Preparation of genitalia

It is essential to examine male genitalia to obtain a positive identification in Agromyzidae. A brief account of a satisfactory method of preparing specimens is outlined. More details of the method can be found in EPPO (2005), Spencer (1981, 1992) and Spencer& Steyskal (1986).

1. Using dissecting needles [made by gluing the blunt end of pointed micro pins ("minuten")  into the end of a wooden match-stick, first making a short hole with a normal pin], carefully separate the abdomen from the rest of the fly.
2. Boil the abdomen in 10% potassium hydroxide (KOH) for 2-4 minutes, or leave in cold 10% KOH overnight.
3. Transfer the abdomen to cold glacial acetic acid for 2-3 minutes to neutralize the KOH.
4. Blot off excess glacial acetic acid and transfer the abdomen to a drop of Hoyer’s medium on a cavity slide.
5. Under a binocular stereoscopic microscope and using fine dissecting needles, carefully dissect out the genital complex from the surrounding membranes, cuticle and associated musculature.
6. Using fine dissecting needles, position the genital complex for lateral viewing under a compound microscope at up to 400X magnification.
7. Reposition the genital complex for ventral viewing of the distiphallus at 400X magnification.
8. To make semi-permanent slides transfer genital complex to a drop of Hoyer’s medium on a clean flat slide. Gently immerse the genitalia in the mountant, then carefully lower a round coverslip over it to evenly spread the mountant.
9. Permanent slides, if required, may be prepared as follows:
   After step 3 above, the abdomen can be transferred to 70% ethanol and, using fine dissecting needles under a binocular stereoscopic microscope, the genital complex can be carefully dissected from the surrounding membranes, cuticle and associated musculature. Transfer the dissected genitalia to absolute ethanol for 2-4 minutes, then to clove oil, where it can be left for any length of time. Gently tranfer the genitalia to a drop of Euparal on a clean flat slide and orient in the mountant. Carefully lower a round coverslip onto the drop, commencing at its edge, then evenly spreading the mountant.
10. Label the slide with adequate data on locality, date of collection, name of collector, common and scientific names of the host.
11. Place the slide in an incubator maintained at 45ºC for drying for two weeks.
12. Mount the remainder of the fly specimen onto a card point with an appropriate label and cross-referenced to its genitalia mounted on slide.

References
EPPO (2005). Liriomyza spp. EPPO Bulletin 35: 335-344.

Spencer KA (1981). A revisionary study of the leaf-mining flies (Agromyzidae) of California. University of California, Division of Agricultural Sciences Publication 3273.

Spencer KA (1992). Flycatcher: memoirs of an amateur entomologist. SPB Academic Publishing, The Hague. 414 pp.

Spencer KA & Steyskal GC (1986). Manual of the Agromyzidae (Diptera) of the United States. Agriculture Handbook 638. United States Department of Agriculture,
Washington DC.

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