2.1. Identification of SOD Genes in C. farreri
In mammals, only three unique and highly compartmentalized SODs have been identified. By contrast, a total of eight SODs, including six Cu/Zn-SODs (SOD1-6) and two Mn-SODs (SOD7, 8), have been found in the C. farreri genome, revealing the expansion of Cu/Zn-SOD. To further test whether other marine animals showed expansion phenomena of the SOD family, we also retrieved SOD genes in oyster C. gigas, snail Biomphalaria glabrata, and sea urchin Strongylocentrotus purpuratus, and our results showed that more than six SOD members were present in these species. It is noteworthy that, just as in C. farreri, most SOD expansion events in these marine animals occurred within the Cu/Zn group. Among Cu/Zn-SODs, SOD2, and SOD3 were found to be located on the same chromosome in C. farreri, indicating that tandem gene duplication may occur. The two Mn-SODs in C. farreri were found respectively localized on chromosomes 2 and 19.
Various exon/intron organization patterns of
SOD genes are presented in
C. farreri (
Figure 1A), and the number of introns were identified to range between three and nine. According to sequence analysis, the ORF lengths of
SOD genes in
C. farreri were from 462 to 3348 bp, and the encoded proteins containing 153 to 1115 amino acids. Most of the SOD proteins were predicted to be stable (instability index ≤40), and all the SODs were found to be hydrophilic based on a grand average of hydropathicity (GRAVY) analysis (the aliphatic index ranged from 44.73 to 88.45) (
Table 1). The
C. farreri Mn-SODs were found to be acidic, whereas the Cu/Zn-SODs showed variable isoelectric point (
pI) values, with two members (SOD3 and SOD4) being basic in character.
For all Cu/Zn-SODs in
C. farreri, Motif 1 or 2 which contained conserved copper ligands and the site involved in a disulfide bond could be detected (
Figure 1B and
Table 2), and they possess the topological signature of β-barrels with ligand clusters localized on the exterior (
Figure 1C). The sub-cellular predictions showed that SOD1 was localized in the cytoplasm; SOD2, SOD3, and SOD4 were mainly transported to the extracellular milieu; and first time in animals, we found two specific nucleus localized Cu/Zn-SODs (SOD5 and SOD6) in
C. farreri. This finding was consistent with the results from the motif analysis that revealed a motif 4 which was classified as a specific ZapB domain (E-value: 1.2e-06) with a nuclear localization signal (score: 10) present in SOD5 and SOD6, and this ZapB domain has been reported to contribute to forming a coiled-coil structure and being involved in cell division [
35] (
Figure 1B). Notably, an unexpected motif 3 which contained manganese/iron ligands and showed conserved “D-x-[WF]-E-H-[STA]-[FY] (2)” Mn/Fe-SOD signature was found to be present at the C terminus of the “extremely long” Cu/Zn-SOD4 (
Table 2). Meantime, a Fe ligand-bound Cys283 was also detected in SOD4 (
Table S1), indicating that SOD4 might be a novel combined or transient SOD type with a complex catalytic metal ion-binding activity in
C. farreri. In addition, SOD4 caught our notice due to its quadruple SOD_Cu domains, which were not found or reported in animals. We further retrieved SOD proteins from several mollusk species, and Cu/Zn-SODs with triple/quadruple SOD_Cu domains were also found in
B. glabrata (XP_013062920.1),
C. gigas (XP_019923318.1; XP_011414606.1),
Pinctada fucata (ALK82329.1), and
Lottia gigantea (V4AP91), which is suggestive of a mollusk-specific SOD type which originated from the common ancestor of these animals.
Mn-SODs in
C. farreri possess both Motif 3 and 5, and SOD7 exhibited mitochondrial localization, while SOD8, for the first time in bivalves, was a SOD demonstrated to be a specific cytosolic type. Similar to bay scallop [
36], the polypeptide chains of
C. farreri Mn-SODs are divided into N-terminal helices and a C-terminal α/β domain, with the active metal ligand in the interior. The metal ion of Mn-SODs in
C. farreri was found to be coordinated in a strained trigonal bipyramidal geometry by four amino acid side chains: His52-His100-Asp185-His189 and His61-His125-Asp214-His218 in SOD7 and SOD8, respectively. In the present study, all 3D models were validated by Ramachandran plot analysis (
Figure S1), and the results showed that residues in the favored region ranged from 87.0% to 98.5%, and less than 3.8% were found in the outlier region, indicating fairly good quality (
Table S3).
