The Ants Chapter 20

CHAPTER 20. COLLECTING, CULTURING, OBSERVING

Introduction
This chapter provides a primer of simple techniques for studying ants for students and a wide variety of field researchers who need to handle material quickly and efficiently. Our exposition is far from an exhaustive account. In the case of the culture of live colonies especially, specialized methods suited to the needs of particular species are often developed as part of research programs, and they can be found in the Materials and Methods section of the respective technical articles. What is offered here is a set of general procedures that we have found to work well over many years of experience, across almost all major groups of ants.

How to collect ants
Collecting ants is simple and straightforward and can be conducted immediately by anyone. We routinely place specimens in 80 percent ethanol or isopropyl alcohol; the latter substance is especially useful because it can be obtained as rubbing alcohol in many parts of the world without a prescription. (An unusual but workable approach was taken by the late astronomer and amateur myrmecologist Harlow Shapley, who used to preserve ants in the strongest spirits of the country he visited. A worker of Lasius niger he placed in vodka while dining with Stalin in the Kremlin is now in the Museum of Comparative Zoology.)  The vials we favor are small and slender, 55 mm long and 8 mm wide, dimensions that allow many to be kept in a small storage space and carried in the pocket or field pack. They are closed with neoprene stoppers, which permits preservation of “wet” material for many years. A few wider bottles, 55 mm long and 24 mm wide, are carried to accommodate the largest ants or exceptionally large series.

Workers should be collected whenever possible. They can be mixed, both as to colonies and species, if the ants are found foraging singly (this fact should be noted on the label). However, if the colony is discovered, a sample of at least 20 workers should be put in a vial by themselves, along with a similar number of queens, males, and larvae if these can be captured. In emergencies, when the supply of vials is running low, these “nest series” can be placed in the same vial and separated from one another by tight plugs of cotton. Up to 4 nest series can thus be accommodated in a typical, 55 x 8 mm vial. In small but clear letters, write a label of the following kind with a sharpened pencil or indelible ink; for example:

FLORIDA: Andytown, Broward Co. VII-16-87. E.O.Wilson. Scrub hammock, nesting in rotting palm trunk.

For picking up the ants, use stiff narrow forceps with pointed (but not needle-sharp) tips. A pair of very sharp watchmaker's forceps, for example Dumont No. 5, can be carried for work with exceptionally small ants. A rapid, efficient method is to moisten the tip of the forceps with alcohol from the vial and touch it to the ant; this fastens the specimen to the forceps long enough to transfer it to the liquid in the vial. Fine flexible forceps can also be carried for the collection of live specimens, if these are needed for behavioral observation.

To conduct a general survey of a particular locality, continue collecting until no new species have been encountered for a period of several days. Work primarily during the day, but search through the same area at night with a flashlight or headlamp to pick up exclusively nocturnal foragers. A good collector can obtain a virtually complete list of the fauna in a site of say, one hectare, within one to three days. However, habitats with dense, complex vegetation, such as those in tropical rain forests, are likely to take much longer and require special techniques such as arboreal fogging with insecticides.

For ordinary arboreal collecting, rake branches and leaves back and forth with a strong sweep net. Then break open hollow dead twigs on the bushes and trees. This will reveal colonies of species not readily discovered in any other way, especially those with nocturnal habits. Often it is possible to make rapid, clean collections by snapping the inhabited twigs into short segments (3 to 6 mm long) and blowing the contents into the vial. An aspirator can also be used to suck up ants rapidly, especially when the nest has just been broken open and the inhabitants are scattering. Care should be used in this technique, however, because many ants (especially dolichoderines and formicines) produce large quantities of formic acid, terpenoids, and other volatile toxic substances. The unwary collector is in danger of contracting “formicosis,” a painful but not fatal irritation of the throat, bronchial passage, and lungs.

