LU Zhibin ,SHI Ce ,LIU Lei ,MU Changkao ,YE Yangfang, ,and WANG Chunlin,

1) Key Laboratory of Applied Marine Biotechnology, Ningbo University, Chinese Ministry of Education, Ningbo 315211,China

2) Collaborative Innovation Center for Zhejiang Marine High-Efficiency and Healthy Aquaculture, Ningbo 315211,China

Abstract Phospholipids are used to improve the growth and survival of Portunus trituberculatus,a widely cultured crab species in China.However,only total phospholipids or several classes are applied in crab diets.In this study,we employed a targeted lipidomic method to investigate the comprehensive phospholipid composition in P.trituberculatus larvae and reveal the changing phospholipid profile over the larval development.Results showed that P.trituberculatus larvae contain 112 phospholipid species belonging to 10 phospholipid classes,in which phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species are the most abundant,and PC,PE,phosphatidic acid,and phosphatidylserine (PS) are with high concentrations.The levels of all phospholipids significantly changed with larval development,which was highlighted by the downward parabolic changes in PE,phosphatidylglycerol,phosphatidylinositol,PS,lysophosphatidic acid,and sphingomyelin levels.In addition,nearly all phospholipid species were depleted at the M stage,which probably contributed to the mass mortality of crab larvae.These findings on the composition and alterations of phospholipids in P.trituberculatus larvae provide novel perspectives for the targeted supplementation of phospholipids in crab diets.Our work also highlights the use of targeted UHPLC-MS lipidomics in understanding the changes of phospholipids during crab development.

Key words Portunus trituberculatus;larval development;phospholipid;UHPLC-MS

1 Introduction

The swimming crabPortunus trituberculatus(Crustacea,Decapoda,Brachyura) is a widely cultured crab species in China with a yield of 113810 tons in 2019 (2020 China Fishery Statistical Yearbook).However,the culture of swimming crabs is still a challenge because of the low survival rate of the larvae (Lim and Hirayama,1991;Wanget al.,2016).The mass mortality of swimming crab larvae usually occurs at the final zoeal stage (Danet al.,2013).Inadequate nutrition is generally considered the probable cause of this problem (Hamasakiet al.,2002;Danet al.,2013).

Phospholipids play essential roles in membrane formation,multiple development processes,and signal transduction (Vance,2008;Shevchenko and Simons,2010;Tanget al.,2011).Phospholipids also function as a surfactant for the efficient emulsification of lipids and a component of high-density lipoproteins for the principle transport of cholesterol (Holmeet al.,2009b).Hence,phospholipids have been applied in crab diets to enhance the survival of cultured crab larvae.For example,the survival ofEriocheir sinensislarvae fed diets supplemented with soybean phospholipids increased about 20% from the megalopa to juvenile stage (Chenget al.,1998).Despite limited information on the role of phospholipids in the survival ofP.trituberculatuslarvae,dietary phospholipids have been proven important in improving growth,survival,and immunity,and reducing the occurrence of precocious puberty.Phospholipids are composed of glycerophospholipids and sphingomyelin (SM).Glycerophospholipids can form phosphatidylcholine (PC),phosphatidylethanolamine (PE),phosphatidylserine (PS),phosphatidylglycerol (PG),phosphatidylinositol (PI),and phosphatidic acid (PA),as well as a range of lysoglycerophospholipids,including lysophosphatidylcholine (LPC),lysophosphatidylethanolamine (LPE),and lysophosphatidic acid (LPA).Traditionally,PC is the active component for enhancing the growth and survival of crustaceans because it is the principal constituent of polar lipids in biomembranes and functions in lipoprotein synthesis and secretion (Coutteauet al.,2000).However,whether other phospholipids are the active components of crustaceans remains unclear (Thompsonet al.,2003).In fact,other phospholipids have specific functions.For example,PE not only serves as the phospholipid substrate and precursor in cellular functions,but also regulates the processing of transcription factors (Vance and Tasseva,2013).Furthermore,PS can act as a ligand for membrane receptor brain-specific angiogenesis inhibitor 1 to promote myoblast fusion (Hochreiter-Huffordet al.,2013),whereas PI is implicated in cell growth and cell division inDrosophilalarvae (Guptaet al.,2013).Each phospholipid class can be further subdivided into individual species according to their chemical structure characteristics,such as carbon chain length and degree of unsaturation.Different chemical structures endow phospholipid species with unique functions.For example,PC(18:0/18:1) can function as an endogenous ligand of peroxisome proliferator-activated receptors (PPARs) (Liuet al.,2013;Kersten,2014),whereas PC(16:0/18:2) can interact with paraoxonase 1 to enhance its uptake by macrophages (Cohenet al.,2014).Furthermore,PA(18:0/22:6) and PA(20:0/22:6) are the direct metabolic precursors in the Kennedy pathway (Carman and Han,2019),and LPC(16:0) plays an essential role in suppressing the sexual differentiation ofPlasmodium falciparum(Brancucciet al.,2017).In this sense,focusing only on the total phospholipids or several classes(Chenget al.,1998;Andréset al.,2007;Holmeet al.,2007,2009a;Suprayudiet al.,2012) cannot thoroughly explore the fundamental requirement of crab larvae for phospholipids.Hence,the qualitative and quantitative phospholipid requirements for crab larvae must be precisely identified.

