Beetle Larvae of the World
Fixation of specimens can be accomplished in a number of ways, but one of the easiest methods is to place the larval specimens in hot water (just below boiling point) for a short time. Other methods involve chemical fixatives which usually contain alcohol and formalin or acetic acid. These are discussed in various books on collecting and preserving insects listed below. I have found hot water with a few drops of acetic acid added is sufficient for most larvae, which are then transferred to 80 percent alcohol. Large larvae should be punctured in several places with a pin to assure penetration of the fixative.
Many structures used in this key can be seen externally on larger larvae which have been prepared in the manner just discussed; however smaller larvae almost always require further preparation. At a minimum, the mandibles should be removed, since most of the important structures cannot be seen when the mandibles are closed and concealed by the labrum-epipharynx above and maxillo-labial complex below. This can usually be accomplished with a hooked pin or pair of fine forceps.
The following procedure may be used to fully prepare a larval specimen for examination:
1. Make a sketch of the larva, if it is the only specimen. Special note should be made of the number of stemmata visible on each side; these usually loose their pigment after maceration and clearing of the cuticle. Also make a note of color patterns.
2. Remove the head with a pin or forceps (a minutin pin may be used for small specimens).
3. Cut the abdomen at about the middle (between segments 4 and 5), so that the larva is now in three pieces.
4. Remove a section of the gut and place it on a slide mount, if it is desirable to determine the food source.
5. Place the three pieces in potassium hydroxide (KOH; usually a 10% solution) and leave overnight cold or warm it for a few minutes on a hot plate or over a flame. The time varies considerably with the size and condition of the specimen.
6. While the pieces are still in the KOH solution, use forceps and-or dissecting pins or hooks to remove soft tissue. The head may be gently squeezed with forceps to force tissue out of the occipital foramen. At this point you might check for sclerotizations of the proventriculus or rectum.
7. Remove the mandibles with a hooked pin. It is usually preferable to remove both of the mandibles to check for strong asymmetry and to make visible the epipharynx above them and hypopharynx below them. Sometimes it is necessary to remove the entire maxillo-labial complex, in order to see structures of the epipharynx.
8. Transfer the pieces into alcohol to which you have added a few drops of acetic acid; this neutralizes the KOH. Then transfer to 80% alcohol.
9. Specimens may be retained in alcohol or transferred again to glycerin for study. Either glycerine or alcohol can be used for permanent storage,
10. Specimens may be further treated with various stains to bring out structures which are normally difficult to see (spiracular details, for instance). Chlorazol black and acid fuchsin are commonly used. Also, a permanent slide mount may be made using a variety of techniques explained in some of the references below or any standard textbook on microtechnique or histology.
Booth, R. G., Cox, M. L. and Madge, R. B. (1990). IIE Guides to Insects of Importance to Man. 3. Coleoptera, vi + 384 pp. International Institute of Entomology: London.
Cogan, B. H. and Smith, K. G. V. (1974). Instructions for Collectors. No. 4a: Insects, vi + 169 pp. British Museum (Natural History): London.
Martin, J. E. H. (ed.) (1977). The Insects and Arachnids of Canada. Part I. Collecting, Preparing and Preserving Insects, Mites, and Spiders, 182 pp. Canadian Department of Agriculture Research Branch: Ottawa; Publication No. 1943).
Steyskal, G. C., Murphy, W. L. and Hoover, E. M. (1986). Insects and Mites: Techniques for their Collection and Preservation. U. S. Department of Agriculture: Washington.
Upton, M. S. (1991). Methods for Collecting, Preserving, and Studying Insects and allied forms, vi + 86 pp. Australian Entomological Society: Brisbane (Miscellaneous Publication No. 3).
Walsh, G. B. and Dibb, J. R. (eds) (1975). A Coleopterist’s Handbook. 2nd Edition (revised by J. Cooter and P. W. Cribb), 142 pp. Amateur Entomologist's Society: Feltham.