Characterization and Functional Analysis of Fads Reveals Δ5 Desaturation Activity during Long-Chain Polyunsaturated Fatty Acid Biosynthesis in Dwarf Surf Clam Mulinia lateralis

Fatty acid desaturases (Fads), as key enzymes in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs), catalyze the desaturation between defined carbons of fatty acyl chains and control the degree of unsaturation of fatty acids. In the present study, two Fads genes, designated MulFadsA and MulFadsB, were identified from the genome of the dwarf surf clam Mulinia lateralis (Mollusca, Mactridae), and their spatiotemporal expression was examined. MulFadsA and MulFadsB contained the corresponding conserved functional domains and clustered closely with their respective orthologs from other mollusks. Both genes were expressed in the developmental stages and all tested adult tissues of M. lateralis, with MulFadsA exhibiting significantly higher expression levels in adult tissues than MulFadsB. Subsequently, the effects of dietary microalgae on Fads expressions in the dwarf surf clam were investigated by feeding clams with two types of unialgal diets varying in fatty acid content, i.e., Chlorella pyrenoidosa (Cp) and Platymonas helgolandica (Ph). The results show that the expressions of MulFads were significantly upregulated among adult tissues in the Cp group compared with those in the Ph group. In addition, we observed the desaturation activity of MulFadsA via heterologous expression in yeasts, revealing Δ5 desaturation activity toward PUFA substrates. Taken together, these results provide a novel perspective on M. lateralis LC-PUFA biosynthesis, expanding our understanding of fatty acid synthesis in marine mollusks.


Introduction
Long-chain polyunsaturated fatty acids (LC-PUFAs), such as n-3 eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and n-6 arachidonic acid (ARA), are essential for human health [1,2].LC-PUFAs play an important role in blood clotting, immune system regulation, neuro-transmitters, cholesterol metabolism, and the structure of membrane phospholipids in the brain and the retina [3].As humans cannot synthesize the precursor fatty acids linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), which are required for the synthesis of EPA, DHA, and ARA, these must be obtained through the diet, primarily from seafood [4].Fish, especially oily fish from cold water, such as cod, tuna, and mackerel, are excellent sources of long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs), predominantly EPA and DHA.Marine fish are a better source of n-3 essential fatty acid (EFA), while freshwater fish are a good source of n-6 EFA [5,6].However, overfishing has significantly reduced the availability of fish as EPA, DHA, and ARA sources, necessitating the exploration of alternative sources.Marine mollusks, mainly bivalves, are emerging as a potential dietary resource, as they are rich in LC-PUFAs [7] and have the ability to endogenously biosynthesize LC-PUFAs [8,9].
LC-PUFA biosynthesis in vertebrates, which involves a series of desaturation and elongation reactions, is mediated primarily via fatty acid desaturases (Fads) and elongases (Elovls); this has been extensively studied [10,11].The complement and catalytic activities of Fads and Elovls, especially for Fads, determine the LC-PUFA biosynthesis ability of organisms.In marine mollusks, Fads with ∆5 desaturation activity were first identified from the cephalopod Octopus vulgaris, which was a key step for the biosynthesis of LC-PUFAs [12].Subsequently, similar ∆5 Fads were also characterized in other mollusks, including abalone (Haliotis discus hannai) [13] and razor clam (Sinonovacula constricta [14].Furthermore, ∆8 Fads and ∆6 Fads were subsequently identified from the noble scallop (Chlamys nobilis) [15] and common cuttlefish (Sepia officinalis) [14], respectively.All these studies have demonstrated that mollusks have different Fads with various desaturation abilities.Additionally, it has been found that the variable marine environment, particularly the availability of LC-PUFA-rich diets, has a great effect on the fatty acid profiles and biosynthetic potential of marine mollusks [16][17][18].Specifically, the expression of genes involved in the LC-PUFA synthesis of marine mollusks is affected by microalgae diets, as the fatty acid contents of microalgae vary.For example, feeding with Chlorella sp. can significantly upregulate the Fads gene expression of the manila clam (Ruditapes philippinarum) [19].
The dwarf surf clam (Mulinia lateralis, 1822), a small filter-feeding bivalve mollusk, is predominantly distributed in estuarine and intertidal zones along the Gulf of Mexico, the West Indies, and the Atlantic coast [20].With a short generation cycle, rapid reproduction, strong environmental adaptability, and ease of artificial breeding, the dwarf surf clam is a potential model organism for bivalves [21,22].At present, we have successfully established an artificial breeding system for M. lateralis, and investigated the effects of different microalgae diets on its development and growth performance [23].The microalgae species with which it is typically fed, including Chlorella pyrenoidesa (Cp), Platymonas helgolandica (Ph), and other several types of microalgae, can be taken up by spats of M. lateralis.There are significant differences in the growth of spat fed with these microalgae.It has also been demonstrated that differences in the nutrient contents of these microalgae result in differences in the growth of spats [24].Recently, we have also found that the varied fatty acid contents of the microalgae diets lead to differences in the fatty acid composition of adult tissues in M. lateralis.However, the composition and function of Fads genes involved in LC-PUFA biosynthesis in M. lateralis and the influence of diet on their expression are still unknown.Moreover, whether M. lateralis, as a potential model organism for bivalves, has the biosynthesis activity of LC-PUFAs, especially with respect to EPA, DHA, and ARA, is still unknown.
Therefore, in this study, we identified the Fads of M. lateralis and analyzed the differential expression patterns of Fads among various tissues and developmental stages.We also investigated the effects of different unialgal diets on Fads expression in M. lateralis.Moreover, we conducted heterologous expression of Fads in yeast to explore their desaturation activity.These findings will contribute to a better understanding of Fads genes and LC-PUFA synthesis in M. lateralis, and may potentially be useful for other mollusks.

