Lipid Profiling of Pacific Abalone (Haliotis discus hannai) at Different Developmental Stages Using Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry

Pacific abalone (Haliotis discus hannai) is a commercially important mollusk; therefore, improvement of its growth performance and quality has been emphasized. During embryonic development, abalones undergo a series of distinct larval stages, including swimming veliger larvae, juveniles, and mature individuals, and their biomolecular composition varies depending on the developmental stage. Therefore, in the present study, we performed untargeted lipid profiling of abalone tissues at different developmental stages as well as the hemolymph of mature female and male abalones using ultrahigh-performance liquid chromatography-tandem mass spectrometry. These profiles can provide meaningful information to understand compositional changes in lipids through abalone metamorphosis and development. A total of 132 lipids belonging to 15 classes were identified from abalone tissues at different developmental stages. Moreover, 21 lipids belonging to 8 classes were identified from the hemolymph of mature abalones. All data were processed following strict criteria to provide accurate information. Triglycerides and phosphatidylcholines were the major lipid components identified in both tissues and hemolymph, accounting for, respectively, 27% and 15% of all lipids in tissues and, respectively, 24% and 38% of all lipids in the hemolymph. Of note, lysophosphatidylcholine was only detected in the tissues of mature abalones, paving the way for further analyses of abalone lipids based on developmental stages. The present findings offer novel insights into the lipidome of abalone tissues and hemolymph at different developmental stages, building a foundation for improving the efficiency and quality of abalone aquaculture.


Introduction
Abalones are large marine gastropods widely distributed in coastal waters worldwide [1]. ese mollusks are well-known for their nutritional benefits and are widely consumed in East Asia, including South Korea and China [2,3]. Among the various abalone species, Pacific abalone (Haliotis discus hannai) is commercially important [4,5]. erefore, substantial efforts have been devoted to improve its quality, and research on the embryology of its aquaculture is paramount [6]. is gastropod undergoes a series of life cycle stages, including the pelagic and benthic phases [7]. e pelagic phase marks a free-living period and includes trochophores, swimming larvae, and swimming veliger larvae. Subsequently, the larvae undergo morphological changes to enter the benthic phase, initiating sedentary life and undergoing further metamorphosis to turn into postlarvae. ereafter, they morph into a young shell or a juvenile abalone, and finally, grow into a sexually mature abalone [8][9][10]. Mature abalones reach their marketable size in approximately 4 years, which is relatively long [11]. Meanwhile, given the substantial expansion of global abalone aquaculture [12], improvement of the nutritive quality of these mollusks within a short period is imperative [13].
Nonetheless, the biomolecular coverage of the commercially important Pacific abalone (H. discus hannai) remains limited [15]. Genomic and transcriptomic analyses have been used to identify meaningful markers for abalone growth [19,20,22,[28][29][30]. A transcriptomic study of sexual maturation in H. discus hannai has uncovered certain pathways related to maturation, with lipid pathways being crucial in this process [19]. In addition, genomic studies aimed at the identification of growth-related single-nucleotide polymorphisms (SNPs) in H. discus hannai have revealed certain lipids involved in abalone growth and proposed genes encoding enzymes that are related to the metabolism of these lipids (e.g., mitochondrial glycerol-3phosphate acyltransferase1, gpam) as reliable candidate markers for growth [22,31].
However, detailed mechanisms that orchestrate the entire developmental process remain to be elucidated, and systematic identification of lipids involved in this process has not been performed in abalone species. Abalone comprises many components, including water, proteins, salts, carbohydrates, and lipids [13]. Although lipids are present in a low proportion, they are the key components of abalone, in that they are part of various biological systems (e.g., membrane) and processes (e.g., development and cellular signaling) of organisms [32,33]. Furthermore, lipids are essential for optimal growth in mollusk species [34]. Given its importance, the lipid composition of abalone tissues has been studied to improve growth and nutritional aspects.
e key factors of lipidomics include lipid coverage, sensitivity, identification, and throughput. anks to recent advances in mass spectrometry (MS) and high-efficiency separation techniques, rapid and sensitive detection of a large number of individual lipids in various biological samples is possible [35]. e lipid profiles of two abalone species have been determined using gas chromatographymass spectrometry (GC-MS) [13]. In addition, lipids in different tissues of H. discus hannai have been identified using liquid chromatography-mass spectrometry (LC-MS) [33]. To date, however, there has been no study on lipidomics at a different developmental stage of abalone.
Here, we explored the synthesis, regulation, and accumulation of lipids through lipid profiling at different developmental stages of H. discus hannai. e knowledge of lipid composition and its changes during development will be important to understand abalone development. Additionally, lipid profiling of the hemolymph of mature females and males will provide meaningful data on lipid homeostasis and abalone maturation.
To this end, in the present study, abalone lipid profiles were analyzed using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) through a nontargeted approach. e objective of the study was to obtain detailed lipid profiles of abalone tissues at different developmental stages and hemolymph of mature females and males. is information will be valuable for improving the growth performance and commercial value of abalone aquaculture.
Artificial fertilization was performed as described previously [36]. Sexually mature H. discus hannai (approximately 5 years old, with a mean SL of 120 mm) individuals were induced to release eggs and sperm using the conventional method of air exposure, followed by ultraviolet (UV)-irradiated seawater treatment. e eggs and sperm were thoroughly mixed to minimize individual variability. Following fertilization, the developing embryos were cultured in filtered and aerated seawater at 17°C. Two groups of H. discus hannai larvae at different developmental stages, including the late veliger stage (4 days postfertilization, dpf ) larvae (also called swimming veliger larvae), identified microscopically, and the juvenile stage (averaging ∼4 mm in SL, 90 dpf ), were collected. All samples were stored in a deep freezer at −80°C to keep them fresh.

