muscles are composed of striated muscle fibers.Furthermore, the posterior adductor muscle has developed in a posterior-dorsal location at this stage. The posterior adductor possesses both of striated and smooth portions,which lie closely apposed to one another but are divided by a connective tissue sheet.

Smooth fibers appear to cling on the surface of the veliger larvae’s digestive mass, which suggests the development of early pediveliger (Fig.2B). The pediveliger stage represents the last larval stage of scallops where both velum and foot are present. Larvae grow and develop an extensible foot (ft) at this stage, which is composed of smooth muscle fibers. The crawling behavior becomes gradually common on available hard surfaces at this stage, although the swimming habit prevails. In addition, a strip of mantle marginparallel muscle is formed along the mantle marginal region and connects with the end of the velum retractor muscles on the left and right valves.

The velum retractor muscles in bivalves are thought to be responsible for the rapid retraction and protraction of the velum into or out of the pallial cavity, which will improve the chances of larval survival, e.g., avoiding predators or unfavorable water conditions (Cragg, 1985). However, the type and number of velar retractor muscles are highly variable among bivalve species. Generally, the crossstriated pattern of velar retractor muscles is observed in scallops (Cragg, 1985; Bellolio et al., 1993; Audino et al.,2015a; this study), oysters (Elston, 1980), and mussels(Odintsova et al., 2007; Dyachuk and Odintsova, 2009),but no striation is found in clams (Altnöder and Haszprunar, 2008). In addition, four pairs of velum retractor muscles in veligers are consistently found in all the studied scallops, such as Nodipecten nodosus, P. maximus and Argopecten purpuratus (Cragg, 1985; Bellolio et al., 1993;Audino et al., 2015a). However, three pairs of velum retractors are usually detected outside Pectinidae, such as oysters (Elston, 1980), mussels (Dyachuk and Odintsova,2009), and clams (Altnöder and Haszprunar, 2008). One of the possible explanations for these morphological distinctions in bivalves might be explained by some selective advantages of velum retraction and protraction during larval dispersal and feeding (Cragg, 1985; Audino et al.,2015a). The contraction of the posterior retractors is predicted to prevent the body fluids from forcing the posterior body wall out between the shell valves (Cragg, 1985).However, the evolutionary consequences of these muscles during larval development are not clear yet (Audino et al.,2015a).

Not surprisingly, the striated and smooth portions of larval adductors appear to share similar functions with adult adductor counterparts during body movement. The striated adductor muscle in scallop adults contracts very quickly, whereas the smooth catch adductor muscle contracts for long periods, keeping shells closed with little expenditure of energy (Chantler, 2016). Similarly, the striated portion of the adductor in veligers makes the shell shut quickly once the velum is retracted in response to predators, and the smooth portion subsequently takes over to keep the shell closed (Cragg, 1985). The sequences of muscle action are thought to have some selective advantages of velum retraction and protraction during larval dispersal and feeding to improve the chances to avoid unfavorable conditions. Furthermore, the appearance of the dimyarian (two adductors) condition in scallop larvae indicates the ancestral state of bivalves, as found in other bivalves, such as mussels, clams and shipworms (Altnöder and Haszprunar, 2008; Dyachuk and Odintsova, 2009; Wurzinger-Mayer et al., 2014).

Foot development prior to the presence of eye-spots is often used as the indicator for putting plastic collectors into the tanks for larvae to set on in hatcheries (Wang and Li, 2010). The unfolded mantle margin in A. irradians veligers is similar to that in the scallop Nodipecten nodosus, which exhibits a single projection beneath the shell margin with no distinct folds (Audino et al., 2015b). This evidence supports the previous hypothesis that an initially unfolded condition of the mantle margin may represent a common feature for bivalve molluscs (Morton and Peharda, 2008).

