Chapter 1
Problems of sclerotized chitin: Softening insect cuticle

1.1 Introduction

The softening and processing of heavily sclerotized specimens for subsequent histological preparations is one of the major problems in insect histology. Many approaches to the solution of this problem have been suggested. Attempts to soften and otherwise alter sections with sclerotized chitin have been incorporated at every procedural level of histological methods. Suggestions have been made for changes in fixation, clearing, mounting, and embedding. Others have also attempted prefixation, postfixation, premounting, presectioning, and so on, as additional steps geared towards improving the quality of sections.

Aside from the more detailed procedures and specific compounds that are recommended in the following pages there are other simple general methods recommended. These techniques represent basic procedures that have been used independently or in conjunction with other methods. One of the most widely used procedures is the treatment of insect specimens with sodium or potassium hydroxide. These chemicals soften sclerotized portions of specimens and dissolve the soft internal tissues. They are generally used either cold or warm at a 10% concentration. These substances are also frequently used in the preparation of insect specimens for taxonomic study.

The use of hypochlorite of soda is another alternate for softening chitin. It is suggested for the preparation of all stages, that is, larvae, pupae, and adults. The insect is usually placed in boiling hypochlorite of soda (about 25% in distilled water). It is usually left in the solution for about 24 hours or more. A third, widely used approach is the use of tenerals or newly moulted specimens. In this way, the specimens are used before the cuticle has hardened.

The elimination of certain chemical agents which tend to harden insect tissues can also be helpful. Occasionally, it is best merely to avoid long exposures to hardening compounds. For example, to avoid excess hardening, short exposures or avoidance of the higher concentrations of ethanol will aid in preventing its hardening effects. The use of n-butyl or t-butyl alcohol as a substitute dehydrating agent may avoid the hardening of tissues. Similarly, prolonged exposure to certain chemicals or fixatives containing chemicals such as acidified dichromate, mercuric chloride or chromic acid is not recommended. Prolonged heating may also cause unwanted brittleness. The choice of clearing agent may also be a key factor in brittleness of tissue preparations. Thus, the use of clearing agents other than xylene or similar compounds will result in improved preparations. Finally, excessively high temperatures and prolonged periods of infiltration in wax may be another source of troublesome tissue hardening.

Fig. 1.1 Beetles have a hardened cuticle. (Source: © Michal Grabowski.http://commons.wikimedia.org/wiki/File:Xylena_exsoleta.jpg#filehistory/CC BY-SA 3.0.)

Fig. 1.2 Components of the cuticle. Procuticle - polysaccaride chitin and cross-linked proteins involved in sclerotization.

Fig. 1.3 The most sclerotized parts of beetles. (Source: Ellis 2000. Reproduced with permission of Elsevier.)

Another widely used procedure involves the puncturing of insect specimens before placing them in a fixative. This allows complete penetration of the fixing agent. Care must always be taken not to damage particular areas of interest on the specimen. The following procedure was suggested as an alternative to the puncturing of specimens.

1.1.1 Gottlieb's technic [1]

Application:

Recommended for the histological preparation of insect larvae, pupae, and adults.

Formula:

1. Solution A: Relaxing fluid
   1. Drosophila Ringer's with magnesium sulphate (4%) added.
2. Solution B: Chrome alum fixative Chrome Alum 3 pt Formaldehyde (40%) 30 pt Proprionic Acid 2 pt Distilled Water 238 pt Dimethyl Sulfoxide 25 pt

Procedure:

1. Rinse specimen in Ringer's solution and place in warmed solution A for 2 to 5 min.
2. Transfer to warmed solution B in a covered dish (on hot plate) for no more than 5 min.
3. Keep specimen in dish and remove from hot plate for 10 to 15 min.
4. Rinse in distilled water and dehydrate.
5. Dehydration must be slow and gentle.
6. Three methods of dehydration are recommended:
   1. Dioxane.
   2. Graded ethanol series with a benzene clearing agent.
   3. Graded tertiary butyl alcohol series.
7. In graded ethanol series a slow transfer to benzene is required before infiltration by using trichloropropane.
8. In the dioxane procedure, the following steps are necessary:
   1. Several changes of 50% dioxane for one day.
   2. Several changes of 100% dioxane for 2 to 3 days.
9. Infiltration is as follows:
   1. Transfer to solutions of increasing paraffin concentrations for 24 to 36 hr.
   2. Transfer to two baths of pure paraffin for 12 hr each.

