Notes from Underground:subants

Notes from
Underground

Methods and Madness

Collecting Ants for Chemical Analysis

Over the past twenty years or so, we have been involved more or less continuously in the chemical analysis of ant exocrine secretions. We have examined ants of numerous genera from Africa, Australia, the Caribbean, India, New Zealand, North America, Puerto Rico, South America, and many other places. Given the sensitivity of modern GC/MS instruments, it is important that the collector who wants the chemistry of his or her collections to be analyzed use the proper materials and techniques. It is not uncommon to detect the DEET from insect repellent, although that is not a problem. What follows are some basic guidelines. For more sophisticated methods, including single ant and single gland analyses, one should consult the elegant work of Professor E.D. Morgan at Keele University.

Solvent:
Over the years we have found that the best solvent is HPLC grade methanol. The 70% ethanol that is used in museum collections is not good at all for the instruments and many compounds may not be soluble in it. In the past we used methylene chloride, but it is extremely volatile, and interestingly, the ants decompose after some time in methylene chloride. We found out that when the legs come off, the taxonomists are not happy. "Wood alcohol" stove fuel, which is mostly methanol, has high percentages of odorants that also make it useless for analysis. The craziest thing that ever happened was having a collection of ants sent to me in rum- a waste of ants and rum!

Vials:
Two or four dram glass vials are usually large enough for this purpose. The important thing is to use the proper caps. Fisher Scientific sells two kinds with their "Qorpack" vials that work well. There are black caps with a small polyethylene cone in them that we used for years. More recently we have found that the green caps that have PTFE lined seals (cat # 03-338-25) work a little better, and there are fewer occasions of leakage during shipment, etc, and virtually no contamination. At all costs foil, paper, and rubber-lined caps should be avoided, as they will make the analysis useless. Each vial should have about 0.5mL of methanol in it.

Collecting:
      We have found that for small ants, a fine camel hair artist's brush works well. A few workers will crawl on it and can be knocked off into the vial. Ten or twenty workers of even a small ant in 0.5mL of methanol should suffice. Of course for the chemist, the more ants the better is the rule. When there is more material the mass spectra of minor components are more reliable, and the statistical foundation of the chemical mixture is stronger. In the past, aspirators made of a test tube with a two-hole stopper and a collecting tube and an "inhaling" tube have been used. Even with a filter, this is hard on the collector's mouth when one is working with species like fire ants. I have used this method to collect large quantities of Monomorium minimum and had some funny twinges even with them. Once the ants are in the test tube, they can be dumped into the vial with methanol in it. Large ants are more difficult, because they are so easy to damage with forceps, which results in large amounts of fatty acids in the methanol and irritated taxonomists!
     Often it is important to find out what glands produce a particular compound, and one of the fastest ways to do this is trisection, with each part of the ant in a different vial. Ten trisected large ants should be enough. The collector should also provide a sample of whole ants for comparison. Dr. Juan Torres in Puerto Rico is absolutely the best "trisector" we have ever received ants from. In the case of the secretive Anochetus kempfi for instance, none of the compounds we detected in the heads were detected in the gaster or thorax samples he sent us.
     It goes without saying that castes should be collected separately. In some species the chemistry is caste-specific and wildly different.

Labels:
Labels can be anything the collector likes as long as they are on the OUTSIDE of the vial. The museum habit of including the label inside the vial has ruined many collections for chemical analysis. Some collectors just have a number and the year and send along a key, and some put everything on the label. Many times the adhesive on labels doesn't last, and Scotch tape is good for holding the labels on. Most collectors who have had chemical analyses done make a duplicate collection in 70% ethanol, on the other hand, many times for species that are difficult to collect, we have removed the methanol from a collection, replaced it with 70% ethanol and returned the ants.

Conclusion:
Chemical analysis of ants obviously has an important place in studies their taxonomy and behavior. Using the alkaloid producing Myrmicines as an example, we have found that while occasionally different species will produce the same compounds, different compounds are not found in different collections of the same species. In this broad example, the relative amounts of compounds may vary with the age of the ants, but not with diet or other conditions. Yet, the question of a dietary source of the compounds always needs to be raised. Additionally, since structure determination usually requires laboratory synthesis, we have often been able to provide samples of synthetic material to those who are studying laboratory colonies of a particular species.
I hope this little introduction is useful. I will be glad to try to answer any questions anyone might have about collecting for chemical analysis.

Tappey H. Jones
email: JonesTH@vmi.edu