Effects of Three Microalgal Diets Varying in LC-PUFA Composition on Growth, Fad , and Elovl Expressions, and Fatty Acid Proﬁles in Juvenile Razor Clam Sinonovacula constricta

: The razor clam Sinonovacula constricta is the ﬁrst marine mollusk demonstrated to possess the complete long-chain polyunsaturated fatty acids (LC-PUFA) biosynthetic pathway. This study explored the impact of different microalgae on growth, Fad and Elovl expressions, and fatty acid (FA) proﬁles in juvenile S. constricta . Results revealed that juveniles fed with Isochrysis galbana (rich in DHA) or Chaetoceros calcitrans (rich in EPA) consistently exhibited higher growth than those fed Chlorella sp. (rich in LA and ALA), underscoring the importance of dietary LC-PUFA in S. constricta ’s development. Expression of most Fad and Elovl in C. calcitrans and I. galbana -fed juveniles were initially up-regulated, then down-regulated, suggesting LC-PUFA demand for faster growth. Although Chlorella sp.-fed juveniles exhibited decreased mRNA levels for most genes, levels were notably higher lately compared to those fed C. calcitrans or I. galbana , hinting at potential LC-PUFA biosynthesis induction. FA proﬁles in S. constricta generally mirrored those in ingested microalgae, implying direct FA accumulation from diets. Some microalgal FA were absent in farmed S. constricta , while others emerged, indicating S. constricta ’s ability to selectively accumulate and synthesize FA. This study enhances the understanding of dietary FA metabolism in S. constricta , valuable for selecting appropriate microalgae in its farming practices.

The razor clam Sinonovacula constricta (Lamarck 1818), also known as the Asian hard clam, is a bivalve mollusk native to the western Pacific coast [20].It holds significant economic and nutritional value, with both farmed and wild-caught razor clams being harvested.In 2016, global production of S. constricta exceeded 823,000 tons, with a total value of USD 1.3 billion [21].This bivalve species is particularly rich in nutrients [22], notably EPA and DHA, each accounting for 10% of the total FA at approximately 3.5 µg•mg −1 (dry weight, DW) [23].Our recent research demonstrated that S. constricta possesses a complete LC-PUFA biosynthetic pathway known as the Sprecher pathway [24].This was confirmed through the functional characterization of key enzymes, including ∆5 Fad_a, ∆5 Fad_b, ∆6 Fad [25], and Elovl2/5, Elovl4_a, Elovl4_b, Elovl_c [26].Importantly, to date, S. constricta is the first and only marine mollusk demonstrated to possess the complete LC-PUFA biosynthetic pathway.As a result, S. constricta serves as a valuable model organism for further investigating the regulation of LC-PUFA biosynthesis in marine mollusks.

Seawater Preparation
The natural seawater (~32 practical salinity units, PSU) was initially passed through a 1 m thick sand filter with a diameter of less than 1 mm.It was then allowed to settle for 2 d and subsequently passed through a 75 µm nylon cribrose sieve before use.The experimental seawater (23 PSU) was prepared by blending natural seawater with freshwater.For microalgae cultivation, the experimental seawater underwent additional filtration (0.45 µm), autoclaving (115 • C, 20 min), and enrichment with a nutritional medium composed of 3 mg•L −1 Fe-citrate•5 H 2 O, 10 mg•L −1 KH 2 PO 4 , 100 mg•L −1 KNO 3 , 0.05 µg•L −1 VB 12 and 6 µg•L −1 VB 1 .Specifically, the culture of C. calcitrans required an additional supplement of 20 mg•L −1 sodium metasilicate.For the cultivation of juvenile S. constricta, the experimental seawater underwent further treatment with hypochlorite sterilization.

Cultivation of Microalgae
The three microalgae species with distinct LC-PUFA compositions (Table 1), including I. galbana, C. calcitrans, and Chlorella sp. were sourced from the Microalgal Culture Laboratory at Ningbo University, Ningbo, China.Initially, the microalgae were cultured in 5 L Erlenmeyer flasks in a microalgal culture chamber at 20 ± 1 • C without aeration.Subsequently, the exponentially growing microalgae were inoculated into 50-L transparent white barrels (at a ratio of 1:1000, v/v) and cultivated in an open greenhouse with continuous aeration at room temperature (~20-29 • C).The microalgae in the stationary phase were then harvested for use as feed for juvenile S. constricta.SFA: total saturated FA; MUFA: total monounsaturated FA; PUFA: total polyunsaturated FA (double bonds ≥ 2); TFA: total FA; "-": not detected.

