# Sterol biosynthesis and phytosterol bioconversion in Crassostrea gigas larvae: new evidence from mass-balance feeding studies

Fiz da Costa, Margaux Mathieu-Resuge, Fabienne Le Grand, Claudie Quéré, Gabriel V. Markov, Gary Wikfors, Philippe Soudant

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Fiz da Costa, Margaux Mathieu-Resuge, Fabienne Le Grand, Claudie Quéré, Gabriel V. Markov, et al.: Sterol biosynthesis and phytosterol bioconversion in Crassostrea gigas larvae: new evidence from mass-balance feeding studies. Biochimie, 2025, 239, Part A, pp.51-59. 10.1016/j.biochi.2025.05.005 hal-05405141

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<figure><img src="image_1.png" /></figure>
# Sterol biosynthesis and phytosterol bioconversion in *Crassostrea gigas* larvae: new evidence from mass-balance feeding studies

<figure><img src="image_2.png" /></figure>

Fiz da Costa<sup>a,*,1</sup>, Margaux Mathieu-Resuge<sup>a,b</sup>, Fabienne Le Grand<sup>a</sup>, Claudie Quéré<sup>a</sup>, Gabriel V. Markov<sup>c</sup>, Gary H. Wikfors<sup>d</sup>, Philippe Soudant<sup>a</sup>

## ARTICLE INFO

**Article history:**

Received 21 August 2024

Received in revised form

28 April 2025

Accepted 15 May 2025

Available online 16 May 2025

Handling Editor: C. Forest

**Keywords:**

Oyster

Larvae

Sterol

Synthesis

Bioconversion

## ABSTRACT

Dietary sterols are important for bivalve larval growth and survival. The aim of the study was to determine quantitatively sterol incorporation and synthesis in Pacific oyster *Crassostrea gigas* larvae by means of a mass-balance approach. The flow-through larval rearing technique allowed accurate quantification of microalgal ingestion and consequently of sterol ingestion. Sterol incorporation was calculated using the sterol composition of the larvae between 2 sample points. Two sets of experiments were done using two cultured microalgae: *Tisochrysis lutea* (T) and *Chaetoceros neogracile* (Cg) as mono- and bi-specific diets (TCg). Accumulation of tissue sterols in oyster larvae, in addition to those present in the diet, indicate that *C. gigas* larvae appear to have the ability to synthetize sterols de novo under low dietary sterol supply, e.g., when fed the T diet. Sterol synthesis was dependent upon sterol dietary supply; larvae fed T exhibited greater sterol incorporation at the pediveliger stage than larvae fed TCg. Larval sterol compositions under the different dietary regimes indicate likely bioconversion pathways modifying dietary sterols. Larvae fed T bioconverted dietary brassicasterol mainly to cholesterol via a 22-dehydrocholesterol intermediate. Brassicasterol was also actively synthetized in larvae fed T and TCg, suggesting a possible metabolic role of this sterol in *C. gigas* larvae. Apparent desmosterol synthesis under all experimental conditions suggests a role as a membrane component or as an intermediate in cholesterol synthesis. Our data also indicate that *C. gigas* larvae require approximately 13 ng cholesterol larvae⁻¹ to achieve competence for metamorphosis. This mass-balance approach will allow the determination of other biochemical requirements in larval nutrition.

© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

Sterols are required for the maintenance of cellular membrane fluidity [1]. Sterol de novo synthesis and bioconversion pathways in mollusks remain incompletely documented, with contradictory data even for the same species (for review see Knauer and

Southgate [2]). Marine mollusks and arthropods, among other invertebrate groups, lack or have a limited ability to synthesize de novo cholesterol, and therefore, these animals must obtain sterols from the diet [3]. As phytoplankton contains little quantities if any of cholesterol, but a great variety of phytosterols, the ability to metabolically modify these phytosterols into cholesterol has been postulated in these group of invertebrates [4].

To our knowledge, until now, the capacity for de novo sterol synthesis has only been studied in bivalves using radiolabeled dietary precursors (for review see Knauer and Southgate [2] and Giner et al. [4]). The study of Salaque et al. [5] was performed with 2-[¹⁴C]-mevalonate and CH₃-[¹⁴C]-L-methionine and lasted for 66–70 h. Such a short experimental exposure may account for the observed lack of sterol incorporation into tissues. Similarly, Walton and Pennock [6] observed no incorporation of sterols after 24–32 h

Abbreviations: GC, Gas chromatography; GC-MS, Gas chromatography–mass spectrometry; Cg, *Chaetoceros neogracile*; LSD, Least Significant Difference; PAR, Photosynthetically active radiation; PVC, polyvinyl chloride; T, *Tisochrysis lutea*; TCg, *T. lutea* + *C. neogracile*.

* Corresponding author.

E-mail address: fiz.dacosta@ieo.csic.es (F. da Costa).

¹ Present address: Instituto Español de Oceanografía (IEO, CSIC), Centro Oceanográfico de Vigo. Subida a Radio Faro, 50, 36390 Vigo, Spain.

<footnote><sup>a</sup>Univ Brest, CNRS, IRD, Ifremer, LEMAR, Plouzané, F-29280, France</footnote>

<footnote><sup>b</sup>Univ Brest, Ifremer, BEEP, Plouzané, F-29280, France</footnote>

<footnote><sup>c</sup>Sorbonne Université, CNRS, Integrative Biology of Marine Models (LBI2M, UMR8227), Station Biologique de Roscoff (SBR), Roscoff, France</footnote>

<footnote><sup>d</sup>Northeast Fisheries Science Center, NMFS, NOAA, 212 Rogers Avenue, Milford, CT, 06460, USA</footnote>
with injected [¹⁴C]-mevalonate in *Cardium edule* and *Mytilus edulis*.