Gene Identification and Sequence Analysis of Fads in M. lateralis
To identify the Fads genes of M. lateralis, the genome database of M. lateralis was searched using 28 Fads amino acid sequences from 11 representative species, including mammals (Homo sapiens and Mus musculus), fish (Danino rerio, Salmo salar, and Oncorhynchus mykiss), and other mollusks (Mercenaria mercenaria, R. philippinarum, S. constricta, Crassostrea gigas, Patinopecten yessoensis, Chlamys farreri, C. nobilis, Aplysia californica, H. discus hannai, O. vulgaris, and S. officinalis).This search was conducted via the TBLASTN algorithm with an E-value cutoff of 1E-05.The orthologous Fads amino acid sequences were downloaded from the NCBI database.Then, the hit target sequences were used to search against a transcriptome database of M. lateralis and sequences that obtained hits with an E-value threshold of 1E-05 were regarded as candidate Fads.To validate these candidates, a BLASTP search against the NCBI non-redundant protein sequence database was performed.To guarantee the completeness and accuracy of the Fads sequences, the ORF finder tool was utilized to predict the nucleotide sequence, and the amino acid sequence was submitted to the SMART database for Fads domain verification.A genetic structure diagram of Fads was generated using the dwarf surf clams' general feature format (GFF) gene annotation file.A conserved domain analysis of M. lateralis Fads sequences was conducted using DNAMAN7.0 [25].

Phylogenetic Analysis of Fads
Fads protein sequences from M. lateralis and representative mammals, fish, and other mollusks (mentioned in Section 2.1) were chosen for phylogenetic analysis.ClustalW2.1 was employed for multiple Fads protein sequence alignment, and MEGA7.0 [26] was used for phylogenetic analysis using the maximum likelihood (ML) method with a bootstrap value of 5000 to ensure the reliability of the constructed evolutionary tree.