Note:

1. Graded alcohols series consist of the following: 10-70% (in 10% steps), to 85% (in 5% steps), to 100% (in 2.5% steps).
2. A complete schedule of dehydration solutions and a timetable is available on Table 1 of Gottlieb (1966).
3. Either t-butyl alcohol or dioxane is recommended to avoid excess hardening.

1.2 General Methods

The following are general methods, fixatives, and softening agents recommended for the softening of histological preparations with extensive sclerotized chitin.

1.2.1 Cox's technic [2]

Application:

Recommended for small insects and insect parts, e.g., beetle elytra.

Formula:

1. Solution A:
   1. Potassium Hydroxide (10%)
2. Solution B:
   1. Acetic Acid (33%)

Procedure:

1. Fix insect specimen.
2. Transfer to solution A for 24 hr.
3. Wash in water for 6 hr.
4. Transfer to solution B for 24 hr.
5. Wash in water for 6 to 8 hr.
6. Dehydrate, embed, and mount.

Fig. 1.4 Components of the cuticle - SEM images of the dry gula (a plate which in most insects supports the basal part of the labium). (a,c,d) Surface of the gula. (b) Cross fracture of the gula cuticle showing the epicuticle (epi), exocuticle (exo) and endocuticle (endo). Fibers of the outer part of the exocuticle are oriented perpendicular to the surface but are parallel in the deeper layers of the exocuticle and in the endocuticle. Pores (pr), dried organic substances (se) and cracks (cr) can be seen on the cuticle surface. Rectangles c and d, indicate parts of the sample magnified in c and d, respectively. (Source: Barbakadze et al. 2006. Reproduced with permission of the authors.)

1.2.2 Eltringham's method II [2]

Formula:

1. Solution A:
   1. Sodium Hypochlorite (6%)
2. Solution B: Fixative Water 250 pt Picric Acid 2.6 pt Nitric Acid 10 pt

Procedure:

1. Fix insect specimen.
2. Wash in running water for 4 hr.
3. Transfer to solution A for 60°C for 36 hr.
4. Wash in water for 4 hr.
5. Transfer to solution B at 60°C for 6 days.
6. Boil in alcohol (70%) for 1 min.
7. Let stand in alcohol (70%) for 1 min.
8. Dehydrate, clear in cedar-oil and mount.

1.2.3 Verdcourt's nitric-ethanol [3]

Application:

Recommended for the softening elytra of Coleoptera.

Formula:

1. Solution A: Ethyl Alcohol 3 pt Nitric Acid (concentrated) 1 pt

1.2.4 Schultze's [3]

Application:

Recommended for the softening elytra of Coleoptera.

Formula:

Note:

1. When warmed, this substance softens in a few minutes, but is harsh and must be watched.

1.2.5 Modified Schultze's [3]

Application:

Recommended for the softening elytra of Coleoptera.

Formula:

Note:

1. Softening occurs within 5 to 6 days, however, after about 12 days specimens become deformed.
2. Wash well in water after use.

1.2.6 Verdcourt's technic [3]

Application:

1. Recommended for the softening and histological preparation of hard chitinous insect tissues.
2. Recommended as particularly useful for softening and preparating slides of the elytra of coleopterans.

Procedure:

1. Place in softening agent (see Verdcourt's Nitric-ethanol, Schultze's...