Sterols can be elongated by C₂₄-alkylation, and sterol breakdown
can occur through C₂₄-dealkylation [2]. Phytosterol alkylation has
been demonstrated in oysters, such as *Crassostrea gigas* and *Cras-
stostrea virginica* [7,8]. It is generally accepted that most bivalves lack
the ability to dealkylate C₂₉-sterols [9]; however, other studies
demonstrated dealkylation activities in bivalves, such as the deal-
kylation of [³H]-fucosterol (C₂₉) to desmosterol and cholesterol
(both C₂₇) in *Ostrea gryphea* and *C. gigas* [7,10]. The study carried out
by Saliot and Barbier [10] with *O. gryphea* using seawater containing
3-[³H]-fucosterol, however, found apparent cholesterol incorpora-
tion after 48 h. Napolitano et al. [11], in their study of incorporation
of dietary sterols by the sea scallop, *Placopecten magellanicus*, fed
live microalgae for 1.5 months, documented low turnover rates of
sterols between bivalve tissues, which may explain why results of
the above studies appeared contradictory. Moreover, Knauer et al.
[7] suggested the importance of using sufficiently long incubation
periods following administration of radiolabelled precursors to
detect labeled sterols. Studies of longer duration are needed to
study sterol bioconversion and synthesis more thoroughly. For
example, sterol bioconversion was demonstrated, but the absence
of *de novo* sterol synthesis was reported, in *Crassostrea gigas* spat fed
[³H]-labeled sterols encapsulated in gelatin-acacia microcapsules
(GAM) [7] for 7 days. Teshima and Patterson [8] observed sterol
synthesis and bioconversion after injection of 2-[¹⁴C]-acetate and
2,3-[³H]-lanosterol in *C. virginica* with incubation periods of 7 and
17 days. More recently, Giner et al. [4] used ¹³C-labeled phytosterols
to supplement the unialgal diet *Rhomonas* sp. to study the
bioconversion of phytosterols to cholesterol by the Northern Bay
scallop *Argopecten irradians* *irradians*. This study showed that scal-
lops efficiently dealkylated Δ⁵ C₂₉ (24-ethyl) sterols to cholesterol,
and the only C₂₈ sterol that was dealkylated efficiently possessed
the 24(28)-double bond. Taken together, the relatively few studies
of sterol metabolism in bivalves support no clear consensus about
the sterol metabolism abilities of bivalve mollusks. This study
examined accumulation of tissue sterols in *C. gigas* larvae fed very
precisely controlled microalgal diets that differed in sterol content
and profile and proposed previously unreported sterol bioconver-
sion and synthesis pathways in this bivalve species.

The flow-through bivalve larval rearing system used in the present study allows the maintenance of a constant food density in culture tanks and, accordingly, precise determination of microalgal consumption, enabling calculation of microalgal sterol incorporation by larvae. A main advantage of our approach is consistent and precise provision of larvae with live microalgae, which contrasted with previous studies related to sterol bioconversion wherein sudden and episodic sterol additions were often done. Indeed, a number of previous studies were performed either with unfed animals, or supplied marked sterols encapsulated in GAM, dissolved in seawater, or injected (e.g. Refs. [7,8,10,12]). Our method also allows the use of several phytosterols extending the limited variety delivered in other sterol metabolism studies (for review see Knauer and Southgate [2]). Lastly, we examined a complete developmental stage (larval stage), contrasting with aforementioned literature.

**2. Materials and methods**

2.1. *Microalgal culture*

Two microalgae were cultured: *Tisochrysis lutea* (Tiso, formerly known as *Isochrysis affinis galbana*, named here T, strain CCAP 927/14) and *Chaetoceros neogracile* (named here Cg, UTEX LB2658). Microalgae were cultivated as described in da Costa et al. [13]. Briefly, microalgae were grown in 6-L glass carboys under

continuous cool-white light. Seawater at 34–35 ppm salinity was
filtered to 1 µm, enriched with sterilized Conway medium [14], and
then autoclaved. Cultures were thermoregulated at 20–23 °C by air
conditioning and pH maintained within a range of 7.5–8.1 by the
supply of a 3 % CO₂-air mixture. Sodium metasilicate (40 mg L⁻¹)
was added as a silica source to *C. neogracile* cultures and salinity
was reduced to 25 ppm by addition of distilled water. Microalgae
were harvested in late-logarithmic phase (after 3–4 days).

## 2.2. Broodstock conditioning and larval culture

Broodstock of the Pacific oyster (*Crassostrea gigas*) were
collected from Aber Benoit (Brittany, France) and transferred to
Argenton hatchery facilities for conditioning. Broostock condi-
tioning and larval culture was carried out as described in da Costa
et al. [13]. Briefly, oysters were maintained in flow-through tanks
at 19 °C for 2 months and fed continuously with a 6 % of oyster dry
weight in dry weight of algae per day of a mixed diet of *T. lutea* and
*C. neogracile*. Gametes were obtained by broodstock gonad strip-
ping, using a pool of 11 males and 4 females. Fertilization was
performed at a ratio of 50 spermatozoans per oocyte. After 2 h,
embryos were transferred for incubation to cylindro-conical tanks
filled with 1-µm-filtered seawater at 22 °C. After 48 h of incuba-
tion, D-larvae were distributed in 5-L translucent methacrylate
cylinders and reared in the flow-through system as previously
detailed by da Costa et al. [15]. Seawater in each experimental tank
was fully renewed each hour by a continuous 1-µm-filtered, UV-
treated seawater flow of 0.87 ml min⁻¹ at the bottom of each
experimental tank. Each diet was delivered by a peristaltic pump
directly to the larval tanks down the seawater line. Temperature
was maintained at 25 °C using a thermo-regulated automatic
valve, and ambient salinity was 34 ppm. In each tank, aeration was
supplied from the bottom at flow of 30 ml min⁻¹. A sieve con-
sisting of a beveled PVC pipe was installed at the outlet of each
tank to prevent larvae from escaping.

Two experiments were performed to study sterol assimilation
by *C. gigas*. In the first, two single diets (T or Cg) and bi-specific diet
(TCg) were compared. In this trial, larvae were reared until they
reached competency (larval duration was different for each dietary
treatment from 16 to 19 days post-fertilization). A second follow-
up trial was performed to confirm and validate results observed in
the first experiment (sterol synthesis and bioconversion) using
only two diets (T and TCg). This second trial was performed until
larvae fed TCg reached competency (15 days post-fertilization).
Each larval diet was evaluated in triplicate. Larvae were fed at a
constant concentration of 1500 µm³ µl⁻¹ at the exit of the rearing
tanks, corresponding to ≈40 algal cells µl⁻¹ (equivalent *T. lutea*
[16]). Twice a day, inlet and outlet seawater were sampled from
each experimental tank to determine phytoplankton consump-
tion. Cell counts were determined by means of an electronic par-
ticle counter (Multisizer 3) equipped with a 100-µm aperture tube.
Survival was calculated every other day from each larval replicate
to determine grazing data, which can be found in da Costa et al.
[15]. Larvae were cultivated at a density of 100 and 50 larvae ml⁻¹
for experiments 1 and 2, respectively, due to the equivalent larval
performance demonstrated previously at both densities in the
flow-through systems used here [17].

