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Preparing Dissections and Slide Mounts of Moths, including Wing Venation, Genitalia, and Whole Bodies

Sangmi Lee and Richard L. Brown

Supplies and Equipment

Forceps, Dumont Microsurgery fine tip, #5, straight, 0.10 x 0.06 mm; #7, curved, 0.17 x 0.10 mm

Hypodermic syringe with a size #30 needle

Camel hair brush, fine tip (size 000)

Vanna spring scissors (optional), angled, tip diameter 0.1 mm., 8.5 cm

Syracuse watch glasses Sorting trays with 24 cells (cell diameter x depth, 16 x 18 mm)

Microscope slides, 3 x 1” (76 x 25 mm); thickness: 0.9 – 1.0 mm

Cover slips, round, 18 mm, No. 2 thickness

Euparal Mounting Medium and Euparal Essence (thinning agent for Euparal mounting medium)

Stains: Eosin Y (E-511), Chlorazole Black E (C.I. 30235)

10% potassium hydroxide (KOH): for 100 ml of KOH solution, use 10 g of KOH and 90 ml of water

Ethyl alcohol, 20%, 70% and 100%

Procedure for Wing Venation Slides

  1. The right pair of wings is separated from the body and wet in 100% ETOH for a few seconds.
  2. Label the pinned specimen from which the wings were removed. The label is prepared in duplicate with one copy affixed to the pin holding the specimen and the other copy accompanying the right pair of wings through the various reagents.  Data from the specimen, species identity, and the slide number should be written into a permanent log. 
  3. The wings are placed in 20% ETOH and denuded with a camel hair brush (size 000) by gently brushing the wing membrane from the wing base towards apex, alternating with gentle tamping of the membrane.
  4. These wings are transferred to a watch glass and stained with 2% Eosin overnight.
  5. The wings are further cleared of scales in 70% ETOH after staining (note:  scales are more easily removed in 70% than 20% ETOH, but the wing membrane is more easily torn).
  6. The wings are dehydrated in 100% ETOH for 2-3 hours before slide mounting.
  7. A microscope slide is cleaned with 100% alcohol, placed on a template that has a circle in middle that matches the size of the coverslip (see description of genitalia regarding cleaning slides).
  8. 3-4 drops of Euparal are added to the slide and spread to cover the area of the template circle.
  9. The wings are placed on the euparal, gently pressed to the bottom of the media, and arranged in correct positions on the slide. The coverslip is brushed with Euparal essence before being placed on top of the wings (see description of genitalia regarding cleaning of coverslips).
  10. Label the slide with a unique number that is also placed on the pinned specimen from which the wings were removed.

Procedure for Preparing Dissections of Male Genitalia [Video] and Female Genitalia [video]