For terrestrial species, collect workers foraging on the ground during both the day and night. It is necessary to look sharply for certain species that are especially small and slow-moving and hence difficult to see. A favorite technique of ours in sampling forest faunas is to lie prone, clear the loose leaves from a square meter of ground to expose the soil and humus, then simply watch for up to a half hour for the most inconspicuous ants.

In open terrain look for crater nests and other excavations and dig down into them with a gardener's trowel in search for colonies. Turn rocks and pieces of rotting wood on the ground to search for the species specialized for nesting in such protected sites. Tear open rotting logs and stumps, looking with special care beneath the bark for the small, inconspicuous species that abound in this microhabitat. Use a ground cloth: take a sheet of white cloth or plastic, one to two meters on the side, and scatter leaf litter, humus, and some top soil over it. Also break up rotting twigs and small tree branches buried within the litter. Where the humus and litter is relatively thick and moist, it is often the locus for a large part of the ant fauna and contains many inconspicuous and still poorly studied species. The following technique has proved especially effective for collecting whole colonies that nest in small rotting logs and branches lying on the ground: pick up a fragment of the decaying wood (say about 50 cm long), hold it above a photographer's developing pan or similar shallow-walled container, and strike the fragment with a trowel several times to shake out portions of the colony. Small pieces of the wood will also fall into the pan, but it is still much easier to locate and collect the ants, including entire colonies, than by ordinary excavation.

Slower but more thorough collecting of terrestrial ants can be accomplished with the aid of Berlese-Tullgren funnels, named after the Italian entomologist, A. Berlese, who invented it and the Swede, A. Tullgren, who modified and improved it. In simplest form the apparatus consists of a funnel topped by a wire-mesh screen onto which soil and litter are placed. As the material dries out, possibly aided by a light bulb or some other heat source placed above, the ants and other arthropods fall or slide down the smooth funnel sides into a collecting bottle partly filled with alcohol and suspended over the lower spout of the funnel. Excellent descriptions of the Berlese-Tullgren methods and other extraction techniques are provided by Southwood (1966) and Mühlenberg (1976).

Preparation for museum work
Ants can be stored indefinitely in alcohol, but it is best to prepare part of the nest series as pinned, dried specimens for convenient museum work. This step is especially important if the ants are to be given to a taxonomist for identification. It is also the best way to store them in museums as voucher specimens, to serve as references for field or laboratory research (all such studies should be taxonomically verifiable with voucher material). The standard method for preparing dried specimens is to glue each ant on the tip of a thin triangle of white, stiff paper. The tip should approach the right side of the ant and touch its ventral body surface beneath the coxae of the middle and hind legs. The droplet of glue should be small enough and placed so as not to obscure any other part of the body except a portion of the coxae and ventral alitrunk surface--which have relatively few features of taxonomic importance. Prior to this “pointing” procedure, an insect pin should be inserted through the broad ends of two or three of the paper triangles, so that two or three ants from the same colony can be mounted one to a triangle on each pin. A rectangular label with the locality data goes beneath the mounted ants, so that when you read the label, the triangles point to the left and the ants point away from you. An effort should be made to get a maximum diversity of castes on each pin: for example, a queen, worker, and male, or a major worker, media worker, and minor worker. In the case of large ants, it may be possible to mount only one or two ants to a pin; and in the case of very large ants, it is sometimes best simply to pass the insect pin directly through the center of the thorax.

