To date, the number of TGF-β superfamily genes has been extensively studied in various animals, showing considerable differences among different organisms [22, 23]. In this study, a comprehensive survey of the TGF-β superfamily was carried out in scallops. Three scallop species had the same number of members in the TGF-β superfamily, which was much lower than that observed in many vertebrates [4]. For example, there are at least 30 in mammals [4, 24]. The number of TGF-β superfamily members in scallops was higher than that in oysters and some other invertebrates, such as fruit flies, leeches, and jellyfish [4]. The expansion of family members probably originated from the duplication of a common ancestral gene and was widely dispersed by chromosomal translocations [2, 25]. Gene duplication has been proposed as a primary mechanism for increasing organismal complexity and generating evolutionary novelty. There has been evidence for two rounds of genome duplication (2R) in vertebrates and additional rounds (3R or 4R) in teleosts [4, 26].
Previous studies have suggested that the first members of the TGF-β superfamily to appear were BMPs/GDFs, which subsequently differentiated into activin/inhibin, while the TGF-βs and LEFTY were more recent, appearing only in deuterostomes [2, 27, 28]. Consistent with previous findings, the current analysis showed that TGF-βs and LEFTY were absent in scallops. BMP, ADMP, GDF, NODAL, activin/inhibin, and AMH were identified in each scallop genome. The PI from the same cluster showed consistency. Most TGF-β superfamily proteins were unstable in nature (INS > 40). According to the aliphatic index, all proteins showed a hydrophilic nature. The characteristics of the TGF-β superfamily in scallops were similar to those in other species in previous studies [29], which showed that the TGF-β superfamily was evolutionarily conserved. This result can be supported by selection pressure analysis. The TGF-β superfamily genes in scallops have evolved under purifying selection. Different branches showed similar selective pressures, and no site was identified for the positive selection test. In general, purifying selection acts to selectively eliminate deleterious mutations, often resulting in a more conservative gene [30, 31]. In two artificially selective A. irradians breeds, the TGF-β type I receptor gene was detected to be selected and no TGF-β superfamily genes were under selection [32]. In general, these results suggested that the TGF-β superfamily was conserved in scallops.
In the current study, TGF-β superfamily genes were specifically expressed at different early developmental stages. BMP5-8-like (CfTGFβ-03 and MyTGFβ-03) were both highly expressed at the 2–8 cell stage. BMP3/GDF10-like (CfTGFβ-05 and MyTGFβ-05) and BMP2/4-like (CfTGFβ-01) showed high expression levels at several developmental stages. BMPs play key roles in gastrulation, mesoderm induction and axial patterning in the embryo [33]. BMP2/4 is a crucial factor for dorsal-ventral patterning in oysters [17]. In jellyfish and leeches, BMP2/4 and BMP5-8 have been implicated in larval axial development [34, 35]. In addition, NODAL-like genes (CfTGFβ-06 and MyTGFβ-06) were specifically highly expressed at the blastula stage. Previous reports have shown that NODAL is required for early cell fate decisions, organogenesis, left-right development [36, 37], anterior-posterior body axis development [38] and the oral-aboral axis in the embryo [33]. In this study, several BMP-like and NODAL-like genes may play important roles in early development, patterning the embryonic body plan and later regulating development and homeostasis.
Previous studies have shown that GDF8/11 plays a critical role in regulating muscle growth [39]. For example, in M. yessoensis, inhibition of myostatin mRNA could increase a combination of hyperplasia and hypertrophy of myosin heavy chain (MHC) II striated myofibers in striated muscle, thereby increasing muscle cellularity [12]. The GDF8 gene is also associated with muscle growth in other scallops [11, 40]. SNPs in the myostatin gene have been developed as candidate molecular markers for selective breeding in C. farreri [10, 41] and the Noble scallop (Chlamys nobilis) [42]. Similar results were obtained in this study, where the GDF8/11-like gene (CfTGFβ-08) showed high expression in striated muscle and smooth muscle. MyTGFβ-08 also showed moderate expression in striated muscle, with low or no expression in other tissues. The results were consistent with previous studies in M. yessoensis [12, 43]. In addition, BMP5-8-like (CfTGFβ-03) and CfTGFβ-10 (activin/INHB-like) showed moderate expression levels in smooth muscle. To date, data on BMP5-8 and activin/inhibin in invertebrates are very limited and have rarely been reported in scallops. Activin/inhibin has been suggested to play an important role in spermatogenesis in mammals [44] and in the regulation of oocyte maturation in fish [45, 46]. Therefore, GDF8/11 can regulate muscle growth in scallops as in other species, and how other TGF-β superfamily genes are involved in muscle development should be further investigated in scallops.
Several TGF-β members have also been identified as sex determination/differentiation genes. In the current study, AMH-like genes (CfTGFβ-12 and MyTGFβ-12) showed specific expression levels in the gonad. “Amh-amhy-amhr2” acts as a master sex-determining gene in teleost fish, regulating germ cells proliferation and gonad development [47, 48]. Interestingly, AMH is duplicated in some fish, such as amhy (AMH on the Y chromosome) in Nile tilapia [49, 50], amhby (Y chromosome-specific copy of AMH) in northern pike [47], and amhy (Y-linked duplicates of AMH) in Patagonian pejerrey [51] and Sebastes rockfish [52]. In these species, a duplicate copy of AMH acts as a master sex-determining gene [48]. In Nile tilapia, loss of amhy function in XY fish resulted in male to female sex reversal, while overexpression of AMH resulted in female to male sex reversal [53]. C. farreri and M. yessoensis are gonochoristic species and the ZW-type sex chromosomes are uncharacterized homomorphic chromosomes [54]. AMH is also required to drive testicular development in a reptilian species, the Chinese soft-shelled turtle, a typical species exhibiting ZZ/ZW sex chromosomes [55]. In scallops, the genes highly expressed in the gonads were from cluster XI. The 6 genes in this cluster shared a high nucleotide identity, and the genes in scallops may have similar functions in other species. Therefore, there may be a duplication of autosomal AMH that was later translocated to the ancestral sex chromosome. The information here provides new insights into the important role of AMH in gonadal growth/maturation in scallops.
The expression profiles of TGF-β superfamily genes under heat stress or hypoxia stress were significantly different from those under heat plus hypoxia stress in scallops. For example, the genes in cluster IV (BMP9/10-like) were both highly expressed under heat plus hypoxia stress in three scallop species. These observations indicated that BMP9/10-like genes may be involved in the combined stress of multiple factors. The genes in cluster I (BMP2/4-like), VII and VIII (GDF8/11-like) were differentially expressed in both C. farreri and M. yessoensis, while there were no significant differences in the expression of these genes only under heat stress or hypoxia stress alone. TGF-β superfamily genes are known to control a wide range of biological processes, including immunosuppression and apoptosis induction. There is evidence that hypoxia stress can induce apoptosis, inflammation, and autophagy in marine bivalves [56]. TGF-β transcription increased in Nile tilapia [57] and rainbow trout [58] during exposure to hypoxia. BMP-4 was significantly downregulated under short-term salinity stress in abalone [59]. However, few studies have reported the function of the TGF-β superfamily in stress tolerance in scallops. In general, this study provided a fundamental clue for understanding the important roles of the TGF-β superfamily in stress tolerance in scallops.