The COMMD (copper metabolism gene MuRR1 domain) family includes ten evolutionarily conserved proteins, namely, COMMD1–10, in the extreme carboxyl terminus of which they share a unique motif known as the COMM domain (Burstein et al., 2005). The COMM domain, with 70–85 amino acids in length and being rich in tryptophan, proline, and leucine, not only defines the gene family but also provides a critical interface for protein–protein interactions with each other (Maine et al., 2007). The hydrophobic residues located in the COMM domain could form two conserved nuclear export signals (NES1 and NES2) (Muller et al., 2009). Except COMMD6, other COMMD members possess an amino terminal region, which is divergent among the subfamilies but is highly conserved within the ortholog proteins (Burstein et al., 2005). COMMD1 is the first identified member, which was initially termed as Murr1 due to its proximity to the U2af1-rs1 locus in mice, and other COMMD members were identified through homologous screening (Nabetani et al., 1997; van De Sluis et al., 2002). A total of ten subfamilies were found in the vast majority of vertebrates (Burstein et al., 2005), while in invertebrates, including insects, worms, and molds, only several COMMD members were reported, and none of the COMMDs were found in unicellular eukaryotic organisms or bacteria (Nabetani et al., 1997). The wide existence as well as the highly conservative characteristic of COMMD homologues imply their critical roles during the metazoan evolution (Riera-Romo, 2018).

As the best-characterized member, COMMD1 may represent a prototype of the family (Burstein et al., 2005). COMMD1 was reported to be able to participate in two distinct activities, control of copper metabolism and regulation of the transcription factor NF-κB (Riera-Romo, 2018). Researchers found that mutations of COMMD1 are responsible for copper toxicosis in Bedlington terrier dogs, resulting in excessive copper accumulation in the liver (Tao et al., 2003). In human, biochemical findings had indicated a direct role for COMMD1 in biliary copper transport, and COMMD1 defect could impair the copper excretion (Tao et al., 2003; Riera-Romo, 2018). Besides COMMD1, various functions were found for other COMMD genes in vertebrates. COMMD4, as one of the protein kinase A targets, was able to interact with myomegalin and inhibit NF-κB activity (Uys et al., 2011). The COMMD3 and COMMD8 complex could selectively recruit GRK6, which induced GRK6-mediated phosphorylation of the receptor and activated the β-arrestin-mediated signaling (Nakai et al., 2019). COMMD3 and COMMD9, which are endogenous regulators, regulate Na⁺ transport through altering ENaC cell surface expression (Liu et al., 2013). COMMD5 affects cell proliferation (Solban et al., 2000), and COMMD6, 7 participates in the invasion and migration regulation of a variety of cancer (You et al., 2017; Yang et al., 2019). Research has reported that COMMD10 is related to phagosomes in murine macrophages (Dill et al., 2015), and in myeloid cells, deficiency in COMMD10 can cause increased NF-κB activation and then aggravate lipopolysaccharide systemic sepsis (Naugler and Karin, 2008; Mouhadeb et al., 2018). In comparison, only the functions of several COMMD genes were reported in invertebrates. For example, COMMD4 is found ubiquitously expressed in amphioxus, with the highest level in gonad, and lipopolysaccharide injection could induce its expression (Jin et al., 2012). Wang et al. have cloned COMMD1 in Crassostrea hongkongensis, and the transcription level of COMMD1 was increased significantly in the gill and hemolymph after salinity stimulation (Wang et al., 2017a).

As benthic filter feeders, bivalve molluscs are well adapted to highly dynamic oceans and freshwater environments since the early Cambrian. Along with intensified human activities in recent decades, the bivalve habitats are subject to various biotic/abiotic stressors, and pathogenic microbes and heavy metals are two of the main stressors. The COMMD genes play critical roles in many vital functions, and exploration of whether these genes participate in heavy-metal or bacterial resistance may help gain a better understanding for the outstanding adaptability of bivalve molluscs. In the present study, a systematic identification and characterization of COMMD genes in four bivalves were conducted, namely, Yesso scallop, Zhikong scallop, Pacific oyster, and dwarf surf clam. Detailed genic structure comparison and spatiotemporal expression analysis provided insights into the potential function of COMMD genes in bivalve molluscs. Further, transcription patterns of different COMMD members under copper ion stress and after Vibrio Anguillarum infection were investigated in the gill and hepatopancreas of M. lateralis. This is the first comprehensive research of the COMMD family genes in bivalves, which notably provided helpful information regarding the classification, evolution, and function of these genes. These findings may assist a better understanding of the bivalves’ adaption to adverse heavy-metal pollutions and bacterial challenge.

