Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' (Formicidae) for Volatilome Analysis by GC-MS and MetaboliteDetector

This method can help to answer key questions in insect metabolomics. The metabolite profiling approach can be used to study the chemical ecology of ants by elucidating the role of the volatiles in the interaction with other organisms. The main advantage of the presented workflow is:that it consists of two distinct parts which can basically be applied independently.

First, the physical isolation of the mandibular gland reservoir and its content, and second, the un-targeted profiling and identification of volatile organic constituence, in the biological sample of interest. Demonstrating the procedure will be Michaela Hoenigsberger, a talented PhD student from my laboratory. Before dissecting the ants, in a fume hood, clean a glass Petri dish and the dissection instruments using paper tissues and a one plus one by volume mixture of methanol and water.

Store the dish at minus 20 degrees Celsius. Then, take the mass of 1.5 milliliter short thread vials including their screw caps which contain silicon PTFE Septa. Store the vials on ice.

Next, place a frozen malleable cold pack into a polystyrene foam box. Then load the Petri dish on top of the cold pack and put a frozen ant into the dish. Now, examine the ant with a stereo microscope.

Check the integrity of its gastral compartments. The ant must have an intact gaster region. Do not use ants with flat gasters or traces of hardened mandibular gland reservoir content on their body surfaces.

Now, isolate the mandibular gland reservoir contents from the ant. Use forceps to grip the propodium and the petiole, then gently pull these body segments apart. Next, take hold of the gaster at tergite four using fine forceps.

With another pair of forceps, grab tergite one and gently remove the tergite by peeling it off. Repeat this process to tear off tergites two and three, so the gastrol portion of the mandibular gland reservoirs is mostly visible. The mandibular gland reservoirs are yellow colored in C.Explodens worker ants, but the color can range from white over yellow, to red in other species.

Next, using a dissection needle, gently remove the wax-like contents of the paired mandibular gland reservoirs by scratching them out of the gastral compartment. Only remove those parts of the mandibular gland reservoir contents that can be isolated without applying pressure on the other glands and body structures present in the ant gaster. It isn't necessary to collect all of the mandibular gland reservoir's contents.

Now, pick up the mandibular gland reservoir contents by gently touching them with the tip of the dissection needle until they stick to it. Then transfer the gland contents to a cold 1.5 milliliter short thread vial. It may be necessary to move the needle tip along the wall of the vial and smear the gland content onto the vial wall.

Keep the collection covered and on ice until it can be analyzed. Between dissections, re-clean the instruments and the dish with the methanol and water mixture. To make one analytical sample, combine the gland reservoir contents of multiple ants.

In this case, the gland contents from five C.explodens worker ants are pooled. For a control, collect samples composed of Dufour's gland which is adjacent to the mandibular gland reservoirs. Before extracting the glandular contents, determine the masses of the analytical samples.

Then, add 14 parts of ice cold high purity ethyl acetate by volume, to one part tissue by mass. Vortex the preparation for five minutes under cooled conditions. Next, centrifuge the samples for 10 minutes and collect the supernatants.

About 55 to 75 microliters of supernatant is expected from a five ant sample. Transfer each supernatant to a pre-cooled 1.5 milliliter short thread vial with a 0.1 milliliter microinsert, and tightly seal the vials using screwcaps containing holes and red rubber PTFE Septa. For a complete GCMS measurement sequence, prepare a 1.5 milliliter short-thread vial with a retention index calibrate mixture, made by diluting commercially available alkane standard solutions in Hexane, to eight milligrams per milliliter per compound.

Then, prepare another vial of pure ethyl acetate to serve as the solvent blank. On the cooled autosampler tray of the gas chromatograph, load first the solvent blank, second, the retention index calibrant mixture, third, the mandibular gland reservoir content extract, and fourth, the Dufour's gland content extract. Put the samples with the tray on ice for transport to the gas chromatograph.

Insert the tray into the cooled autosampler. For each sample, inject a one microliter aliquat into the GC mass spec for chromatographic separation on a column suitable for the target compounds. Such as an HP-5MS UI column.

Next, set the appropriate GC parameters. For example, use helium at the carrier gas with a constant column flow of one milliliter per minute. Use an oven temperature ramp starting at 40 degrees Celsius for two minutes, followed by a ramp of 10 degrees Celsius per minute up to 330 degrees Celsius.

Followed by a seven minute hold. With these parameters, set the inlet temperature to 270 degrees Celsius. Now, if needed, adjust the split ratios for the GC mass spec.

injection so that they provide adequate peak shapes and intensities for the compounds of interest. Set the auxiliary temperature to 350 degrees Celsius. For the mass spec.

use the following parameters. An MS source temperature of 230 degrees Celsius, an MS quad temperature of 150 degrees Celsius, a scan range from low mass 30 to high mass 500, to result in a scan rate of about three scans per second. And a solvent delay of 4.1 minutes for the solvent blank and the experimental samples, or a solvent delay of 6.1 minutes for the retention index calibrant mixture.

When the measurements are completed, open the GCMS data analysis software, and export the data as an AIA project file, onto a portable storage device to facilitate the data transfer to a computer with 64-bit Linux-based operating system. To analyze the data, follow the protocol text description of the use of the metabolite detector software. In the field, ants were collected and frozen at minus 20 degrees Celsius for two days, and then stored at minus 80 degrees Celsius until they were analyzed.

Mandibular gland reservoir contents from five C.explodens worker ants were pooled, and measured by GCMS. Data analysis revealed dozens of putative gland content compounds. The two dominant metabolites in the mandibular gland reservoir content extract caused column overloading.

Which is why the same sample was analyzed again at a higher split ratio of 50 to one. Cross-contamination of the mandibular gland reservoir content by constituence of the Dufour's gland was investigated by having Dufour's gland samples to examine. Such contaminants exhibited late retention times, starting around 29 minutes.

The detected putative mandibular gland reservoir content metabolites were identified based on a combination of spectrum and retention index similarity to an in-house library. This way it was possible to confirm the identity of about 10%of the detected metabolites, some of which are listed as compounds one to five. After watching this video, you should have a good understanding of how to isolate, extract, and determine, one a time metabolites, by choosing MS measurement.

Once mastered, the isolation, solvent extraction, and data acquisition, of the same sample, can be done in about two hours if it is performed properly. Depending on the complexity of the volatile organic compound profile, the time needed for data evaluation will vary. While attempting this procedure, it's important to remember to keep the sample permanently cooled, from collection until analysis.

Moreover, before the section, it has to be verified, that the ant's gaster is physically intact. This procedure can also be combined with comparative or absolute quantification of the metabolites, in order to answer additional questions, like the metabolic composition, and the different environmental conditions, or absolute amounts of canned constituence. Conveniently, the extraction, measurement, and data evaluation steps of this protocol, can also be applied to multiple biological systems, such as other insects, micro-organisms, or plants.