Experimental Design
2.3.1. Lipid Extraction from Tissues. Before lipid extraction, fresh frozen abalone tissues in liquid nitrogen were individually pulverized using a Covaris CP02 cyro-prep instrument (Covaris, Woburn, USA). For lipid extraction from tissues, a two-step method involving neutral and acidic extraction was used. First, in neutral extraction, 1 mL of chloroform:methanol (1 : 2, v/v) was added to the cell pellets [37]. e samples were incubated for 10 min on ice and centrifuged at 13,800 × g for 2 min at 4°C. en, 950 μL of the supernatant was transferred to a fresh clean tube. Next, in acidic extraction, 750 μL of chloroform:methanol:HCl (1N, 37%) (40 : 80 : 1, v/v/v) was added to the remaining samples. After incubating for 15 min at room temperature, 250 μL of cold chloroform and 450 μL of cold 0.1 M HCl were added to the sample. e mixture was vortexed for 1 min and centrifuged at 6,500 × g for 2 min at 4°C. The lower organic phase was collected and combined with the prior extract. e sample was then dried using the Scan Speed 40 centrifugal evaporator (1,800 rpm, 3 h; Labogene, Denmark). Before starting the lipid analysis, the sample was dissolved in 50 μL of mobile phase solvent A:solvent B (2 : 1, v/v).

Lipid Extraction from Hemolymph.
Lipids from the hemolymph samples were extracted according to the Folch method [38] using a mixture of chloroform and methanol (2 : 1, v/v). e samples were vortexed and incubated on ice for 30 min. After the addition of chloroform:methanol:water (8 : 4:3, v/v/v), the samples were incubated on ice for an additional 10 min and centrifuged at low speed (2,000 rpm) for 5 min at 4°C. e lower organic phase was transferred to a fresh tube, and the aqueous layer was re-extracted with 1 mL of chloroform: methanol (2 : 1, v/v). e organic phases were combined and dried using the Scan Speed 40 centrifugal evaporator (1,800 rpm, 3 h; Labogene). e overall experimental workflow is presented in Figure 1. e HESI parameters were set as follows: sheath gas flow rate, 5 arb; auxiliary gas flow rate, 5 arb; sweep gas flow rate, 0; spray voltage, 4.0 kV; capillary temperature, 320°C; and S-lens radio frequency (RF), 50%. All samples were analyzed in technical triplicate.

