Marine molluscs, such as oysters and scallops, secrete calcified shells as a supporting frame for their bodies and for the protection from predators (Zhang et al., 2012; Hendriks et al., 2015). The biosynthesis of the larval shells is originated from a peculiar group of ectodermal cells in the early developmental stages of embryos, which are called shell gland (Bevelander and Nakahara, 1969; Kniprath, 1979). When the ectodermal cells are sunken to construct the shell gland, the surrounding cells begin to generate the outmost matrix shell layer, the periostracum (Eyster and Morse, 1984). The cells of shell gland region are then turned over from inside to outside, transforming into the mantle epithelium of larvae (Kniprath, 1979; Bielefeld and Becker, 1991). During this process, the periostracum spans the whole shell field epithelial surface, and only then, the crystalline calcium carbonate appears for the first time (Eyster, 1986). The onset of shell mineralization occurs during the trochophore larval stage, which is usually about 20 h post fertilization (Kniprath, 1981; Nudelman et al., 2006), and the shell is called the prodissoconch I (PDI) (Weiss et al., 2002). The bivalve PDI is enlarged until the embryo is entirely enfolded and able to close its two valves. Transformation into the motile D-shape larvae then occurs. The D-shape larvae enlarges PDI to form prodissoconch II (PDII) which is fully calcified (Carriker and Palmer, 1979; Waller, 1981).

In the past few decades, these crucial processes have been severely inhibited by ocean acidification (OA) (Orr et al., 2005; Waldbusser and Salisbury, 2014). The oceans absorb the excess CO₂, and cause changes in oceanic chemistry, including increased levels of dissolved CO₂, a reduction in pH from pre-industrial levels of 8.2 to a projected 7.8 in 2100, and a reduction in the saturation state of biologically useful forms of CaCO₃, especially aragonite and calcite (Sabine et al., 2004; Kerr, 2010). Compared with the adults, molluscan larvae are especially sensitive to OA during the hours to days-long bottleneck when initial shell (PDI) is formed during embryogenesis (Waldbusser et al., 2013; Talmage and Gobler, 2009). During calcification of PDI, the calcification surfaces are in greater contact with ambient seawater than during the following shell stages (Waldbusser et al., 2014). Before PDI shell formation, the larvae mainly depend on the maternal energy from eggs for survival. Failure of forming complete shell before exhausting the maternal energy reserves would result in eventual mortality of larvae (Barton et al., 2012). Therefore, a better understanding of how OA suppresses the formation of calcified shell during larval development of oysters will not only contribute to illustrate the stress response patterns of marine calcifiers upon OA, but also provide new insights into the shellfish ecological aquaculture industry in a fast-changing environment.

Chitin synthase and chitinase, the crucial enzymes for the metabolism of chitin, have been proved to be important in shell biosynthesis of bivalve larvae because the calcified shell is deposited at early D-shape larvae stage to cover the chitin shell of trochophore larvae (Weiss and Schonitzer, 2006; Zhang et al., 2012). For example, a homologous gene for chitin synthetase was identified from mussel Mytilus galloprovincialis, which was expressed in the early embryo and the shell forming tissue of larvae (Weiss et al., 2006). It was speculated that chitin synthesis contributed via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types (Schonitzer and Weiss, 2007). Meanwhile, it was found that the chitinase of pearl oyster Pinctada fucata might possess key function in the biomineralization of shell (Li et al., 2017). Similar results were also reported in C. gigas that chitinase could regulate the shell formation in larvae under the modulation of dopamine (Liu et al., 2018). However, the correlation between OA and the expressions of chitinase and chitin synthetase has never been reported during the formation of calcified shell in oyster larvae.

In the present study, a chitinase (Cgchit4) and a chitin synthase (Cgchs1) were identified from the Pacific oyster C. gigas with the major objectives to (1) explore the alteration of their expression levels during larval development stages as well as under acidification treatments, (2) illustrate their involvements in the biosynthesis of calcified shell during larval development, and (3) reveal the underlying mechanisms of how acidified seawater influence the formation of oyster calcified shell.