2.2. Phylogenetic Analysis of SODs
Based on polygenetic analysis (
Figure 2), distinct evolutionary paths for Mn- SODs and Cu/Zn-SODs with varying degrees of protein conservation were observed. In addition, SOD members with different subcellular locations diverged from each other at early stages of evolution, prior to the differentiation of invertebrates and vertebrates, suggesting the rapid sequence divergence of SODs. All mitochondrial Mn-SODs across 14 species were clustered together, and two branches formed by vertebrates and invertebrates could be tracked, indicating highly conserved protein structures and evolutionary lineages. Prior to mitochondrial Mn-SODs, the cytosolic Mn-SODs near the phylogenetic root were clustered together firstly, indicating that these two types of Mn-SODs diverged long ago.
All the Cu/Zn-SODs in eukaryotes formed a large clade and comprising three subgroups, highly consistent with the subcellular predictions. Obviously, the cytosolic Cu/Zn-SODs (indicated by an orange color) detected across all species were clustered together, except in
L. vannamei and
E. sinensis since they do not have cytosolic Cu/Zn-SOD in their genome. Aquatic crustaceans have been reported to usually lack cytosolic Cu/Zn-SODs, and the relatively ancient cytosolic Mn-SODs might be linked to the fluctuation in copper metabolism induced by the special copper-dependent oxygen carrier protein hemocyanin [
37,
38,
39,
40]. For extracellular Cu/Zn-SODs with single functional domain, a clear branch for vertebrate Cu/Zn-SOD3 was detected, with conserved vertebrate-specific residues being found (
Figure S2). In this context, the branch represented in light green (96% bootstrap value) was restricted to mollusk-specific extracellular Cu/Zn-SODs, which is attributed to the multiple tandem SOD_Cu domains. In addition, the potential ancient nuclear Cu/Zn-SODs (purple color) were only found from the phyla Molluska (
C. farreri,
C. gigas) and Echinodermata (
S. purpuratus). The complex phylogenetic relationship of Cu/Zn-SODs may be due to their flexible plastic N- and C-termini decorated with localization signal peptides, and Cu/Zn-SODs may have evolved independently multiple times after the divergence of different lineages [
41,
42], indicating the differential interspecific evolution rates as well as rapid intraspecific sequence divergence of these proteins.
2.3. Spatiotemporal Expression Profiles of Scallop SODs During Development and in Adult Organs/Tissues
During embryonic and larval development, the temporal activation of
SOD genes and their expression patterns could be clearly distinguished in
C. farreri (
Figure 3). In multicellular stage, a set of
SOD transcripts, including cytosolic
Cu/Zn-SOD1, extracellular
Cu/Zn-SOD4, and mitochondrial
Mn-SOD7, were detected at the very beginning of fertilization and exhibited high expression until blastula formation, suggesting their maternal origin to play protective roles and to help maintain a redox balance during fertilization and cell cleavage. Afterwards, dominant expression was observed for extracellular
Cu/Zn-SOD2 and
Cu/Zn-SOD3 during gastrulation, from which more than 200-fold elevated mRNA level was detected, and their high expression was maintained until D-stage veliger formation. When get into umbo larvae development, the expression levels of
SOD1 and
SOD7 were respectively enhanced 3.3-and 12.2-fold again, together with a significant activation of cytosolic
Mn-SOD8. Nevertheless, only
Mn-SOD8 could exhibit persistent high expression in creeping larvae as well as in juvenile scallops, during which the expression of nuclear
Cu/Zn-SOD5 and
Cu/Zn-SOD6 was remarkedly increased. The participation of
SODs in gastrulation and metamorphosis has been found by several lines of evidence, including in prawn [
43], seabass [
44], frog [
45], fruit fly [
46], and chicken [
47], which may due to the elevation of oxygen consumption to meet the high demands of energy reserve utilization during organ initiation and structural remodelling. Studies in mouse embryos also found that, regardless of whether fertilization had occurred in vivo or in vitro, addition of SOD led to a protective effect against oxidative stress on both sperm viability and fertilized embryos [
48]. Thus, the explicit temporal expression patterns of
SODs observed in the present study may suggest their important roles in key processes during development, indicating the indispensability of