2.3. *Biochemical analysis*

Each microalgal diet (250-300 × 10⁶ cells) was sampled
throughout larval rearing as described in da Costa et al. [13]. Thus,
dietary composition data represent means and variances of sterol
measurements during all larval culture (diets were temporally
replicated, n = 5 and n = 7 for experiments 1 and 2, respectively).
Sterol profiles of the diets can be found in da Costa et al. [15]. Larval samples were collected for all treatments at day 2 (D-shaped larvae, start of feeding, ~200,000 larvae per sample), on day 8 (~50,000 larvae per sample), and when the positive control (TCg) reached the pediveliger stage (day 16 or 15, for experiments 1 and 2, respectively, ~20,000 larvae per sample). In the first trial, larval replicates were also sampled when larvae fed other diets reached the pediveliger stage (day 18 or 19, for larvae fed T or Cg, respectively, ~20,000 larvae per sample). Samples (diets and larvae) were washed with 3.5 % ammonium formate to remove salt and collected on 450 °C pre-combusted GF/F glass fiber filters (Whatman, diameter 47 mm). Lipids were extracted in 6 ml chloroform-methanol (2:1, v/v) according to Folch et al. [18], sealed under nitrogen, and stored at -20 °C.

Sterols in microalgae and larvae were hydrolyzed with sodium methoxide (MeONa) for 90 min at room temperature as described by Soudant et al. [19]. The sterols were extracted in hexane and injected directly into the gas chromatograph (GC) Chrompak CP 9002 (Varian Inc., Walnut Creek, CA, USA) equipped with a Restek RTX65 fused silica capillary column (15 m × 0.25 mm, 0.25 µm film thickness) using an on-column injection system. Hydrogen was used as the carrier gas with a temperature gradient from 160 to 280 °C. The sterols were identified by comparison of their retention times with commercial standards and in-house standard mixtures from marine bivalves, and cholestane was used as an internal standard (2.3 µg; SUPELCO, Lyon, France) for sterol quantification. Sterol identification in larvae was also confirmed by GC mass spectrometry (GC-MS). A 1-mL aliquot of the total lipid extract was saponified and silylated as described in Mathieu-Resuge et al. [20]. Then, derived sterols were injected in a Shimadzu QP2010-SE GC-MS equipped with a Restek RTX65 column (30 m × 0.25 mm ID, 0.25 µm). The GC temperature was programmed as follows: from 60 °C to 250 °C at 60 °C/min, then to 280 °C at 2 °C/min and maintained for 5 min. The GC was equipped with a splitless injector slated at 270 °C; whereas, the mass spectrometer was equipped with an ion source programmed at 250 °C, and an interface at 250 °C. Mass spectra were generated with the scan mode, from 50 to 1000 m/z. Trivial and systematic names of the detected sterols were: 22-dehydrocholesterol (cholesta-5,22E-dien-3β-ol) (C₂₇H₄₄O), Dihydrocholesterol (cholestan-3β-ol), (C₂₇H₄₈O), Cholesterol (cholesta-5-en-3βol) (C₂₇H₄₆O), Brassicasterol (ergosta-5,22E-dien-3β-ol) (C₂₈H₄₆O), Desmosterol (cholest-5,24-dien-3β-ol) (C₂₇H₄₄O), Campesterol (campest-5-en-3β-ol) (C₂₈H₄₈O), 24-methylenecholesterol (24-methylcholesta-5,24-dien-3β-ol) (C₂₈H₄₆O₂), Stigmasterol (stigmasta-5,22E-dien-3β-ol) (C₂₉H₄₸O), 4-α-methyl poriferasterol (4-α-methyl-24-α-ethylcholesta-22-en-3β-ol) (C₃₀H₅₂O), β-Sitosterol (stigmast-5-en-3β-ol) (C₂₉H₅₀O), Fucosterol (24E-ethylidene-cholest-5-en-3β-ol) (C₂₉H₄₈O) and Isofucosterol (24Z-ethylidene-cholest-5-en-3β-ol) (C₂₉H₄₈O). A representative chromatogram of the sterols detected can be found in Suppl. Fig. 1.

## 2.4. Data treatment and statistical analysis

In the present work, we focused on sterol metabolism between day 8 and days 15–16 because the first 5–6 days of larval life in C. gigas correspond to a period during which larvae feeding changes from lecithotrophic to exotrophic. During the transition (mixotrophic) period, larvae are still relying upon the energy and material reserves contained within the embryo itself as exogenous feeding is low due to the gradual activation of digestive enzymes in early larvae [21,22]. Moreover, total sterol contents decrease during the mixotrophic phase in bivalves, specially steryl esters which are fully depleted [23]. Accordingly, both processes may mask sterol bioconversion and synthesis. We also report sterol

incorporation during the entire larval phase, between day 2 and competence for metamorphosis (Experiment 1: day 16 for TCg; day 18 for T and day 19 for Cg; and experiment 2: day 15 for TCg), to determine cholesterol requirement for the entire larval phase.

We compared the quantity of ingested sterols (ng larvae⁻¹) with the accumulated counterpart by larvae between two sampling dates. For each experiment, sterol ingestion was calculated using mean sterol composition for each diet (n = 5 or n = 7 for experiments 1 or 2, respectively) and microalgal consumption for each diet treatment. When a sterol was present in larvae but absent from the diet, we interpreted this to indicate that this sterol was of endogenous origin (through biosynthesis de novo or bioconversion of another dietary sterol). When the accumulated amount of a sterol by larvae was above 100 % of the ingested amount, we expected that this sterol had both exogenous and endogenous origins.

Data were analysed using STATISTICA software (Stat Soft, Inc., Tulka, OK, USA, version 12). Data normality was evaluated using the Shapiro-Wilk test. Then, one-way analysis of variance (ANOVA) for significant differences was used to test differences among treatments for the sterols ingested and incorporated. Homogeneity of variances was checked by means of the Barlett test. When necessary, post hoc analyses with the Least Significant Difference (LSD) test were applied. Percentage data were arcsine-transformed to normalize variance [24]. Differences were considered statistically significant if $P \le 0.05$.