In this study, the COMMD family in four bivalve molluscs was identified and characterized based on the genomic and transcriptomic data. Similar to the human COMMD proteins, these bivalve COMMD proteins contain the conserved COMM domain in the extreme carboxyl terminus, which could mediate the interaction of COMMD-COMMD proteins and the formation of the COMMD polymer (Burstein et al., 2005). The amino terminal of COMMDs shares a low homology among members of the family but has highly conserved sequences with their ortholog proteins, which may contribute to the functional diversity of different subfamilies (Maine and Burstein, 2007). Besides four bivalve molluscs in our study, a 5-intron pattern of COMMD9 was also discovered in human and zebrafish, which may imply that COMMD9 has retained the ancestral exon–intron structure during the evolution. Other COMMD subfamilies of four bivalve molluscs have inconsistent intron patterns. Previously, researchers have found that intron insertion and loss may be associated with selective splicing, encoding the untranslated RNAs and enhancing the levels of mRNA transcription (Jin et al., 2012). The frequent occurrence of intron insertion and deletion among the COMMD family except for COMMD9 may be to a certain extent related to the function of introns.

Phylogenetic analysis showed that four bivalve mollusc COMMD genes always clustered together in the invertebrate clades, consisting of their evolution status and conservativeness. Previous findings show that COMMD genes are highly conserved throughout vertebrate evolution, and COMMD1 and COMMD9 seem to be restricted to vertebrates (Burstein et al., 2005). In the present study, we further retrieved COMMD genes from 26 metazoan species ( Figure 3 ). We found that both COMMD1 and COMMD9 could be identified in the Lophotrochozoa, Deuterostomia, Cnidaria, and Sponge groups, while they were absent in the Ecdysozoa group. Besides COMMD1, 9, COMMD6, 7, 8 were also absent in most ecdysozoans and that nematodes only have the COMMD4 subfamily, which may attribute to the fact that the Ecdysozoa genomes are rapidly evolving (Telford et al., 2008). Besides, COMMD1, 6, 8 have been lost in ciona (Jin et al., 2018), while 10 intact single-copy COMMD genes were found in amphioxus as well as in most vertebrates. Species in the Mollusca, Deuterostomia (except Urochordata), Brachiopoda, Cnidaria, and Sponge groups almost have ten intact single-copy COMMD family genes; only several species were found to have loss of one the COMMD subfamily members. However, it remains unclear whether this phenomenon is due to genome assembly fragmentation or they have been lost during evolution. An across-Metazoa comparison implied that COMMD members diverged from each other at early stages of evolution and the integrated COMMD family may already exist in the metazoan last common ancestors.

Evolutionary analysis have shown that purifying selection dominated the evolution of COMMD genes (Jin et al., 2018). For four bivalves, a total of 25 sites under potentially positive selection were identified which may provide a support for the structural and functional diversity of the COMMD family members. Similarly, the selective pressure analyses of COMMD family genes in amphioxus showed that there were 16 positive selective sites detected, although the COMMD family genes have undergone very strong purifying selection during evolution (Jin et al., 2018).