Data Processing and Statistical
Analysis. MS data were acquired and processed using Xcalibur (v4. 1. 31.9, ermo Fischer Scientific, San Jose, CA, USA). e obtained raw data were uploaded and processed using Lipid Search 5.0 ( ermo Fischer Scientific, San Jose, CA, USA) for lipid profiling under the following conditions: product search; 5.0 ppm precursor ion mass tolerance; 8.0 ppm product ion tolerance; 1.0% intensity threshold; and top rank, main isomer peak, and FA priority filters. To filter inaccurate identification, lipids that were graded A, B and C were selected [39,40]. In addition, lipids with odd-numbered fatty acid chains, commonly called bacterial fatty acids, were filtered out to accurately consider animal lipids [41][42][43]. Lipids with % rsd values exceeding 50 were also removed to minimize error [44,45]. For statistical analysis, Metabo Analyst 5.0 (https://www.metaboanalyst.ca/) was used [46]. Multivariate statistical analyses, including principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and heat map clustering, were used.

Overview of H. discus hannai Lipid
Profiling. To achieve reproducible sample extraction, liquid nitrogen was used to homogenize the tissue of abalone. Different modified extraction methods based on chloroform and methanol were used to extract lipids from tissue and hemolymph, for increased coverage of lipid species in the sample [47]. Optimized mobile phases A and B were used for efficient ionization and separation in liquid chromatography. Each lipid class was well separated showing identical elution tendency as the previous research results on lipid separation in LC [47,48].
For data processing, rather than using the entire data set, we applied strict criteria and filtered the data to ensure high reliability. Only features that can confirm MS 2 data were used. As shown in Table S1, the ppm value for all lipid species was <0.01.
Additionally, lipid species containing odd-numbered fatty acids have been identified in several previous studies. In the present study, such lipid species were removed, as they are not common because of fatty acid metabolism and synthesis.
In this context, the following are the strengths of the present study: (1) To homogenize the tissues and efficiently extract the lipids, cyro-prep using liquid nitrogen followed for extraction in the first step. (2) Instead of using GC-MS, which can identify only a few central lipids, UHPLC-Quadrupole-Orbitrap-MS was used for scan-type lipidomics, which is appropriate for the analysis of phospholipids, neutral lipids, or sphingolipids [47]. (3) Greater coverage of lipidome was attained using cutting-edge software (Lipidsearch 5.0) and a comprehensive database. (4) A filter for lipids with oddnumbered fatty acids was added during data processing.