Cultivation of Juvenile S. constricta
Juvenile S. constricta were sourced from Fujian Baozhi Aquatic Science and Technology Co., Ltd., Zhangzhou, China.Initially, juveniles of similar size (9.35 ± 0.49 mm in shell length) were subjected to a 3 d fasting period to acclimate and empty their stomachs.Subsequently, 900 individuals were evenly distributed on the bottom of nine pre-prepared barrels, with 100 individuals per barrel to enhance sampling randomness.Each barrel had a volume of 500 L and a bottom diameter of 100 cm.They were pre-filled with 5 cm thick fresh sea mud (previously filtered through a 75 µm cribrose nylon sieve) and filled with seawater to a depth of 45 ± 1 cm.Given S. constricta's benthic and burrowing behavior, sea mud was provided.The nine barrels were grouped into three sets of three barrels, each allocated to supply Chlorella sp., C. calcitrans, and I. galbana, respectively.The final microalgae concentration was maintained at ~40-60 cells•µL −1 , a level empirically determined to satisfy the feeding requirements of juvenile S. constricta.Microalgae concentrations were calculated using a hemocytometer.Juveniles were fed twice daily at 7:00 a.m. and 5:00 p.m. to ensure a continuous supply of food in the water.Continuous aeration was provided to ensure sufficient oxygen levels in the water.Notably, before the second feeding each day, half of the cultured seawater volume was renewed to maintain water quality.The sea mud was replaced every 8 d to prevent deterioration due to fecal deposition in the relatively closed barrels.The seawater depth was consistently maintained at 45 ± 1 cm.The entire experiment was performed in an open greenhouse at room temperature (~20-29 • C).The experiment lasted for 24 d.

Sampling and Processing
Sampling was conducted every 8 d, coinciding with the renewal of sea mud.It is worth noting that before each sampling event, the juveniles underwent a 1 d fasting period to facilitate digestion of ingested microalgae.After taking growth measurements of all juveniles, six individuals were randomly selected from each barrel and subjected to the following steps.
The shells were carefully removed using medical dissecting scissors and forceps.The dissected tissue was divided into two primary parts.The first part, referred to as the visceral mass, comprises the intestine, digestive glands, and gonads.These tissues are the primary sites for LC-PUFA synthesis in S. constricta [25,26].The second part of the sample was designed as muscular tissues, encompassing the foot, siphons, labial palps, and gills.Subsequently, the visceral mass from six individuals in each barrel was combined, homogenized, and utilized for the analysis of Fad and Elovl expression.Additionally, the muscular tissues from six individuals in each barrel were pooled, homogenized, and used to analyze the FA composition.Furthermore, the FA composition of the visceral mass of juvenile S. constricta at both the beginning and end of the experiment was also analyzed.

