Unravelling polar lipids dynamics during embryonic development of two sympatric brachyuran crabs (Carcinus maenas and Necora puber) using lipidomics

Embryogenesis is an important stage of marine invertebrates with bi-phasic life cycles, as it conditions their larval and adult life. Throughout embryogenesis, phospholipids (PL) play a key role as an energy source, as well as constituents of biological membranes. However, the dynamics of PL during embryogenesis in marine invertebrates is still poorly studied. The present work used a lipidomic approach to determine how polar lipid profiles shift during embryogenesis in two sympatric estuarine crabs, Carcinus maenas and Necora puber. The combination of thin layer chromatography, liquid chromatography – mass spectrometry and gas chromatography – mass spectrometry allowed us to achieve an unprecedented resolution on PL classes and molecular species present on newly extruded embryos (stage 1) and those near hatching (stage 3). Embryogenesis proved to be a dynamic process, with four PL classes being recorded in stage 1 embryos (68 molecular species in total) and seven PL classes at stage 3 embryos (98 molecular species in total). The low interspecific difference recorded in the lipidomic profiles of stage 1 embryos appears to indicate the existence of similar maternal investment. The same pattern was recorded for stage 3 embryos revealing a similar catabolism of embryonic resources during incubation for both crab species.

The European green crab Carcinus maenas and the velvet swimming crab Necora puber are two sympatric brachyuran crabs that commonly occur in estuarine habitats of Western Europe 1 . Carcinus maenas is a keystone species commonly used as a model in ecological studies, whereas N. puber is a commercially important species for coastal fisheries. Brachyuran crabs commonly display a bi-phasic life cycle, with a pelagic larval phase developing in open ocean waters and a benthic post-larval phase that occurs in coastal and estuarine habitats 2 . Decapod embryos are incubated in the abdomen of females (with the exception of penaeiod shrimps); being lecithotrophic throughout their embryonic development, they solely rely on the catabolism of yolk reserves originating from maternal investment for energy and organogenesis 3 .
Lipid reserves catabolism is the main energetic pathway that fuels embryonic development in decapod crustaceans 4 . However, most studies performed so far on crustacean embryos solely focus their fatty

Results
The limited number of samples processed per crab species and embryonic stage (n= 3) appeared to have no influence on our results given the little variation recorded among replicates. All significant differences recorded on the relative content of each PL class, molecular species and FA are highlighted in the figures presented, along with accurate P values for each statistical test performed.

Identification of PL classes and quantification by TLC. The fractioning of total lipid extracts in
TLC plates showed two different profiles for each stage of embryogenesis (Fig. 1a,b). The separation by TLC enabled to identify phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM) as the PL classes present in the initial stage of embryogenesis (Fig. 1a). LysoPC was observed at Scientific RepoRts | 5:14549 | DOi: 10.1038/srep14549 stage 1 embryos of C. maenas, but not on N. puber. At the end of embryonic development (stage 3) the separation by TLC revealed two new classes: phosphatidylinositol (PI) and cardiolipin (CL) (Fig. 1b)

Identification of molecular profile in PL classes by hydrophilic interaction liquid chromatography mass spectrometry (HILIC-ESI-MS).
In general, the HILIC-ESI-MS analysis confirmed the lipidomic profile revealed by TLC. However, this technique allowed to identify a new PL class in the embryos of N. puber at stage 1 which had not been previously detected by TLC: lysophosphatidylethanolamine (LysoPE) (see supplementary Fig. S1). A total of 98 molecular species were identified in the seven PL classes detected. In the next sections, the composition of the most abundant molecular species, namely the assignment of polar head groups and fatty acyl chain composition (determined by MS/MS 12 ), is explained in detail. MS/MS spectra of each PL class can be seen in supplementary Fig. S2 for C. maenas and Fig. S3 for N. puber. The number of carbon atoms (C) and double bonds (N) (C:N) are described for all molecular species recorded, as well as the FA side chains of the diacyl, plasmanyl or plasmenyl species (whenever possible) (total molecular species identified are listed in supplementary Table S4).  (Fig. 2b).