Spatiotemporal Expression of Fads in M. lateralis
According to RNA-seq datasets of M. lateralis obtained in our laboratory, the expression profiles of MulFads were analyzed.The expression level was described by transcripts per kilobase per million mapped reads (TPM) from the RNA-seq datasets, including 11 developmental stages (egg, zygote, 2-4 cells, 8-16 cells, multicellular, blastula, gastrula, trochophore, D-shaped larvae, umbo larvae, and juvenile) and 4 adult tissues (mantle, foot, digestive gland, and gonad).The samples representing various developmental stages were obtained through artificial fertilization from a cohort of fifty mature adults, followed by larval cultivation.Additionally, various tissues from three distinct adult individuals were selected for RNA extraction and subsequent transcriptomic sequencing.

Expression Analysis of Fads in M. lateralis under Two Unialgal Diets
The transcriptomic databases of M. lateralis in response to two unialgal diets, C. pyrenoidesa (Cp) and P. helgolandica (Ph), were constructed (unpublished data) and the expression levels of MulFads were detected.Specifically, the feeding trials were conducted with three-monthold M. lateralis over a period of two weeks.For each trial, 20 individuals were housed in an aquarium tank measuring 30 cm × 40 cm × 35 cm and were fed with CP or Ph twice daily, at 10:00 a.m. and 10:00 p.m.The water temperature in all tanks was consistently maintained at 22 ± 1 • C, with a salinity level of 27 ± 1 ppt.After the two-week period, three individuals from each feeding group were randomly selected for sample collection.Four types of tissues (mantle, foot, digestive gland, and gonad) were harvested for RNA extraction and subsequent transcriptomic sequencing.The expression levels of MulFads in response to Cp and Ph diets were described by transcripts per kilobase per million mapped reads (TPM) from these RNA-seq datasets.

Functional Characterization via Heterologous Expressions of M. lateralis Fads Open Reading Frame in Yeasts
The MulFadsA gene was chosen for constructing the recombinant plasmid pYES2-MulFadsA using the pYES2.0yeast expression vector (Coolaber, Beijing, China), which was synthesized by Sangon Biotech Shanghai Co., Ltd (Sangon Biotech, Shanhai, China).This recombinant plasmid was transformed into Saccharomyces cerevisiae INVSc1 competent cells (Coolaber, Beijing, China), and yeast harboring pYES2-MulFadsA was selected on SCMMuracil minimal medium (Coolaber, Beijing, China).The selected yeast was subsequently cultivated in SD-U liquid medium (Coolaber, Beijing, China) until an OD400 of 1.0 was reached, followed by concentration and resuspension in 2 mL SG-U induction medium (Coolaber, Beijing, China).The concentrated yeast was added to 100 mL of SG-U induction medium to adjust the OD400 to 0.4.
To investigate the desaturation activity of Fads in the synthesis of LC-PUFAs in the clam, yeast transformed with pYES2-MulFadsA was cultured with exogenous supplementation 18:3n-3 (Aladdin, Shanghai, China) and 18:2n-6 (Solarbio, Beijing, China) for the ∆6 desaturase substrate fatty acids, 20:3n-3 (Aladdin, Shanghai, China) for the ∆8 desaturase substrate fatty acids, and 20:3n-6 (Aladdin, Shanghai, China) for the ∆5 desaturase substrate fatty acids, at a final concentration of 0.5 mM.The yeast culture was induced for 72 h, after which the yeast cells were centrifuged and freeze-dried for subsequent fatty acid analysis.

Fatty Acid Analysis Using GC-MS
The analysis of fatty acids in yeast powder was performed by Qingdao Kechuang Quality Testing Co., Ltd.(Qingdao, China) using a Thermo (race1310 ISQ, Thermo, Waltham, MA, USA) gas chromatograph.The conversion rate of fatty acid substrate to desaturated product was determined using the equation [product area/(product area + substrate area)] × 100%.

Statistical Analyses
Statistical analyses were conducted using SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA).Data were analyzed with one-way ANOVA and Newman-Keuls tests.A p-value of <0.05 was deemed statistically significant.