Analysis of Fad and Elovl Expressions by Quantitative Real-Time PCR (qRT-PCR)
RNA was extracted from the homogenized visceral mass using the MiniBEST Universal RNA Extraction Kit (Takara, Shiga, Japan).Subsequently, RNA quality was assessed using a 1% agarose gel, and the quantity of RNA was determined using NanoDrop ® ND-1000 (NanoDrop, Waltham, MA, USA).To prepare the cDNA template for qRT-PCR, 1 µg of RNA was transcribed into cDNA using the PrimeScript RT Master Mix Kit (Perfect Real Time) (Takara, Shiga, Japan).The qRT-PCR was conducted using a quantitative thermal cycler (Mastercycler ep realplex) (Eppendorf, Hamburg, Germany) with SYBR Premix Ex Taq (Tli RNaseH Plus) (Takara, Shiga, Japan) and the primers listed in Table 2. Briefly, after an initial denaturation step at 95 • C for 30 s, the amplifications were carried out with 35 cycles at a melting temperature of 95 • C for 5 s, an annealing temperature of 55 • C for 15 s, and an extension temperature of 72 • C for 20 s, followed by a melting curve analysis from 58 to 95 • C with an increment of 1.85 • C•min −1 .Finally, the relative expressions of Fad and Elovl were calculated with the housekeeping gene of β-actin using the 2 −∆∆CT method [28].The expression of each gene was normalized to that of the initial sample.
Fishes 2023, 8, 484 5 of 15 2.6.Analysis of FA Profiles by Gas Chromatography-Mass Spectrometry (GC-MS) The preparation of fatty acid methyl esters (FAMEs) was conducted following the procedure outlined by Xu et al. [18].Initially, the homogenized tissues were lyophilized and subsequently ground to a fine powder.Next, 30 mg of the powder sample was dissolved in 4 mL CHCl 3 /CH 3 OH/H 2 O (1:2:0.8, v/v/v) for crude lipid extraction.The solution was vigorously shaken for 10 min.To this mixture, 0.3 mL of HCl (8%, w/v) in methanol/water (in a ratio of 85:15, v/v), 1.5 mL of methanol, 0.2 mL of toluene, and 15 µL of nonadecanoic acid 19:0 (1 µg•µL −1 , serving as the internal standard) (Cayman Chemicals, Ann Arbor, MI, USA) were added.This mixture was then incubated for 1 h at 100 • C to yield FAMEs. Subsequently, the FAMEs were extracted into 1 mL hexane-chloroform (in a ratio of 4:1, v/v), dried under nitrogen, re-diluted into 1 mL of chromatography-grade hexane (filtered through a 0.22 µm ultrafiltration membrane), and loaded onto a GC-MS platform (7890B/7000C) (Agilent, Santa Clara, CA, USA).
The GC-MS analysis was conducted using a CD-2560 capillary column (100 m × 250 µm × 0.2 µm, CNW, Frankfurt, Germany).Helium, of high purity, served as the carrier gas at a constant flow rate of 0.81 mL•min −1 .A 1 µL sample was injected in split-less mode.The initial precolumn pressure was 30.36 psi.The injector temperature was set at 250 • C. Initially, the GC oven temperature was maintained at 140 • C for 5 min and then increased to 240 • C over 20 min at a rate of 4 • C•min −1 .The MS ion source temperature was 230 • C, the quadrupole temperature was 150 • C, and the transmission line temperature was 255 • C. The mass spectrometer scanned from 40 to 600 m/z, and the collision energy was set at 70 eV.
FA were identified by matching the mass spectral data with the NIST 14.L mass spectral database, using characteristic mass spectrometry fragments and the relative retention times of commercially available FA standards.The composition of FA content was calculated using the following formula: (area of specific FA/area of the internal standard 19:0) × 15 µg of 19:0/weight (mg) of the lyophilized sample.

Statistical Analyses
Statistical analyses of S. constricta growth, relative expressions of Fad and Elovl, and FA composition were conducted using one-way ANOVA, followed by multiple pairwise comparisons using Tukey's test (SPSS 22.0, Stanford, CA, USA).When the data did not meet the assumptions of normality (Kolmogorov-Smirnov test) or homoscedasticity (Levene's test), log10 transformation was employed.Differences were considered statistically significant when p < 0.05.
To provide an overview of the changes in FA composition in S. constricta throughout the experiment, FA were treated as dependent data and subjected to the projection to latent structures with discriminant analysis (PLS-DA) using SIMCA-P+ software package (version 14.1, Umetrics; Umea, Sweden).Additionally, to further explore the correlation of FA composition between S. constricta and microalgal diets, FA were treated as dependent data and analyzed by ORIGINPRO ® 2021 (OriginLab, Northampton, MA, USA) to generate a Correlation Plot.

Growth of Juvenile S. constricta
As depicted in Figure 1, the growth of juvenile S. constricta fed with Chlorella sp.consistently showed significantly lower values compared to those fed with C. calcitrans or I. galbana throughout the experiment.Specifically, at the end of the experiment, the shell length of the juveniles fed with Chlorella sp. was 13.99 ± 1.41 mm, in contrast to their initial measurement of 9.35 ± 0.49 mm.In contrast, the shell length of the juveniles fed with C. calcitrans and I. galbana reached 19.42 ± 1.49 mm and 20.29 ± 0.95 mm, respectively.Notably, no statistically significant differences in growth were observed between the juveniles fed with I. galbana and those fed with C. calcitrans throughout the course of the experiment.
length of the juveniles fed with Chlorella sp. was 13.99 ± 1.41 mm, in contrast to their initial measurement of 9.35 ± 0.49 mm.In contrast, the shell length of the juveniles fed with C. calcitrans and I. galbana reached 19.42 ± 1.49 mm and 20.29 ± 0.95 mm, respectively.Notably, no statistically significant differences in growth were observed between the juveniles fed with I. galbana and those fed with C. calcitrans throughout the course of the experiment.