## 2.5. Comparative genomics analysis

To determine the known presence of sequences coding for enzymes involved in sterol metabolism, protein sequences for candidate Δ24 sterol reductase (DHCR24) were retrieved from previous studies [25,26], and the dataset was completed by targeted BLAST searches on the NR database on NCBI. A maximum phylogenetic tree was built using the phyML version implemented in Seaview [27], using the LG model with a gamma law and an approximate likelihood ratio test (aLRT) to assess branch robustness.

# 3. Results

## 3.1. Experiment 1

Larvae receiving T and TCg incorporated more total sterols than their dietary consumption between day 8 and 16 (8.2 vs 3.5 and 17.1 vs 12.2 ng of total sterol incorporation vs ingested per larva in T and TCg respectively: Table 1). Larval sterol contents corresponded to 233 and 139 % of total supplied sterol, respectively. In contrast, total sterols incorporated by larvae fed Cg were lower than sterols ingested (6.3 vs 11.3 ng of total sterol per larva: Table 1). Brassicasterol incorporation by larvae fed T and TCg exceeded brassicasterol ingested by larvae (6.6 vs 3.5 and 6.3 vs 4.9 ng of total sterol per larva in T and TCg, respectively: Table 1). Larvae fed Cg incorporated only 3.8 ng of cholesterol per larva of the 6.4 ng ingested, thus representing 58 % assimilation (Table 1). Similarly, only a half of the 24-methylene-cholesterol ingested was incorporated by larvae receiving Cg (0.6 vs 1.0 ng per larva: Table 1). Incorporation of fucosterol in larvae fed Cg was even lower, 30 % (Table 1). Cholesterol incorporated by larvae fed TCg was 1.5-fold the quantity supplied in the diet (6.8 vs 4.4 ng larva⁻¹: Table 1).

Larvae fed T accumulated cholesterol (0.4 ng larva⁻¹) when none was present in the diet, implying de novo synthesis. These larvae also appear to have synthesized significant quantities of desmosterol, β-sitosterol, 22-dehydrocholesterol, 24-methylene-cholesterol, campesterol, and stigmasterol (Table 1). Desmosterol
<caption>**Table 1**</caption>

Sterol ingestion and incorporation (expressed in ng larva⁻¹) by *Crassostrea gigas* larvae fed *Tisochrysis lutea* (T), *Chaetoceros neogracile* (Cg) and the mixed diet (TCg) between days 8 and 16 in experiment 1 (mean ± S.D.).

<table><thead><tr><th rowspan="2"></th><th colspan="3">Ingested</th><th colspan="3">Incorporated</th></tr><tr><th>T</th><th>Cg</th><th>TCg</th><th>T</th><th>Cg</th><th>TCg</th></tr></thead><tbody><tr><td>22-dehydrocholesterol</td><td>—</td><td>—</td><td>—</td><td>0.2 ± 0.0<sup>A</sup></td><td>0.1 ± 0.0<sup>B</sup></td><td>0.1 ± 0.0<sup>B</sup></td></tr><tr><td>Cholesterol</td><td>0.0 ± 0.0<sup>c</sup></td><td>6.4 ± 0.2<sup>a</sup></td><td>4.4 ± 0.2<sup>b</sup></td><td>0.4 ± 0.1<sup>c</sup></td><td>3.8 ± 0.7<sup>B</sup></td><td>6.8 ± 0.6<sup>A</sup></td></tr><tr><td>Brassicasterol</td><td>3.5 ± 0.3<sup>b</sup></td><td>0.0 ± 0.0<sup>c</sup></td><td>4.9 ± 0.2<sup>a</sup></td><td>6.6 ± 1.5<sup>A</sup></td><td>0.1 ± 0.0<sup>B</sup></td><td>6.3 ± 0.6<sup>A</sup></td></tr><tr><td>Desmosterol</td><td>—</td><td>—</td><td>—</td><td>0.1 ± 0.0<sup>c</sup></td><td>0.5 ± 0.1<sup>B</sup></td><td>1.2 ± 0.1<sup>A</sup></td></tr><tr><td>Campesterol</td><td>—</td><td>—</td><td>—</td><td>0.1 ± 0.0<sup>A</sup></td><td>0.0 ± 0.0<sup>B</sup></td><td>0.1 ± 0.0<sup>A</sup></td></tr><tr><td>24-methylene-cholesterol</td><td>0.0 ± 0.0<sup>c</sup></td><td>1.0 ± 0.0<sup>b</sup></td><td>1.1 ± 0.0<sup>a</sup></td><td>0.2 ± 0.1<sup>c</sup></td><td>0.6 ± 0.1<sup>B</sup></td><td>0.8 ± 0.0<sup>A</sup></td></tr><tr><td>Stigmasterol</td><td>—</td><td>—</td><td>—</td><td>0.1 ± 0.1<sup>A</sup></td><td>0.0 ± 0.0<sup>B</sup></td><td>0.0 ± 0.0<sup>B</sup></td></tr><tr><td>β-sitosterol</td><td>—</td><td>—</td><td>—</td><td>0.2 ± 0.1<sup>A</sup></td><td>0.1 ± 0.0<sup>B</sup></td><td>0.2 ± 0.0<sup>A</sup></td></tr><tr><td>Fucosterol</td><td>0.0 ± 0.0<sup>c</sup></td><td>3.9 ± 0.1<sup>a</sup></td><td>2.5 ± 0.1<sup>b</sup></td><td>0.1 ± 0.0<sup>B</sup></td><td>1.1 ± 0.2<sup>A</sup></td><td>1.3 ± 0.1<sup>A</sup></td></tr><tr><td>Total</td><td>3.5 ± 0.3<sup>c</sup></td><td>11.3 ± 0.3<sup>b</sup></td><td>12.2 ± 0.5<sup>a</sup></td><td>8.2 ± 2.0<sup>B</sup></td><td>6.3 ± 1.1<sup>B</sup></td><td>17.1 ± 1.3<sup>A</sup></td></tr></tbody></table>

Values with same superscript letters in the same row for ingestion (lower case letters) and incorporation (capital letters) are not significantly different at *P* > 0.05. -: sterols not present in the diet and not ingested.

was also presumably synthesized in both larvae receiving Cg and TCg (0.5 and 1.2 ng larva⁻¹, respectively, Table 1). Secondarily, larvae fed Cg produced brassicasterol and dihydrocholesterol, with 5-fold lower synthesis activity compared to desmosterol synthesis (Table 1). Larvae fed TCg also accumulated a small amount of β-sitosterol (0.2 ng larva⁻¹: Table 1).

