SPB & Invasive Insects - Antennal Preperation
We have found that the preparation method detailed below works well for antennae of most insects we have examined, and it provides a high degree of reproducibility and a longer antennal life than other methods we have attempted so far. It has been used successfully with Coleoptera (6 species), Hymenoptera (7 species) and Lepidoptera (1 species). It is our standard method for making “first attempts” with a new insect species because of its reliability and simplicity. Its major advantage is that the tip of the antenna does not need to be cut or punctured, reducing the number of steps required while simultaneously improving the usable life of the antenna. When surfactant is included in the saline (0.02% v/v Triton X-100 in Beadle-Ephrussi ringers solution) the signal/noise ratios obtained with this method are equal to those obtained using cut antennae. The basic procedure is outlined in the Syntech ® manual (“Electroantennography: A practical introduction” 1998 version, p. 7) but we have elaborated on it.
The steps are illustrated here with a preparation of the clerid bark beetle predator, Thanasimus dubius:
1) The insect's head is excised with a #11 scalpel blade on a pad of filter papers moistened with distilled water. The tip of one saline-filled electrode is broken at a point where its diameter is slightly less than the foramen (the opening between head and thorax) and then inserted into the foramen until the head is firmly secured.
2) The pipette/head is attached to the indifferent electrode holder while a second saline-filled electrode is secured to the recording electrode holder positioned opposite.
3) The tip of the recording electrode is broken off at a point where its internal diameter is slightly less than the maximum diameter of the antenna's (or the antennal club's) distal-most segment. The pipette break should be as smooth and symmetrical as possible. Clipping or crushing the pipette tip with a forceps invariably leaves a jagged edge or produces an opening of inappropriate size. For this reason we first gently scratch (“score”) the glass at the point where we want to break it using a piece of ceramic wafer secured in a wooden dowel (left; also see “NIFTY TOOLS”). Once scratch marks are visible on the glass, we very gently grasp the pipette tip with a sharp forceps just beyond the scratches and twist slowly in the direction opposite the scratches (right). In most cases, the pipette will break cleanly leaving a smooth, circular opening (below).
4) The cut pipette tip is maneuvered with a micromanipulator until the saline just makes contact with the tip of the antenna (left and below). If the antenna is oriented perpendicularly or away from the tip, a small wire probe (see NIFTY TOOLS) can be used to guide the tip of the antenna into the saline meniscus. Avoid any contact between the saline and the sides of the antenna. Once contact with the saline is achieved, the surface tension of the saline will maintain the antennal tip in contact with the electrode while the recording electrode is repositioned. (For most antennae, it is essential that the saline contain some wetting agent—such as Triton X-100 as described above—for the uncut antennal tip to adhere to the saline.)
5) The recording electrode is moved away from the head to straighten the antenna and then moved in a roughly circular motion to gradually work the tip of the antenna into the pipette tip (right). When finished, the antennal tip should effectively “plug” the opening of the pipette with its circumference, leaving a minimal amount of saline from the pipette in contact with the air (below).
Below are some additional examples of this technique applied to antennae of other insects.
(Above) A hymenopterous parasitoid of bark beetles, Coeloides pissodis (Braconidae)
(Right) The southern pine beetle, Dendroctonus frontalis (Scolytidae). The antennal club is disk shaped, as is the case with many bark beetles. The opening of the pipette is made slightly smaller than the circumference of the club, and club is laid flat against the opening so that the most of the surface on one entire side is in contact with the saline. (This procedure was suggested to us by Dr. Qing-He Zhang of USDA-ARS Beltsville). In the specific case of D. frontalis , the preparation works better if no surfactant is added to the saline.
Mention of trade names is for information purposes only and does not imply an endorsement by the USDA. Questions, comments, and corrections are welcome and should be sent to Brian Sullivan at briansullivan@fs.fed.us .