Discussion
The lipidomic approach employed in the present study allowed an unprecedented insight on the lipid dynamics during embryonic development in decapod crustaceans. TLC analysis revealed the biochemical  The important role that PC plays during embryogenesis was reflected in our results, with their decrease in relative abundance from stage 1 to 3 embryos agreeing with data from other marine species 14,15 . PC is recognized as the major component of biological membranes 16 , therefore of paramount importance on cells biochemistry and physiology 17,18 . Additionally, PC also has a similar role in the transport of yolk components in developing embryos 15 . Although the catabolism of PC may provide a secondary energy source when TAG are depleted 4 , it is most likely related with the provision of phosphate and choline. PCs likely play an important function as a source of EFAs for developing embryos 15 . PCs are rich in EPA and DHA, which are both required for cell differentiation and membrane formation during embryogenesis 19 . In line with the above, a possible consequence of excessive or even complete catabolism of PLs for energy would be the loss of important HUFA. However, studies carried out on fish embryos and larvae showed a selective retention of the DHA liberated by the catabolism of PCs in neutral lipids and/ or PEs 15,20 . Such EFAs are also known to play a major role during late embryonic development and early larval life in brachyuran crabs 21 . The low variability in the relative abundance of PEs between embryonic stages, in both crab species, suggests a structural function of this PL class. PC and PE were the major components of polar lipids. As already referred above for PC, PE is also a key component of membrane bilayers. Moreover, the FA composition of the molecular species of PEs showed a high level of unsaturation, which is known to contribute to membrane fluidity, as this feature is largely determined by the degree of FA unsaturation and n-3 HUFA 22 . Specifically, the role played by DHA and EPA on membrane flexibility has already been documented 22 . In both C. maenas and N. puber, the molecular species of this class that display significant differences between stage 1 and 3 embryos (m/z 762.4 and m/z 808.4) possess EPA, DHA and/or arachidonic acid (ARA, 20:4n-6) in their composition. The selective retention of these FAs in the last stages of embryonic development may reflect a preparation for planktonic life. In decapod crustaceans, those EFAs are known to be decisive for larval fitness during early planktonic life stages 3 . These molecules influence early neural development and functions 23 , hatchability at the end of embryonic development 24 , larval growth 25 and larval resistance to stress 26 . Other molecular species of LysoPEs include C 20 and C 22 HUFA, important FAs, such as ARA, EPA, DHA, docopentaenoic acid (DPA, 22:5) and 12,15-epoxy-13,14-dimethyl-eicosadienoate (Fig. 5c), which are known to be precursors of key biomolecules. ARA and EPA are the precursors of highly bioactive derivatives, the eicosanoids 7 , whereas ARA is the precursor of prostaglandins, which are known to be involved in a number of vital pathways (e.g., reproduction, digestion, respiration, membrane permeability and fat dissolution) 27 .
PIs and CLs were not recorded in early stage embryos and thus must originate from the catabolism of yolk reserves provided through maternal investment. PIs are considered as key signalling molecules and are involved in secretory events, as well as intercellular signalling events 28 . The metabolism of inositol lipids is involved in the signal transduction of many hormones, neurotransmitters and growth factors 29,30 . Qualitative requirements suggest that PC may be more important for growth, while PI may be relevant for organogenesis and tissue differentiation 7 . These features may explain their occurrences in different periods of embryogenesis (e.g., PCs are already present in newly extruded embryos).
In general, the analysis of the lipidomic profiles of developing embryos of C. maenas and N. puber did not reveal the existence of major interspecific differences. In the sampling site, these two species share the same estuarine habitats, which allow us to infer that they may have access to the same dietary resources. The scarce differences in the lipidomic profiles of embryos in the beginning of their development (stage 1) demonstrate a similar maternal investment in both crab species. Embryos of C. maenas and N. puber also appear to be programed to catabolize embryonic resources in a similar pattern during the incubation period, as the incubation environment (in terms of female brooding chamber) is similar for both species 31,32 . A follow up study surveying ovigerous females from these two species where they no longer