Beetle immatures, especially larvae and pupae, are generally found in the same habitats as their respective adults. Yet, beetles spend the vast majority of their lives feeding and interacting with the environment as larvae. Unfortunately, this is a life stage of most beetles for which we little or no information. As such, whenever your collecting provides you an opportunity to obtain immatures, do so. Always examine closely the microhabitat that you are searching. If you find adults and immatures of beetles together, always collect both and keep them together for examination by a technician, curator, or specialist. Usually, associated immatures and adults are of the same species. Since the immatures of most beetles are even more poorly studied than adults, discovering such associations is an excellent lead to new scientific conclusions.
Bioprospecting has its greatest potential with the larvae of many beetles. Thus, it is important to record as much information as possible on the association, microhabitat, and ecological conditions of your discovery. This information will be invaluable for recollecting specimens, and determining methods and procedures for rearing immatures. All of these information pieces are necessary steps for bioprospecting evaluation. If possible, take photographs or make sketchs or descriptions of the collecting site, and note your sampling method, host identification, and any other observations.
Retaining specimens of beetle immatures requires different methods than for adults. Your first decision or question should be: preserve the specimen(s) or keep them alive? If specimens are numerous, immediately preserve exemplars and retain the remainder for rearing. With immatures, experience shows that there is rarely the problem of too many specimens of larvae or pupae!
Preservation methods of beetle immatures can be kept simple and still yield high quality specimens for taxonomic evaluation. Some methods are acceptable while on the trail, others require a bit more time and patience, and should be done in the "lab." All methods employ some ethanol or amended ethanol solution for final preservation.
Proper field preparation of larvae and pupae for preservation requires "fixing" the specimens. "Fixing" specimens means, among other aspects, that the microflora of bacteria, protozoans, and digestive enzymes in the gut of the insects need to be killed completely and rapidly. Attempting to preserve many beetle larvae without fixing them first will result in internally blackened and partially decayed specimens that are nearly useless. Examples of families with larvae that are particularly prone to internal decay are Scarabaeidae, Passalidae, Cerambycidae, Buprestidae, and other bulky pale-colored larvae with large volumes of body fat. "Fixing" is necessary because standard ethanol solutions will not penetrate the body tissues rapidly enough to kill the gut microflora and enzymes.
Three methods are acceptable for field preparation of beetle larvae: 1) boiling, 2) direct immersion in acid-alcohol, 3) direct immersion into KAA. In all cases, the best specimens will be obtained by replacing all fluids with 75-80% ethanol after 24-48 hours, and very large immatures will need a second ethanol change after another 48-72 hours. The exception to these methods is for larvae of aquatic species, which can be placed directly into ethanol, and then the ethanol changed after 12-24 hours.
Basically, ethanol is a preservative, not a fixative. It is an excellent fluid for preserving specimens after they have been fixed with boiling water, acid-alcohol or KAA, but is terrible for fixing immatures, except aquatic species. Changes of alcohol or acid-alcohol solutions are necessary because dilution from body fluids will affect preservation and may permit decay.
The best field method for fixing specimens is to collect them alive and return them to camp or laboratory, and cook them in near boiling water. The method is simple.
If circumstances do not allow such treatment, the next best method is to place your specimens into an acid-alcohol solution or KAA. Specimens killed in acid-alcohol may be retained in this solution for long periods, or even preserved and stored in this solution. However, immatures killed in KAA should have the solution replaced with ethanol within 24-48 hours.
Acid-alcohol is a mixture of 80% ethanol with glacial acetic acid at a ratio of 19 parts alcohol solution to 1 part glacial acetic acid. KAA is a solution of kerosene, glacial acetic acid, and 80% ethanol mixed at the same ratio as acid-alcohol, but with the addition of one-half part purified kerosene.
KAA is a generally useful chemical for many insect immatures, but particularly larvae of Coleoptera, Hymenoptera, and Lepidoptera. The kerosene causes distension of the body so that the appendages and mouthpart structures are more easily examined. However, both KAA and acid-alcohol are less convenient to work with because of the chemical odors and the fact that the acetic acid soaks into the skin very quickly and may cause allergies for some people.
This manual is not the place to cover in detail the wide diversity of methods and techniques for dealing with the immatures of a biologically diverse beetle fauna as that in Costa Rica. Therefore, we recommend that the parataxonomist attempt only the most basic effort for rearing, and preferably in consultation with the appropriate scientific specialist. Consequently, we will provide only rudimentary techniques for those persons interested in attempting limited rearing.