Gene Identification and Sequence Analysis of Fads in M. lateralis
Two Fads genes were identified based on the transcriptome and genome of M. lateralis; these were named MulFadsA and MulFadsB.As shown in Table 1, the cDNA lengths of MulFadsA and MulFadsB were 1175 bp and 4510 bp, encoding 366 amino acids and 438 amino acids, respectively.Both MulFadsA and MulFadsB were composed of 10 exons and 9 introns (Figure 1A).Multiple sequence alignment of Fads protein sequences from M. lateralis and other species revealed that the aligned sequences encompass three conserved histidine-rich motifs (H***H, H**HH, and Q**HH), a putative cytochrome b5-like domain, and a conserved N-terminal heme-binding domain (HPGG) (Figure 1B).Meanwhile, the deduced amino acid sequences of MulFadsA and MulFadsB exhibited 63.76% homology to Fads of the hard clam (M.mercenaria), razor clam (S. constricta), Pacific oyster (C.gigas), and Yesso scallop (P.yessoensis).

Phylogenetic Analysis
An ML phylogenetic tree was constructed using the amino acid sequences of MulFads and Fads members from other species.All the mollusk front-end desaturases were classified into two distinct clades identified as clade A and clade B (Figure 2) according to the nomenclature proposed by Surm et al. [27,28] and Ramos-Llorens et al. [29].Specifically, clade A contained all functionally characterized ∆5 Fads and Fads-like sequences of other mollusks (not functionally characterized, denoted with *).MulFadsA, together with another two Fads from R. philippinarum and M. mercenaria, were clustered into this clade.Clade B contained more Fads-like sequences of mollusks that were not functionally characterized, except for ∆6 Fads sequences functionally characterized from S. constricta, and MulFadsB were clustered into this clade.

Phylogenetic Analysis
An ML phylogenetic tree was constructed using the amino acid sequences of MulFads and Fads members from other species.All the mollusk front-end desaturases were classified into two distinct clades identified as clade A and clade B (Figure 2) according to the nomenclature proposed by Surm et al. [27,28] and Ramos-Llorens et al. [29].Specifically, clade A contained all functionally characterized Δ5 Fads and Fads-like sequences of other mollusks (not functionally characterized, denoted with *).MulFadsA, together with another two Fads from R. philippinarum and M. mercenaria, were clustered

Spatiotemporal Expression of MulFads
RNA-seq datasets for different developmental stages and adult tissues of M. lateralis were analyzed to detect the spatiotemporal expression patterns of MulFadsA and MulFadsB genes (Figure 3).Specifically, the expression of MulFadsA in the early developmental periods was low, with TPM below X, and then gradually increased from blastula stage (Figure 3A).MulFadsB showed the opposite expression pattern, with high expression levels in eggs and during early developmental periods and low expression levels from the trochophore stage (Figure 3A).During complete developmental stages, MulFadsA showed lower expression levels compared with MulFadsB.In adult tissues of M. lateralis, the overall expression level of MulFadsA was higher than that of MulFadsB.MulFadsA had significantly higher expression levels in the mantle, gonad, and foot compared to the digestive gland (Figure 3B).The expression level of MulFadsB was highest in the gonad, followed by the mantle, digestive gland, and foot (Figure 3B).

Spatiotemporal Expression of MulFads
RNA-seq datasets for different developmental stages and adult tissues of M. lateralis were analyzed to detect the spatiotemporal expression patterns of MulFadsA and MulFadsB genes (Figure 3).Specifically, the expression of MulFadsA in the early developmental periods was low, with TPM below X, and then gradually increased from blastula stage (Figure 3A).MulFadsB showed the opposite expression pattern, with high expression levels in eggs and during early developmental periods and low expression levels from the trochophore stage (Figure 3A).During complete developmental stages, MulFadsA showed lower expression levels compared with MulFadsB.In adult tissues of M. lateralis, the overall expression level of MulFadsA was higher than that of MulFadsB.MulFadsA had significantly higher expression levels in the mantle, gonad, and foot compared to the digestive gland (Figure 3B).The expression level of MulFadsB was highest in the gonad, followed by the mantle, digestive gland, and foot (Figure 3B).