Fad and Elovl Expressions in Visceral Mass of Juvenile S. constricta
Figure 2A-G depicts the expressions of Fad and Elovl in the visceral mass of juvenile S. constricta throughout the experiment.When fed with Chlorella sp., the expression of Δ5 Fad_a significantly increased on the 8th d compared to the initial sample, followed by a significant decrease on the 16th d, and no significant difference on the 24th d.Similarly, the expressions of Δ5 Fad_b and Elovl2/5 exhibited no significant differences on the 8th d compared to the initial sample, then significantly decreased on the 16th d, and exhibited a notable increase on the 24th d.In contrast, the expressions of Δ6 Fad and Elovl4_b remained relatively stable throughout the experiment, while Elovl4_a and Elovl_c showed no significant differences on the 8th d compared to the initial sample but decreased significantly on the 16th and 24th d.When fed with C. calcitrans, the expressions of Δ5 Fad_a, Δ5 Fad_b, Δ6 Fad, Elovl2/5, and Elovl_c significantly increased on the 8th and 16th d compared to the initial sample, followed by a significant decrease on the 24th d.Conversely, the expressions of Elovl4_a and Elovl4_b showed no significant differences on the 8th and 16th d compared to the initial sample, followed by a significant decrease on the 24th d.Finally, when fed with I. galbana, the expressions of Δ5 Fad_a, Elovl2/5, and Elovl4_a significantly increased on the 8th d compared to the initial sample, followed by a significant decrease on the 16th and 24th d.However, the expressions of other genes showed no significant differences on the 8th d compared to the initial sample and then decreased significantly on the 16th and 24th d.

Fad and Elovl Expressions in Visceral Mass of Juvenile S. constricta
Figure 2A-G depicts the expressions of Fad and Elovl in the visceral mass of juvenile S. constricta throughout the experiment.When fed with Chlorella sp., the expression of ∆5 Fad_a significantly increased on the 8th d compared to the initial sample, followed by a significant decrease on the 16th d, and no significant difference on the 24th d.Similarly, the expressions of ∆5 Fad_b and Elovl2/5 exhibited no significant differences on the 8th d compared to the initial sample, then significantly decreased on the 16th d, and exhibited a notable increase on the 24th d.In contrast, the expressions of ∆6 Fad and Elovl4_b remained relatively stable throughout the experiment, while Elovl4_a and Elovl_c showed no significant differences on the 8th d compared to the initial sample but decreased significantly on the 16th and 24th d.When fed with C. calcitrans, the expressions of ∆5 Fad_a, ∆5 Fad_b, ∆6 Fad, Elovl2/5, and Elovl_c significantly increased on the 8th and 16th d compared to the initial sample, followed by a significant decrease on the 24th d.Conversely, the expressions of Elovl4_a and Elovl4_b showed no significant differences on the 8th and 16th d compared to the initial sample, followed by a significant decrease on the 24th d.Finally, when fed with I. galbana, the expressions of ∆5 Fad_a, Elovl2/5, and Elovl4_a significantly increased on the 8th d compared to the initial sample, followed by a significant decrease on the 16th and 24th d.However, the expressions of other genes showed no significant differences on the 8th d compared to the initial sample and then decreased significantly on the 16th and 24th d.
In terms of the difference in Fad and Elovl expressions at the same sampling time point: on the 8th d, no significant differences were observed in ∆5 Fad_a expression among all samples.However, the expressions of other genes were significantly or noticeably lower in the samples of juveniles fed with Chlorella sp.Additionally, there were no significant differences between the samples of juveniles fed with C. calcitrans and I. galbana, except for Elovl4_a, which showed significantly higher expression in the sample of juveniles fed with I. galbana.On the 16th d, the highest expressions of all Fad and Elovl genes were found in the sample of juveniles fed with C. calcitrans.There were no significant differences observed between the samples of juveniles fed with Chlorella sp. and I. galbana, except for ∆6 Fad and Elovl4_a, which showed significantly higher expressions in the sample of juveniles fed with Chlorella sp.compared to those fed with I. galbana.On the 24th d, it was observed that the expressions of ∆5 Fad_a, ∆6 Fad, Elovl2/5, and Elovl4_b were significantly higher or noticeably higher in the sample of juveniles fed with Chlorella sp.compared to those fed with C. calcitrans or I. galbana.However, no significant differences were found in the expressions of other genes among all the samples.The PLS-DA results for the FA composition in the muscular tissues of S. constricta over the experiment are shown in Figure 3A.A remarkable 97.2% of X variables were correlated with sample classification, demonstrating a high value of R 2 Y (0.816) and Q 2 (0.537).These values underscore the reliability of the data analysis.Each point on the graph represents the FA composition in a specific sample.Notably, the points are distributed across different areas of the plot, indicating that the FA compositions in the muscular tissues of S. constricta, when fed with different microalgae, were notably distinct from each other throughout the experiment.Furthermore, samples fed with the same microalgae tend to cluster closer together.Similarly, the FA compositions in the visceral mass of S. constricta fed with different microalgae also exhibited significant differences, as demonstrated by the scattered distribution of points in Figure 3B.
and I. galbana, except for Δ6 Fad and Elovl4_a, which showed significantly higher expressions in the sample of juveniles fed with Chlorella sp.compared to those fed with I. galbana.On the 24th d, it was observed that the expressions of Δ5 Fad_a, Δ6 Fad, Elovl2/5, and Elovl4_b were significantly higher or noticeably higher in the sample of juveniles fed with Chlorella sp.compared to those fed with C. calcitrans or I. galbana.However, no significant differences were found in the expressions of other genes among all the samples.