## 3.2. Experiment 2

Total quantities of sterols accumulated within larvae fed T and TCg between days 8 and 15 were higher than the quantities ingested (5.9 vs 1.8 and 22.3 vs 13.7 ng of total sterol per larva in T and TCg, respectively: Table 2). Accumulation of total sterols by larvae fed T and TCg were 323 and 163 % of the ingested sterol amounts. Similarly, brassicasterol accumulation by larvae fed T and TCg surpassed 2.7 and 1.8 times the quantities ingested, respectively (Table 2). Cholesterol was presumptively synthesized in larvae fed TCg (6.3 ng per larva ingested vs 9.3 ng per larva incorporated: Table 2). In contrast, incorporation of 24-methylene-cholesterol and fucosterol was lower than ingestion in larvae fed TCg (Table 2).

Larvae fed T, which supplied brassicasterol only, mainly synthesized cholesterol (0.3 ng larva⁻¹), and in a lesser extent desmosterol (0.2 ng larva⁻¹: Table 2). Desmosterol was actively synthesized de novo (no known pathway from brassicasterol) by larvae fed TCg (2.1 ng larva⁻¹: Table 2).

<caption>**Table 2**</caption>

Sterol ingestion and incorporation (expressed in ng larva⁻¹) by *Crassostrea gigas* larvae fed *Tisochrysis lutea* (T) and the mixed diet (TCg) composed by *T. lutea* (T) and *Chaetoceros neogracile* (Cg) between days 8 and 15 in experiment 2 (mean ± S.D.).

<table><thead><tr><th rowspan="2"></th><th colspan="2">Ingested</th><th colspan="2">Incorporated</th></tr><tr><th>T</th><th>TCg</th><th>T</th><th>TCg</th></tr></thead><tbody><tr><td>22-dehydrocholesterol</td><td>—</td><td>—</td><td>0.1 ± 0.0<sup>B</sup></td><td>0.1 ± 0.0<sup>A</sup></td></tr><tr><td>Cholesterol</td><td>0.0 ± 0.0<sup>b</sup></td><td>6.3 ± 0.3<sup>a</sup></td><td>0.3 ± 0.1<sup>B</sup></td><td>9.3 ± 1.1<sup>A</sup></td></tr><tr><td>Brassicasterol</td><td>1.8 ± 0.3<sup>b</sup></td><td>4.6 ± 0.2<sup>a</sup></td><td>4.9 ± 0.6<sup>B</sup></td><td>8.4 ± 1.0<sup>A</sup></td></tr><tr><td>Desmosterol</td><td>—</td><td>—</td><td>0.2 ± 0.0<sup>B</sup></td><td>2.1 ± 0.3<sup>A</sup></td></tr><tr><td>Campesterol</td><td>—</td><td>—</td><td>0.1 ± 0.0<sup>A</sup></td><td>0.0 ± 0.0<sup>B</sup></td></tr><tr><td>24-methylene-cholesterol</td><td>0.0 ± 0.0<sup>b</sup></td><td>0.6 ± 0.0<sup>a</sup></td><td>0.1 ± 0.1<sup>B</sup></td><td>0.5 ± 0.1<sup>A</sup></td></tr><tr><td>Stigmasterol</td><td>—</td><td>—</td><td>0.2 ± 0.1</td><td>0.3 ± 0.0</td></tr><tr><td>β-sitosterol</td><td>—</td><td>—</td><td>0.0 ± 0.0</td><td>0.1 ± 0.1</td></tr><tr><td>Fucosterol</td><td>0.0 ± 0.0<sup>b</sup></td><td>2.2 ± 0.1<sup>a</sup></td><td>0.0 ± 0.0<sup>B</sup></td><td>1.5 ± 0.2<sup>A</sup></td></tr><tr><td>Total</td><td>1.8 ± 0.3<sup>b</sup></td><td>13.7 ± 0.7<sup>a</sup></td><td>5.9 ± 0.5<sup>B</sup></td><td>22.3 ± 2.9<sup>A</sup></td></tr></tbody></table>

Values with same superscript letters in the same row for ingestion (lower case letters) and incorporation (capital letters) are not significantly different at *P* > 0.05. -: sterols not present in the diet and not ingested.

## 3.3. Sterol contents within competent larvae

At competence, total sterol accumulation substantially exceeded sterol ingestion in larvae fed T (17.0 vs 6.3 ng total sterol incorporation vs ingested per larva, data calculated between days 2 and 18; Table 3). Incorporation of brassicasterol was twice dietary ingestion in larvae receiving T (Table 3).

In contrast, at competency, larvae fed Cg incorporated only 66 % of total sterol supplied in the diet (33.0 vs 21.3 ng total sterol per larva, calculated between days 2 and 19: Table 3).

Larvae fed Cg achieved competence on day 19, exhibiting 74 and 55 % of cholesterol and 24-methylene-cholesterol incorporation, respectively (Table 3). Fucosterol was also poorly incorporated by larvae fed Cg (35 % accumulation of ingested sterols (Table 3)).

In competent larvae fed TCg in experiment 1, a higher incorporation of total sterols was also observed (18.4 vs 13.5 ng total sterol per larva between day 2 and day 16: Table 3). Cholesterol and brassicasterol accumulation by larvae receiving TCg in experiment 1 were 148 and 129 %, respectively (Table 3). The incorporation of 24-methylene-cholesterol and fucosterol observed in larvae fed TCg in experiment 1 and 2 were similar (73–83 and 51–64 %, respectively between day 2 and day 16: Table 3). Similarly, larvae fed TCg in experiment 2 exhibited greater total sterol accumulation than sterol supply at competence (23.4 vs 15.0 ng total sterol per larva between day 2 and day 16: Table 3). In experiment 2, cholesterol and brassicasterol incorporation by larvae fed TCg was also greater than dietary supply (Table 3).

## 3.4. Comparative genomics analysis

A clear ortholog of the delta-24 sterol reductase widely conserved across eukaryotes was identified in oysters, as well as in two other molluscs (Fig. 1). Interestingly, the DHCR24 genes is duplicated in arthropods, and the DHCR24-1 paralog, closer in sequence to the molluscan candidate DHCR24, is known to be the most efficient in bioconversion of dietary sterols into cholesterol [28,29]. Therefore, the oyster protein is a clear candidate for performing the bioconversion of isofucosterol into sitosterol and desmosterol into cholesterol (Fig. 2). Using the same methodology, a search for other candidate enzymes to perform the dealkylation or 22–23 hydrogenation and C22-desaturation steps was also carried out on oyster candidate proteins but led to no conclusive result. In particular, no clear ortholog was found in the oyster predicted proteome for the sterol dealkylase Des24 described in the ciliate *Tetrahymena* [30], in spite of the presence of numerous
<caption>**Table 3**</caption>

Sterol ingestion and incorporation (expressed in ng larva⁻¹) by *Crassostrea gigas* larvae fed *Tisochrysis lutea* (T), *Chaetoceros neogracile* (Cg) and the mixed diet (TCg) between days 2 and competent larvae in experiment 1 and 2 (mean ± S.D.).