Lipidomic approaches play important roles in such an analysis.For example,ultra-performance liquid chromatography-triple time of flight mass spectrometry (UPLCTriple TOF-MS/MS) has been applied to characterize lipid profiles,including 14 classes,in four shellfish species (Liet al.,2020).Notably,UPLC-Q-Exactive Orbitrap/MS has been used to identify 310 marine phospholipid species,including 83 PCs,50 PEs,33 LPCs,24 PIs,17 LPEs,7 PAs,21 SMs,and 22 PSs from shrimp heads,codfish roes,and squid gonads (Liet al.,2018).Moreover,LC/MS has been used to detect 102 PC-containing lipid species,including 10 lyso-PCs,36 diacyl-PCs,7 P-PCs,5 O-PCs,and 13 SMs in rat lungs (Leeet al.,2018).In addition,an ultrahighperformance liquid chromatography system coupled with triple-quadrupole mass spectrometry (UHPLC-MS) has been established and proven to be potent in identifying lowabundance phospholipids and achieving the high-throughput and quantitative detection of phospholipids (Huanget al.,2019).More than 160 phospholipid species have been identified in A549 cells,human plasma,and rat liver.

In the present study,we identified and quantified the phospholipid molecular species inP.trituberculatusextracts during larval development by using a UHPLC-MSbased lipidomic approach.The objectives are to investigate the overall and specific changes in the phospholipid compositions ofP.trituberculatusduring larval development.The findings from the lipidomic investigation are useful for gaining insights into the phospholipid requirements ofP.trituberculatuslarvae for survival.

2 Materials and Methods

2.1 Chemicals and Reagents

HPLC-grade ammonium acetate,methanol,and formic acid were purchased from Sigma-Aldrich (St.Louis,MO,USA).HPLC-grade chloroform was obtained from Duksan Pure Chemicals (Seoul,Korea).Deionized water was prepared with an Elix Advantage system (Waters,Millipore,MA,USA).Nine internal standards PC(17:0/14:1),PE(17:0/14:1),PG(17:0/14:1),PS(17:0/14:1),PA(17:0/14:1),PI(17:0/14:1),LPC(17:1),SM(17:0),and LPA(17:1) were purchased from Avanti Polar Lipids (Alabaster,AL,USA).The internal standards were prepared at 10 μg mL-1and then stored at -20℃ for further analysis.The nomenclature of phospholipids was described by LIPID MAPS (http://www.lipidmaps.org/).

