Identification and Expression Characterization of the Smad3 Gene and SNPs Associated with Growth Traits in the Hard Clam (Meretrix meretrix)

It has been demonstrated that the sekelsky mothers against decapentaplegic homolog 3 (Smad3) plays an important role in the growth and development of vertebrates. However, little is known about the association between the Smad3 gene and the growth traits of mollusks. In this study, Smad3 from the hard clam Meretrix meretrix (Mm-Smad3) was cloned, characterized, and screened for growth-related single nucleotide polymorphisms (SNPs) in its exons. The full-length cDNA of Mm-Smad3 was 1938 bp, encoding a protein with 428 amino acid residues. The protein sequence included an MH1 (27–135 aa) and MH2 domain (233–404 aa). Promoter analysis showed that the promoter sequence of Mm-Smad3 was 2548 bp, and the binding sites of Pit-1a, Antp, Hb, and other transcription factors are related to the growth and development of hard clams. The phylogenetic tree was divided into two major clusters, including mollusks and vertebrate. The expression level of Mm-Smad3 was predominantly detected in the mantle and foot, while extremely less expression was observed in the digestive gland. The low expression level of Mm-Smad3 was detected at the stages of unfertilized mature eggs, fertilized eggs, four-cell embryos, blastula, gastrulae, trochophore, and D-shaped larvae, whereas an opposite trend was observed regarding the highest expression at the umbo larvae stage (p < 0.05). In the mantle repair experiment, the time-course expression profiles showed that compared to the expression level at 0 h, Mm-Smad3 significantly decreased at 6 h (p < 0.05) but increased at 12 and 48 h. Further, the association analysis identified 11 SNPs in the exons of Mm-Smad3, of which three loci (c.597 C > T, c.660 C > T, c.792 A > T) were significantly related to the growth traits of clam (p < 0.05). Overall, our findings indicated that Mm-Smad3 is a growth-related gene and the detected SNP sites provide growth-related markers for molecular marker-assisted breeding of this species.

Sex ratio, gonadal and condition indexes of the Asiatic hard clam, Meretrix meretrix in Marudu Bay, Malaysia

Duisan L, Salim G, Ransangan J. 2021. Sex ratio, Gonadal and Condition indexes of the Asiatic Hard Clam, Meretrix meretrix in Marudu Bay, Malaysia. Biodiversitas 22: 4895-4904. Asiatic hard clam, Meretrix meretrix is one of the important shellfishery resources in Marudu Bay, Sabah, Malaysia. It is among the most popular clam species being widely traded in the local wet markets around Sabah, Malaysia. Unfortunately, the shellfishery management for this species has not been well established. In addition to overexploitation, habitat destruction is also one of the significant threats to this species due to the extensive land use of the coastal areas in Sabah. Hence, conservation and breeding efforts for this species are greatly required. Therefore, the current study was conducted to examine the sexual maturity of the clam with respect to shell length classes for artificial seed production purposes. For this study, a total of 86 clam specimens were randomly collected from mudflats in Marudu Bay. The specimens were utilized for gonad histological and condition analyses. The clams were grouped into three shell length classes; (3.00-4.99) cm, (5.00-6.99) cm, and (7.00-8.99) cm prior to the analyses. Results showed the natural stock of the Asiatic hard clams in Marudu Bay was dominated by females (1.39:1) over males with no hermaphroditism observed. The gonadal index was recorded higher among clams with shell lengths between 5.00 and 6.99 cm. The condition index analysis also recorded high (>4.0) for clams in all the shell length classes. The findings of this study suggest that the clams with shell lengths between 5.00 cm and 7.00 cm are already fully matured and can be utilized as a broodstock candidate for an artificial breeding program in the hatchery.