3.3 Musculature in Post-Metamorphic Spats

Metamorphosis of molluscan larvae results in great changes in larval anatomy and behavior. The process of metamorphosis involves changes in loss of some organs,greater development of foot, gill and mantle margin, and relocation of mouth and foot (Wanninger et al., 1999; Cragg,2016; Chantler, 2016). For instance, two main organs are lost at metamorphosis, including velar retractor muscles and the anterior adductor. The mouth moves from its postero-ventral larval location to the adult antero-dorsal position, whereas the foot becomes ventral rather than posterior, and the posterior adductor migrating to the centre of the valve. The gill filaments increase in length and number after metamorphosis, which are capable of suspension feeding (Hodgson and Burke, 1988). The shape of the foot becomes better developed and functional after metamorphosis (Gruffydd, 1975). The presence of cilia on the mantle region represents the precursor of the adult inner mantle fold (Sastry, 1965).

In the present study of A. irradians, we find three crucial modifications take place prior to the metamorphosis in the muscle system of the larval body, including the loss of the velum, the development of the mantle margin and the foot,and the remodeling of the adductor muscle system (Fig.4).The schematic drawing for the spat of A. irradians is displayed in Fig.5. The light and confocal images for 16-dayold spat musculature indicate that the retractor muscles (e.g.,velum retractor muscles and posterior retractor muscles)have completely vanished after metamorphosis of A. irradians (Fig.4B).

Fig.4 Light and confocal microscopic images of 16-day-old spats of the bay scallop, A. irradians. A, light microscopic image of spat; B, spat musculature demonstrated by phalloidin staining for the same specimen; C, the striated and smooth components of spat adductor muscle. vent, ventral; dors, dorsal; ant, anterior, post, posterior; pa, posterior adductor; ft,foot; mmp, mantle margin-parallel muscle; mrm, U-shaped mantle retractor muscle; frm, foot retractor muscle; st, striated muscle; sm, smooth muscle. Scale bar: 50 μm.

Fig.5 The schematic drawing of the spat of A. irradians.vent, ventral; dors, dorsal; ant, anterior, post, posterior; ft,foot; mmp, mantle margin-parallel muscle; mrm, U-shaped mantle retractor muscle; st, striated muscle; sm, smooth muscle.

Two more strips of mantle margin-parallel muscles (mmp)in the mantle margin appear along the inner side of mantle region (Fig.4B; Fig.5). The inner surface of each shell valve forms two folds at the valve rim, which is served as a bridge between the valves at the anterior or posterior regions close to the hinge region (Fig.4B; Fig.5). The mantle margin-parallel muscles (mmp) line the shells, and extend between the shell valves, having a brush border of short microvilli at the apical surface. Some of them are parallel to the edge, while other bundles are branching retractors that run towards the mantle margin (Fig.4B). Furthermore,U-shaped mantle retractor muscles (mrm) are evenly distributed along the mantle margin, running perpendicular to the mantle margin-parallel muscles (mmp; Fig.5). All of the three strips of mantle margin-parallel muscles are composed of smooth fibers, whereas the U-shaped mantle retractor muscles seem to have striated fibers according to the phalloidin staining (Fig.4B; Fig.5).

Moreover, another important change in the larval muscle system of bivalves after metamorphosis is the remodeling of the adductor muscle system from dimyarian (two adductors in veligers) to monomyarian condition (one adductor in juveniles/adults). For the posterior adductor in the umbo veligers, muscle bundles grow larger and thicker,while a remarkable shrinkage occurs in the anterior adductor until it disappears after metamorphosis. Finally, the posterior adductor appears as the most dominant feature as in the adult body of the animal.

Similarly, the transitory role of the velum and the retractor muscles is also observed in other bivalves, which sheds lights on the primitive condition in the Bivalvia(Cragg, 2016; Wurzinger-Mayer et al., 2014; Audino et al.,2015a). Together with the previous study on Nodipecten nodosus, the present investigations of A. irradians provide clear evidence for the emergence of pallial musculature in metamorphic larvae, including mantle retractor muscles and margin-parallel bundles (Audino et al., 2015a). These findings support the conclusion that the three-folded condition of mantle musculature is a typical characteristic in scallops, which includes a secretory outer mantle fold, a sensorial middle fold, and a muscular inner fold (Yonge,1983; Audino et al., 2015b). In contrast, the two-fold mantle margin is present in arks and oysters (Waller, 1980;Waller, 1981; Morton, 1982; Morton and Peharda, 2008).As suggested, heterochrony may be responsible for the significant anatomical and evolutionary changes in bivalves(Stanley, 1972). However, 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 ancestral functions (e.g., shell secretion), the growth of sensorial organs(e.g., tentacles and eyes), and development of neuronal activity (Audino et al., 2015b).

