Ammonia solution

PL standards, including phosphatidylcholine (12:0/12:0 PC), phosphatidylethanolamine (18:0/18:1 PE, ), phosphatidylserine (16:0/16:0 PS), phosphatidylinositol (18:0/18:0 PI), and triacylglycerol (16:0/16:0/18:1 TG) were purchased from Avanti Polar Lipids, Inc. (Alabaster, USA). Isopropanol and n-hexane (MS grade) were purchased from Honeywell Riedel-de Haen (MO, USA). Ammonia solution, triethylamine, and formic acid were purchased from Sig-ma-Aldrich (MO, USA). The ultrapure water was obtained using a Milli-Q water purification system (Millipore, MA, USA). Chloroform and methanol (analytical grade) were purchased from Vekton (St. Petersburg, Russia).
Total lipids were extracted with a Chloroform/methanol mixture (1:2 v/v) according to procedure of Bligh and Dyer^{50 (link)}. A sample (~ 200 mg) of crab eggs, larvae and juveniles was homogenized on a disperser (IKA T25 digital Ultra-Turrax, Germany) in 2 mL of a Chloroform/methanol mixture (1:2 v/v) and filtered. The residues were extracted twice with 2 mL of a Chloroform/methanol mixture (1:2 v/v) and three times of Chloroform (2 ml). The extracts were combined, mixed water (6 mL), and then left overnight for phase separation at 4 °C. Then the near-bottom layer was separated and evaporated evaporated under reduced pressure, weighed, dissolved in Chloroform, and stored at − 40 °C. Internal standards 12:0/12:0 PC and 16:0/16:0/18:1 TG were added to each lipid sample for further quantitative analysis.
To analyze the content and structure of the molecular species of phosphorus-containing lipids, total lipids were separated on a Shim-Pack diol column (4.6 × 50 mm, particle size 5 μm) (Shimadzu, Japan) using a Nexera-e chromatography system (Shimadzu, Japan). Solvent system A (2-propanol / hexane / H₂O / HCOOH / 28% NH₄OH / Et₃N, 28/72/1.5/0.1/0.05/0.02, v/v) and solvent system B (2-propanol / H₂O / HCOOH / 28% NH₄OH / Et₃N, 100/1.5/0.1/0.05/0.02, v/v) were used as eluents. The content of System B was programmed as follows: 0% (8 min), 0 to 20% (7 min), 20 to 100% (5 min), 100% (15 min), 100 to 0% (0.1 min), and 0% (12 min). The elution rate was 0.2 mL/min. To detect lipids, a LCMS-IT-TOF high-resolution tandem mass spectrometer (Shimadzu, Japan) was used. The analysis was performed under the electro-spray ionization (ESI) mode with simultaneous registration of signals of positive and negative ions. Scanning was performed in a m/z range of 100–1400. Source voltage was − 3.5 kV in the case of negative ion formation and 4.5 kV in the case of positive ion formation. The temperature of the ion source was 200 °C; the dry gas (N₂) pressure, 150 kPa; the flow rate of nebulizing gas (N₂), 1.5 L/min. Argon (0.003 Pa) was used in the collision chamber of the mass spectrometer. The structural identification of each lipid molecular species was carried out by liquid chromatography–mass spectrometry (LC–MS). For this, the retention times, ion forms, and specific fragmentation behaviors of the phospholipid subclasses were compared to those of the commercially available lipid standards. The identification procedure was described in detail earlier^{51 (link)}. Percentages of the individual molecular species of each lipid subclass were calculated on the basis of peak area of negative ions [M–H]^‒, except for PC that were estimated on the basis of peak area of negative ions [M + HCOOH]^‒. The peak areas of lipid molecular species were inferred from mass spectrometry data with an accuracy of 5–7 ppm. The difference in the phospholipids ionizations was taken into account using a correction coefficient. The coefficients were calculated as the ratio of the areas of individual PL standards to the area of the internal standard (PC 12:0/12:0). The peak areas of the molecular species of analyzed crab lipids were calculated using the obtained coefficients. The PL subclass content was calculated as sum of contents of all molecular species of each PL subclass.