Changes in FA Composition in S. constricta and Correlation with Dietary FA
The PLS-DA results for the FA composition in the muscular tissues of S. constricta over the experiment are shown in Figure 3A.A remarkable 97.2% of X variables were correlated with sample classification, demonstrating a high value of R 2 Y (0.816) and Q 2 (0.537).These values underscore the reliability of the data analysis.Each point on the graph represents the FA composition in a specific sample.Notably, the points are distributed across different areas of the plot, indicating that the FA compositions in the muscular tissues of S. constricta, when fed with different microalgae, were notably distinct from each other throughout the experiment.Furthermore, samples fed with the same microalgae tend to cluster closer together.Similarly, the FA compositions in the visceral mass of S. constricta fed with different microalgae also exhibited significant differences, as demonstrated by the scattered distribution of points in Figure 3B.The correlation results for FA composition between S. constricta and microalgae diets are shown in Figure 4. Consistently, the FA composition of S. constricta exhibited a significantly higher correlation coefficient with that of the corresponding dietary FA.Notably, the FA composition in the muscular tissues of S. constricta fed with I. galbana also displayed a relatively higher correlation with that of Chlorella sp.

FA Composition in Muscular Tissues of Juvenile S. constricta
A total of 21 FA was identified in the muscular tissues of juvenile S. constricta (Table 3).The contents of nearly all types of FA increased to varying degrees in the three trials when compared to those of the initial sample.In particular, the content of total fatty acids (TFA) in the initial sample was 18.34 ± 1.02 µg•mg −1 .However, in the samples fed with Chlorella sp., C. calcitrans, and I. galbana, TFA fluctuated from ~25.04 to 28.09 µg•mg −1 , ~33.99 to 42.72 µg•mg −1 , and ~23.84 to 30.05 µg•mg −1 , respectively.Furthermore, the FA composition was primarily characterized by a predominance of polyunsaturated fatty acids (PUFA) of ~7.04-18.28µg•mg −1 , followed by saturated fatty acids (SFA) of ~7.53-13.54µg•mg −1 .Monounsaturated fatty acids (MUFA) were present at lower levels, ranging from ~3.43 to 11.46 µg•mg −1 .The correlation results for FA composition between S. constricta and microalgae diets are shown in Figure 4. Consistently, the FA composition of S. constricta exhibited a significantly higher correlation coefficient with that of the corresponding dietary FA.Notably, the FA composition in the muscular tissues of S. constricta fed with I. galbana also displayed a relatively higher correlation with that of Chlorella sp.