Lipid Profiling of Abalone Tissue.
To observe the overall changes in lipid composition and distribution during H. discus hannai development, we extracted lipids from swimming veliger larvae, juveniles, and mature abalones and  (Figure 2(a)). Total ion current (TIC) chromatograms of tissue samples are provided in Figure S1 and detailed data (Rt, m/z, ppm, adduct ion, area, and % rsd) for each of the 132 lipid species are presented in Table S1. TGs, DGs, PGs, PIs, and Co were detected as [M + NH 4 ] + adducts, while the remaining lipids were identified in the protonated form [M + H] + . Our results are consistent with lipid adduct ions identified in previous studies [49]. e [M + NH 4 ] + ion was likely produced by ammonium formate in the LC mobile phase. e [M + Na] + and [M + K] + ions are produced during the extraction process, HPLC valve operation, or ESI; however, based on the previous data [33], this was judged as noise and not considered for lipid identification. e highest number of TG types was eluted between 17 and 25 min, and the highest number of PC types was between 6 and 20 min. erefore, the longer the alkyl chain and saturated lipid, the longer the retention time. is result is consistent with previous reports on abalone lipidomics, in which TGs accounted for a high proportion among lipid classes; as such, these TGs may be derived from the viscera or gonads, rather than foot tissues [50]. e relative abundance of the normalized average peak area of lipid classes in abalone tissues according to the developmental stage is represented in Figure 2(c) and the list of normalized peak areas of each lipid species is shown in Table S4. A total of 15 classes were identified, and PCs and TGs accounted for the highest proportion of lipids at all stages. In juveniles, most lipid species were present at lower proportions than in swimming and mature individuals, except for DG, which was nearly two times more abundant in juveniles. Interestingly, one characteristic feature of the intersample lipid profile is that LPCs were only detected in mature individuals (Table S1) [51]. LPCs were eluted between 6 and 20 min and were detected as [M + H] + adducts. Lysophospholipids, as intermediates of glyceride metabolism, act as mediators in several neural pathways [52]. Lysophospholipids are produced by the action of phospholipase (PL)A 1 and PLA 2 and are either hydrolyzed by lysophospholipase or used to regenerate phospholipids through the remodeling pathway [53]. In the remodeling pathway of phospholipid synthesis, lysophospholipids are transiently produced by the action of PLA 2 but rapidly acylated to acyl-CoA through the diacylation reaction cycle for the maintenance of normal and essential neuronal membrane composition [54]. A previous study on the transcriptome of H. discus hannai demonstrated that the MtsPLA 2 gene related to PLA 2 was highly expressed in mature abalone [19]. In the present study, as LPCs were detected only in mature abalones, additional attention should be paid to the dynamics of these lipids during the development of this mollusk in the future. Specifically, integrated genomic, transcriptomic, and proteomic analyses are warranted to identify, among the profiled lipid species, useable candidates for the rapid growth and successful aquaculture of abalones.

Lipid Profiling of Mature Abalone Hemolymph.
To the best of our knowledge, the present study is the first to analyze lipid profiles of the hemolymph of H. discus hannai [13,33,55]. Information on the lipid profiles of the hemolymph of aquatic mollusks is limited to TGs and cholesterol in the studies of Pomacea canaliculata [56], Mercenaria mercenaria [57], and Achatina fulica [58]. We performed LC-MS/MS analysis of lipids extracted from the hemolymph of mature female and male abalones and identified a total of 21 lipids belonging to 8 classes (note that this refers to the sum of lipids from both female and male abalones). 8 PC types (38.1%), accounted for the highest proportion of all lipids, followed by 5 TG types (23.8%), 2 DG types (9.5%), and 2 MG types (9.5%). Cholesterol, PE, PS, and SPH types were identified in only one sample, accounting for 5% of the total lipids (Figure 2(b)). Overall, the observed lipid profile of the hemolymph of abalone, characterized by abundant PCs and TGs, is similar to that of other living blood samples [59]. TIC chromatograms of hemolymph samples are provided in Figure S2 and detailed data (Rt, m/z, ppm, adduct ion, area, and % rsd) of each of the 21 lipid species are presented in  [60,61]. In hemolymph analysis, [M + Na] + and [M + K] + ions were considered background noise, similar to that in tissue analysis. e relative percentage of the normalized average peak area of lipid classes in the hemolymph of female and male abalones is shown in Figure 2(d) and the list of normalized peak areas of each lipid species is shown in Table S4. Compared with tissue, the hemolymph exhibited a higher proportion of SPHs. Specifically, SPH was the most abundant lipid in hemolymph, and its proportion was over three times the proportion of DG, which was the second most abundant lipid in the hemolymph. SPH is involved in the regulation of various cellular functions, including cell growth, and constitutes part of the lipoprotein particles circulating in animal blood [62]. e percentage of peak area of DGs was followed by that of PCs and TGs, and their levels were comparable to those recorded in the human blood [63]. Although PCs and TGs were the most diverse species, they were relatively less abundant, and this result is contrary to the finding that these  Journal of Analytical Methods in Chemistry are the major lipids in human blood [59]. Lipid species abundance differed between females and males. While PE and PS were identified only in males, their average peak area was relatively small, indicating no significant differences.