In previous studies, the expression profile of COMMD family genes has been reported in vertebrates (van De Sluis et al., 2002; Klomp et al., 2003). However, such research remains lacking in invertebrates. To better understand the characteristic and function of COMMD genes in molluscs, extensive transcriptome resources were used to profile the temporal and spatial expression patterns of COMMD genes in four bivalve molluscs ( Figure 5 ). The COMMDs were reported to play a vital role during mouse embryonic development, and COMMD-knockout mice are embryonically lethal and die at different stages of embryogenesis (Semenova et al., 2003; van de Sluis et al., 2007; Bartuzi et al., 2013). Besides, researchers found that COMMD1 has a regulatory role in the cell cycle of HEK293 cells (Jiang et al., 2019). Our results showed that bivalve COMMD1 is highly expressed during multicellular cleavage and its transcription level declines rapidly from blastula. It may also implicate that COMMD1 is involved in the regulation of cell proliferation. Bivalve COMMD7s were found to enhance their transcription exponentially from the D-shaped veliger stage and sustained a high level of transcription during late larval development. Previous studies have found that COMMD7 promoted cell proliferation, migration, and invasion processes but suppressed cell apoptosis (Devlin et al., 2003; Zheng et al., 2018). Therefore, we speculate that COMMD7 may be involved in the regulation of organogenesis during embryonic larval formation. Among different adult tissues, high transcription levels of most COMMDs were found in the hepatopancreas. As filter-feeding animals that mainly feed on microalgae, bivalves could accumulate hazardous substance produced through diet, and the hepatopancreas is the main organ for processing and accumulating the incoming hazardous substance (Lian et al., 2019). Therefore, the relatively higher transcription levels of COMMDs in the hepatopancreas may assist with toxin tolerance in bivalves. COMMD1, 9, 10 are involved in the regulation of cell proliferation, migration, and cell-cycle progression (Yang et al., 2017; Zhan et al., 2017). We found that COMMD1, 6, 10 from two scallops were highly expressed in the female gonads, suggesting that these three COMMD members may contribute to the ovarian cell homeostasis maintenance to assist with oogenesis in scallops.

The COMMD genes play key roles in regulating copper homeostasis and innate immune response (Bartuzi et al., 2013; Jin et al., 2018; Mouhadeb et al., 2018). To investigate the possibly biological functions of the MlCOMMDs, their mRNA expression levels were measured at different time points under copper ion stress and bacterial stress. MlCOMMD1, 4, 9 transcriptions exhibited a significant alteration after copper ion stress in both assayed organs of dwarf surf clam, and MlCOMMD9 showed the most drastic upregulation, suggesting their functional relation with cellular copper ion metabolism. It was reported that COMMD1 regulates the endosomal sorting of the copper transporter (Phillips-Krawczak et al., 2015), and COMMD9 may be an endogenous regulator of the epithelial sodium channel (ENaC) to regulate Na⁺ transport, which could indirectly alter intracellular Cu flux (Handy et al., 2002; Liu et al., 2013). Besides, it was previously found that deficiency of COMMD1 or COMMD9 can result in hepatic copper accumulation under high-copper diets (Singla et al., 2021). Our V. Anguillarum infection experimental results revealed that MlCOMMD4 was significantly upregulated in both the gill and hepatopancreas of dwarf surf clam. Previous studies reported that COMMD4 has the ability to inhibit NF-κB, the key regulator of both innate and adaptive immune responses (de Bie et al., 2006; Maine and Burstein, 2007; Hayden and Ghosh, 2008; Naugler and Karin, 2008);, while whether bivalve COMMD4 can regulate NF-κB needs further more detailed studies.

In this study, 10 COMMD genes were respectively identified from the four bivalves, namely, Yesso scallop, Zhikong scallop, Pacific oyster, and dwarf surf clam. They possessed conserved COMM domains and comprised ten subfamilies. Purifying selection of six subfamilies was revealed, with the strongest selection on COMMD4. The expression profiling during embryonic development and in adult organs provided valuable implications for exploring the function of the bivalve COMMD gene. After exposure to two different stresses, MLCOMMDs exhibited different regulation patterns in different tissues or organs. The responses of COMMD members under stresses were found more sensitive in the hepatopancreas than in the gill, and MlCOMMD9 and MlCOMMD4 might be the good candidate stress indicator genes respectively for copper ion stress and V. Anguillarum infection. This study comprehensively describes the first genome-wide characterization of the COMMD gene family in bivalves, and our work will be helpful in better understanding the function and evolution of COMMD family bivalve molluscs.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

SL and JH conceived and designed the study. CX and NH performed the experiments. LL and FS participated in the data analysis. XC, SL, LZ, SW, and ZB wrote the manuscript. All authors contributed to the article and approved the submitted version.

We acknowledge the grant support from the Project of Sanya Yazhouwan Science and Technology City Management Foundation (SKJCKJ-2019KY01), the Key R&D Project of Shandong Province (2020ZLYS10, 2021ZLGX03), and the China Agriculture Research System of MOF and MARA.

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.

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