  1. Label the pinned specimen from which the abdomen is to be removed.  The label should include the sex of the specimen, a unique identifying number and the name of the dissector, e.g., "male (using male symbol) genitalia slide; MEM 5566, Sangmi Lee."  A colored paper should be used, we use green, for labels so that these can be easily detected in the currated collection.  The dissection number is duplicated and is carried with the abdomen through the various reagents.
  2. The abdomen is detached from the body by using forceps to press up on the venter of the end of the abdomen.
  3. The abdomen is placed in 10% potassium hydroxide (KOH) overnight at room temperature.  For rapid dissections, the abdomen in KOH can be placed on a hot plate at a low temperature for 1-2 hours, although this latter method is less satisfactory for digesting extraneous fat bodies and tissues and subsequent cleaning of scales.
  4. After digestion in KOH, the abdomen is transferred to a Syracuse watch glass with distilled water and gently cleaned hairs, scales, and the debris from within the abdomen with a fine camel hair brush and a pair of curved forceps for holding the abdomen in place while brushing.
  5. When the preliminary cleaning is completed, and while the genitalia are still attached, transfer the abdomen to a watch glass with 2% Eosin for four hours.  Chlorazol black may be used as a second stain to differentiate membranous from sclerotized structures, but the abdomen should be left in chlorazol black for less than ten seconds after it is stained with eosin.  The time required for adequate staining varies depending on the concentration of the stain as well as the taxon.  Other common stains include saffranine, merbromin (mercurochrome), and orange-G.  Merbromine is the quickest stain, but it does not differentiate between sclerotized and membranous structures.  In confocal laser microscopy is used for imaging, stains with eosin and saffranine provide the most fluorescence and best images, whereas use of chlorazol black reduces the quality of the images (Lee and Brown, 2009).
  6. The abdomen is transferred from the stain into a watch glass with 20% ETOH and further cleaned.
  7. The abdomen is then transferred to a watch glass with 70% ETOH for final cleaning and separation of the genitalia from the abdomen.  Note:  Higher concentrations of ETOH allow easier cleaning of scales, but the disadvantage is that these higher concentrations make the specimen more brittle and subject to tearing.  The male genitalia are separated from the abdomen by tearing the intersegmental membrane between the last abdominal segment and the genitalia. In males the phallus may need to be removed from the genitalia by holding the base of the valvae with one pair of forceps and extracting the phallus with another pair. In females the intersegmental membrane between the 6th and 7th segment is cut, or torn apart with two pairs of forcepts – while being careful not to break the ductus  bursae.  In some families of moths, the genitalia (and ovipositor) are separated by a cut between the 7th sternum and the ostium bursae.   Vanna spring scissors can be used to cut the segments apart, or two pairs of forceps pulling in opposite directions will also work.  A small slit in the corpus bursae can be made and a hypodermic needle used to remove any remaining parts of the spermatophore.  This hypodermic needle also can be used for removing extraneous material from other parts of the male and female genitalia that are difficult to extract with forceps.
  8. The abdominal pelt and genitalia are transferred to a watch glass in 100% ETOH, and quickly positioned and held in place with small chips of a glass slide, cut with a dimond cutter.  Genitalia of most Lepidoptera are positioned with the valvae spread and held open by the glass chip, but some taxa have male genitalia that are mounted laterally
  9. Mount in Euparal on a clean microscope slide.  Although purchased slides are advertised as "pre-cleaned", we transfer the slides to a container of 100% ETOH and wipe them dry with cheesecloth (which has no lint) before use.  Coverslips are also held with a container of 100% ETOH and are wiped with cheesecloth before use.  The coverslip is brushed with Euparal essence before being placed on top of the parts of abdomen and genitalia.
  10. Label the slide so that it can be connected to the pinned specimen from which the abdomen was removed.

Procedure for Preparing Whole Body Mounts of De-scaled Specimens

Note:  One option for examining endo- and exoskeletal features is to follow steps 1-9 below, and then preserve the body parts in glycerin in a microvial.  This option has an advantage of viewing the structure at different angles, in contrast to slide mounted structures.  However, slide mounts of whole bodies have the same advantages as those of genitalia, e.g., the ability to store, retrieve, examine, and compare structures very quickly and efficiently.  One slide can be placed on top of another slide – separated by glass chips if the mounting medium is not dry, for quick comparison of two taxa.  Additionally, slide mounted specimens can be viewed and imaged with compound or confocal microscopes.  More information on this technique can be found in Lee and Brown (2006).