<table><thead><tr><th rowspan="2"></th><th colspan="4">Ingested</th><th colspan="4">Incorporated</th></tr><tr><th>T-Exp 1</th><th>Cg-Exp 1</th><th>TCg-Exp 1</th><th>TCg-Exp 2</th><th>T-Exp 1</th><th>Cg-Exp 1</th><th>TCg-Exp 1</th><th>TCg-Exp 2</th></tr></thead><tbody><tr><td>22-dehydrocholesterol</td><td>–</td><td>–</td><td>–</td><td>–</td><td>0.3 ± 0.0<sup>A</sup></td><td>0.2 ± 0.0<sup>B</sup></td><td>0.1 ± 0.0<sup>C</sup></td><td>0.1 ± 0.0<sup>BC</sup></td></tr><tr><td>Cholesterol</td><td>0.0 ± 0.0<sup>d</sup></td><td>17.9 ± 0.7<sup>a</sup></td><td>4.8 ± 0.2<sup>c</sup></td><td>7.0 ± 0.4<sup>b</sup></td><td>1.0 ± 0.1<sup>C</sup></td><td>13.1 ± 3.0<sup>A</sup></td><td>7.1 ± 0.5<sup>B</sup></td><td>9.7 ± 1.2<sup>B</sup></td></tr><tr><td>Brassicasterol</td><td>6.3 ± 0.2<sup>a</sup></td><td>0.0 ± 0.0<sup>c</sup></td><td>5.3 ± 0.2<sup>b</sup></td><td>5.0 ± 0.3<sup>b</sup></td><td>13.3 ± 1.0<sup>A</sup></td><td>0.3 ± 0.1<sup>D</sup></td><td>6.9 ± 0.6<sup>C</sup></td><td>8.9 ± 1.1<sup>B</sup></td></tr><tr><td>Desmosterol</td><td>–</td><td>–</td><td>–</td><td>–</td><td>0.3 ± 0.0<sup>A</sup></td><td>1.6 ± 0.4<sup>B</sup></td><td>1.3 ± 0.1<sup>B</sup></td><td>2.2 ± 0.3<sup>A</sup></td></tr><tr><td>Campesterol</td><td>–</td><td>–</td><td>–</td><td>–</td><td>0.2 ± 0.0<sup>A</sup></td><td>0.0 ± 0.0<sup>C</sup></td><td>0.1 ± 0.0<sup>B</sup></td><td>0.0 ± 0.0<sup>C</sup></td></tr><tr><td>24-methylene-cholesterol</td><td>0.0 ± 0.0<sup>d</sup></td><td>2.7 ± 0.1<sup>a</sup></td><td>1.2 ± 0.0<sup>b</sup></td><td>0.6 ± 0.0<sup>c</sup></td><td>0.4 ± 0.0<sup>C</sup></td><td>1.5 ± 0.4<sup>A</sup></td><td>0.9 ± 0.0<sup>B</sup></td><td>0.5 ± 0.1<sup>C</sup></td></tr><tr><td>Stigmasterol</td><td>–</td><td>–</td><td>–</td><td>–</td><td>0.3 ± 0.0<sup>A</sup></td><td>0.0 ± 0.0<sup>B</sup></td><td>0.0 ± 0.0<sup>B</sup></td><td>0.3 ± 0.1<sup>A</sup></td></tr><tr><td>β-sitosterol</td><td>–</td><td>–</td><td>–</td><td>–</td><td>0.4 ± 0.0<sup>A</sup></td><td>0.2 ± 0.0<sup>C</sup></td><td>0.3 ± 0.0<sup>B</sup></td><td>0.1 ± 0.1<sup>D</sup></td></tr><tr><td>Fucosterol</td><td>0.0 ± 0.0<sup>c</sup></td><td>10.9 ± 0.4<sup>a</sup></td><td>2.8 ± 0.1<sup>b</sup></td><td>2.4 ± 0.1<sup>b</sup></td><td>0.3 ± 0.1<sup>C</sup></td><td>3.9 ± 0.9<sup>A</sup></td><td>1.4 ± 0.1<sup>B</sup></td><td>1.5 ± 0.2<sup>B</sup></td></tr><tr><td>Total</td><td>6.3 ± 0.2<sup>d</sup></td><td>31.5 ± 1.2<sup>a</sup></td><td>13.5 ± 0.5<sup>c</sup></td><td>15.0 ± 0.9<sup>b</sup></td><td>16.9 ± 1.1<sup>B</sup></td><td>20.9 ± 4.7<sup>AB</sup></td><td>18.3 ± 1.3<sup>AB</sup></td><td>23.4 ± 3.0<sup>A</sup></td></tr></tbody></table>

Values with same superscript letters in the same row for ingestion (lower case letters) and incorporation (capital letters) are not significantly different at *P* > 0.05. -: sterols not present in the diet and not ingested.

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Maximum-likelihood tree of eukaryotic candidate 24-sterol reductases.</figcaption></figure>

proteins from the fatty acid hydroxylase superfamily in the Pacific oyster genome. Regarding C22-desaturation, the canonical enzyme in plants and yeast is assumed to be secondary lost in animals, red and brown algae, but one promising alternative candidate is the DES22 A/B enzyme characterized in *Tetrahymena* [31]. However, this one belongs to an highly multigenic fatty acid hydroxylase/desaturase family, that would require further extensive investigation in order to identify specific oyster candidates. Candidate C24 sterol methyltransferases (C24-SMTs) were not searched since a recent study concluded that they are absent from molluscan genomes [32].

The oyster candidate sequence is highlighted in bold, and groups with two other bivalve sequences, and more widely with

other animals (cnidarians, deuterostomes and arthropod DHCR24-1). Branch support values (aLRT) are shown when superior to 0.95. Colour code is as follows: blue for animals, green for viridiplantae, red for red algae, orange for haptophyte and brownish yellow for diatom.

