The following procedures have been successful for general use by specialists in the Systematic Entomology Laboratory. Dissecting, staining, and mounting Lepidoptera genitalia are highly specialized procedures that are not included here. Many other procedures are well adapted for general use, but the simplicity and dependability of the following make them preferred by many specialists.

4.1 - Preparation and Storage of Genitalia

The structures at the end of the insect abdomen in both sexes are the postabdomen, terminalia, or genitalia, although the last term is more restrictive and refers morphologically only to certain organs of the ninth abdominal segment. These structures, sometimes extending to modifications of many segments of the abdomen, are of great identification importance. Many insects cannot be identified to species without critical examination of these parts, and even then can only be identified in one sex. In some insects, these parts are seen easily without special preparation; in others, just a little special positioning of the genitalia at the time the insects are pinned is sufficient. But in many insect species, these structures are so withdrawn or folded that, for critical examination, the abdomen or a large part of it must be removed and the genitalia specially prepared as follows:

(1) Carefully remove the abdomen by grasping it with forceps as close as possible to the thorax. Bending it slightly upward, then downward, usually will break it free of the specimen. It is well to perform this operation over a small dissecting dish containing water or 70 percent ethanol into which the part can fall. If the specimen is in a fluid and therefore soft, the abdomen may be severed with fine scissors.

(2) Place the severed abdomen in a small beaker or crucible containing three pellets of sodium hydroxide (NaOH) in about 10 ml of water. Then set the container on a hotplate at a temperature a little above that needed to boil the solution. It is well to place a cover loosely over the container to prevent the specimen from being thrown out and lost if the solution 'bumps' when heating, or to use a copper-mesh screen between the hotplate and crucible to eliminate 'bumping.' Allow the solution with the specimen in it to boil for 1 minute. Great care must be taken to avoid contact of hot or cold NaOH or caustic with your skin. If that should happen, wash it off immediately with plenty of water.

 

Figure 32. A point-mounted specimens with a genitalia vial

(3) Remove the specimen with forceps and return it to the dissecting dish. Examine it to see that muscles and most internal organs have been dissolved. If not completely so, return the specimen to the solution and heat it a little more.

(4a) When the specimen is well macerated, take a pair of No. 1 stainless steel insect pins, glued in wooden handles with a drop of epoxy, and pry the genitalia into an extended position. Clear away unwanted parts or debris, or if much unwanted material is present, transfer the specimen to a clean dish of 70 percent ethanol. Water or dilute alcohol is better than glycerin in which to examine small, colorless specimens, partly because fine structures are more clearly visible. The water may be tapwater, but it should be boiled before use to remove dissolved gases that may collect on and in the specimen and be very difficult to remove. The specimen then may be examined and identified or, if necessary, it may be placed in an aqueous solution of a few grains of dry Mercurochrome for staining (see p. 38). The specimen may be held at various angles with a bent piece of minuten and even sketched. If it is to be preserved for permanent reference, the decision must be made whether to store it in a microvial or to mount it on a microscope slide.

(4b) Another useful method when several specimens are to be identified simultaneously includes using a 'spot,' 'well,' or 'depression' plate, which is a white or black ceramic dish generally with 12 wells on the surface. The same number of wells are utilized as the number of specimens to be identified. (When using more than one specimen, be absolutely certain that the abdomens are properly associated with the correct specimens. To insure this, place the abdomens in the wells in the same order or configuration as the specimens are arranged in their holding container, and mark one side of the plate to indicate its orientation.) To each well add water and one pellet of NaOH with forceps. After the NaOH has dissolved, place one abdomen or part of it in each depression, and warm the plate gently under an incandescent bulb for about 1 hour. After some of the water has evaporated, replace it with fresh distilled water. Also at this time, examine the abdomens and press out any large air bubbles trapped within that might prevent penetration of the caustic. Then reposition the plate under the bulb. A thin stream of macerated tissues soon will be seen to issue from the abdomens into the fresh solution. After an hour or so, depending on the degree of maceration desired, transfer the abdomens for a few minutes to the wells of a second plate that you have filled with 70-80 percent alcohol to which has been added a small amount of acetic acid to neutralize the caustic. While in these wells, the abdomens may be gently manipulated to remove any remaining tissues. Wash and dry the first plate, place 2 drops of glycerin in each well, top with 70-80 percent alcohol, and transfer the abdomens to this plate, using care to keep them in the proper order. The abdomens may now be examined or left in a clean open place for several days if necessary. The glycerin will not evaporate. If the genitalia are to be permanently preserved, place the parts in a microvial as described on page 40.