The molecular species of TG were separated by supercritical fluid chromatography on a Nexera UC system (Shimadzu, Japan) using two sequential Shim-pack XR-ODSII (2.0 × 150 mm) columns (Japan) under isocratic elution (0.6 mL min^–1) with supercritical CO₂ supplemented with 35% methanol; the maximum column pressure was 50 MPa; and the column temperature was 17 °C. To measure amounts of the molecular species of TG, an ELSD LT II (Shimadzu, Japan) light scattering detector was used (the evaporating tube temperature was 40 °C; the spraying gas (N₂) pressure was 0.4 MPa). The structures of the molecular species were determined on a LCMS-IT-TOF high-resolution tandem mass spectrometer (Shimadzu, Japan); HCOOH (0.1% in MeOH) was added to the eluent flowing out of the chromatographic column using a pump-through post column micromixer (0.2 mL min^–1). The analysis was performed under atmospheric pressure with chemical ionization (APCI) in the positive ion mode. Spectra were recorded at m/z 150–1100. The interface temperature was 350 °C; the desolvation line temperature, 200 °C. The rates of nebulizing (N₂), heating (air), and dry (N₂) gas supplies were 3, 10, and 10 L/min, respectively. Mass spectra were interpreted according to the fragmentation schemes published previously^{52 (link)}. To measure amounts of the molecular species of TG was used 16:0/16:0/18:1 TG internal standard. TG content was calculated as sum of contents of all molecular species.

Dye-labelled silica particles were produced by adapting the Stöber method.^{71 (link)} Labelling of silica particles with dye was achieved by reacting a N-hydroxysuccinimide (NHS) ester bearing fluorescent dye with an amine-terminated organosilane (APTES). 2.3 mg of sulfo-cyanine5 (Cy5) NHS ester (Lumiprobe, Germany) was dissolved in 200 μL dimethylsulfoxide (DMSO) (>99.9%, sterile-filtered, Sigma Aldrich, Germany) to which 3 μL of (3-aminopropyl)triethoxysilane (APTES) (99%, Sigma Aldrich, Germany) was added and allowed to react overnight. For small (120 nm) silica particles, 29 mL H₂O, 81 mL ethanol (>99.8%, analytical grade, VWR, United Kingdom) and 2.9 mL of ammonia solution (25% for analysis, Merck, Germany) were mixed in a reflux condenser and heated to 60 °C. Subsequently 10 mL of tetraethylorthosilicate (TEOS) (>99.0%, Sigma Aldrich, Germany) was added and stirred for two minutes after which 200 μL of Cy5–APTES was added and the mixture was allowed to react for five hours. The suspension was centrifuged three times at 2000g for 40 minutes and dispersed in ultrapure water. Large (360 nm) silica particles labelled with Cy5 were prepared with 13.5 mL H₂O, 174 mL ethanol and 45 mL ammonia solution were heated to 60 °C. Following, 22.5 mL of TEOS was added to the reaction vessel and reacted for two minutes prior to the addition of the dye-conjugated silane and reacted for three hours before three centrifugation cycles at 2000g for 20 minutes and redispersion in ultrapure water. Green BODIPY fluorescent dye NHS ester (BDP) (Lumiprobe, Germany) was conjugated to APTES overnight in DMSO. BDP-labelled silica was prepared by mixing 13.5 mL H₂O, 174 mL ethanol and 40.9 mL ammonia solution. The reaction mixture was brought to 55 °C and equilibrated for 30 minutes. Then, 22.5 mL of TEOS was added and stirred for three minutes, before addition of 200 μL of BDP–APTES. Mixture was allowed to react for three hours followed by three centrifugation cycles at 2000g for 20 minutes each.