Slide preparation of Encarsia

Slide mount of a female specimen of Encarsia whittieri Girault

(after Noyes 1982, with modifications)

Dry mounted specimens

• Attach specimen with ventral surface to a small piece of card board with a very small drop of water soluble glue. Wings must remain free of glue. Specimens stored in ethanol have to be dried first by placing them for a short time on a tissue.
• Write sample/specimen number on card board.
• Glue labels on the slide and write sample/specimen number on label. The labels should be made of 1-1.5 mm thick white card board.

• Remove wings with insect pin. This should be done carefully to avoid removing diagnostically important setae from the submarginal and marginal veins and is best achieved by moving the wings at the point where they are attached to the body. Preferably the fore wing comes off with the tegula attached to them.
• Put a small drop of Canada balsam at the location of the slide that is designated for the wings (i.e., top left). (Note: wings, body, and head should be oriented upside down so that the specimen is in upright position when examined with a compound microscope).
• Transfer wings to drop of Canada balsam and arrange them. Leave slide on a hot plate (60° C) for several hours to fix their position.

• Add a drop of water to the specimen glued on the piece of card board. After a short while the specimen will dislodge and float.
• Mazerate specimen in 10% KOH, either by heating in an Eppendorf tube for 5-7 minutes at 95° C using a block heater, or by leaving in an excavated block (covered with lid) for about 12-24 hours at room temperature.
• Remove the KOH with a pipette. For the following steps, the excavated block should be covered with a lid.
• Add 5 drops of glacial acetic acid and leave for 2 minutes.
• Remove acetic acid and add 5 drops of distilled water. Leave for at least 30 minutes, longer (e.g. 1-2 hours) is better and helps to avoid collapsing at later steps during the procedure (in particular if specimen was preserved in ethanol).
• Add 5 drops of 70% ethanol and leave for 2 min.
• Remove liquid and add 5 drops of 70% ethanol. Leave for 2 minutes.
• Add 5 drops of absolute ethanol and leave for 2 minutes.
• Remove liquid and add a few drops of absolute ethanol so that specimen is just covered. Leave for 2 minutes.
• Add 3 drops of clove oil and leave excavated block uncovered for 15 minutes or until the ethanol is evaporated.
• If specimen add 3 drops of a mixture of 60% clove oil and 40% Canada balsam. Leave for 5 minutes.
• Place three drops of Canada balsam on the slide at the appropriate locations for body, antennae, and head.

• Transfer specimen from clove oil (or mixture of clove oil/Canada) to bottom left drop of balsam on the slide.
• Separate the head from the body with an insect pin. This is easiest if the specimen is placed on its side.
• Carefully transfer the head (with the antennae attached!) with insect pin to the top right drop of balsam.
• Detach the antenna from head with an insect pin. This is easiest if the head is in upright position and the outermost tip of the pin is used to remove the antenna at its very base. The radicle should be attached to the antenna and not to the head.
• Carefully transfer head with an insect pin to the bottom right drop of balsam.
• Arrange the body so that the legs are spread and the body is not tilted.
• After all parts have been arranged place the slide on a hot plate or in an oven at 50-55°C for about 24 hours.
• Add a small drop of Canada balsam to each of the four positions with body parts and cover with round cover slips (6mm diameter). Care should be taken to assure that the cover slips are horizontal.
• Place immediately on a hot plate (50-55°C) for several hours.
• Store in an oven until Canada balsam is sufficiently hardened.
• The slide should be labelled with locality information of the left and species identification of the right.

Specimens mazerated in lysis buffer

DNA of specimens used for molecular studies is usually extracted using a destruction-free voucher recovery protocol that leaves the specimens intact for subsequent morphological studies. These specimens can be slide mounted straight from ethanol (100%) using the same procedure as described above, with the following differences. The specimen is placed in an excavated block with some ethanol and a few drops of clove oil are added. After evaporation of the ethanol the specimen is transferred to a drop of Canada balsam on a microscope slide. The wings are then removed with a pin and transferred to a drop of Canada balsam in the top left part of the slide. The following procedure is the same as with dry mounted specimens.