Mount the specimen on a microscope slide only if it is relatively flat and all needed characters can be seen in the final position. For example, the ovipositors of fruit flies (Tephritidae) are flat enough that they may be fully extended and the ovipositor and sheath, including spermathecae, can be mounted on a slide with all necessary characters well displayed. The postabdomens of the male tephritids, however, are ill suited to such treatment because they are about as thick as they are wide and must be examined in profile as well as in ventral and posterior view.

(5a) If the specimen is to be mounted on a slide, place it in a small dish of 95 percent ethanol for a short time (1 minute is usually sufficient), then add a drop or more as needed of Euparal on a slide. Remove the specimen from the ethanol and immediately place it in the desired position in the Euparal. Break any large bubbles present before carefully lowering the cover glass. If insufficient Euparal is present to run to the entire circumference of the cover glass, add a little more at the edge of the cover glass until a light pressure on the top of the specimen through the cover glass brings the Euparal to the entire edge. Label the slide and allow it to cure (see p. 40) overnight in a warm oven or for a few days in a clean open place to make it usable. Small bubbles will disappear, and the specimen will become a little more transparent.

With the aid of an ocular grid in the microscope, the genitalia may be examined and even sketched when lying in a small dish of water, which gives more contrast than glycerin to delicate structures that may be difficult to see. The object may be held in place with a minuten bent in the shape of the letter 'L' which is laid over it or pierces it at a convenient place. A bit of petroleum jelly will hold a preparation in place, but the jelly must be dissolved before the specimen is replaced in a microvial or mounted on a slide.

(5b) If the specimen is not suitable for mounting on a slide, it may be kept in a microvial. The best microvials are made of transparent plastic with neoprene stoppers. Those with an inner lip are particularly desirable. The former practice was to use glass microvials with cork stoppers, but the tannin in the cork is injurious both to the specimen and, when wet with the glycerin in which the specimen is kept, to the pin on which the preparation is held. Whatever kind of microvial is used, before placing the specimen in the vial, add just enough glycerin to the bottom to cover the specimen completely. A throwaway injection syringe is excellent for this purpose. It may be kept filled with enough glycerin for many preparations. A small container of squeezable plastic with a fine tubular nozzle is made for modelmakers to dispense plastic cement. It is also an excellent glycerin dispenser. After placing the specimen in the vial, add the stopper. A dull-pointed pin inserted between the stopper and vial allows pressure to escape and prevents droppage of the vial from the stopper, which is to be held by an insect pin, preferably the same pin carrying the specimen from which the genitalia preparation was made (fig. 32). The specimen may be removed from the microvial and reexamined in water or ethanol solution at any time and then replaced.

Reference: Gary & Marston 1976; Robinson 1976 (slide-mounting genitalia of Lepidoptera).

4.2 - Mounting Wings

Wings of many kinds of insects can be mounted on microslides for detailed study or photography. Those covered with scales, such as wings of Lepidoptera and mosquitoes, must first have the scales removed or at least bleached for study of the venation.

Wings are bleached by immersion in an ordinary laundry bleach (sodium hypochlorite solution). Wetting them first with ethanol will activate the bleach. Immersion in the bleach for 1-3 minutes is usually sufficient. As soon as the veins become visible, remove the specimen or part from the bleach and wash it in plain water. It is frequently desirable to remove the scales under water by brushing the wings carefully with a fine brush or with the tip of a small feather. The descaled wing may then be stained, if desired, in eosin-Y or in an aqueous solution of Mercurochrome for a few to several hours and then washed again. The wing is then ready for mounting as described here, or it may be allowed to dry on a slide, then placed under a cover slip, and the cover slip ringed with fingernail polish or ringing compound to hold it in place.

Wings not needing descaling may be removed from a fresh specimen or one that has had a drop of household ammonia (containing detergent) placed at the base of the wing and allowed to stand in a closed receptacle for about an hour. The wing may be removed with finepointed forceps by piercing the body cuticle surrounding the wing base and then pulling the wing loose. In this way, one may be assured of obtaining the complete wing, even with basal sclerites if desired. The wing is then wet with 70 percent ethanol and placed in plain water for about 10 minutes to soften it. It is often desirable, if the wing is from a dried specimen, to place it in water that is then carefully heated until it barely starts to boil. This will aid in removing air from the larger veins. While the wing is in the water, carefully remove any dirt that may be present with a fine brush, but avoid removing fine hairs and setae. Also remove any unwanted parts of body cuticle and muscles at the base of the wing.