There are two general ways of associating larvae or pupae with their respective adults. One is to find these stages together in a single habitat, and the other, better method is rearing. Rearing may be done from larva to adult, or by collecting adults, having them mate and oviposit in the lab, and then raise the larvae. The former method is by far the easiest, as it is often difficult to simulate the natural habitat (and factors that may stimulate ovipsotion) in the laboratory. The rearing of adult beetles from collected larvae provides: 1) taxonomically-significant series of species that have heretofore been considered uncommon, or are only rarely encountered by other collecting methods; 2) natural history information, including basic host plant data and host ranges, larval development time, stratification of niche and substrate use, feeding guild composition, and competitor species; 3) parasitoid information; 4) collection of larvae directly associated with parent stock through F1 breeding (as desired); 5) photographs of life history stages and materials for WWW and other publication products. All of these products and data may be provided within the context of a single, simple methodology for many taxa.
The single most important aspect for successful rearing is to maintain larval environments as close as possible to the natural elevational pressures, temperatures and humidity regimes. Often, this is difficult to do, especially in the absence of specialized equipment, growth chambers, incubators, etc. Important scientific contributions are still possible with even the most basic of supplies and equipment.
Essentially any container that will prevent escape of the larva and maintain necessary humidity levels is usable. For these purposes, simple plastic boxes and tubs will generally work, but the container must conform to the normal "habitat" of the larva. As an example, larvae that normally exist in wood should have section of the wood placed into an elongate container to adequately fit the substrate section. If you are attempting to rear larvae that are in seeds, fruits, flowers, etc., then a simple quadrangular or round container, or even a plastic bag, would work.
Rearing from dead wood primary The rearing of beetles from sound wood usually consists of the taking of a length an occupied branch or other piece of wood. The sample may require sectioning it into manageable pieces, but consideration must be given to the needs of the beetle larvae. The volume or number of sections is determined by container capacity, so a single large diameter piece may be sufficient, while several smaller sections and many twigs will be included in a single sample, depending on availability and needs. In all cases, larval specimens must be taken for preservation so that the correlation can be made with adults that emerge from the samples.
Branches can be gathered and transferred to rearing chambers and observed. As adult specimens emerge from branches, they can then be collected, and data recorded for emergence date, date wood cut, wood species and branch class. Parasitoids and other insect groups may also may emerge from branches, and these will be collected into alcohol; data recorded must include the same information as above, with a code to indicate the probable beetle host association (determination of the beetles may not be done for some time after the emergence cycles).
During the period of larval maturation, the rearing chambers for often examined for signs of larvae, adults, moisture levels, and mold growth. Occasional soaking of drying wood with water will be required to maintain moisture levels in the rearing chamber, and chamber walls should be allowed to dry as the wood is checked. Excessive mold growth should be wiped off surfaces with a moist rag. Thick pieces of sound wood may be moistened by submerging the branches in a barrel of water for 1 or 2 minutes, once every two weeks.
Usually, most larvae develop within a few months. At the end of year one, samples should be broken apart and examined for additional larvae, and larval galleries photographed as appropriate. Wood material may be returned to the forest for additional decomposition and nutrient recycling.
Many beetles, such as species of Tenebrionoidea, Scarabaeoidea, and Curculionoidea, have larvae that develop in wood that is already in an advanced state of decomposition. Moisture and temperature levels need to be closely monitored when containerizing rotting wood, so that excessive mold, high temperatures, and accelerated decay does not kill the larvae.
Rearing beetles from leaves, buds, seeds and galls
Rearing beetles from leaves, buds, seeds and galls involves a wide
variety of beetles, but especially larvae of Chrysomelidae,
Bruchidae, Curculionidae, Mordellidae, and many others. Taking of
plant samples consists of searching for potential or
evidently-infested hosts, taking locality data, identifying the host,
and rearing the specimens in plastic bags. Moisture in the bags may
be maintained by the addition of leaves with high water content, or
by adding damp paper toweling. However, water droplets in the bags
indicates excessive moisture that permits the growth of mold and
death of the larvae. Host materials for leaf and flower feeders may
require changing every 3 - 4 days, or as determined by observation of
conditions. A pad of moist paper towels may be wadded into the bottom
of the bags to provide a pupation substrate for species which abandon
the host to metamorphose.