2.2 Broodstock and Larval Culture

Wild ovigerous females of swimming crabs were reared in an indoor 30-m3cement pond sanded with approximately 10 cm thickness at the Yonggan Nursery Farm (Ningbo,China).Water temperature and salinity were controlled at 24℃ ± 1℃ and 25 ± 1,respectively.Broodstock crabs were fed daily with two razor clams (Sinonovacula constricta) after the removal of feces and food debris by siphoning.Every morning,a small number of eggs were examined by microscopy to assess egg development.Once the eggs developed into the heartbeat stage,six berried females were separately placed into plastic baskets and evenly placed into another three 30 m3cement ponds for hatching.Seawater was aerated and circulated through the basket.The berried females were not fed until the larvae hatched.Once the zoea 1 (Z1) larvae were released,the ovigerous crabs were immediately returned to the original cement pond.The larval culture was carried out following the conventional protocol with some modifications (Wuet al.,2014).In brief,microalgae (Skeletonema costatuma)at a density of 5×104– 1×105mL-1and fairy shrimp (Artemia nauplii) at a density of 2–3 individuals per mL were used to feed Z1 larvae every 12 h.The feeding amount increased gradually with larval development from zoea 2(Z2),zoea 3 (Z3),zoea 4 (Z4) to megalopa (M).No water was changed throughout zoea culture,whereas 1/3 of the water in each pond was renewed once at the megalopa stage.

2.3 Larva Sampling

In determining the phospholipid composition,the newly hatched Z1,Z2,Z3,Z4,and M larvae and the newly settled first-stage crabs (C1) were sampled from three ponds when 70%– 80% of the population had molted to the desired development stage to ensure that the collected larvae were in the same development stage (Wuet al.,2014).The samples were rinsed with filtered seawater.Each sample was divided into two portions with approximately 714,555,222,123,47,and 7 individuals per 100 mg portion for the Z1,Z2,Z3,Z4,M,and C1 groups,respectively.Thus,three biological replicates and two technical replicates were used in this study.All samples were snap-frozen in liquid nitrogen and then stored at -80℃for further analysis.

2.4 Phospholipid Extraction and UHPLC-MS Analysis

Phospholipid extraction was performed as previously described (Huanget al.,2019).Approximately 20 mg of crab individuals with nine internal standards (100 ng of each)were used for quantitative analysis,whereas approximately 300 mg was used for qualitative analysis.UHPLC-MS spectra were obtained on a 1290 ultrahigh-performance liquid chromatography system coupled to a 6470 triple-quadrupole mass spectrometer equipped with a dual AJS electrospray ionization (ESI) source and a ZORBAX Eclipse Plus C18 (2.1 mm × 100 mm,1.8 μm) column (Agilent Technologies,Inc,USA).The ESI positive and negative ion modes were used to collect phospholipid data in separate runs.UHPLC-MS conditions were set and acquired as previously described (Huanget al.,2019).Furthermore,the relative standard deviation was calculated by assessing the reproducibility of the peak area and retention time to evaluate the precision and accuracy of the method.

2.5 Statistical Analysis

All multiple reaction monitoring (MRM) spectra and MS/MS spectra were processed manually using Mass Hunter Qualitative software (Agilent,B.06.00) and Mass Hunter Quantitative software (Agilent,B.06.00).The concentration of each phospholipid species was calculated based on its relative abundance and internal standard.The obtained concentration was further normalized to the wet weight of the corresponding crab sample.The quantitative data of phospholipids obtained from different crab groups were imported into SIMCA-P+software (v15.0,Umetrics,Sweden) for principal component analysis (PCA) with mean-centered scaling.The general trend and sample outliers were detected in this unsupervised multivariate data analysis.The quantitative data of phospholipids were further analyzed using univariate data analyses,including oneway-ANOVA and Mann-Whitney U test with the false discovery rate (FDR) approach of SPSS (v22.0,SPSS,Chicago),to screen the significantly changed phospholipids due to larval development.Values are presented as means± SD (standard deviation).The difference will be considered significant whenP<0.05.