occur in sympatry (C. maenas present in the inner regions of the coastal lagoon, which are more strongly influenced by fresh water runoffs, and N. puber located in a typically marine environment, the outer regions of the pier protecting the inlet of the coastal lagoon) will allow us to determine if the trends recorded in maternal investment and lipid catabolism during embryogenesis remain similar under contrasting environmental conditions. Furthermore, the analysis of FA classes revealed scarce differences between both crab species. Decapod larvae generally exhibit a limited ability to introduce double bonds into the n-6 and n-3 position of C 18 , C 20 , and C 22 FAs 33 . The presence of EPA and DHA in newly hatched larvae is known to originate from maternal lipids transferred at oogenesis, as decapod embryos are lecitothrophic and cannot synthesize these FAs de novo 34,35 . The selective retention of essential HUFA through embryogenesis may thus reduce the nutritional vulnerability of newly hatched larvae to suboptimal conditions they may experience in the early stages of their planktonic life 3 . High levels of HUFA and PL improve the osmoregulation process by optimizing membranous lipid composition and/or structure of the gills 8 , a key feature for larvae as the ones of C. maenas and N. puber that may hatch in estuarine systems prone to salinity shifts. Since HUFA are essential components in the nutrition of decapod larvae, the lipid composition of last embryonic stages may serve as an indicator of the physiological condition of larvae about to hatch. Additionally, lipid content in embryos can potentially determine early larval success and optimal development 36 . Higher lipid content in pre-hatching embryos is commonly interpreted as sign of superior tolerance by larvae to longer periods of starvation before first feeding 37 . It must be highlighted that to date no study has ever tried to link the performance of newly hatched larvae to the lipidome displayed by developing embryos. Therefore, until these studies are made available, any assumption on the superior/inferior quality of newly hatched larvae solely based on embryos lipidomics remains speculative and probably misleading. The unprecedented level of resolution achieved on polar lipids dynamics using lipidomics opens a new research window for studying maternal investment and resource partitioning in marine organisms. Methods Sampling. Ovigerous females of C. maenas and N. puber (carapace width, average ± SD, 50.2 ± 2.5 mm) were collected in the mussel beds of Mira Channel, Ria de Aveiro (Portugal) (40°38′26.30"N, 8°43′58.90"W) during March (early spring) 2012. Embryos were classified according to Rosa et al. 5 : stage 1 (newly extruded embryos) -uniform yolk, no cleavage or eyes; stage 3 (embryos ready to hatch in < 48 h) nearly no yolk present and embryo fully developed. Three females carrying embryos at stage 1 and three carrying embryos at stage 3 were selected for each species and their egg mass removed with fine forceps. All collected samples were freeze-dried and stored at -32 °C for biochemical analysis.
Lipid extraction. The Bligh and Dyer method 38 was used to isolate total lipids from embryos. Samples were resuspended in glass centrifuge tubes using 1 mL of water and 3.75 mL of chloroform/methanol (1:2, V/V) and incubated on ice for 30 min. The samples were centrifuged at 2000 rpm for 10 min at room temperature to resolve a two-phase system: an aqueous upper phase which contained the non-lipid components and an organic lower phase from where the lipids were recovered. The extraction was repeated twice. The organic phases were dried under a nitrogen stream. Lipid extracts were preserved at − 20 °C for further analysis.
Quantification of PL by phosphorus assay. Quantification of PL in the total lipid extract and in the spots separated by TLC was performed according to Bartlett and Lewis 39 . Samples were put on acid-washed glass tubes and resuspended in 0.650 mL of perchloric acid (70%, m/V). Glass tubes were incubated for 60 min at 180 °C in a heating block. After incubation, 3.3 mL of water, 0.5 mL of ammonium molybdate (2.5%, m/V) and 0.5 mL of ascorbic acid (10%, m/v) were added. After each addition, the mixture was well homogenized in a vortex mixer and incubated during 5 min at 100 °C in a water bath. Standards from 0.1 to 3.0 μ g of phosphate (standard solution of NaH 2 PO 4 . 2H 2 O, 439 mg L −1 of water, i.e. 100 μ g of phosphorus mL −1 ) underwent the same treatment as the samples. Absorbance of standards and samples was measured at 800 nm, at room temperature, in a microplate UV-vis spectrophotometer. The relative abundance of each PL class was calculated by the relation of the amount of phosphorus in each spot to the phosphorus amount of the total lipid extract in the sample applied in the TLC spot. (Alabaster, AL, USA), applied on the TLC plate. Spots coincident with the migration of standards were scraped into glass tubes and quantified as described above.
