Gas-chromatography and UV-spectroscopy of Hymenoptera venoms obtained by trivial centrifugation

This paper summarises gas-chromatography (GC–MS) and preliminary UV-spectroscopy analyses data of fresh, unmodified venom of aculeate hymenopterans (ants, bees, wasps), mainly focusing on red imported fire ants. No solvents nor fractionation were used at any point, which is a novel approach to describing integral toxins cocktails as proposed by Fox et al. (2018a) [1] 10.1016/j.toxicon.2018.02.050 where these results are discussed in deeper details. Herein we focus on further characterising the obtained venom extracted through a novel approach. Pertaining raw data is accessible from Fox et al. (2018b) [2] 10.17632/cpnscw2gkc.1 including further relevant information regarding the used insects, machinery settings, chemical standards.


Subject area
Immunology and Microbiology, Chemistry, Biology, Entomology, Toxinology More specific subject area Animal Toxins of Medical Importance, Bioassays of Natural Products, Immunotherapy Type of data Table, image (GC-MS chromatogram) How data was acquired Pure venoms were obtained by centrifugation using a simple adaptation described in cited references. The venoms were injected directly into Agilent gas chromatographs according with settings described in references. The resulting chromatogram files were analysed using the software OPENCHROM v. 1

Value of the data
The enclosed chromatograms of natural-state venoms are novel, and such data is rarely published as raw chromatograms are seldom made available by researchers in this research field.
A micro-volume spectrophotometre is employed for the first time for the chemical analysis of venomous secretions.
Raw chromatograms available from the referenced database [2] relate with future publications, and the described methods are open for critical evaluation and revisitation by peers interested in the chemistry of natural products.
The methods proposed can facilitate the identification of novel compounds of animal origin.

Data
The presented results are general analyses of GC-MS chromatograms obtained from crude venoms without any chemical or physical manipulation (e.g. chromatography, solubilisation). Such information is still rare in the scientific literature of venom toxins, as authors usually obtain modified venom fractions from dissecting animals and/or soaking venom-containing tissues in solvents. Herein venom collected by centrifugation of two species of honeybees (Fig. 1) , one social wasp (Fig. 2) , and three castes of red imported fire ants are presented (Figs. 3-5) . All pertaining raw files are publicly available at http://dx.doi.org/10.17632/cpnscw2gkc.1.
The venoms of fire ants are analysed and discussed in further details at [1].

Experimental design, materials and methods
Aggressive aculeate insects used were obtained around the university campus of South China Agricultural University. They were anesthesized with either CO2 or ethyl acetate (details at [1]).   Table 1. Raw spectra available at [2].
Isolated living venom-containing body parts were amassed at the numbers of 3-10 inside an adapted basket made of either glass wool or fine metal mesh in a glass vial insert. The glass insert was inserted into a trivial centrifuge tube and allocated into a centrifuge Eppendorf 5417R set to 28°C and centrifuged at short 30-60 s cycles to 2000-6000 g. The same was done with mutilated alitrunks from fire ant males.
You will see that liquids collect at the bottom of the glass insert during centrifugation. Gently relocate the tissues inside the basket in order to change their orientation as to push out more liquids (e.g. venom or hemolymph) through the stopper mesh. Centrifuge again. After ca. 8 cycles the amount of collected liquid at the bottom of the glass insert stabilised (collected amounts presented in [1]).
The obtained venoms and hemolymph were injected directly without dilution to Agilent GC-MS systems according with the method described in [1] and available directly from [2].
Furthermore, venom of fire ants, synthetic solenopsin alkaloids, and hemolymph were submitted to spectrophotometric analysis using a Nano-300 AllSheng micro-spectrophotometer. The equipment was blanked empty, and about 1.0 μl of liquid extract was applied to the reading pedestal, and scanned 4 3 times to ensure readings are stable. Pedestals were cleaned with acetone and distilled water between each use. The same solvents were also used as controls to test for reading stability and consistency between samples.   5. Spectrophotometric UV analysis of synthetic and natural fire ant extracts. A relative purity check for alkaloids was attempted following the same principle used with DNA and protein extracts, as described in [3], assuming maximum absorbance of solenopsin alkaloids at OD232 and of proteins at OD280. A -Synthetic isosolenopsin A, OD232:OD280 ¼ 4.24; B -Venom obtained by gentle centrifugation of venom glands apparatuses of S. invicta minor workers, OD232:OD280 ¼ 3.42; C -Whole-gaster extract obtained by high centrifugation of S. invicta minor workers, OD232:OD280 ¼ 1.66; D -Thoracic hemolymph obtained by high centrifugation of S. invicta virgin males, OD232:OD280 ¼ 1.19.) Table 1 Tentative identification of differential peaks from Fig. 1