3 Results and Discussion

3.1 Phospholipid Analysis of P.trituberculatus Larvae

A total of 112 phospholipid species (positive and negative modes) belonging to 10 phospholipid classes were identified fromP.trituberculatuslarvae using MRM and MS/MS spectra.These phospholipid species comprised 32 PCs,23 PEs,2 PAs,7 PSs,3 PGs,16 PIs,15 LPCs,6 LPEs,2 LPAs,and 6 SMs.Their molecular species,MRM,and retention times are listed in Table 1.Among them,five pairs of phospholipid species still cannot be precisely identified mainly because of the similar chemical character and the same ion pair for MS quantification,including PC(16:0/18:3) and PC(16:1/18:2),PC(14:1/20:1) and PC(16:0/18:2),PC(18:1/20:3) and PC(20:1/18:3),PC(18:1/22:6) and PC(20:2/20:5),and PE(18:0/18:0) and PE(20:0/16:0).In our knowledge,this study is the first one to perform a comprehensive analysis of phospholipids inP.trituberculatus.

Table 1 The molecular species,MRM and retention time of phospholipids in P.trituberculatus

(continued)

In all ten detected phospholipid classes of swimming crab larvae,PC and PE species were the most abundant,which is consistent with typical biological samples,including shellfish,rat,and human samples (Huanget al.,2019;Liet al.,2020).By contrast,PA and LPA had the fewest species,with only two species in each class within larvae.In terms of quantity,the amount of total phospholipids in Z1 was 3254.48 ± 262.70 nmol g-1.Among them,PA was the most abundant with a content of 1036.94 ± 194.06 nmol g-1.PC,PS,and PE were lower in quantity than PA with the contents of 771.26 ± 71.08,662.76 ± 119.69,and 398.55 ± 29.41 nmol g-1,respectively.These four phospholipid classes accounted for 88.17% of the total amount of phospholipids(Fig.1).These results showed apparent differences from the phospholipid composition ofDrosophila melanogasterand shellfish (Huanget al.,2018;Liet al.,2018,2020).For instance,PC and PE generally have high abundances in most aquatic animals (Liuet al.,2017;Liet al.,2020) but are not predominant in zoea 1 ofP.trituberculatus.Notably,PA and PS were considerably low in shellfish but were considerably high inP.trituberculatuslarvae.Phospholipid composition is highly related to feeding or prey (Coutteauet al.,1997).Therefore,the difference in phospholipid composition between animal species might be attributed to different diets.PC has been widely used as a supplement in diets for crab culture (Wuet al.,2014;Wanget al.,2016;Linet al.,2020,2021).However,nutritional studies on other phospholipids,such as PE,PA,and PS,remain lacking.The high abundance of PE,PA,and PS in zoea strongly suggests that they play essential roles in the growth and survival of swimming crab larvae.

Moreover,these phospholipids play essential roles in different biological processes.For example,PE serves as a substrate for critical posttranslational modifications,which can influence membrane topology and promote cell fusion,oxidative phosphorylation,mitochondrial biogenesis,and autophagy (Thomaset al.,2010).PA can function as a critical intermediate in glycerophospholipid synthesis and is closely associated with diacylglycerol generation (Wanget al.,2006).PA can also act as a second messenger molecule in the diacylglycerol/protein kinase C signal transduction pathway (Buckland and Wilton,2000).PS is involved in programmed cell death,and sufficient PS can promote muscle formation and cell differentiation (Hochreiter-Huffordet al.,2013).These functions probably drive swimming crab larvae to accumulate high levels of PE,PA,and PS.In addition to these phospholipid classes,PI,SM,and LPC were also relatively abundant with contents ranging from 70.49± 8.64 to 177.36 ± 13.46 nmol g-1.Moreover,LPE,PG,and LPA were detectable but much less abundant in zoea 1 with 23.35 ± 4.58 nmol g-1or less than 10 nmol g-1.These phospholipids play vital roles in signal transduction and cell growth (Mikiet al.,2002;Birgbauer and Chun,2008;Daiet al.,2015;Shenget al.,2015).Whether these lipids contribute to swimming crab survival and growth remains unknown,but the results obtained here provide a potential basis for a broader nutrition study of this crab species.