Then place the wing for about half a minute in 95 percent ethanol while adding a few drops of Euparal (or balsam) to a slide. Remove the wing from the ethanol and immediately place it in the Euparal (or balsam) on the slide. Position the wing as desired, turning it over if necessary and making sure that its basal part is well stretched out. Alternatively, especially with very delicate wings, it is usually better to arrange the wet wing on the bare slide first, then pour the mounting medium on top. Carefully apply a cover glass, touching it to one side of the Euparal first at a slight angle from horizontal to avoid entrapping bubbles. Press the cover glass down on the wing carefully to expand it as much as possible and to force bubbles out of the basal veins and elsewhere. Then cure the slide in a warm oven overnight or in the open, clean air for a few days. Always excercise caution when dealing with recently mounted slides. While the medium may appear dry at the edges, the interior of the slide may remain liquid for some time and tilting or placing the slide on its side may result in movement of the cover slip.

4.3 - Mounting Larvae of Diptera, Coleoptera, Lepidoptera, and Other Groups

The study of the immature stages of many insects is of great importance for identification purposes, but special techniques are usually needed because of their soft cuticle. Immature insects of most groups are seldom suitable for preservation in a dry condition. A method given here for preparing dipterous larvae may also be used for immatures of some other groups. Dipterous larvae, especially those of the higher Diptera, have mouthparts, a cephalopharyngeal skeleton, anterior and posterior spiracular structures, anal plates, cuticular spicules, and other features that are important for their systematic study, but these parts usually must be examined at high magnification and require special treatment. The larvae of Diptera, Coleoptera, Lepidoptera, and many other groups are best killed in boiling water because it leaves them in good condition for critical examination.

For cursory examination of the internal cephalopharyngeal skeleton, place the larva with no more fluid than will adhere to it in a dissecting dish. Pierce the cuticle in a few places near the anterior end of the larva and apply a few drops of pure liquid phenol there. Be careful not to get any phenol on your skin; wash with water if you do. In a short time the tissues will become as clear as glass. The larva may be returned to 75 percent ethanol after examination, when the tissues will again become opaque.

For more detailed and permanent preparation of larvae, place the larva in water in a dissecting dish and cut the cuticle with fine dissecting scissors along one side, starting close to the anterior end, passing below the lateral spiracle, and continuing almost to the posterior end. Then place the larva in an NaOH solution and boil as described on page 38. When the larva is well macerated, remove the body contents, almost separate the posterior spiracular area from the remainder of the skin, and pull the cephalopharyngeal skeleton a short way out of the body. Place the skin in 95 percent ethanol while adding a few drops of Euparal on a slide. Then put the skin in the Euparal, opened outward so that the cephalopharyngeal skeleton with the mouth hooks lies away from the skin and the posterior spiracular area lies with both spiracles upward. Apply the cover glass and carefully press it into place. This should give a clear view under high magnification of the cephalopharyngeal skeleton in lateral view, the anterior spiracles, all structures of dorsum and venter of one side, anal plates, and posterior spiracles. The last, often somewhat domed or on conical protuberances, may be distorted, but the sunray hairs and relationships of one spiracle to the other should be easily observed.

As with the genitalia, the larval skin is sometimes best preserved in glycerin in a microvial.

Other parts of the insect body, such as antennae, legs, and palpi, may be mounted on slides in Euparal in the same manner as described for the genitalia, wings, and larvae.

The references cited here concern specialized procedures for making slide mounts of lepidopterous genitalia (Hardwick 1950) and methods using Canada balsam (Noyes 1982; Richards 1964) for aphids, scale insects, parasitoids, and various other small insects. For methods to use with mites (Acarina) (Furumizo 1975; Lipovsky 1951, 1953). Further procedures are also given in the Appendix.

References: Hardwick 1950; Richards 1964; Wirth & Marston 1968.

To have any scientific value, specimens must be accompanied by a label or labels giving, as a very minimum, information about where and when the specimen was collected, who collected it, and, if pertinent, from what host or food plant. During preparation and mounting, specimens should bear temporary labels with this information, and any time a sample is subdivided, the label must be copied so that every specimen continues to be accompanied by the data. Many collectors keep a field notebook to record more detailed information, such as general ecological aspects of the area, abundance and behavior of the specimens, and any other observations noted at the time of collection.