Culturing ants
The culture and study of ants in the laboratory is a.relatively simple operation. For many years we have used an economical arrangement that serves for a majority of species, for both mass culturing and behavioral observation. The newly collected queenright colony is brought into the laboratory (preferably with some of the original nest material) and placed in plastic tubs chosen for size according to the size of the ants and numbers of workers in the colony. For example, fire ant colonies (Solenopsis spp.) with populations up to 20,000 are readily maintained in tubs about 50 cm long, 25 cm wide, and 15 cm deep. In order to prevent the ants from escaping, various means are used depending on the humidity of the room in which the ants are to be kept. The sides are coated with petroleum jelly, heavy mineral oil, talcum power, or preferably Fluon® (Northeast Chemical Co., Inc., Woonsocket, Rhode Island, USA), a water-based material that is both effective (providing a silky smooth surface) and long-lasting (but not satisfactory under humid conditions). The colony is allowed to settle into test tubes (15 cm long with inner diameters of 2.2 cm) into which water has been poured and then trapped at the bottom with tight cotton plugs, leaving about 10 cm of free air space from the plug to the mouth of the tube. The 10-cm segment is surrounded by aluminum foil to darken the air space and encourage the ants to move in (most do so promptly). It can be removed later to allow behavioral studies. To this end, most ant species adapt well to light at ordinary room intensities, carrying on brood care, food exchange, and other social activities in an evidently normal manner. The tubes are stacked at one end of the tub prior to the placement of the colony, leaving most of the bottom surface of the tub bare to serve as a foraging arena.

The nest tubes can also be placed in closed plastic boxes, making it easier to keep the ambient air of the foraging arena moist and hence better suited for forest-dwelling species. The following dimensions are roughly correct for ant species with different sized workers:

Small. 11 x 8.5 cm on the side and 6.2 cm deep. Very small ants such as Adelomyrmex, Cardiocondyla, Leptothorax, small Pheidole, and Strumigenys. These species can also be cultured readily in small, round petri dishes (10 cm diameter, 1.5 cm depth).

Medium. 17 x 12 cm on the side and 6.2 cm deep. For example, Aphaenogaster, Conomyrma, and Formica. Smaller colonies of Camponotus, Messor]], and Pogonomyrmex''.

Large. 45 x 22 cm on the side and 10 cm deep. For example, larger colonies of Pheidole, Pogonomyrmex, and Solenopsis.

Variations on the elementary test-tube arrangement can be adapted to ant species with unusual nesting habits. For example, colonies of arboreal stem-dwelling ants such as Pseudomyrmex and Zacryptocerus can be induced to move into glass tubes 10 cm long with diameters 2 to 4 mm, the latter dimensions varied according to the size of the workers. The tubes are closed at one end with cotton plugs. The plugs can be kept moist, but in many cases this is not necessary, because stem-dwelling ants are often adapted to dry nest interiors, and a small dish of water placed nearby is an adequate source. Each set of tubes containing a colony is then placed in a tub of the kind just described. Or they can be bound horizontally in rows on a rack or potted plant, in order to simulate the natural environment.

Colonies of small fungus-growing ants such as Apterostigma and Trachymyrmex can be maintained easily in moistened tubes in tubs. Large fungus-growers, in other words leafcutter ants of the genera Acromyrmex and Atta, are better kept by the following technique due to Neal A. Weber (1972). Newly inseminated queens or incipient colonies are collected in the field and transferred to a series of closed, clear plastic chambers each about 20 cm x 15 cm and 10 cm deep (ordinary refrigerator food receptacles with transparent walls serve very well). The chambers are connected by glass or plastic tubes 2.5 cm in diameter, allowing the ants to move readily from one chamber to the other. Foraging workers are permitted to collect fresh vegetation (possibly supplemented by dry cereal) from either empty chambers or from an open tub lined with Fluon or surrounded by a moat containing water or mineral oil. As the colony expands in size, the ants fill one chamber after another with the characteristic sponge-like masses of processed substratum, through which the symbiotic fungus grows luxuriantly. Except in the driest laboratory environments, no special water supply is needed, because the ants obtain all of the moisture required from the vegetation. Leaves from a wide range of plant species are accepted by the ants. In the northeastern United States, we have used basswood (linden), oaks, maples, and lilac most frequently; the latter two are especially attractive to the foragers. The colonies deposit the exhausted substrate in some of the chambers, which should be removed and cleaned from time to time.