At the species level,the most abundant phospholipid species was PA(18:0/22:6),with a content of 673.50 ±132.14 nmol g-1.Other phospholipid species,including PA(20:0/22:6),PS(18:0/18:1),PS(18:0/20:5),PC(16:0/18:1),and PE(18:0/20:5),were also relatively abundant with contents of 363.44 ± 64.68,250.13 ± 68.14,162.80 ± 22.87,133.94 ± 13.50,and 101.56 ± 7.13 nmol g-1,respectively.These six species,accounted for 51.79% of the total amount of phospholipids in Z1 larvae.In recent years,the functions of some phospholipid species have been explored.For example,PS(18:0/18:1) and PS(18:0/20:5) act as regulatory factors for signal transduction and cell growth in bacterial and mammalian cells (Liuet al.,2017;Sanset al.,2017;Ledvinaet al.,2018).Considering the high abundance,these six species probably play vital roles in larval survival and growth,which merits further study.

Similar to the metabolic profile,the phospholipid profile ofP.trituberculatuslarvae is expected to shift over development (Shiet al.,2019).Our results clearly showed differences in the concentrations rather than the types of phospholipid classes/species among the larval stages.The total phospholipids rapidly accumulated to 5311.10 ± 973.61 nmol g-1wet weight larvae at Z2 stage and presented a fluctuating downward trend to 2621.73 ± 611.52 nmol g-1wet weight larvae at C1 stage.This total phospholipid level of C1 was comparable to that ofD.melanogasterin the first day (152.61 ± 4.92 nmol mg-1dry weight) (Huanget al.,2018).Nevertheless,the obvious changes in the total phospholipid level may indicate level differences for a large number of phospholipid class/species.Hence,the detailed phospholipid differences at the classes/species level for these six developmental stages were obtained through multivariate and univariate data analyses.

3.2 Phospholipid Changes with Larval Development

An unsupervised PCA was constructed using the phospholipid quantification datasets to determine the global phospholipid changes during larval development.The first two principal components (PC1 and PC2) jointly explained 92.3% of the total variance (Fig.2).The scores plot showed a clear separation between Z1 and the other five stages but a slight separation among groups from Z2 to C1,broadly presenting a development-dependent change trend.Thus,the main change in phospholipid composition occurs during the early development of crab larvae.ANOSIM was used to further corroborate this pattern,and results revealed a significantly different phospholipid composition in each group pair (Table 2).Univariate data analysis was further used to characterize such differences in phospholipid compositions due to larval development.At the class level,a significant change in larval development was observed at the levels of all phospholipid classes (Fig.1).For PC,the changing trend followed a downward parabola.In detail,a significant increase occurred from Z1 to Z3 with no significant change between Z3 and Z4.The peak level of PC was 1684.21 ± 201.98 nmol g-1at the Z4 stage.Then,PC was markedly depleted from Z4 to M and from M to C1 with a recovery to the Z1 level at the C1 stage.Notably,the PC levels accounted for the highest proportion of phospholipids from the Z3 stage,indicating its critical role in the growth and development ofP.trituberculatus(Wanget al.,2018).However,the amount of PCviaendogenous synthesis usually cannot meet the requirements of most crustaceans during early development (D’Abramoet al.,1982).Our results,manifested by the marked decrease in the PC level from M stage,further support this view.Thus,dietary PC is needed forP.trituberculatuslarvae,especially at the megalopa stage.However,no information is available on the dietary PC inP.trituberculatuslarvae,although studies have been conducted onP.trituberculatusjuveniles(Liet al.,2014;Wanget al.,2016) and other crabs (Wuet al.,2007,2010,2011,2014).

