Julieta Brambila

USDA-APHIS-PPQ
September 2010

Instructions for the preparation and dissection of moths from bucket

traps from the Autographa gramma (Lepidoptera: Noctuidae) surveys

Introduction

The purpose of this handout is to guide you in the preparation and dissection of
specimens obtained during surveys for Autographa gamma. These instructions will
help you distinguish Autographa gramma from native species, some of which are
strongly attracted to the lures, including Rachiplusia ou and Autographa californica.
You can adapt the preparation techniques to the materials you have available in your
work area.

This handout presents in detail how to prepare specimens for dissection. It

includes a summary of the preparation technique, which can be posted by your
preparation area, and instructions on how to make a 10% potassium hydroxide
solution.
 Part 1: Preparing Specimens for Dissection

1. Heat. Turn an electrical heating block on. Use of a fume hood is

recommended.
Note 1. Other heating units may be used such as a cup warmer, electric cooking
plate, electric pan, or gas range. Heating blocks are recommended because they
provide even heat.
Note 2. If you plan to use a cold-clearing technique you can skip steps 1, 2, 8 and
10. The cold technique takes many hours, especially for large moths. You can leave
the specimens in cold 10% KOH overnight.

2. Water bath. Place on the heating minutes for the specimens to be ready
block a metal cooking pan or a for dissection.
Pyrex glass dish with about ½ inch
of tap water. A lid is optional.
The water should not reach boiling
point. Use the same temperature
each time specimens are
processed. Use a thermometer to
establish over time the desired
temperature. A recommended
temperature is 70°C, but any place
between 40 and 70°C is
appropriate. At 70°C it takes 45 Start warming the water.

3. Specimens. Open the box or bag of specimens and drop them on a white
plastic tray, sheet of paper, or clean table top. If the field samples look dry
and dusty, work inside a fume hood, or at least wear a dust mask, to avoid
breathing in moth scales. Count the specimens and write the number on the
specimen report. Process a single sample at a time (a sample is a set of
specimens collected at the same site and date).

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 Clean work area It is important to work at a fume hood.
Dust mask use is recommended when
processing dusty samples if a fume hood is
not available.
4. Removing abdomens. Remove the abdomen from each moth with blunt
forceps. If the specimens are dry and brittle, break each abdomen off at the
base where it meets the thorax by pushing the abdomen to a side with the
forceps while holding the thorax down with a finger or forceps. If the
specimens are soft, separate the abdomen by first pressing down at the base
with broad forceps, then pulling it off carefully from near the base, not from
the apex. You can find where the abdomen begins by examining the area
near the base of the hind wings, where you may notice two large tympana,
which look like windows, one on each side of the abdomen. The abdomen
begins after the tympana (=ear membranes).

tympanum

Autographa gamma
Helicoverpa zea

Hold the specimen with forceps or with Break the abdomen off where
your fingertips. the line above indicates

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5. Labeling. Place all abdomens of glass vials can be used, as
from a single trap in a glass vial well as small open glass or
and add a small paper label ceramic containers.
written with pencil or
waterproof ink. Make matching
notes on the specimen reports.
For samples larger than 30
specimens, divide them and label
label them, for example, 1a, 1b,
1c and 2a, 2b, 2c, 2d, each with
between 20 and 30 abdomens.
Twenty-ml screw-top Keep track of samples with small labels.
scintillation glass vials with a
wide mouth and screw top lids
are recommended. Other types

6. Moistening: Add alcohol to each vial to moisten the abdomens so they

readily sink into the KOH in the next step. Keep them in alcohol less than
5 minutes. Isopropyl (=rubbing alcohol) is fine for our purposes but
ethanol is better for research purposes that include the preparation of
permanent slides. If this step is skipped the abdomens will tend to float in
the KOH*.

7. KOH*. Wear lab gloves and eye protection. Remove the alcohol and add
10% KOH solution (see instructions on how to make the KOH* solution).
Fill each vial about halfway, that is, about 10 ml; you should use more for
large samples but do not fill the vials to the top. Close the vials with the
screw top lids so that you reduce chances of a spill, but do not close the
vials tightly so that hot air is let out.

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 Prepare your KOH* solution in advance.
Fill half of the scintillation vial with 10%
KOH*. Close the vial loosely with a lid.
Wear lab gloves and eye protection.

* Attention: Hot KOH can irritate skin

and permanently damage the eyes

8. Heating KOH*. Place all vials with labels and lids inside the metal pan
with the warm water and cover (although the cover is optional). Make sure
the heat is set below the boiling point of the water (that is, less than 100
degrees Celsius); you can try 70°C (it takes about 45 minutes to process
specimens at this temperature).