The development of two adductor muscles in a sequential manner is also found in other scallops, such as N.nodosus (Audino et al., 2015a) and P. maximus (Cragg,1985). Despite that the presence of one or two adductor muscles is a common feature in bivalves, muscle components of bivalve adductors are distinctive among species.The components of striated and non-striated fibers are both present in scallop adductors, while only non-striated,smooth fibers are found in non-swimming bivalves, such as mussels, clams and shipworms (Odintsova et al., 2007;Altnöder and Haszprunar, 2008; Wurzinger-Mayer et al.,2014). This might be partially explained by the evolutionary consequences of the swimming ability in adults.Most scallop species are active swimmers, and they can propel themselves by the valve clapping through the rapid contraction of the striated portion (Chantler, 2016). In contrast, most other species that do not belong to Pectinidae lose their swimming ability during metamorphosis, which may result in the disappearance of striated components in their bodies. For example, the mussel larvae have initially a striated pattern of muscle filaments, but it is then replaced by a smooth muscle pattern with a diffuse distribution of muscle proteins (Odintsova et al., 2010). In contrast to the mussel larvae, the musculature of mussel adults is simple and consists entirely of smooth muscle cells with an unusual structure of their thick filaments (Dyachuk and Odintsova, 2009). Moreover, the striated portion appears in the posterior adductor muscle of oyster C. gigas larvae, but it is consists of several muscle bundles rather than two parts in the scallop larvae (Li et al., 2019).

Myogenesis in bivalves seems to have a highly dynamic and potentially variable process, which is proved to be adaptive changes of life style from planktonic to benthic state (Odintsova et al., 2010; Li et al., 2019; Sun et al.,2019). The myogenesis during larval stages is consistently found in Yesso scallop P. yessoensis and the bay scallop A. irradians (this study), but it is slightly different from Pacific oyster C. gigas (Li et al., 2019; Sun et al., 2019).For instance, three pairs of velum retractors are found in the umbo veligers of Pacific oyster C. gigas, whereas four pairs of velum retractors are consistently detected in the scallops. Therefore, the morphological changes, including the development and loss of four velum retractors, sequential development of anterior and posterior adductor muscles,formation of mantle margin and foot organ, suggest the adaptive changes of myogenesis and its functional consequences for the musculature in scallops.

4 Conclusions

In conclusion, we report here the developmental dynamics of the larval muscle system in bay scallop (A. irradians) for the first time. The muscle system is well developed in the umbo veligers, but it appears the loss of velum retractor muscles, the greater development of mantle margin and foot, and the remodeling of adductor muscle system after metamorphosis. The crucial modifications of the larval muscle system after metamorphosis suggest the adaptive changes of the larval muscle system from planktonic stages to benthic life. These findings highlight that the adaptive changes in larval muscle system are the typical feature in scallops. The present knowledge on the developmental dynamics of myogenesis in bay scallop will not only improve our understanding of the phenotypic diversity of larval myoanatomy in bivalves, but also provide some useful information on larval culture in hatcheries.

Acknowledgements

We would like to thank Dr. Rihao Cong (Institute of Oceanology, Chinese Academy of Sciences) for his assistance in larval sampling in Haiyi Hatchery. This study was supported by research grants from the National Key R&D Program of China (No. 2018YFD0900104), the National Natural Science Foundation of China (No. 3160 2153), the Central Public-Interest Scientific Institution Basal Research Fund, YSFRI, CAFS (No. 20603022019 005), and Qingdao People’s Livelihood Science and Technology Project (No. 18-6-1-110-nsh).