Fig.2 PCA scores plot constructed from the UHPLC-MS spectra of larval extracts of P.trituberculatus at zoea 1– 4(Z1– Z4,circles were getting larger over the development),megalopa (M,triangles),first juvenile crab (C1,squares).

Table 2 Community dissimilarity test of phospholipids in P.trituberculatus based on analysis of similarity using weighted UniFrac distance

A downward parabolic trend was also observed in PE,PG,PI,PS,LPA,and SM (Fig.1).For PE,a sharp rise occurred from 398.56 ± 29.42 nmol g-1at the Z1 stage to 816.77 ± 44.35 nmol g-1at the Z2 stage.A significant decrease was observed from Z2 to Z3 and from Z4 to C1 with no significant change between Z3 and Z4.The first juvenile crab had the lowest PE level among the six developmental stages.For PG and PI,a marked rise occurred from Z1 to Z2 with no significant change from Z2 to Z4;a significant decrease occurred from Z4 to C1.For PS,no significant change occurred during the zoeal stage,but a significant decrease occurred at M and C1.For LPA and SM,an overall rise occurred from Z1 to Z4,and a significant decrease occurred from M to C1.All these changing trends indicate that the M stage is a critical threshold for swimming crab larvae to maintain sufficient PE,PG,PI,PS,LPA,and SM for survival.Another notable phospholipid class is PA because of its high abundance and marked depletion at the Z3 and M stages.More than 70% of PA was consumed at the M stage.However,no available information focuses on this phospholipid class in crustaceans.The mass mortality of crab larvae at the final zoeal stage is probably directly attributed to the significant shortage of these phospholipids.Dietary phospholipids may be essential to helpP.trituberculatuslarvae pass through this critical stage.Moreover,targeted dietary supplementation with phospholipid classes may be much more precise in maintaining the nutrient balance of swimming crab larvae than total phospholipids.In fact,targeted supplementation has been proposed and conducted by replacing crude total phospholipids with purified PC in the diets of crab (P.trituberculatusjuveniles) (Wanget al.,2016),shrimp (Camaraet al.,1997),and fish (Kenariet al.,2011).These studies indicate that purified PC significantly improves larval growth compared with PE and PI.In this study,LPC and LPE presented an increasing trend.A similar change in LPC was found inE.sinensisjuveniles fed diets supplemented with phospholipids (Linet al.,2020).In mammals,LPC is produced from PCviaa deacyl-reacyl pathway and plays a potential role in the delivery of long-chain polyunsaturated fatty acids,such as docosahexaenoic acid (22:6n-3,DHA),to the fetus (Ferchaud-Roucheret al.,2019).DHA is an important nutrient for the growth of swimming crabs(Yuanet al.,2019).In this study,the increased LPC level likely suggests a role of transferring DHA inP.trituberculatuslarvae.