For close behavioral studies, more elaborate artificial nests are often required. One that works well for the great majority of ant species can be prepared as follows. Plaster of Paris is poured to a depth of 2 cm in the bottom of a tub, the size of which is chosen to suit the worker size and population of the colony under study (thus for minute ants such as thief ants or Brachymyrmex the container may be only 10 x 15 cm and 10 cm deep). As the plaster of Paris sets, carve 10-20 chambers into its surface that are roughly similar in size and proportions to the natural nest chambers of the colony to be cultured. In the case of some medium-sized Pheidole living in pieces of rotting wood, the chambers are typically ovoid or circular in shape and 1-4 cm across; hence chambers should be excavated that are about 2 x 3 cm and 1 cm deep. The artificial nest chambers are connected by galleries 5 mm wide and deep and covered tightly by a rectangular glass plate. Two to four exit galleries are cut from the outermost chambers to the remainder of the plaster of Paris surface, which serves as the foraging arena. Fragments of decaying wood and leaves from the vicinity of the original nest can be scattered over the surface in order to add to the “naturalness” of the microenvironment.

Additional culturing techniques adaptable to almost every known ant species are to be found in the works by Wheeler (1910a), Skaife (1961), and Gösswald (1985). Freeland (1958) invented an excellent vertical observation nest for Myrmecia and other very large ants. Wilson (1962b) designed a plastic nest that requires minimal maintenance and serves for the simultaneous observation of large colonies, for example those of fire ants, inside and outside the nest during foraging activity.

A completely defined synthetic diet for ants has been invented by Ettershank (1967). Diets and several mass culturing techniques for various ant species have been reviewed by Carney (1970). We employ the Bhatkar diet (Bhatkar and Whitcomb, 1970), which is prepared as follows:

Ingredients: l egg 62 ml honey 1 gm vitamins 1 gm minerals and salts 5 gm agar 500 ml water

Dissolve the agar in 250 ml boiling water. Let cool. Mix 250 ml water, honey, vitamins, minerals and egg until smooth with egg beater. Add to this mixture, stirring constantly, the agar solution. Pour into petri dishes to set (0.5 to l cm deep). Store in refrigerator. The concoction fills the bottoms of 15-cm diameter petri dishes, and is jelly-like in consistency.

Most insectivorous ant species thrive on this diet when fed three times weekly along with fragments of freshly killed insects, such as mealworms (Tenebrio), cockroaches (Nauphoeta), and crickets offered in small quantities. If the ants are also predators, they do especially well when allowed access to bottles containing Drosophila cultures, preferably flightless mutants. Alternatively, the Drosophila adults can be frozen and sprinkled onto the foraging arenas for the ants to discover.

Very often the food habits of ants newly brought to the laboratory are unknown. In the case of predatory species we have successfully employed the “cafeteria” method (e.g., Wilson, 1953, 1955b; Hölldobler and Wilson, 1986). Large numbers of arthropods and other potential prey are collected from the original nest vicinity by aspiration or Berlese funnelling into moistened traps. These are released onto the foraging area of the captive colony amidst some soil and leaf litter, where they can find shelter. A record is then kept of the proportions of various species captured by the ants and brought into the brood chamber. The method has proved very successful in detecting prey specialization. For example, Belonopelta and Prionopelta were discovered to be specialists on campodeid diplurans and many species of Smithistruma and small dacetines were proved to be specialists on entomobryomorph collembolans.

Transporting colonies
Colonies can be kept for days or weeks at a time in bottles or other tight containers providing certain elementary procedures are followed. The first, absolute rule is that the ants must be given a moist area into which to retreat: not soaking wet, with films or drops of water that might entrap the ants, but containing a zone with moist surface and saturated ambient air. The ideal retreat is part of the nest material itself, placed directly into the container with a portion of the colony preferably in it. A large piece of moistened (but not soaking) cotton wool or paper toweling should be added as a back-up. The rest of the container can be filled with nest material or loose-fitting paper toweling or other neutral material to prevent the colony from being knocked about excessively.