## 4. Discussion

In multicellular animals, cholesterol is an essential cell-membrane component. Vertebrate animals satisfy this requirement by *de novo* synthesis of cholesterol from simple precursors with what is known as the squalene-mevalonate pathway. Metabolic pathways that provide cholesterol in other animal phyla are
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Possible pathways for sterol bioconversion in *C. gigas* larvae. 1. In the heterotrophic protists *Oxyrrhis marina* and *Gyrodinium dominans* [33]. 2. In the oyster *Ostrea gryphea* [10]. 3. In the oyster *Crassostrea gigas* [7]. 4. In the prawn *Penaeus japonicus* [34]. 5. In the prawn *P. japonicus* [35]. 6. In the Northern Bay scallop *Argopecten irradians* [4]. 7. In photosynthetic eukaryotes [36], mammals or insects. In blue, the two possible places where the candidate sterol 24 reductase may act. Sterol substrates are highlighted in bold. The chemical transformation is also highlighted.</figcaption></figure>

less thoroughly understood. Phytophagous insects, perhaps the best-studied animal models outside vertebrates, have been shown to metabolically modify some but not all dietary phytosterols to cholesterol [37]. Bivalve mollusks, including important seafood species, have been studied less methodically. As hatchery production of shellfish seed for aquaculture becomes more widely practiced, a better understanding of what live-feed microalgae are necessary to constitute a complete diet for young stages is becoming more urgent. Sterol nutrition and metabolism of larval shellfish remains a topic in need of refined knowledge for effective expansion of shellfish farming.

This is the first study determining the incorporation of cholesterol and other dietary sterols using a flow-through larval rearing system in bivalves. This larval culture system allows accurate quantification of algal cells, and their component sterols, ingested by larvae. It represents a step forward for the study of nutritional requirements of bivalve larvae, because the incorporation of individual sterols can be calculated using a mass-balance approach, and optimum levels of dietary sterols can be determined.

Total sterol accumulation in larvae fed T surpassed sterol ingested between day 8 and day 15–16 in both experiments (238 and 325 % of total ingested sterol in experiments 1 and 2, respectively), implying *de novo* synthesis. Similarly, larvae fed TCg likely synthetized *de novo* sterols, because total sterol accumulation in larvae was 137 and 156 % of total ingested sterols at competence (day 15–16) in experiments 1 and 2, respectively. Conversely, total sterol accumulation in larvae fed Cg was only 65 % of total ingested sterols, possibly because higher dietary sterol supply by Cg than T (132 vs 17 fg cell⁻¹ [15]). Teshima and Patterson [8] reported that adult *Crassostrea virginica* biosynthesized *de novo* cholesterol, desmosterol, isofucosterol, and 24-methylenecholesterol from injected

[2–¹⁴C]-acetate through squalene, probably using the lanosterol pathway. Moreover, *Ostrea gryphea* was capable of incorporating sterols when 3-[³H]-fucosterol propionate was dissolved in seawater [10]. Conversion of [2–¹⁴C]-mevalonate to cholesterol, desmosterol, 22-dehydrocholesterol, and 24-methylenecholesterol was tentatively demonstrated in *Mytilus edulis* [38]. In contrast, *C. gigas* spat were unable to synthetize *de novo* sterols when fed gelatin-acacia microcapsules containing ³H-squalene [7]. Sterol synthesis from [¹⁴C]-acetate was not detected in *C. virginica* heart tissue culture [39]. These authors, however, did not exclude the possibility of sterol *de novo* synthesis in other oyster tissues and/or life stages. Moreover, homologs from the enzymes encoding the classical cholesterol biosynthesis pathway are partially absent in oyster, with only the most downstream part of the pathway being conserved [40]. Despite the inconsistent literature, our data suggest that sterol *de novo* biosynthesis may take place in *C. gigas* larvae under low sterol dietary supply, which would imply that it either goes through different biosynthetic intermediates in the upstream part, or involves enzymes that are not orthologous to the classical eukaryotic ones. We also note that this sterol *de novo* synthesis may be species- and stage-dependent.

A key finding of this study is that, when *C. gigas* larvae were supplied with low-sterol diets (i.e., with T and TCg), they were able to partially bioconvert dietary phytosterols. Larvae fed T accumulated significant amounts of cholesterol in both experiments (0.28–0.46 ng larva⁻¹), with this sterol absent from the microalgae fed. We attribute this to sterol bioconversion or synthesis *de novo* of sterol. Brassicasterol (C₂₈) can be bioconverted into cholesterol (C₂₇) in the heterotrophic protists *Oxyrrhis marina* and *Gyrodinium dominans* [33,41]. Brassicasterol (C₂₈) is dealkylated to 22-dehydrocholesterol (C₂₇) by the removal of a methyl group at C24 and subsequently 22-dehydrocholesterol is hydrogenated at
C22 to cholesterol (C27) [33] (Fig. 2). 22-dehydrocholesterol was also detected in oyster larvae fed T, ranging from 0.1 to 0.2 ng larva⁻¹. Brassicasterol transformation to cholesterol in the crustacean *Artemia salina* has also been reported [42].

Cholesterol incorporation in larvae fed Cg (74 % of ingested cholesterol during the entire period of larval development), which is rich in cholesterol, is in accordance with general food-assimilation efficiency reported in bivalves, which is approximately 70 % [43]. In larvae fed TCg, we observed incorporation of cholesterol exceeding 100 % of ingested cholesterol in both experiments (139–148 %). Consequently, its synthesis is likely, consistent with a lower dietary supply in the mixed diet TCg as compared to Cg alone. Our data allowed us to propose an optimal supply of 13 ng cholesterol per C. gigas larvae to reach competence. Further studies with varying cholesterol dietary contents, however, should allow establishment of more accurate cholesterol requirements. Moreover, this approach could also be used to determine any dietary requirements for other metabolites or sterols.