S. constricta Can Modulate FA Composition in Addition to Direct Accumulation
As shown in Table 3, there were significant differences in the FA composition of the muscular tissues among S. constricta fed with different microalgae, a trend also evident in the PLS-DA result (Figure 3A).Furthermore, the FA compositions in the muscular tissues of juvenile S. constricta (Table 3) closely mirrored those present in the respective dietary microalgae (Table 1), as indicated by the relatively high correlation coefficient in Figure 4.These observations align with previous studies on other marine mollusks [14][15][16][17][18][19], which also showed similar correlations between dietary microalgae and tissue FA compositions.To elaborate the results of the present study further, it was observed that when fed with Chlorella sp.(which is richest in LA but lacks 14:0), the samples displayed the highest levels of LA except for on the 16th d and the lowest levels of 14:0 on all sampling days.When fed with C. calcitrans (abundant in 16:1n-7 and EPA but lacking ALA), the samples exhibited the highest levels of 16:1n-7 and EPA, while maintaining the lowest levels of ALA across all sampling days.Finally, when fed with I. galbana (which is richest in 18:1n-9, 18:4n-3, and DHA), the samples consistently showed relatively higher levels of 18:1n-9, 18:4n-3, and DHA on all sampling days.Those results underscore the tendency of marine mollusks to accumulate FA directly from their diets.Therefore, maintaining an appropriate FA composition in the diet, especially LC-PUFA, is critical for optimizing the growth of marine mollusks.
However, the result also suggested that juvenile S. constricta possesses a certain ability to modulate the FA composition through selective accumulation and endogenous synthesis (Table 3).For example, despite Chlorella sp.containing relatively high levels of 20:0, 16:2n-6, and 16:3n-4 (Table 1), these FA were not detected in the samples of juveniles fed with Chlorella sp., suggesting their negligible impact on S. constricta development.Moreover, the juveniles fed with Chlorella sp.accumulated a relatively higher level of 18:0, which might compensate for the absence of 14:0 in their diet.Furthermore, even though Chlorella sp.lacks LC-PUFA, the samples of juveniles fed with Chlorella sp.displayed relatively higher levels of ARA, 22:5n-3, EPA, and DHA (Table 3), indicating that S. constricta has the capacity for significant endogenous LC-PUFA biosynthesis.When fed with C. calcitrans, the juveniles exhibited the highest levels of 18:1n-7 and 20:1n-7, which might result from the abundant precursor, 16:1n-7, in their diet.Additionally, the juveniles fed with C. calcitrans accumulated the highest levels of 20:4n-3, likely because its product, EPA, was obtained in sufficient quantities directly from diets.Notably, despite the significantly lower DHA levels in the juveniles fed with C. calcitrans compared to those fed with I. galbana (except for on the 24th d), no significant differences were observed in their growth (Figure 1).This suggests that EPA may effectively compensate for the physiological functions typically attributed to DHA.Although I. galbana contains a notably high level of 18:4n-3 (50.35 ± 6.01 µg•mg −1 ), its concentration in the juveniles fed with I. galbana was very low (~0.16-0.54µg•mg −1 ), implying a minor role of 18:4n-3 in S. constricta physiology.In contrast, although ARA was exclusively detected in C. calcitrans (2.49 ± 0.47 µg•mg −1 , Table 1), it was present at relatively high levels in all samples (~0.77-1.81µg•mg −1 , Table 3), highlighting a potential essential role for ARA in S. constricta development.

Selective Retention and Incorporation of FA Exist in S. constricta
Likewise, the FA composition in the visceral mass (Table 4) also clearly mirrors that of the dietary microalgae (Table 1, Figure 4).Nevertheless, some FA found in microalgae were absent in both the visceral mass and the muscular tissues.This includes FA like 20:0, 16:2n-6, and 16:3n-4, which have relatively high levels in Chlorella sp. at 5.23 ± 0.19 µg•mg −1 , 4.92 ± 0.09 µg•mg −1 , and 11.12 ± 1.50 µg•mg −1 , respectively (Table 1).Conversely, some FA were exclusively detected in the visceral mass but not in the muscular tissues, such as 16:3n-3, 18:3n-6, and 20:3n-7.These findings indicate that the dietary FA is selectively retained by S. constricta and further incorporated into its tissues.Additionally, some FA were uniquely detected in the farmed juveniles but not in the dietary microalgae.These include FA like 18:2n-3, 20:1n-7, 20:2n-6, 22:2(5,13), 22:4n-6, and 22:5n-3.These FAs may be synthesized as intermediate precursors of LC-PUFA or play specific physiological roles in S. constricta.The variations in FA composition between S. constricta and the dietary microalgae may be attributed to species-specific factors, suggesting that certain FA may be necessary for one species but not for another.