As the exoskeletons for most molluscan species, shells have been extensively studied in both adults and larvae for their evolutionary and economic importance. The larval shell emerges early in embryogenesis of molluscs, and chitin biogenesis is crucial for this process (Peters, 1972). The ongoing CO₂-induced ocean acidification has been reported to severely threaten the shell formation of molluscan larvae and cause mass mortality (Kurihara et al., 2007; Miller et al., 2009; Kroeker et al., 2010; Talmage and Gobler, 2010; Parker et al., 2012). Previous studies have already demonstrated that the early development of bivalves including oyster C. gigas (Kurihara et al., 2007; Barros et al., 2013), blue mussel Mytilus edulis (Gazeau et al., 2010) and barnacle Amphibalanus amphitrite (McDonald et al., 2009) is negatively affected by acidified conditions. In the present study, a chitin synthase (Cgchs1) and a chitinase (Cgchit4) were identified from C. gigas. Their responses against acidification treatment during larval development stages were investigated to illustrate the impact of OA on the shell formation of oyster larvae by modulating the biogenesis of chitin.

Chitin synthase and chitinase were reported to be employed in many biological and physiological processes such as digestion (Mali et al., 2004), host defense (Boot et al., 1995, 2001), and most importantly, in biomineralization (Schonitzer and Weiss, 2007). In order to understand the modulation effect of chitin synthase and chitinase in shell formation, in the present study, a chitin synthase gene (Cgchs1) and a chitinase gene (Cgchit4) were identified from C. gigas. The length of Cgchs1 sequence was of 813 bp, encoding a putative peptide fragment of 271 amino acids with the theoretical molecular weight of 30.16 kDa. There was a Chitin_synth_2 domain in Cgchs1, which was the key domain to catalyze the chemical reaction (Sburlati and Cabib, 1986). Chitin synthases belong to the members of glycosyltransferases and consist of a transmembrane protein family with at least three transmembrane helices. The N- and C-terminal residues of chitin synthetase possibly differ even in the isoforms from the same species, but these two lower complexity motifs contain plenty of acidic or basic residues which are the main characteristics for various molluscan chitin synthase. In addition, the catalytic site of intracellular is highly conserved which possesses the function of catalytic chitin synthesis and plays a key role in the precisely controlled mineral deposition process of other mollusk as well as Pacific oyster C. gigas (Marin et al., 2008; Schonitzer et al., 2011). As for Cgchit4, the full length of cDNA sequence was of 2118 bp, encoding a putative polypeptide of 706 amino acids with the theoretical molecular weight of 80.34. A Glyco_18 domain was included in the Cgchit4, which was important for the hydrolysis of the glucosidic bond of carbohydrates (Renkema et al., 1995). Cgchs1 shared high sequence similarity (42–44%) with chitin synthase from other vertebrates and invertebrates. Phylogenic analysis also showed that Cgchs1 was close to chitin synthase from invertebrates such as B. glabrata, M. yessoensis (Bulawa, 1992; Luo et al., 2015). Cgchit4 exhibited high sequence similarity (41–52%) with chitinase from other vertebrates and invertebrates, and it was clustered with chitinases from invertebrates such as P. fucata and H. cumingii (Gao et al., 2014; Li et al., 2017). These results indicated that Cgchs1 and Cgchit4 might belong to the family of chitin synthase and chitinase in molluscs, respectively.

In order to investigate the possible function of Cgchs1 and Cgchit4 in shell formation of oyster larvae, their mRNA expression levels and localization were detected by quantitative real-time PCR and WMISH, and the larval morphological characteristics were observed by SEM. The expression level of Cgchs1 mRNA was extremely lower at D-shape larvae compared with that at trochophore and early D-shape larvae, while the expression level of Cgchit4 mRNA in early D-shape larvae was distinctly higher than that at trochophore and D-shape larvae. As shown in Figure 5, a uniformly calcified shell was successfully formed from trochophore to D-shape larvae at control group. Similar results have also been reported from other molluscan species. It was found that the chitin synthase could be detected in the shell margin of larval M. galloprovincialis which was the region involved in shell extend in early developmental stage (Weiss et al., 2006). Chitin synthase and chitinase are the key enzymes for chitin shell biosynthesis in molluscs. Chitin synthase is in charge of the synthesis of chitin, while chitinase is responsible for the degradation of chitin (Weiss and Schonitzer, 2006; Weiss et al., 2006). Results of this study suggested that synthesis of chitin was down-regulated and the degradation of chitin was prompted at the stages from trochophore to early D-shape larvae, which might cause the content reduction of chitin in the shell of oyster larvae. These results were in consistent with previous findings that biomineralization was activated from trochophore to D-shape larvae in molluscs, resulting in the organic-mineral composite (mainly CaCO₃) shell layer to cover the chitin framework one.