Effects of Different Unialgal Diets on Fads Gene Expression in M. lateralis
To examine the expression patterns of MulFads genes in response to different unialg diets, RNA-seq datasets of four tissues from M. lateralis under two types of unialgal diet including C. pyrenoidosa (Cp) and P. helgolandica (Ph) with a distinct LC-PUFA compositio [23], were used for analysis.According to our previous research [24] and a recent stud (unpublished), the Cp diet has a positive effect on the growth and fatty acid compositio of M. lateralis as compared to the Ph diet.The feeding effects of the two microalgae die on the expressions of MulFads are shown in Figure 4. Specifically, MulFadsA expressio levels were significantly higher in the mantle, gonad, and foot of the Cp group compare to those in the Ph group, while no significant difference was found for the digestive glan between the two groups.MulFadsB showed a similar expression pattern between the tw groups but with a notably lower expression level than MulFadsA (Figure 4).

Effects of Different Unialgal Diets on Fads Gene Expression in M. lateralis
To examine the expression patterns of MulFads genes in response to different unialgal diets, RNA-seq datasets of four tissues from M. lateralis under two types of unialgal diets, including C. pyrenoidosa (Cp) and P. helgolandica (Ph) with a distinct LC-PUFA composition [23], were used for analysis.According to our previous research [24] and a recent study (unpublished), the Cp diet has a positive effect on the growth and fatty acid composition of M. lateralis as compared to the Ph diet.The feeding effects of the two microalgae diets on the expressions of MulFads are shown in Figure 4. Specifically, MulFadsA expression levels were significantly higher in the mantle, gonad, and foot of the Cp group compared to those in the Ph group, while no significant difference was found for the digestive gland between the two groups.MulFadsB showed a similar expression pattern between the two groups but with a notably lower expression level than MulFadsA (Figure 4).

