Glycerides

Fly lipids were extracted according to Bligh and Dyer [36] (link). Five flies per replicate were homogenized in 150 µl methanol, 75 µl chloroform and 60 µl water in a Bioruptor sonifier (15 min with alternating 45 sec on/off intervals, low intensity setting; www.diagenode.com) or in the peqlab Precellys 24 instrument (10 sec 5000 rpm) using 1.4 mm ceramic beads (peqlab 91-PCS-CK14S). Lipids were extracted from the homogenates for 1 hour at 37°C before 75 µl chloroform and 75 µl 1 M KCl were added. Phase separation was achieved by centrifugation (Eppendorf 5417C; 2 min 3000 rpm) and the chloroform phase solvent was evaporated in a SpeedVac concentrator (Thermo Savant ISS110). Lipid pellets were resuspended in 60–70 µl chloroform/methanol (1∶1). For fat extraction after CCA the samples were extracted with 500 µl methanol and 250 µl chloroform for 15 min at 37°C before adding 250 µl chloroform and 250 µl 1 M KCl and lipid recovery as described above. Lipid extracts from CCA samples were separated by TLC as described below using 20 µg each of triolein, pentadecanoin and stearic acid as lipid standards.
Lipids extracted from 1 mg fly wet weight were separated on high performance thin layer chromatography (HPTLC) plates (Merck 105633) using n-hexane/diethylether/acetic acid (70∶30∶1, v/v/v; Merck) as liquid phase along with the following standard lipids: triolein (TAG; Sigma T7140), pentadecanoin (DAG; Sigma D8508), stearic acid (FA; Fluka 85679). Plates were air dried, dipped into 8% (w/v) H₃PO₄ containing 10% (w/v) copper (II) sulfate pentahydrate and charred for 10 min at 180°C on a hot plate (Gerhard H22 electronic). Fly lipid classes were quantified by photodensitometry (FujiFilm LAS-1000 and Image Gauge V3.45) scaled to a dilution series of the corresponding lipid standard (5–80 µg triolein; 1–16 µg pentadecanoin).
Depicted in Fig. 3A are representative experiments with average values of triplicate measurements and corresponding standard deviations. Experiments were repeated at least twice. Shown in Fig. 3B are average values of triplicate measurements of two independent experiments.
To determine the glyceride composition of fly homogenates the TAG and DAG content of flies was determined by TLC and the free glycerol content by CCA. Relative abundance of the glyceride classes was calculated using the following (average) molecular weights: glycerol (92,1 g/mol), triglycerides (844,96 g/mol), diglycerides (562,5 g/mol) and expressed as nmol/ mg fly wet weight.

All QM single-point energies,
angle scans, and torsion scans were performed using Gaussian 09.¹⁵ Angle and torsion scans on model hydrocarbon
molecules were performed at the MP2/cc-pVTZ level. Angles were scanned
from 90 to 150° in 1° increments. Torsions were scanned
over 360° in 10° increments. Single-point energy calculations
on model glyceride, ceramide, and phosphatidylcholine molecules were
performed at the MP2/cc-pVDZ level, with application of the polarizable
continuum model to create an implicit solvent environment for phosphatidylcholine
only. To obtain relevant glyceride, ceramide, and phosphatidylcholine
conformations, POPC and PSM simulations were performed for 100 ns
using initial Lipid21 parameters, and 1000 random lipid structures
were extracted. Prior to the QM single-point energy calculation, each
molecule was minimized using AMBER20¹⁶ for
1000 steps, with the first 500 steps using the steepest descent and
the final 500 steps using the conjugate gradient method,¹⁷ with restraints of 5000 kcal/mol/rad on each
of the torsions being fitted. The GBn generalized Born model (igb
= 7)^{18 (link)} was used during minimization of
model phosphatidylcholine molecules only. We repeated this process
on separate trajectories to create a test set, used for parameter
validation. Parameters for the model glyceride, ceramide, and phosphatidylcholine
molecules consisted of initial Lipid21 parameters and partial charges
derived at the MP2/cc-pVDZ level after optimization of a single molecular
conformation, allowing a two-stage RESP fit.^{19 (link)}