HILIC-ESI-MS of the total lipid extracts. HILIC analysis of total lipid extracts was performed on a Waters Alliance 2690 HPLC system (Waters Corp., Milford, MA, USA) coupled to a Finnigan LXQ electrospray linear ion trap mass spectrometer (Thermo Fisher, San Jose, CA, USA). Mobile phase A consisted of 50% acetonitrile, 25% methanol, and 25% water with 10 mM ammonium acetate, and mobile phase B consisted of 60% acetonitrile and 40% methanol with 10 mM ammonium acetate. The lipid extracts (25 μ g) were diluted in mobile phase B (90 μ L) and 10 μ L of the reaction mixture was introduced into an Ascentis Si HPLC Pore column (150 mm × 1.0 mm, 3 μ m; Sigma-Aldrich). The solvent gradient was programmed as follows: gradient started with 0% of A and 100% of B, linearly increased to 100% of A in 20 min, and isocratically held for 35 min, returning to the initial conditions in 5 min. The flow rate through the column was 7.5 μ L min −1 obtained using a pre-column split (Accurate, LC Packings, San Francisco, CA, USA). PL internal standards were purchased from Avanti ® Polar Lipids, Inc. (Alabaster, AL, USA) and used without further purification: 1′ ,3′ -bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol (CL); 1,2-dimyristoyl-sn-glycero-3-phosphocholine (dMPC); 1-nonadecanoyl-2 -hydroxy-sn-glycero-3-phosphocholine (LysoPC); 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (dMPE); 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′ -myo-inositol) (dPPI); and N-(heptadecanoyl)-sphing-4-enine-1-phosphocholine (SM). Polar lipid analysis was carried out by negative-ion electrospray ionization mass spectrometry (ESI-MS) on the Finnigan LXQ linear ion trap mass spectrometer. The electrospray voltage was 4.7 kV, the capillary temperature was 275 °C, and the sheath gas total lipid extracts (20 μ g of total PL). FAMEs were prepared using a methanolic solution of potassium hydroxide (2.0 M) according to the previously described method 43 . FAMEs were resuspended in 40 μ L of hexane, with 2 μ L of this hexane solution being used for GC-MS analysis on an Agilent Technologies 6890N Network (Santa Clara, CA) equipped with a DB-1 column with 30 m of length, 0.25 mm of internal diameter, and 0.1 μ m of film thickness (J&W Scientific, Folsom, CA). The GC was connected to an Agilent 5973 Network Mass Selective Detector operating with an electron impact mode at 70 eV and scanning the range m/z 40-500 in a 1s cycle in a full scan mode acquisition. The oven temperature was programmed from an initial temperature of 90 °C, standing at this temperature for 0.5 min and following a linear increase to 220 °C at 20 °C/min, a linear increase at 2 °C/min to 240 °C, and 5 °C/min until reaching 250 °C. The injector and detector temperatures were 220 and 230 °C, respectively. Helium was used as the carrier gas at a flow rate of 1.7 mL/min. FAME identification was performed by comparing their retention time and mass spectrum, which was analysed with MS spectra of commercial FAME standards (Supelco 37 Component FAME Mix) and confirmed by comparison with the chemical database Wiley and the spectral library "The AOCS Lipid Library" 44 . FA profile was classified in six FA classes: SFA, MUFA, PUFA, HUFA, Branched FA (BrFA)), Cyclic FA (CyFA) and Epoxy FA (EpFA). While polyunsaturated FAs are commonly defined as all FAs with ≥ 2 double bonds, in the present study we distinguish between PUFA (FAs with 2 or 3 double bonds) and HUFA (FAs with ≥ 4 double bonds).

Statistical analysis.
For PL quantification by LC-MS, the area of each molecular species in the spectra was transformed in their relative abundance, using the area of the PL internal standards as reference. The relative content of each PL class and FA was calculated as percentage of total PL and FA profiles, respectively. Differences in the relative abundance of PL classes, FA classes and molecular species of PL, were determined by 2-way ANOVA with interaction (factor crab species: C. maenas and N. puber; factor stage: stage1 and stage 3). Post hoc Tukey HSD test was used when ANOVA results revealed significant differences (P < 0.05). Statistical analyses of PL classes and their molecular species present in only one embryonic stage were performed using pairwise comparisons (Student´s t-test) between crab species. The sample size was identical in all treatments: n = 3. Prior to analysis, we tested for deviations from normality in the response variable with the Shapiro test and homogeneity of variance with the Levene's test. The level of statistical significance was P < 0.05. The statistical analyses were performed using the statistical package R version 2.13.2 45 .