45 minutes
70°C

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9. Clearing. Keep specimens in the warm KOH* solution for about 45
minutes. The potassium hydroxide macerates the tissues, and proteins and
fats are removed, leaving chitinous structures such as the skin of the
abdomen and the valves. If you do not want to use warm KOH*, you can
leave the abdomens (or even whole specimens) in cold KOH* overnight or
for 5 to 8 hours. If you leave specimens for several days in the KOH*
solution, they will be damaged or destroyed.

10. Cooling: Wear eye protection

because of the caustic hot
KOH*. Remove vials from the
warm water wearing leather or
cotton gloves, paper towels, or a
cloth. Let the vials cool down
slowly, on the counter, at least
between 10 and 30 minutes.
After cooling, specimens should
not be left in the KOH*.

Remove vials from the warm water. Gloves

are optional but recommended.

* Attention: Hot KOH can irritate skin

and permanently damage the eyes

11. Rinsing. Remove the cooled KOH* solution with a plastic pipette or a
glass dropper and discard the used KOH* (it may be reused if the liquid is
clear). Wear eye shields (goggles or glasses) and lab gloves. Rinse the
specimens with alcohol (or water) two or three times with a plastic or glass
pipette. Leave the specimens in alcohol. After this step the specimens are
ready for dissection.

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 Notice safety
glasses and gloves

Remove KOH* and rinse samples.

* Attention: Hot KOH can irritate skin

and permanently damage the eyes

Acknowledgements: I thank Kayla Brownell (USDA) for allowing me to photograph her.

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 Steps for Preparing Moth Specimens

1. Heat. Turn on the heating block.

2. Water bath. Place a metal cooking pan with ~ ½ inch of tap water on the
heating block.

3. Sorting specimens. Sort and count specimens.

4. Removing abdomens. Remove the abdomen from each specimen with

blunt forceps.

5. Labeling. Place all abdomens from a single trap in a vial and add a small
paper label.

6. Moisten: Add alcohol to each vial to moisten the abdomens. Keep them in
alcohol less than 5 minutes.

7. Adding KOH*. Add 10% KOH* solution. Close the vials loosely.

8. Heating KOH*. Place all vials in the pan with warm water.

9. Clearing. Keep specimens in the warm KOH* solution for 45 minutes.

10. Cooling: Remove vials from the warm water. Let vials cool down slowly,
between 10 and 30 minutes.

11. Rinsing. Remove KOH* with a plastic pipette. Rinse with alcohol or water
2 or 3 times. Add alcohol in the last step. The specimens are ready for
dissection.

* Attention: Hot KOH can irritate skin

and permanently damage the eyes.
Keep the MSDS at hand. Keep it
attached to this handout.

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 Preparing the 10% KOH solution
KOH* – potassium hydroxide

Option 1: Purchase 10% KOH* solution.

Option 2: Prepare your own fresh 10% solution.

How to prepare the 10% KOH* solution

-wear lab gloves and eye protection.
-add 100 ml (milliliters) of water to a plastic Nalgene or glass bottle.
Plain tap water can be used for screening purposes, but distilled or deionized
water is recommended for permanent preparations (slides).
-place a small plastic or glass dish on a balance.
-turn the balance on and select ‘grams’ for weight units.
-weigh 10 gr (grams) of pure KOH* pellets (don’t touch the pellets!).
-add the pellets to the plastic Nalgene or glass bottle with water, close
with a lid, and gently swirl. Let this mix rest at least for 15 minutes before using
the solution.
-other quantities you can prepare are 20 gr KOH* pellets to 200 ml of
water, or 15 gr KOH* pellets to 150 mls of water. Each ml of water weighs one
gram, so you can also weigh the water in the balance. For example, you can
measure 25 gr of pellets and add them to 250 gr of water.
-Label the plastic or glass bottle with “10% KOH*” to avoid accidents.

*Caution: KOH is very caustic. Do not grab or hold the solid pellets
with your bare fingers. Always transfer the pellets by shaking them into a dish
or vial, if necessary using a metal or plastic spatula, or use forceps. Use gloves,
especially when handling warm KOH solution. If some of the 10% KOH
solution touches your skin, rinse your skin thoroughly with water. A splash of
warm KOH solution can do permanent damage to your eyes. So, for eye
protection, wear safety glasses especially when handling warm or hot KOH
solutions. Read the MSDS (material safety data sheet) information for KOH
that arrives when you order this chemical. Know where the closest emergency
eye rinsing station is located and have emergency eye rinse bottles at hand.