Discussion
The Fads gene encodes the crucial rate-limiting enzyme in the synthesis of LC-PUFAs through desaturation, which is a front-end desaturase [33].Typical front-end desaturases of animals in the biosynthesis of LC-PUFAs include ∆4, ∆5, ∆6, and ∆8 fatty acid desaturases.Various Fads genes demonstrating a range of desaturation activity, such as ∆4, ∆5, ∆6, ∆8, ∆5/6, and ∆6/8, have been identified in mammals and teleost fish [30,[34][35][36][37][38].In addition, a series of studies have demonstrated that Fads with ∆5, ∆6, or ∆8 activity appear to be widely distributed among mollusks, including cephalopods [12,39], gastropods [13], and bivalves [14,15,19,40].Generally, it is accepted that mollusks possess the ability to facilitate PUFA biosynthesis [41].However, such capability appears to vary among species depending on the complement of rate-limiting enzymes involved in this process.The expanding availability of genomic data from various invertebrate taxa now enables detailed investigations of the LC-PUFA biosynthetic pathways at a molecular level, focusing on the characterization of crucial biosynthesizing enzymes.In our study, with the help of the whole genomic and transcriptomic database of M. lateralis, we systematically conducted the identification and phylogenetic analysis of two MulFads genes, examined their spatiotemporal expression profiles, and investigated their responses to two types of unialgal diets.Then, we determined the desaturation activity of MulFadsA enzymes via a yeast heterologous expression assay, which confirmed that ∆5 desaturation activity was characterized.
Two Fads genes, MulFadsA and MulFadsB, were successfully identified in the genome of M. lateralis.Both MulFadsA and MulFadsB showed high homology with Fads sequences from other representative vertebrates and invertebrates and typically have the structural characteristics of front-end desaturases, including a cytochrome b5-like domain, three typical histidine boxes (H***H, H**HH, and Q**HH), and a heme-binding motif, i.e., HPGG (Figure 1B), indicating the conserved functional domains of Fads during evolution [39] and the potential desaturation activity of M. lateralis Fads.In addition, the phylogenetic analysis revealed that MulFadsA was clustered together with ∆5 functionally characterized Fads from other marine mollusks, indicating the potential corresponding ∆5 Fad activity of MulFadsA (Figure 2).Meanwhile, MulFadsB was clustered together with ∆6 Fads from other marine mollusks (Figure 2), which is consistent with the phylogenetic results with respect to Fads from R. philippinarum [19] and other mollusks [27].These results indicate that both ∆5 and ∆6 Fads might exist in M. lateralis and other marine mollusks [14,19].
Furthermore, spatial and temporal expression analyses of MulFadsA and MulFadsB revealed different expression trends across developmental stages and in adult tissues.During the developmental process of M. lateralis, MulFadsB was expressed in eggs and subsequently primarily expressed in the early embryonic developmental stages (Figure 3A).This result suggests that MulFadsB transcripts in the eggs and early larvae are maternally derived rather than endogenously expressed [42].A similar maternal expression pattern of Fads has also been reported in Danio rerio [42], Common Carp Cyprinus carpio [38], and noble scallop C. nobilis [40].While the MulFadsA transcript was low at the early developmental stage, it increased dramatically from blastula stages to juvenile stages, indicating that ∆5 Fads may be a critical enzyme in the biosynthesis of PUFAs that are needed in normal development during the early stages in mollusks [40] and other animals [42].This result indicates that the maternal MulFadsB transcript mainly regulates the metabolism of fatty acids and participates in the synthesis of HUFAs in the early embryonic developmental stages.After the blastula stage, as the lower maternal MulFadsB transcripts and higher MulFadsA transcripts, both MulFadsA and MulFadsB are involved in the synthesis of PUFAs.Based on our preliminary observations in this study, we hypothesize that these two genes may be involved in LC-PUFA synthesis and may be functional at different stages of M. lateralis embryonic development.