AEs and SEs were first screened using GC fitted with a flame ionization detector (FID) for quantification and general screening of preservation. An Agilent 7890A Series gas chromatograph was fitted with a DB1-high temperature (HT) column (15 m × 0.32 mm × 0.1 µm). One microlitre of the extract was injected via a splitless injector maintained at a temperature of 300°C. The temperature of the column was kept at 100°C for 2 min and then increased by 20°C every minute until a final temperature of 325°C was reached. A temperature of 325°C was then held for 2 min. Helium was used as the carrier gas at constant flow. The detector was kept at 300°C with hydrogen flow of 30 ml min⁻¹. For SEs, the temperature of the column was kept at 50°C for 2 min and then increased by 10°C every minute until a final temperature of 375°C was reached. A temperature of 375°C was then held for 10 min.
To identify molecular profiles, all extracts were analysed using GC-MS. For analysis of AEs and ABEs, the GC component was an Agilent 7890A series attached to an MS Agilent 5975 Inert XL mass selective detector with a quadrupole mass analyser (Agilent Technologies, Cheadle, UK). A DB-5MS (5%-phenyl)-methylpolysiloxane column (30 m × 0.250 mm × 0.25 µm; J&W Scientific, Folsom, CA, USA) was used. The GC column was inserted directly into the ion source of the mass spectrometer. One microlitre of sample was injected via a splitless injector maintained at a temperature of 300°C. Helium at constant flow was used as the carrier gas. The ionization energy of the spectrometer was 70 eV and spectra were obtained by scanning between m/z 50 and 800. The temperature of the column was kept at 50°C for 2 min and then increased by 10°C every minute until a final temperature of 325°C was reached. A temperature of 325°C was then held for 15 min. For SEs, a HT column and programme were used to detect the presence of tri-, di and mono-acylglycerols (TAGs, DAGs and MAGs) and wax esters. A DB5-HT column (30 m × 0.25 mm × 0.1 µm) was used, and the temperature of the column was kept at 50°C for 2 min and then increased by 10°C every minute until a final temperature of 375°C was reached. To target ions specific to alkylresorcinols SEs were analysed using the same chromatographic conditions with the mass spectrometer in SIM mode. The ions m/z 73, 268, 464, 492, 520, 548, 576, 604 and 632, corresponding to alkylresorcinols with cyclic carbon chain lengths C₁₇ to C₂₅, were monitored.
AEs were also analysed using an Agilent 7890A series chromatograph attached to an MS Agilent 5975 Inert XL mass selective detector with a quadrupole mass analyser (Agilent Technologies, Cheadle, UK) equipped with a DB-23 (50%-Cyanopropyl)-methylpolysiloxane column (60 m × 0.250 mm × 0.25 µm; J&W Scientific, Folsom, CA, USA). The temperature of the column was kept at 50°C for 2 min and then increased by 10°C every minute until 100°C. The temperature increased then until 140°C by 4°C every minute, then until 160°C by 0.5°C every minute and finally until 250°C by 20°C every minute. A SIM mode was used to target different groups of ions. These groups were: m/z 74, 105, 262, 290, 318 and 346 for the detection of ω-(o-alkyl phenyl)alkanoic acids of carbon lengths C₁₆ to C₂₂ (APAA_16–22), m/z 74, 87, 213, 270 for TMTD, m/z 74, 88, 101, 312 for pristanic acid, m/z 74, 101, 171, 326 for phytanic acid and m/z 74, 105, 262, 290, 318, 346 for the detection of ω-(o-alkyl phenyl)alkanoic acids of carbon lengths C₁₆ to C₂₂ (APAA_16–22).

Following common protocol for the extraction of absorbed residues [2 (link)], each ceramic sample was first ‘cleaned’ by removing the outer layer of the ceramic surface before collecting approximately 2 g of ceramic powder by drilling into the pot. An acidified methanol extraction (AE) method was applied to all samples following common procedure [32 (link)]. In brief, a mixture of methanol (4 ml) and sulphuric acid (800 µl) was added to 1 g of ceramic powder alongside an internal standard (10 µl of C_34:0) before heating at 70°C for 4 h. After centrifuging, the supernatant was extracted and transferred to a clean labelled hatch tube. The lipid extract was separated from the acid by adding 2 ml of n-hexane, removing the supernatant and passing through a filter pipette to neutralize the sample with potassium carbonate (K₂CO₃). This process was repeated three times. The samples were dried under a gentle stream of nitrogen, and then dissolved in n-hexane and transferred to an auto-sampling vial with a micro insert. A second internal standard (10 µl of C_36:0) was added before analysis by GC techniques.
A solvent extraction (SE) was undertaken on the majority of samples to extract and identify tri-, di- and mono-acylglycerols and/or wax esters, which do not remain intact after AE. Briefly, a mixture of dichloromethane-methanol (DCM 2 : 1, v/v) was added to the remaining 1 g of ceramic sample. The samples were sonicated for 15 min and then centrifuged. The supernatant was transferred to a clean labelled hatch tube. These steps were performed three times. The solution was then dried to completion under a gentle stream of nitrogen.
The ceramic powder remaining after SE was then submitted to acidified butanol extraction (ABE) [15 (link),16 (link)]. Here, 2 ml of n-hexane and 1 ml of boron trifluoride solution (BF₃) in butanol were added to the sample. After heating for 2 h at 80°C, the samples were centrifuged. The supernatant was then transferred to a clean labelled hatch tube and neutralized using a saturated solution of sodium carbonate. To ensure no more sample was trapped in the ceramic powder, 2 ml of DCM was added to the sample, vortexed, centrifuged and the supernatant transferred to the same extract. After centrifugation, the organic phase was transferred to a new clean labelled hatch tube. The aqueous phase was extracted twice more with DCM. The combined organic phases were washed twice with water, and dried under nitrogen. The dried extracts from SE and ABE were derivatized by adding N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethyl-chlorosilane and heated for 1 h at 70°C. n-hexane was added to redissolve the extract before transferring to an auto-sampling vial which contained 10 µl of the second internal standard C_36:0 for GC analysis.