ࡱ> ܥhc e_[ _Z lZ llZ lZ lZ lZ lZ Z Z Z Z Z Z Z Z Z JZ Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z X>[ ZZ lZ Z Z Z Z Z Z Z lZ lZ Z Z Z Z Z Z lZ Z lZ Z Z !4Z Z lZ lZ lZ lZ Z Z Z Z A SIMPLE TECHNIQUE FOR PREPARING AND PHOTOGRAPHING MOTH GENITALIA Winter (2000) gave detailed instructions for preparing, mounting, and photographing the genitalia of Lepidoptera. I have been using a simpler method for preparing, storing, and photgraphing moth genitalia for many years. With practice it is possible to obtain a mounted preparation in as little as 5 minutes hands-on time. Equipment 1] A perferctly dry moth, either pinned or in envelope. It is difficult/impossible to obtain a decent genitalia preparation with anything other than a perfectly dry specimen. 2] 2 pairs of fine straight forceps (e.g., BioQuip Swiss Style Forceps #4531, or equivalent). #4 are fine, #5 are superfine. 3] 5% solution of Potassium Hyroxide (KOH) in water (tap water is OK). Note: KOH is caustic, will burn skin and clothing; wear rubber gloves and eye protection. 4] Glass vials, any size but just large enough to hold an abdomen is all that is needed. I use 1/2 dram shell vials (e.g., BioQuip #8620 or equivalent) 5] A system for storing vials upright. I use Egg Crate, sold as 2x4 sheets as a diffuser for ceiling mounted fluorescent lights. The square holes are 0.5 on side and I cut the sheet up to give a grid of 10x10 squares. As the grid is only 3/8 deep, I stack 2 pieces to give square holes that are 3/4 deep. 6] Glycerine 7] Eppendorf centrifuge tubes. 8] Dissecting microscope or magnifying lens. 9] Camera set-up if photographs are required Technique 1] Label the moth (Fig 1) and an empty vial (Fig 2 top) with a unique number if more that 1 moth is being processed. I find it convenient to work with 10 or 20 moths at one time. The labelled emptied vial accompanies the abdomen throughout.  Fig 1. Labelled moth, male Melanolophia sp. either canadaria or signataria 2] Break the abdomen off the moth (Fig 2 bottom); pushing down or lifting up the tip of the abdomen will usually snap it off cleanly at the junction with the thorax.  Fig 2. Labelled vial, KOH vial, detached abdomen 3] Place the abdomen in a vial and add sufficient 5%KOH to just cover the abdomen (Fig 3). The abdomen will initially float on the surface but will eventually sink into the solution. Leave for about 12 hrs at about room temp (20 C, 68F) in egg crate with labelled companion vial (Figs 4 & 5).  Fig 3. Companion labelled vial, detached abdomen in 5% KOH  Fig 4. One-half inch Egg Crate, fluorescent light diffuser; double thickness  Fig 5. Companion labelled vial and KOH vial containing abdomen in egg crate 4] Pour off the KOH, discard. Rinse the abdomen in clean water (tap water is OK), about 3-5 changes. 5] Transfer the abdomen to a dish of water beneath a microscope (or lens) (Fig 6)  Fig 6. Abdomen after 12 hr soaking in cold 5% KOH and after rinsing in tap water 6] Grab the dorsal surface (tergum) of the abdomen at the broken end (segment 1) with 1 pair of forceps. Grab the broken end of the ventral surface (sternum) with the other pair of forceps. Pull apart, the abdomen will unzip along the 2 lateral membranes between the terga and sterna. The genitalia will be in a capsule at the posterior end of the abdomen. The male genitalia are especially tough and one can be reasonably rough in removing the parts of the abdomen and fat bodies from the genitalia. Female genitalia are far more delicate. Practice on males before attempting to dissect a female. 7] Rinse the male genitalia in water to remove debris and hairs; use either a fine brush, a pipette to squirt water, or place the genitalia back into a clean vial and shake gently. 8] Tranfer the clean genitalia to a dish or Micro Cavity Slide (e.g., BioQuip 6330A or equivalent). Remove any restraining membranes with forceps. Rinse with water if necessary (Fig 7)  Fig 7. Male genitalic capsule removed from abdomen. (Note that male Melanolophia are somewhat odd in that they have a lateral organ or double comb between abdominal segments 7 & 8. This is visible in Fig 6 immediately anterior to the genitalia, and the lower image in Fig 7. It is possible to separate canadaria from signataria by the structure of this double comb). 9] Flatten the genitalia by spreading the valves apart from the ventral side (similar to opening a book) (Fig 8). Remove the water with a pipette or paper towelling and flood genitalia with a very dilute solution of gycerine in water; about a 5% solution. Leave for as long as it takes for the water to evaporate, gentle warming will accelerate the evaparation. You cannot over evaporate. The idea is to get the genitalia into a pure solution of gylcerine. If the genitalia are plunged directly into glycerine from water the osmotic pressure may distort the genitalia (particularly with female genitalia).  Fig 8. Male genitalia, final preparation in glycerine. This is Melanolophia signataria. The asymmetry is diagnostic, the process of the left sacculus is significantly longer that that of the right 9] I rarely find it necessary to stain genitalia. Coloured ink squeezed from an empty ink jet cartridge will mix with glycerine. Soak the genitalia in this coloured solution and the genitalia will absorb the dye within a matter of hours. Frequent checking is necessary so as not to overstain. (This works with Canon ink jet cartridges). 10] Cut the cap off an Eppendorf tube (Fig 9), trim off the excess plastic. Place genitalia in cap, add 1 drop of clean gycerine (Fig 10), pin beneath moth. The genitalia will not dry out and are readily accessible for examination .  Fig 9. Complete Eppendorf centrifuge tube  Fig 10. Trimmed cap from Eppendorf tube with genitalia in glycerine. In reality, this will be placed on the same pin as the moth Photography Place the genitalia in glycerine in a Micro Cavity Slide. Place the slide on a light table (I use a slide viewer) on a piece of black paper with 3/4 diameter hole. I use a slave flash to illuminate the genitalia from beneath. Use the slide viewer lamp to focus the genitalia and use the cameras built-in flash to trigger the slave; no need to turn off the slide viewer light (Fig 11).  Fig 11. Genitalia in a Micro Canvty Slide, on black paper with hole cut out beneath cavity, on top of a slide viewer. The cordless slave flash has the flash head beneath the plastic top of the slide viewer and the triggering light above. The built-in flash on the camera triggers the slave and this illuminates the genitalia from beneath (see Fig 8 for result). Reference Winter, W.D. Jr. 2000. Basic Techniques for Observing and Studying Moths & Butterflies. Chapter 9. Preparing Genitalia, pp.265-276. Memoirs of the Lepidopterists Society No. 5. 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