The tissue distributions show that both MulFadsA and MulFadsB exhibited significantly high expression levels in the gonad, and particularly high expression of MulFadsA was found in the mantle and foot (Figure 3B).Similar results with respect to tissue distributions of ∆5 Fads have also been observed in other marine mollusks [12,14,43].For example, O. vulgaris Fads exhibit higher expression in the digestive gland and gonad [12].∆5 Fads in S. constricta also exhibit high expression levels in the gonad [14].The expressions of MulFadsA and MulFadsB in the digestive gland were lower than those in other organs, which may indicate that different tissues have different requirements for long-chain fatty acid metabolism.Furthermore, the expression of MulFadsA in most tissues was higher than that of MulFadsB; it is speculated that MulFadsA plays a stronger role and is more efficient in the process of HUFA synthesis than MulFadsB in adult M. lateralis.
To provide insights into the function of MulFads in response to a microalgae diet, the expression of MulFads was investigated in four adult tissues under two different unialgal diets.Multiple factors regulate Fads gene expression, with diet being a significant factor [44].In general, in most tissues of M. lateralis, the expressions of MulFadsA and MulFadsB were significantly upregulated in the Cp group compared to those in the Ph group (Figure 4).Specifically, feeding with C. pyrenoidosa has a more pronounced effect on MulFadsA expression in the mantle, gonad, and foot.This is similar to the findings reported by Wu et al. [19] in respect of the Fads of R. philippinarum.The expression levels of Fads in R. philippinarum fed with Chlorella sp. were significantly higher compared to those fed with Chaetoceros calcitrans and Isochrysis galbana.This result may be related to the lower content of LC-PUFAs in Chlorella sp., especially EPA and ARA, compared to P. helgolandica [40], resulting in upregulated expression of desaturase genes to promote the rapid synthesis of LC-PUFAs to meet the needs of growth and development.Similar findings have also been observed in crustaceans, such as Cherax quadricarinatus [45] and Scylla paramamosain [46], and in teleost fishes, such as Siganus canaliculatus [47], S. salar [48], and O. mykiss [49]; these studies showed that the expression levels of Fads were significantly upregulated in response to dietary DHA and EPA deficiency.
In terms of functional verification, our study shows that the expression products of MulFadsA can exhibit ∆5 Fads activity when using C20:3n-6 as the substrate, which is consistent with our phylogenetic results (Figure 2), indicating that MulFadsA clusters together with ∆5 Fads from other mollusks.Notably, we observed no desaturation when ∆6 substrates (C18:2n-6 and C18:3n-3) or ∆8 substrates (C20:3n-3) were used, indicating that this enzyme lacks ∆6 or ∆8 desaturase activities (refer to Figure 5 and Table 2).These findings imply that MulFadsA, with its ∆5 desaturase function, may play a crucial role in converting 20:3n-6 into ARA.In previous studies, Octopus Fads was the first enzyme to be shown to exhibit ∆5 desaturation activity in marine mollusks [12].Subsequently, ∆5 Fads desaturation activity was verified in abalone (H.discus hannai) [13], noble scallop (C.nobilis) [40], common cuttlefish (S. officinalis) [39], razor clam (S. constricta) [14], and pearl oyster (Pinctada fucata martensii) [50].In our study, the conversion rate of M. lateralis MulFadsA from C20:3n-6 to C20:4n-6 was 26% (Table 2), which was higher than that of S. constricta (10.41-13.71%)[14], H. discus hannai (about 15%) [13], and C. nobilis (about 17%) [40], but lower than that of S. officinalis [39] and O. vulgaris [12] (up to 39%).This may be due to the various desaturation activities among different mollusks.In addition, MulFadsB was clustered with ∆6 Fads that have functional properties in S. constricta, suggesting that MulFadsB may exhibit ∆6 desaturation activity (Figure 2); this needs to be further verified.Fads enzymes are pivotal in regulating the unsaturation levels of PUFAs, facilitating the initial steps of desaturation [10].Specifically, ∆6 and ∆5 desaturases are essential for the synthesis of ARA from linoleic acid (LA, C18:2n-6), and eicosapentaenoic acid (EPA, C20:5n-3) from linolenic acid (LNA, C18:3n-3), respectively.Combined with the results of the functional verification of MulFadsA and the phylogenetic tree of MulFadsA and MulFadsB, it is speculated that MulFadsB may potentially exhibit ∆6 activity.As a result, it is speculated that M. lateralis might have the capacity to biosynthesize ARA and EPA/DHA from LA and ALA substrates, respectively.In future studies, we will select more diverse substrates to further verify the desaturation activity of MulFadsA and MulFadsB.We may also examine the activity of fatty acid elongases involved in LC-PUFA biosynthesis, thus allowing a better understanding of the LC-PUFA synthesis ability of M. lateralis.

