Lipid Incorporation During Experimental Decay of Arthropods - Biopolymers: A Molecular Paleontology Approach - page 108

Electron microscopy was performed on the original, 4 week- and 44 week- decayed
shrimp, cockroach and scorpion cuticles without any chemical treatment and using a
Philips XL 30 environmental scanning electron microscope.
Specimens were photographed before incubation and on recovery from the
experiment to record the state of decay. Samples of tissue and cuticle were trans-
ferred to glass vials and frozen at −60 °C for subsequent analysis. Three posterior
segments in the cockroach, segments 5-7 of the pre-abdomen in the scorpion and
two posterior segments in the shrimp were targeted for chemical analysis, but in the
last stage of decay in the shrimp available cuticle was sampled. For each analysis
the cuticle of a single segment was cleaned of any adhering exogenous matter and
internal soft tissue with tweezers and scalpel and then subjected to ultrasonication
in deionised water for 30 min. The isolated cuticle was then extracted in 2:1
dichloromethane:methanol, for 30 min three times, to remove solvent-soluble,
organic components.

Chemical Analysis

The insoluble residue remaining at each sample stage was analysed using a CDS
5150 Pyroprobe by heating at 650 °C for 20s (py-GC-MS: mass spectral identifi ca-
tions were based on model compounds—see Gupta et al. 2008a ). Compound detec-
tion and identifi cation were performed with on-line GC-MS in full scan mode using
a Hewlett Packard HP6890 gas chromatograph interfaced to a Micromass AutoSpec
Ultima magnetic sector mass spectrometer. GC was performed with a J&W
Scientifi c DB-1MS capillary column (60 m × 0.25 mm ID, 0.25 m fi lm thickness)

using He as carrier gas. The oven was programmed from 50 °C (held 1 min) to
300 °C (held 28 min) at 8 °C min −1 . The source was operated at 250 °C and 70 eV
ionization energy in the electron ionization (EI) mode. The AutoSpec full scan rate
was 0.80 s-decade over a mass range of 50-700 Da and a delay of 0.20 s. The origi-
nal, 1 month and 44 week decayed shrimp cuticle samples were also subjected to
thermodesorption at 310 °C prior to pyrolysis at 650 °C, to compare the macromo-
lecular moieties obtained after pyrolysis of the solvent extracted residue with those
obtained after thermodesorption.

μ

13 C NMR Analysis

Solid state 13 C nuclear magnetic resonance spectroscopy ( 13 C-NMR) was conducted
on the fresh and 44 week decayed shrimp and scorpion, after crushing the solvent
extracted residue in liquid nitrogen followed by freeze drying to remove excess
water. All 13 C solid state NMR experiments were performed on a Varian-
Chemagnetics Infi nity spectrometer located at the W. M. Keck Solid State NMR
Facility at the Geophysical Laboratory. The static fi eld of this instrument is 7.05 T;