Detailed Characterization of Altered Lipids of Each Group in Tissue and
Hemolymph. Intratissue and intertissue sample variations were visualized using PCA, as shown in Figure S3(a), which revealed marked differences among the three groups. Juvenile samples were grouped into the most compact clusters, with the smallest 95% confidence interval. PC1 explained 54.8% of the variance, while PC2 explained 40.7% of the variance. Each sample group was clearly separated. Furthermore, PLS-DA, which is a supervised statistical method [64], was applied for the classification of the three groups, as shown in Figure 3(a). Similar to that in the PCA biplot, juvenile samples formed a more compact cluster, and each group was clearly separated, indicating distinct differences in lipid profiles among the groups. In the PLS-DA score plot, components 1 and 2 explained, respectively, 51% and 44% of the total variance.
Among the 132 lipids, the VIP score plot of the top 10 lipids selected based on the PLS-DA model is shown in Figure 3  recorded VIP values exceeding 1.5 [65]. PC (36 : 1) was the most significant lipid in abalone tissues at different developmental stages, achieving a score close to 8.0, and it was also abundant in swimming veliger larvae. PC (34 : 5) was the most abundant in mature individuals, followed by juveniles and swimming veliger larvae, indicating that its content increases with the progression of abalone development.
Next, intrasample and intersample variations in hemolymph were visualized by PCA and PLS-DA, as shown in Figure S3(b) and Figure 3(b). On the PCA biplot, the two groups were well separated; PC1 explained 88.2% of the variance and PC2 explained 11.6% of the variance. Likewise, in the PLS-DA score plot, clear separation between female and male groups was observed, implying differences in lipid composition. Among the 21 lipids, the VIP score plot of the top 10 lipids based on the PLS-DA model is shown in Figure 3(d). e lipid species that contributed the most to the discrimination were DG (34 : 0) (VIP score > 3.0), followed by SPH (d16 : 0), and MG (16 : 0) (VIP score > 1.5).
e heatmap in Figure 4 shows the tendencies of lipids identified in the tissue and hemolymph. Detailed data are reported in Figure S4.

Notable Lipid Synthesis Pathways.
A schematic of the notable lipid biosynthesis pathways explored in the present study is presented in Figure 5 [66,67]. In the glycerophospholipid synthetic pathway, glycerolipids (MG, DG, and TG), phospholipids (PC, PE, PG, PI, and PS), and lysophospholipids (LPC, LPE, LPG, and LPI) were identified. e sphingolipid and sterol biosynthetic pathways are also part of the lipid synthesis pathway in abalone. In addition, acylcarnitine (AcCa) and coenzyme 10 are directly related to the mitochondria. AcCa is involved in fatty acid transport to the mitochondria, and Co participates in electron transfer [16]. e lipid biosynthetic pathways and species emphasized in the present study ( Figure 5) will extend our understanding of H. discus hannai lipidome and can be applied in future studies. Based on these lipid biosynthetic pathways, the overall molecular mechanism of H. discus hannai development can be unveiled by assessing the expression of genes or proteins involved in lipid metabolism and synthesis (e.g., fatty acid synthase, carnitine palmitoyltransferase 1, acetyl-CoA carboxylase, and 3-hydroxy-3-methyl-glutaryl coenzyme A reductase) [68].  Table S1: List of identified lipids in tissue of abalone, Haliotis discus hannai at different developmental stages (swimming veliger larvae, juvenile, mature). Table S2: List of identified lipids in hemolymph of female and male abalone, Haliotis discus hannai. Table S3: List of the normalized area of lipids in tissue of abalone, Haliotis discus hannai at different developmental stages (swimming veliger larvae, juvenile, mature). Table S4: List of normalized area of lipids in hemolymph of female and male abalone, Haliotis discus hannai. Figure S1 Figure S3: PCA score plots of (a) tissues in three different developmental stages, (b) female and male hemolymph. Figure S4: e detailed heatmap of abalone tissue. (Supplementary Materials)  (ii) Composition of unsaponifiable lipids (4 sterol species) was examined (iii) Enzyme-assisted organic solvent method (iii) irteen saponifiable fatty acids were identified (iv) Percent composition depends on the extraction method Journal of Analytical Methods in Chemistry 9