At the species level,all phospholipid species presented a significant change during the larval development (Fig.3).Detailed information on the phospholipid species changes due to larval development was obtained from the Mann-Whitney U test with an FDR controlling technique (P≤ 0.05)of phospholipid composition of two neighboring developmental stages.Compared with that of Z1,the phospholipid composition of Z2 was highlighted by higher levels of 26 PCs,21 PEs,9 PIs,2 PSs,3 PGs,PA(18:0/22:6),11 LPCs,5 LPEs,2 LPAs,and 3 SMs and lower levels of 5 PCs,8 PIs,2 PSs,4 LPCs,and 2 SMs (Fig.4).Notably,the levels of PC(18:1/20:2),PC(20:1/18:1),PC(18:3/20:5),PC(18:1/20:3) &PC(20:1/18:3),PE(18:1/20:1),PE(18:1/20:2),PE(18:3/18:1),PE(18:3/20:5),PI(18:3/20:5),PI(20:3/20:5),and LPE(18:0) in Z2 remarkably accumulated to above 300% of those in Z1.Furthermore,the levels of 16 PCs,7 PEs,5 LPCs,3 LPEs,3 PIs,2 PGs,5 LPCs,3 LPEs,2 LPAs,and SM(d18:0/24:1) accumulated to above 50% of those in Z1.Simultaneously,the levels of PC(14:0/16:0),PC(22:2/22:2),PC(20:5/22:6),PA(18:0/22:6),and LPC(22:6)in Z2 reduced by more than 50% compared with those in Z1.Compared with that of Z2,the development to Z3 significantly increased the levels of 11 PCs,4 PEs,3 PIs,PG(20:1/16:0),PA(20:0/22:6),LPC(22:0),3 LPEs,and 2 LPAs,and significantly decreased the levels of 8 PCs,5 PEs,2 PIs,PS(18:0/20:5),PG(16:0/18:1),PA(20:0/22:6),12 LPCs,5 LPEs,and 2 SMs.The increase was highlighted by a high accumulation in the levels of PC(16:0/18:3) &PC (16:1/18:2),PC(18:1/18:3),PC(18:1/18:2),PC(18:1/20:3) &PC(20:1/18:3),PC(18:3/14:0),PC(18:3/16:1),PC(18:3/20:5),PE(18:2/16:1),PE(18:3/18:1),PI(18:1/18:1),and PI(18:1/18:2) to above 50% those in Z2.Conversely,the levels of PA(20:0/22:6),PA(18:0/22:6),LPC(20:5),and LPC(22:6)presented an over 50% decrease compared with those in Z2.Compared with Z3,Z4 presented higher levels of 12 PCs,10 PEs,7 PIs,4 PSs,2 PGs,2 PAs,13 LPCs,LPE16:0,and 4 SMs and lower levels of 8 PCs,9 PEs,7 PIs,3 PSs,PG(16:0/18:1),and 2 LPCs.Notably,the levels of PC(14:0/20:5),PC(18:0/20:5),PC(20:5/20:5),LPC(20:4),and LPC(20:5) remarkably accumulated to above 50% of those in Z3.In addition,the levels of PC(22:2/22:2) and PI(18:1/18:1) decreased by more than 50% of those in Z3.However,more phospholipids were significantly depleted in the megalopa stage compared to Z4,including 30 PCs,23 PEs,12 PIs,6 PSs,3 PGs,PA(20:0/22:6),3 LPCs,5 LPEs and SM(d18:0/24:1).Simultaneously,8 LPCs,4 LPEs,and 2 SMs significantly accumulated in the megalopa stage compared with Z4,which was highlighted by the remarkable accumulation of LPC(16:1),LPC(18:2),LPC(20:4),LPC(20:5),LPE(18:1),LPE(20:1),LPE(20:4),and LPE(20:5)to above 50% those in Z4.By contrast,a sharp decrease above 50% of the levels of PC(22:2/22:2),PE(18:3/20:5),PE(22:6/20:4),PE(22:6/20:5),PG(16:0/20:2),PG(20:1/16:0),and PS(18:0/18:1) was observed.Compared with the megalopa stage,the first juvenile crab exhibited significantly higher levels of PA(20:0/22:6),8 LPCs,2 LPEs,and SM(d18:0/20:2),and significantly lower levels of 23 PCs,17 PEs,9 PIs,PS(18:0/22:6),3 PGs,4 LPEs,and 4 SMs compared with the megalopa stage.However,only the levels of LPC(22:0) and LPE(18:0) decreased to more than 50% of those in C1.