  1. Select the specimen for a whole body mount that has all appendages and complete wing patterns.
  2. Data labels are removed from the selected specimen and placed on a second pin with an additional label providing a unique slide number.  Each slide number label is prepared in duplicate with one copy affixed to the pin holding the specimen and the other copy accompanying the right pair of wings through the various reagents.
  3. The left pair of wings is separated from the body for dry preservation of the color pattern and is set aside for future slide mounting.
  4. The right pair of wings for studying venation is detached from the body and is set aside for future cleaning, staining, and slide mounting.
  5. The rest of the specimen, while still on the pin, is submerged in 100% ETOH for a few seconds and then is placed in 10% potassium hydroxide (KOH) overnight at room temperature.
  6. After the body is cleared overnight, it is transferred to a watch glass with water and the head, thorax, and abdomen are separated and initially cleaned by removing as many scales as can easily be removed by tamping and brushing the structure with a camel hair's brush, size 000.  These portions of the body then are transferred to a separate container with 2% Eosin for staining.  Chlorazol black may be used as a second stain to differentiate membranous from sclerotized structures, although this latter stain is not preferred for imaging with confocal microscopy.  The time required for adequate staining varies with the taxon, but typically staining with chlorazol black requires less than ten seconds and Eosin requires as much as four hours.
  7. After staining, the separated portions are transferred to 20% ETOH, and antennae, labial palpi, and one half of the proboscis are detached from the head capsule, the thoracic segments are separated, and the genitalia are separated from the abdomen.  These parts are further cleaned with the camel's hair brush and the abdomen is tamped flat such that the dorsal tergites are opposite the dorsal tergites.  Scales are removed from the appendages by holding the structure with a curved forceps and lightly brushing the appendage towards its apex.  It is important to hold the structure with forceps near the area that is being brushed in order to prevent damage to the structure.  Any remaining internal contents of the head, thorax, and abdomen are removed with forceps or a hypodermic syringe with a size #30 needle. 
  8. The separated portions of the body transferred to 70% ETOH and removal of remaining scales and other non-sclerotized tissue is completed.  Scales are more easily removed in 70% ETOH than in water or 20% ETOH, but the higher concentrations of ETOH make the specimen more brittle, and more care is needed to avoid damaging the structure. 
  9. All body parts except wings are dehydrated about 8 hours in 100% ETOH before being mounted in Euparal on a clean microscope slide, following the methods given for genitalia.
  10. The left pair of wings with intact wing pattern is dry mounted on the middle of the slide using clear nail polish around the edge of a coverslip.
  11. The right pair of wings wet in 100% ETOH for a second and is transferred to a watch glass with Eosin overnight.
  12. After staining, the wings are transferred to 20% ETOH to be denuded.
  13. The wings are further cleared of scales in 70% ETOH and dehydrated in 100% ETOH before being mounted in Euparal on a clean microscope slide.
  14. Prepare two slides for mounting the whole body.
  15. Slide “A” (labeled “A” with the dissection number) includes the whole body parts except the wings.  The head and its appendages, prothorax, and mesothorax are mounted under one coverslip in the middle of the slide, and the metathorax, abdomen and genitalia are mounted under a second coverslip on the right side of the slide.  A label with species identification, sex, selected collection data, mounting medium, and slide number is attached to the left side of the slide.
  16. Slide “B” includes the wings.  The left pair of wings is dry mounted on the middle of the slide using clear nail polish around the edge of a coverslip.  The right pair of wings is mounted in Euparal on the right side of the same slide.  The coverslip is brushed with Euparal essence before being placed on top of the wings.
  17. For large specimens three or more slides may be needed for mounting the entire body.

References

Clarke, J.F.G. 1941.  The preparation of slides of the genitalia of Lepidoptera.  Bulletin of the Brooklyn Entomological Society 36: 149-161.

Huemer, P. 1987. Eine modifizierte Genitalpräparationstechnik für die Gattung Caryocolum. Mitteilungen der Schweizerischen Entomologischen Gesellschaft 60: 207-211.

Lee, S.M. and R.L. Brown. 2006. A new method for preparing slide mounts of whole bodies of microlepidoptera. J. Asia-Pacific Entomology, 9(3): 249-253.

Lee, S. and R.L. Brown. 2009.  Use of confocal scanning microscopy in systematics of insects with a comparison of fluorescence from different stains.  Systematic Entomology 34: 10-14.

Pitkin, L.M.  1986.  A technique for the preparation of complex male genitalia in microlepidoptera.  Entomologist’s Gazette 37: 173-179.

Robinson, G.S. 1976.  The preparation of slides of Lepidoptera genitalia with special reference to the microlepidoptera.  Entomologist’s Gazette 27: 127-132.

Zimmerman, E.C. 1978.  Insects of Hawaii.  Volume 9.  Microlepidoptera, Part I, Monotrysia, Tineoidea, Tortricoidea, Gracillarioidea, Yponomentoidea, and Alucitoidea.  881 pp + 8 pls. [see “notes on the preparation of dissections of microlepidoptera…,” pp. 73-91]