Specimen Preparation

SPECIMEN PREPARATION

Note that many elements of the protocols described below were videotaped in a workshop entitled “Advanced Taxonomic Training: Fruit Flies”, University of Florida IFAS Extension Online Learning conducted at the University of Florida in 2019, which is available at: https://ifas-taxonomic-training.catalog.instructure.com/courses/fruit-flies-under-the-microscope.

Preservation of larvae

Rinse live larvae in cool, distilled water with a drop of mild dishwashing detergent.
Drop live larvae into very hot water (65°-70°C) and “cook” for 2 minutes or more. Allow specimens to cool to room temperature for 2-5 minutes.
Transfer larvae to 35% ethanol for a few hours, then 50% for a few hours, and finally preserve in 70% ethanol.
Label with complete collection data (locality, date, collector, host plant or lab colony and number of generations in culture) and store for further study.

This procedure minimizes discoloration, retraction of head, splitting or shriveling, and hardening of specimens. We have not found that sonication improves specimen quality.

Preservation of eggs and pupae

Eggs and pupae do not need to be cooked. They can go straight into 70% ethanol.

DNA subsample

It is useful to preserve a specimen subsample for DNA extraction and sequencing to corroborate morphological identification. The middle abdominal segments do not have much value for morphological identification. Therefore, abdominal segments 4, 5, or 6 can be cut out, preserved in 95-100% ethanol, and separately processed for molecular analysis.

Dissecting microscope observation techniques

Remove larva from alcohol, place on damp tissue, allow surface moisture to evaporate. Take care to prevent specimen from drying/shriveling.

Record facial features, count oral ridgesoral ridges:
several rows of ridges on each side of the mouth opening which may be entire, incised, emarginate, serrate, scalloped, dentate, or fringed on their lower (posterior margin) edge <a href="https://idtools.org/fsm/tools/index.cfm?pageID=3385" target="_blank">(anatomy: figures 2, 7.8.9)</a>
, make length/width measurements of exposed mouthhooks (MH), and prothoracic and posterior spiracles (PS); record shape of prothoracic spiracles and number of lobes, distribution of dorsal cuticular spinules, development of tubercles and presence/absence of pigmented line on posterior segment, shape of anal lobes (AL).

Compound microscope techniques

The facial maskmask:
the area on the head surrounding the antenna, maxillary palp, and part of the mouth <a href="https://idtools.org/fsm/tools/index.cfm?pageID=3385" target="_blank">(anatomy: figure 2)</a>
can be observed on an intact, alcohol-preserved specimen by placing the larva on a piece of tissue paper on a glass slide. When the alcohol evaporates away, the oral ridgesoral ridges:
several rows of ridges on each side of the mouth opening which may be entire, incised, emarginate, serrate, scalloped, dentate, or fringed on their lower (posterior margin) edge <a href="https://idtools.org/fsm/tools/index.cfm?pageID=3385" target="_blank">(anatomy: figures 2, 7.8.9)</a>
and other facial features can be seen clearly under 100x magnification.

For detailed examination of the cephaloskeletoncephaloskeleton:
the internal sclerotized region of the pseudocephalon, which consists of three main parts: mouthhooks, intermediate sclerite, and basal sclerite; has also been called cephalopharyngeal skeleton. <a href="https://idtools.org/fsm/tools/index.cfm?pageID=3385" target="_blank">(anatomy: figure 10)</a>
(CS) and spiracles and greater magnification of cuticular features such as dorsal spinules, the specimen must be macerated, and slide mounted in either of the two configurations shown here:
[insert config1 photo]

Detach the MHs from the external oral cavity using a minuten pin.
Make a small lateral cut through the cuticle of thoracic segments (during cooking and preserving, many specimens split naturally along the ecdysial line of weakness laterally on the thorax). The CS will be removed through this slit after maceration.
Make incisions on caudal segmentcaudal segment:
abdominal segment 8 <a href="https://idtools.org/fsm/tools/index.cfm?pageID=3385" target="_blank">(anatomy: figure 13)</a>
dorsally and on right side and between posterior spiracles and anal lobes so the caudal segmentcaudal segment:
abdominal segment 8 <a href="https://idtools.org/fsm/tools/index.cfm?pageID=3385" target="_blank">(anatomy: figure 13)</a>
will lay flat when mounted on a slide.
Remove internal tissues and preserve in 95-100% ethanol for DNA extraction and sequencing, if desired.
Macerate specimen in hot 10% NaOH (a coffee cup warmer works well) for ca. 15 minutes (more for larger specimens) or overnight at room temperature.
When digestion is complete, rinse specimen in distilled water and gently flush out tissue debris or remove with fine forceps. (Careful: the anal lobes are easily lost!).
Gently force the CS into the body cavity and through the lateral slit in the thorax to completely dislodge and remove it from the cuticle.
Transfer the cuticle and CS to glycerin on a glass slide, and gently brush the anterior spiracle forward to provide a good lateral view.
Add a cover slip and view/photograph/make measurements under a compound microscope.
Record measurements on CS and prothoracic and posterior spiracles; number of lobes on prothoracic spiracles, distribution of dorsal cuticular spinules; count trunks and branches of posterior spiracular processes.
Return to glycerin or alcohol for long-term storage.
Alternatively, permanent mounts can be made in euparol or Canada balsam according to standard procedures. However, great care must be taken to position the spiracles and CS correctly.

[Configuration 2 image]

Prepare as for Configuration 1, except
2. Make lateral incision along the complete length of right side of the body.
3. Make additional incisions around the prothoracic spiracles and caudal segmentcaudal segment:
abdominal segment 8 <a href="https://idtools.org/fsm/tools/index.cfm?pageID=3385" target="_blank">(anatomy: figure 13)</a>
as shown, so caudal segmentcaudal segment:
abdominal segment 8 <a href="https://idtools.org/fsm/tools/index.cfm?pageID=3385" target="_blank">(anatomy: figure 13)</a>
will lay flat.

Differential Interference Contrast (DIC) and Phase Contrast lighting is very useful to see certain features. A good quality microscope will allow you to distinguish many of the characters that are even more clearly seen with SEM.

Scanning electron microscope techniques

Choose preserved larvae for SEM that have the head fully exserted.

If no tissue is needed for DNA extraction, poke one or two holes in right side of whole larvae with a minuten pin to facilitate exchange of fluids during dehydration.

Dehydrate:
Place anterior and posterior ends (or whole larva) into 85% ethanol for >1 hour (longer may be necessary for whole specimens to allow complete perfusion of internal tissues).
Place larva or larval ends in 95% ethanol for >1 hour.
Place larva or larval ends in ethyl acetate for >1 hour.
Remove larva from ethyl acetate, allow to dry for a few seconds on a piece of tissue.
(Critical point drying is an alternative to ethyl acetate treatment)

Mount on SEM stub:
A single, whole specimen is best mounted in lateral view (right side-down to conceal minuten holes), which provides a good overall view of most external features. If multiple specimens are prepared, they may be mounted in various orientations to facilitate observation of all exterior features on dorsal, lateral, ventral, and caudal surfaces.
Mount specimen on a stub with carbon tape. Only use conductive carbon paste if specimen fails to stick to stub or is loosely attached to tape. Carbon paste may spread unevenly over the surface or within the specimen and obscure important features.

Allow mounted specimens to air-dry for a few hours, then place in a desiccator overnight before sputter-coating. SEM observations are best made at about 10-15 kV, depending upon the specimen quality and model/make of SEM used.