Larvae fed Cg could have transformed cholesterol to brassicasterol by the reverse aforementioned pathway (Fig. 2), as suggested by Lund et al. [33]. Strikingly, the high level of brassicasterol accumulation in oyster larvae suggest that it is likely that brassicasterol was also synthetized de novo in larvae fed T (190–270 % of incorporation) and TCg (130–182 % of incorporation), even with a high dietary supply in T. Brassicasterol is synthesized in higher plants, microalgae, and fungi from squalene [44,45]. There is controversy in the presence of C-24 sterol methyltransferase (C24-SMT) in different invertebrate groups. Whereas de novo sitosterol synthesis has been demonstrated in some annelids using a non-canonical C24-SMT [46], to our knowledge brassicasterol synthesis was never previously reported in mollusks due to the absence of canonical C24-SMT genes in mollusks [32]. Such brassicasterol synthesis in larvae fed T (i.e., diet lacking cholesterol) and TCg support the assertion of Voogt [47] who highlighted the importance and possible role of brassicasterol in bivalves, which might replace cholesterol in some of its structural and/or physiological functions. Another possible explanation may be linked to membrane fluidity. Sterol chain length and unsaturation (bonded at C20) can influence membrane microviscosity [23]. Brassicasterol (28C5,22) created a more rigid structure in the membrane than cholesterol in the freshwater mollusk *Diplodon delodontus* [48]. Da Costa et al. [15] reported that C. gigas larvae fed T actively synthesized de novo the essential fatty acids 20:4n-6 and 20:5n-3 and non-methylene-interrupted fatty acids from fatty acid precursors found in the diet. The increase in unsaturated fatty acids in the membranes of C. gigas larvae fed T may have increased the fluidity of membranes and thus the incorporation of brassicasterol in membranes may be a compensating mechanism to maintain homeostasis of membrane fluidity.

Fucosterol was supplied by *C. neogracile* in larvae fed Cg and TCg, but its incorporation was low after 16 days of larval development (27 and 51 % in experiment 1, respectively). This could be attributable to fucosterol (C29) bioconversion to cholesterol (C27) by dealkylation at C24 as described in the oyster *O. gryphea* [10] (Fig. 2). The isomers isofucosterol/fucosterol can be bioconverted to cholesterol by the scallop *Argopecten irradians* [4].

In contrast to low fucosterol incorporation, desmosterol (C27) appeared to be actively synthetized (1.3–2.2 ng larva⁻¹ in larvae fed TCg at day 15–16). Desmosterol is an intermediate in bioconversion of fucosterol to cholesterol in *C. gigas* [7] and *O. gryphea* [10] and it is also a late intermediate in animal *de novo* cholesterol synthesis [3]. Desmosterol is reduced by Δ24-sterol reductase to cholesterol (Fig. 2), as demonstrated in the prawn *Penaeus japonicus* and the oyster *C. gigas* [7,48]. In the present study, genomic

analysis reveals a clear ortholog of Δ24-sterol reductase in oysters. Desmosterol was also actively synthetized in larvae fed Cg (0.6 ng larva⁻¹, between days 2 and 16) and T (0.15 ng larva⁻¹, between days 2 and 15–16 in both experiments). Desmosterol synthesis observed under all experimental conditions suggests the importance of desmosterol as an intermediate in sterol bioconversion to cholesterol in *C. gigas* larvae. This agrees with the observation by Giner et al. [4] who found relatively large amounts of labeled desmosterol in all cases in which the test sterol was converted into cholesterol.

Assimilation of 24-methylene-cholesterol in larvae fed Cg was lower (55 %) than those receiving TCg (73 %) at competence. Bioconversion to cholesterol may contribute to relatively low 24-methylene-cholesterol level in TCg-fed larvae by the dealkylation of 24-methylene-cholesterol (C28) to desmosterol (C27), as previously demonstrated in adult *C. gigas* [7] (Fig. 2).

Beyond the identification of the clear ortholog of Δ24-sterol reductase. Our attempts to confirm other sterol metabolic pathways within the genome of the Pacific oyster provided inconclusive results. Annotation efforts are ongoing, and we recommend that attention be given to sequences coding for enzymes involved in the specific pathways implied by our findings and those of others investigating sterol metabolism in bivalves.

In conclusion, the use of a flow-through larval rearing system coupled with a mass-balance approach allowed the calculation of incorporation and metabolic modification and synthesis of dietary sterols. Our results highlight the complexity of sterol metabolism in oyster larvae as a result of the varying levels of assimilation of dietary sterols depending on dietary sterol profiles and quantities. This study represents a step forward in the knowledge of the incorporation of dietary components in bivalves and links to biological performance. The present work has established foundations for the determination of essential or important biochemical components in bivalve larvae nutrition that may limit larval development, such as growth, survival and competency to morphophose to postlarval stage. Future studies using this methodology should be carried out to determine sterol requirements, deficiencies in dietary supply and sterol bioconversion to cholesterol. Moreover, the present technique could be coupled with [¹³C]-labeled microalgae using GC-c-IRMS to investigate sterol bioconversion pathways in different bivalve species and life stages. Our findings suggest that *C. gigas* larvae are capable of accomplishing not only sterol bioconversion, but also *de novo* sterol synthesis, using a combination of well-conserved enzymes, such as the DHCR24, and other ones in the downstream part of the pathway and lineage-specific enzymes in the upstream part. Cholesterol and brassicasterol synthesis were observed and desmosterol could be a metabolic intermediate in sterol synthesis or an important membrane component.

### CRediT authorship contribution statement

**Fiz da Costa:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. **Margaux Mathieu-Resuge:** Writing – review & editing, Validation. **Fabienne Le Grand:** Writing – review & editing, Validation. **Claudie Quéré:** Writing – review & editing, Methodology, Investigation. **Gabriel V. Markov:** Writing – review & editing, Formal analysis. **Gary H. Wikfors:** Writing – review & editing. **Philippe Soudant:** Writing – review & editing, Validation, Supervision, Conceptualization.

### Data availability

The dataset is available in the database figshare [28832252.v1].
The dataset is freely available to use with appropriate citation under Creative Commons Attribution 4.0 International Public License.

## Funding

This work was carried out during the EU funded ReproSeed project (FP7-KBBE 245119), which contributed to its functional outlays.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

Fiz da Costa acknowledges the Fundación Juana de Vega (Spain) for the postdoctoral fellowship at Ifremer. Special thanks to Dr. René Robert who was the postdoctoral supervisor of Fiz da Costa during his stage at Ifremer. Recently retired, he refused to be one of the co-authors despite his active involvement in the work that led to this manuscript. The authors would like to acknowledge the technical staff of the experimental mollusk hatchery of Argenton, Bruno Petton. Christian Mingant, Isabelle Quéau and Luc Lebrun for assistance in broodstock conditioning, larval rearing and phytoplankton production. The authors would like to thank Jean Girard and Loïc Harrault who participated in the implementation of the sterol analysis method in GC-MS.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biochi.2025.05.005.

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