Conclusions
In summary, this study highlights the critical role of dietary FA composition in the development of S. constricta, directly impacting the FA composition of this bivalve.Additionally, the findings demonstrated the ability of S. constricta to regulate its FA composition through the significant modulation of Fad and Elovl expressions.Overall, the combination of Fad and Elovl expressions data with changes in FA composition provides valuable insights into the metabolism of the dietary FA in S. constricta.These results hold significance for the selection of appropriate dietary microalgae and the formulation of artificial feeds in the farming industry of this bivalve species.

Figure 1 .
Figure 1.Growth of juvenile S. constricta fed with different microalgae during the experiment.Values (mean ± SD, n = 3) within the same sampling time sharing a common superscript are not significantly different (p ≥ 0.05).

Figure 1 .
Figure 1.Growth of juvenile S. constricta fed with different microalgae during the experiment.Values (mean ± SD, n = 3) within the same sampling time sharing a common superscript are not significantly different (p ≥ 0.05).

Figure 2 .
Figure 2. Relative expressions of Fad and Elovl in the visceral mass of juvenile S. constricta fed with different microalgae during the experiment.Panels (A-G) indicate the relative expression of Δ5 Fad_a, Δ5 Fad_b, Δ6 Fad, Elovl2/5, Elovl4_a, Elovl4_b, and Elovl_c, respectively, in the samples.Values (mean ± SD, n = 3) of the same color sharing a common superscript are not significantly different (p ≥ 0.05).Meanwhile, values within the same sampling time sharing an asterisk are significantly different (p < 0.05).

Figure 2 .
Figure 2. Relative expressions of Fad and Elovl in the visceral mass of juvenile S. constricta fed with different microalgae during the experiment.Panels (A-G) indicate the relative expression of ∆5 Fad_a, ∆5 Fad_b, ∆6 Fad, Elovl2/5, Elovl4_a, Elovl4_b, and Elovl_c, respectively, in the samples.Values (mean ± SD, n = 3) of the same color sharing a common superscript are not significantly different (p ≥ 0.05).Meanwhile, values within the same sampling time sharing an asterisk are significantly different (p < 0.05).

Fishes 3 .
Changes in FA Composition in S. constricta and Correlation with Dietary FA

Figure 3 .
Figure 3. PLS-DA score plots for FA compositions in muscular tissues (A) and visceral mass (B) of S. constricta over the course of the experiment.Cs, Cc, and Ig represent the diets of Chlorella sp., C. calcitrans, and I. galbana, respectively.X_Yd indicates the FA composition in the muscular tissue of S. constricta fed with the corresponding microalgae diet (X) sampled on Y d.X_final denotes the FA composition in a visceral mass of S. constricta fed with the corresponding microalgae diet (X) sampled at the end of the experiment.The different colored shapes (circles, squares, triangles) correspond to the data regarding the dependent FA composition of the samples, respectively.

Figure 3 .
Figure 3. PLS-DA score plots for FA compositions in muscular tissues (A) and visceral mass (B) of S. constricta over the course of the experiment.Cs, Cc, and Ig represent the diets of Chlorella sp., C. calcitrans, and I. galbana, respectively.X_Yd indicates the FA composition in the muscular tissue of S. constricta fed with the corresponding microalgae diet (X) sampled on Y d.X_final denotes the FA composition in a visceral mass of S. constricta fed with the corresponding microalgae diet (X) sampled at the end of the experiment.The different colored shapes (circles, squares, triangles) correspond to the data regarding the dependent FA composition of the samples, respectively.

Figure 4 .
Figure 4. Correlation analysis of FA composition between S. constricta and microalgae diets.The color shades represent the numerical magnitude of the correlation coefficient.Asterisks denote the level of significance: * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.The nomenclatures used are consistent with those in Figure 3.

Figure 4 .
Figure 4. Correlation analysis of FA composition between S. constricta and microalgae diets.The color shades represent the numerical magnitude of the correlation coefficient.Asterisks denote the level of significance: * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.The nomenclatures used are consistent with those in Figure 3.

Table 3 .
FA composition (µg•mg −1 ) in muscular tissues of juvenile S. constricta fed with different microalgae during the experiment.Values (mean ± SD, n = 3) within the same sampling time sharing a common superscript are not significantly different (p ≥ 0.05).

Table 4 .
FA composition (µg•mg −1 ) in the visceral mass of juvenile S. constricta before and after experiment.Values (mean ± SD, n = 3) within the same sampling time sharing a common superscript are not significantly different (p ≥ 0.05).