The colony should be uncrowded, in no case occupying more than one percent to the container volume. The lid of the container should be tightly fitted. Unless the colony is unusually active or aggressive, it is not necessary to punch holes in the lid in order to aerate the interior; in fact, this procedure risks excessive drying. Once or twice a day the lid can be removed and the container waved gently back and forth to freshen the air. The colony can be provided drops of sugar water and fragments of insects or other foods if the duration of the journey is more than several days. If ants appear dead after remaining in a closed container too long, they may be just narcotized by carbon dioxide instead. Expose them to the open air for a few hours to see if they can recover.

Because many countries have restrictions on the importation of live insects, it is prudent to check with the appropriate government agencies before collecting live colonies abroad. In the United States, for example, a permit must be obtained from the United States Department of Agriculture (Animal and Plant Health Inspection Service, Plant Protection and Quarantine, Plant Importation and Technical Support), which has first been approved by the appropriate state officials. The entire procedure usually requires six to eight weeks. The permit is then presented to the appropriate customs officer upon reentry into the United States.

An increasing number of countries restrict the export of preserved and living specimens, including insects, and a special export permit may be required. The local regulations should always be consulted and respected.

Breeding new colonies
Reproductive forms can be easily reared in the laboratory, but those of most species cannot be induced to mate under the culturing conditions ordinarily employed. The reason is that the virgin queens and males must engage in extensive nuptial flights under an exacting regimen of temperature and humidity before they will copulate. However, the rule is not absolute. A few polygynous species mate in or close to the nest, so that laboratory colonies can be maintained and multiplied indefinitely. Examples include pharaoh's ant Monomorium pharaonis (see Peacocke and Baxter, 1950, and Berndt and Eichler, 1987), a species of []Solenopsis (Diplorhoptrum) from Ecuador we have had in culture for over twelve years, Xenomyrmex floridanus (Hölldobler, 1971d), several species of Cardiocondyla (Robin Stuart, personal communication), the slavemaking myrmicine Harpagoxenus sublaevis and many other parasitic ants (Buschinger, 1972b, 1976a), the Argentine ant Iridomyrmex humilis (Smith, 1936a), and Paratrechina longicornis. Within these species it is possible to make careful studies of reproductive behavior, including the bioassaying of sex pheromones. In fact Karl Gösswald and his co-workers exploited this form of mating behavior to mass-produce queens. Colonies were then started in forests, where the workers protect the trees against pest insects (see Plate 4).

Buschinger (1975a) has furthermore gone so far as to conduct a genetic analysis by breeding experiments in the case of the ergatogynic locus of Harpagoxenus sublaevis, but in general such studies are handicapped by the relatively long time (sometimes several years) to rear a mature colony from a newly inseminated queen. It is likely that future genetic studies will depend a great deal on electrophoretic separation of enzymes and amino acid and nucleotide sequencing.

The nuptial flight could conceivably be short-circuited and fertile queens produced if queens were readily inseminated. This has been achieved in the case of the fire ant Solenopsis invicta by Cupp et al. (1973). These authors kept reproductive forms in a warm (32°C), humid environment with an 18-hour photoperiod. They decapitated the male (an action that disinhibits copulatory movements), pinned it through the thorax, and set it in a parafilm mount with the abdomen pointing upward. The female was anesthetized with carbon dioxide. Her wings were grasped with jeweler's forceps and her abdomen stroked against the body in a front-to-rear motion until copulation occurred. Genetic analysis by breeding experiments could be greatly accelerated if artificial insemination were combined with precocious production of sexual forms through the treatment of young colonies with appropriate levels of disinhibitors or juvenile hormone. This technology, however, remains to be proven.

'''Hölldobler, B. and Wilson, E. O. 1990. The Ants. Cambridge, Mass. Harvard University Press. Text used with permission of the authors.'''

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