Conclusions
In our comprehensive study on M. lateralis Fads, encompassing both MulFadsA and MulFadsB, we explored the expression patterns during embryonic development and across various tissues of M. lateralis.This investigation offers valuable insights into the mechanisms of LC-PUFA biosynthesis in mollusks.Our findings reveal that MulFadsA exhibits lower expression levels in the early stages of embryonic development, with an increase observed in the mid to late stages.Conversely, MulFadsB displays a contrasting pattern of expression.Additionally, when M. lateralis was fed with C. pyrenoidosa, a significant upregulation of MulFads expression was observed in the mantle, gonad, and foot, suggesting that the dietary absence of EPA and ARA triggers the upregulation of MulFads.The functional characterization of MulFadsA highlights the potential of the model bivalve M. lateralis to endogenously synthesize LC-PUFAs, and to at least convert C20:3(n-6) into ARA.This capability is not only pivotal in understanding the molecular evolution of the Fads family but also underscores the significance of LC-PUFA biosynthesis from fatty acid substrates in mollusks and other invertebrates.This study sheds light on the regulatory mechanisms of Fads in M. lateralis, contributing to our broader understanding of fatty acid metabolism in marine organisms.

Figure 1 .
Figure 1.Sequence analysis of Fads.(A) Gene structure of the MulFadsA and MulFadsB of M. lateralis.The blue boxes and polylines indicate the exons and introns, respectively.The numbers in the boxes and under the line indicate the lengths of exons and introns, respectively.(B) Multiple sequence alignment of Fads sequence in M. lateralis.The cytochrome-b5 like domain is underlined with a solid line, the heme-binding motif of HPGG is highlighted with a short bold line, and the three histidine boxes are denoted with frames.An asterisk indicates any amino acid in the domain.

Figure 1 .
Figure 1.Sequence analysis of Fads.(A) Gene structure of the MulFadsA and MulFadsB of M. lateralis.The blue boxes and polylines indicate the exons and introns, respectively.The numbers in the boxes and under the line indicate the lengths of exons and introns, respectively.(B) Multiple sequence alignment of Fads sequence in M. lateralis.The cytochrome-b5 like domain is underlined with a solid line, the heme-binding motif of HPGG is highlighted with a short bold line, and the three histidine boxes are denoted with frames.An asterisk indicates any amino acid in the domain.

Figure 2 .
Figure 2. Phylogenetic tree of Fads proteins from M. lateralis and other representative species.The tree was constructed using MEGA7.0 via the maximum-likelihood (ML) method with a bootstrap value of 2000.The numbers represent the frequencies with which the tree topology presented was replicated.The scale bar indicates a branch length of 0.05.An asterisk indicates Fads sequences of M. mercenaria, R. philippinarum, C. gigas, P. yessoensis, C. farreri, and A. californica that have not been functionally characterized.The black solid triangle represents the sequence validated in this experiment.

Figure 2 .
Figure 2. Phylogenetic tree of Fads proteins from M. lateralis and other representative species.The tree was constructed using MEGA7.0 via the maximum-likelihood (ML) method with a bootstrap value of 2000.The numbers represent the frequencies with which the tree topology presented was replicated.The scale bar indicates a branch length of 0.05.An asterisk indicates Fads sequences of M. mercenaria, R. philippinarum, C. gigas, P. yessoensis, C. farreri, and A. californica that have not been functionally characterized.The black solid triangle represents the sequence validated in this experiment.

Figure 3 .
Figure 3. Expression pattern of MulFads from M. lateralis.(A) Expressions of MulFadsA an MulFadsB in M. lateralis at 11 developmental stages based on the transcripts per kilobase per millio mapped reads (TPM).(B) Expressions of MulFadsA and MulFadsB in M. lateralis adult tissue SPSS28.0 was used for one-way ANOVA analysis of the data, and the Duncan method was used test the significance of the difference between the values of each group.Different letters indica significant differences among experimental groups (p < 0.05).

Figure 3 .
Figure 3. Expression pattern of MulFads from M. lateralis.(A) Expressions of MulFadsA and MulFadsB in M. lateralis at 11 developmental stages based on the transcripts per kilobase per million mapped reads (TPM).(B) Expressions of MulFadsA and MulFadsB in M. lateralis adult tissues.SPSS28.0 was used for one-way ANOVA analysis of the data, and the Duncan method was used to test the significance of the difference between the values of each group.Different letters indicate significant differences among experimental groups (p < 0.05).

Table 1 .
Characteristics of the Fads genes in M. lateralis.

Table 2 .
Functional characterization of M. lateralis MulFadsA in S. cerevisiae.

Table 2 .
Functional characterization of M. lateralis MulFadsA in S. cerevisiae.