Dynamic Regulation of Ions and Amino Acids in Adult Asian Hard Clams Meretrix lusoria Upon Hyperosmotic Salinity

The dynamic regulation of ions and amino acids in the gills and mantle of the Asian hard clam, Meretrix lusoria, following the exposure to a hyperosmotic environment was hitherto unclear. The present study revealed that the osmolality as well as the Na+ and Cl– concentrations in the hemolymph were significantly increased 3 h after transferring the clams from an environment with the salinity of their natural habitat (brackish water; BW; 20‰) to one with hyperosmotic salinity (seawater; 35‰). In addition, we found that the specific activities of Na+/K+-ATPase, a key enzyme that plays a significant role in cell osmoregulation, in the gills and mantle of clams were significantly increased at 72 and 12 h post-transfer, respectively, during acclimation to hyperosmotic salinity. Similarly, the contents of free amino acids (FAAs) such as taurine, alanine, and glycine were significantly elevated during hyperosmotic salinity acclimation. Previous research indicates that taurine is the most abundant FAA in the gills and mantles of Asian hard clams and that the taurine transporter (TAUT) plays an important role in taurine accumulation. The present study showed that TAUT mRNA and protein expression were significantly and transiently increased in the mantle of Asian hard clams following exposure to seawater; although the expression of TAUT mRNA in the gills of Asian hard clams was also transiently stimulated by exposure to hyperosmotic salinity, the relative TAUT protein abundance decreased only at later stages. Accordingly, the findings of this study improve our understanding of the dynamic processes of ion and amino acid regulation in the peripheral tissues of bivalves under hyperosmotic stress.

Effect of a Novel Microwave-Assisted Induction Heating (MAIH) Technology on the Quality of Prepackaged Asian Hard Clam (Meretrix lusoria)

The microwave-assisted induction heating (MAIH) method—an emerging thermal technique—was studied to heat the prepackaged raw hard clam (Meretrix lusoria). The cooking effects on microbial and physiochemical qualities of clam were investigated. After the heating of the clam meat samples, the aerobic plate count (APC), psychrotrophic bacteria count (PBC), and total volatile basic nitrogen (TVBN) levels decreased with increasing heating time, but the shucking ratio, area shrinkage, and texture (hardness, cohesiveness, and chewiness) increased. In addition, the L* (lightness) and W (whiteness) of the clam meat samples increased significantly at the beginning of the heating period, whereas they decreased significantly with extended heating time. However, a* (redness) had the opposite trend. This study found that when clams were heated for more than 120 s at 130 °C or 150 s at 90 °C, they displayed obvious shrinking and a yellow-brown appearance, indicating that they are overcooked. After heating by MAIH for at least 110 s at 130 °C or 130 s at 90 °C, the samples were cooked well and gains a completely shucking, along with no microbial count detected. Therefore, the results indicated that the optimum heating conditions for prepackaged hard clams subjected to an MAIH machine were 130 °C for 110 s or 90 °C for 130 s.

Characterization and Function Analysis of β, β-carotene-9′, 10′-oxygenase 2 (BCDO2) Gene in Carotenoid Metabolism of the Red Shell Hard Clam (Meretrix meretrix)

The relationship between carotenoid and shellfish shell color has gained increasing attention. β, β-carotene-9′,10′-oxygenase 2 (BCDO2) is a key enzyme in animal carotenoid metabolism, and its accumulation affects the change in body color, as demonstrated in mammals, birds, and fish. However, it is unclear whether BCDO2 is involved in the formation of the red shell color of clam. To explore the molecular structure and biological function of BCDO2 gene in the process of carotenoids accumulation, in this study, the BCDO2 from hard clam Meretrix meretrix (designated as Mm-BCDO2) was cloned and characterized, and the single-nucleotide polymorphisms (SNPs) associated with shell color were detected. The results of qRT-PCR indicated that Mm-BCDO2 gene was expressed in all six tested tissues, and the expression of mantle was significantly higher than other tissues (P &lt; 0.05). The association analysis identified 20 SNPs in the exons of Mm-BCDO2, among which three loci (i.e., c.984A &gt; C, c.1148C &gt; T, and c.1187A &gt; T) were remarkably related (P &lt; 0.05) to the shell color of clam. The western blot analysis revealed that the expression level of Mm-BCDO2 in the mantle of red shell clams was stronger than that of white shell clams (P &lt; 0.05). Further, the immunofluorescence analysis indicated that the single-layer columnar cells at the edge of the mantle were the major sites for the Mm-BCDO2 secretion. This study explored the potential impacts of BCDO2 gene on the shell color of M. meretrix, which provided a theoretical basis for a better understanding of the important role of BCDO2 in carotenoid metabolism.