SODs for organ/tissue initiation and maturation in
C. farreri.
We further investigated the transcriptional profiles of
SOD genes in 14 organs/tissues of adult scallop (
Figure 4). Clearly,
SOD1 was the only
Cu/Zn-SOD showed widespread expression in all the examined organs/tissues with significantly higher read per kilobase of exon model per million mapped reads (RPKM) values (
p < 0.001) than most of the other
SODs; relatively high levels were detected in kidneys, muscles, and ganglions. Similar widespread tissue expression was observed for cytosolic
Mn-SOD8, while the transcript amount was much lower than that of
SOD1. Hepatopancreas and ganglions showed higher expression of
Mn-SOD8 than other organs/tissues. The expression of extracellular
Cu/Zn-SOD2 and
Cu/Zn-SOD3 was much lower than other
SOD genes, with the male gonad exhibiting a high level of
SOD2 and the eye showing a high expression for both
SOD2 and
SOD3. Similar results have been reported during investigation of mammal extracellular
SODs, which revealed the protective function of extracellular
SOD in the corneal endothelium [
49,
50] and on the Sertoli/germ cell surface in testicles [
51]. Of note, the dominant transcript mRNA of extracellular
Cu/Zn-SOD4 was found in the foot/byssus of
C. farreri. Similarly, the same extracellular
Cu/Zn-SOD (ALK82329.1) with a quadruple SOD_Cu domain was identified from the distal thread region of the byssus in
P. fucata, and the researchers proposed that extracellular
SOD could be required for prevention of the degradation of threads within the oxidative seawater environment [
52]. The tissue expression of nuclear
Cu/Zn-SOD5 and
Cu/Zn-SOD6 caught our attention due to their outstanding hepatopancreas-specific expression, with more than 900-fold enhancement compared with other tissues, indicating a specialized tissue-specific function. Interestingly, the mitochondrial
Mn-SOD7 in
C. farreri showed rather high levels in the striated muscle and smooth muscle, the primary organs associated with energy and mobility in scallops [
25], implying this gene play important roles against oxidative stress in muscle. Previous study of mice found that conditional knockout of
Mn-SOD targeted to type IIB skeletal muscle fibers not only can lead to oxidative stress enhance, but also is sufficient to reduce contractile muscle force and alter aerobic exercise capacity [
53].
2.4. Diversified Expression Regulation of SODs in Response to PSTs Producers
Previous studies have documented that SOD activities were induced in bivalves when exposed to toxic algae [
31,
32], but the expression of underlying
SOD genes has not been revealed. Our previous study indicated that the hepatopancreas and kidney in
C. farreri are both toxin-rich organs containing the highest concentrations of PSTs [
25]. To gain a deeper understanding of the defensive mechanism of bivalve
SOD genes in response to PST-producing algae challenge, expression regulation of the