Fig.3 Contents of phospholipid species in P.trituberculatus larvae at different development stages.Z1,zoea 1;Z2,zoea 2;Z3,zoea 3;Z4,zoea 4;M,megalopa;C1,first juvenile crab;PC,phosphatidylcholine;PE,phosphatidylethanolamine;PG,phosphatidylglycerol;PI,phosphatidylinositol;PS,phosphatidylserine;PA,phosphatide acid;LPC,lysophosphatidylcholine;LPE,lysophosphatidylethanolamine;LPA,lysophosphatidic acid;SM,sphingomyelin.

Fig.4 Different phospholipid species between two neighboring development stages of P.trituberculatus larvae illustrated by volcano plots.Z1,zoea 1;Z2,zoea 2;Z3,zoea 3;Z4,zoea 4;M,megalopa;C1,first juvenile crab.

Despite this detailed changing information on phospholipid species with the larval development,limited information is available on the functions of these phospholipid species in crustaceans.Previous studies demonstrated the roles of some phospholipid species.For example,PC(16:0/16:0) has unique characteristics of alveolar surface tension reduction (Langet al.,2005),whereas PC(18:0/18:1) and PC(16:0/18:1) participate in the pulmonary surfactant system (Schmidtet al.,2007).Furthermore,PC(18:0/18:1) can serve as an endogenous ligand for PPARs,which inversely regulate cell differentiation,glucose homeostasis,and lipid metabolism during embryonic development (Kersten,2014;Wuet al.,2017).In crabs,PPARs can protectE.sinensisfrom polychlorinated biphenyl exposure by regulating anti-inflammatory and antioxidative activities (Fenget al.,2019).In addition,PC(18:0/18:1) and PC(16:0/18:2)exert a stimulatory effect on paraxonase 1,which is associated with atherosclerosis (Cohenet al.,2014).In this study,all detected PC species changed significantly with larval development,suggesting the possibility of functional variation.Considering that purified PC is still a mixture of various PC species,a more precise dietary regime of PC at the species level may be necessary for swimming crab larvae.For another example,PS(18:0/18:1) is the major PS species in many cell types (Roget al.,2016) and plays an important role in membrane function (Wilson and Bell,1993).The interleaflet coupling of cellular membranes contributes to the interdigitation between PS(18:0/18:1) in the inner leaflet and SM in the outer leaflet (Skotland and Sandvig,2019).In swimming crabs,the metamorphosis from the Z4 to the megalopa stage is accompanied by great changes in morphogenesis,including muscle formation and bone development.Sufficient and adequate PS exposure is necessary for the fusion of developing myoblasts (Eijndeet al.,2001).Hence,a significant reduction in the levels of almost all detected PS species,especially PS(18:0/18:1) from the M stage,probably contributes to the mass mortality of crab larvae.

4 Conclusions

This study revealed the comprehensive phospholipid profile ofP.trituberculatusand its succession over the larval development cycle for the first time by using a targeted UHPLC-MS analysis.In total,112 phospholipid species belonging to 10 phospholipid classes were identified inP.trituberculatuslarvae.High concentrations of PC,PE,PA,and PS were observed in crab larvae,while PC and PE species were the most abundant,All of these phospholipids significantly changed with larval development in quantity,which was highlighted by the downward parabolic changes in PE,PG,PI,PS,LPA,and SM levels.The marked depletion of almost all phospholipid species in the M stage may contribute to the mass mortality of crab larvae.These findings reveal a detailed phospholipid composition ofP.trituberculatusand its changing characteristics over the larval development cycle and provide novel perspectives for the targeted supplementation of phospholipids in crab diets.Our work also highlights the use of targeted UHPLC-MS lipidomics in understanding the phospholipids changes during crab development.

Acknowledgements

Financial supports from the National Natural Science Foundation of China (Nos.41673076,32073024),the Collaborative Promotion Program of Zhejiang Province Agricultural Technology of China (No.2020XTTGSC03),the China Agriculture Research System– CARS48 and K.C.Wong Magna Fund in Ningbo University are acknowledged.