According to previous study, there are several hypotheses trying to explain how shell formations in oyster larvae are affected by OA. Some researchers considered that OA lowered the calcium carbonate saturation state, which caused oyster larvae harder to form the complete calcified shell. It was also believed that acidification upset the acid-base balance between the marine organism and environment, and even the inner acid-base balance of organism (Zhao et al., 2017), which influenced the energy metabolism and homeostasis in oyster larvae (Lannig et al., 2010). We are inclined to a hypothesis that OA might disturb the biosynthesis of chitin and finally invite the failure of shell formation in oyster larvae. In the present study, the expression of Cgchs1 and Cgchit4, and the morphological characteristics of oyster larvae under acidification treatment were explored. It was found that in the moderate acidification treatment (pH 7.8) group, the expression level of Cgchs1 mRNA was decreased slightly in early D-shape stage, while that of Cgchit4 mRNA was obviously decreased in early D-shape stage in the severe acidification treatment (pH 7.4) group. These results suggested that the synthesis of chitin was significantly inhibited by severe acidification treatment (pH 7.4), while the degradation of chitin was dramatically activated. As shown in Figure 5, the shell formation in oyster larvae was indeed significantly suppressed by severe acidification treatment. Chitin is proverbial as a crucial component in molluscan shell and nacre biosynthesis. Chitin constructed the initial shell basis and supplied for other macromolecular and mineral deposition that visibly guide the biomineralization process, even in the system of crystal polymorphism (Levi-Kalisman et al., 2001; Zentz et al., 2001). Results in current research implied that the inhibition of biosynthesis of chitin caused by acidification conditions might be the reason for the failure of shell formation from trochophore to D-shape larvae during ontogenesis of oyster.

Moreover, the structural organization of chitin synthesizing complex in oyster larvae was further explored under normal and acidification conditions with WMISH. As shown in Figure 6, the Cgchs1 and Cgchit4 mRNA was mainly expressed in the beneath of newly formed shells. In trochophore stage, the positive signal was mainly located on the margin of shell field. In D-shape larvae stage, the positive signal was found on the edge of periostracum. These consequences were consistent with the anterior studies that chitin synthase could be detected in the shell gland region and related to shell biosynthesis of M. galloprovincialis larvae, and the inhibition of chitin synthase through Nikkomycin Z could dramatically alter the shell structure and function, for instance the bivalve hinge and the shell margin (Schönitzer and Weiss, 2007). Another previous study reported that the calcification taken place on the margin of the shell, among mantle tissue, periostracum and the shell itself (Marin et al., 2012). Therefore, all these results collectively suggested that the shell biosynthesis process and intricate framework compounds structure in oyster larvae were located close to the edges of shell. Acidification treatment could severely inhibit the biosynthesis of chitin, resulting in the failure of biomineralization in oyster larvae.

In conclusion, a Cgchs1 and a Cgchit4 gene were identified from C. gigas, which belonged to chitin synthase and chitinase family, respectively. During the key developmental stages for shell formation, the chitin biosynthesis was decreased from trochophore to D-shape larvae since the down-regulated mRNA expression of Cgchs1, while the decomposition of chitin was accelerated due to the up-regulated mRNA expression level of Cgchit4. When oyster larvae suffered from CO₂-induced acidification, the formation of calcified shell was severely inhibited, and the expression of Cgchs1 was significantly suppressed while that of Cgchit4 was dramatically activated, resulting in a decrease in chitin content on the edges of the shell. These results collectively suggested that OA might inhibit shell formation in oyster larvae by suppressing the biosynthesis of chitin.

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

This study was carried out in accordance with the recommendations of the Ethics Committee of Dalian Ocean University. The protocol was approved by the Ethics Committee of Dalian Ocean University.

YZ, ZL, XS, SH, and LW conceived and designed the experiments. YZ, ZL, and XS performed the experiments. YZ and ZL analyzed the data. LW and LS contributed reagents, materials, and analysis tools. YZ, ZL, LW, and LS wrote the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.