SOD gene family in these two vulnerable organs of
C. farreri challenged with PST-producing algae
A. minutum (strain AM-1) and
A. catenella (strain ACDH) were analyzed.
In scallop hepatopancreas which is the main organ for PST uptake from algae, all the six
Cu/Zn-SODs showed significant alterations after
A. minutum exposure, with
SOD1,
2,
3,
5, and
6 being upregulated and
SOD4 being downregulated, while no significant change was detected in
Mn-SODs (
Figure 5A). Notably, the most dramatic upregulation was observed in
SOD6, and the fold changes reached 39.01 and 17.10, respectively, on days 5 and 15. Chronic induction was also observed in
SOD1 and
SOD5 at 15 days post exposure, while
SOD2 and
SOD3 showed acute up-regulation on day 1. For
SOD4, chronic suppression was detected on day 15. After exposure to
A. catenella, up-regulation was observed in
SOD2,
4,
6, and
8, while
SOD7 was down-regulated. As shown in
Figure 5B, significant acute induction of
SOD6 (10.82-fold) was observed on day 1, and chronic induction was found in
SOD2 (day 15),
SOD4 (day 15), and
SOD8 (day 10). Acute and chronic suppression of
SOD4 (on day 1 and 3) and
SOD6 (on day 10 and 15) was also observed, respectively. In addition, acute and chronic suppression of
SOD7 was detected on day 3 and day 10, respectively. Taken together,
Cu/Zn-SOD6 showed the most dramatic induction for both
A. minutum and
A. catenella exposure, implying that
SOD6 plays an important role in the antioxidant protection during PST accumulation in the hepatopancreas and may be a promising hepatopancreatic indicator gene during toxic dinoflagellate challenge in
C. farreri. Furthermore, as the two algae contained different PST members, with
A. minutum mainly synthesizing GTX1-4 and
A. catenella synthesizing C1-2, the chronic response of
SOD6 for
A. minutum exposure and its acute response for
A. catenella exposure suggests that the activation of
SOD6 for antioxidant defense is dependent on the species or toxicity of the PSTs accumulated. Similar phenomena were also observed for
SOD4, from which we could detect its chronically suppressed expression after
A. minutum exposure, while acute suppression followed by chronic stimulation of
SOD4 was observed after
A. catenella exposure. These findings all indicate dinoflagellate-dependent responses of
SOD members in hepatopancreas. Meanwhile, we further found that expression of
SOD3 and
SOD5 was negatively correlated (
p < 0.05) for both
A. minutum and
A. catenella exposure (
Figure S3A,B), suggesting their complementary or substitutionary function in scallop hepatopancreas to cope with PSTs producing algae exposure.
In the kidney, where the ingested PSTs are transformed to more toxic analogs [
25], all the
SODs except
SOD1 showed significant alteration at least at one time point (
Figure 5C,D). Among
Cu/Zn-SODs, after
A. minutum exposure, rapid elevation of
SOD4 (2.47-fold),
SOD5 (12.89-fold),
SOD6 (26.58-fold), and
SOD8 (2.81-fold) expression was observed, and significant activation was maintained until day 15 for
SOD6. In addition, acute suppression of
SOD2 and
SOD7 was observed, and
SOD7 was supressed at all the time points examined. After
A. catenella exposure, all the up-regulated members were from
Cu/Zn-SODs, including
SOD2,
3,
4,
5, and
6, and except for
SOD5, the highest fold change of these genes was present at 15 days after exposure. Like the regulation pattern during
A. minutum exposure,
SOD6 was up-regulated and
SOD7 was down-regulated at all the sampling time points after the
A. catenella challenge. Meanwhile, in the kidney,
SOD6 showed the highest fold change among all the
SODs after the challenge of both algae, similar to the results in hepatopancreas. In addition, highly positive correlation was observed between the expression of
SOD3 and
SOD6 (
p < 0.01) in scallop kidney, for both
A. minutum and
A. catenella exposure, with the coefficients of 0.67 and 0.74, respectively (
Figure S3C,D), indicating their co-regulation in response to PSTs producing algae challenge in kidney.
Overall, in scallops after exposure to different toxic algae, SOD up-regulation mainly occurred in the expanded Cu/Zn-SOD group, and SOD6 could be the promising indicator gene due to its highest fold change among all the SODs and being up-regulated under all PST-producer challenge scenarios. These findings may indicate the importance of Cu/Zn-SODs, especially SOD6 in protecting scallop from the stress of PSTs. In addition, diversified responsive patterns of SOD genes were detected in two toxin-rich organs after a A. minutum or A. catenella challenge according to the present data, with some members being up-regulated, some down-regulated, and some other members showing different regulation directions at different sampling times, depending on the examined organs and ingested algae. The diverse regulation pattern of SODs provides important information for understanding the mechanism of SOD enzymes in protecting scallop organs/tissues against PSTs accumulation, as the enzyme activities were determined by the expression regulation of all the SOD genes. Our results suggest the diverse function of scallop SODs during development and in response to PST-producing algae challenge, and the expansion of Cu/Zn-SODs might be implicated in the adaptive evolution of scallop or bivalve with respect to antioxidant defense against the ingested toxic algae.