United States

Department of

Agriculture

Combined Forest Pest

Research and

Development Program

Agriculture Handbook

No. 520

Douglas-Fir

Tussock Moth

Handbook

Rearing the

Douglas-fir

Tussock Moth

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Rearing the Douglas-Fir

Tussock Moth

by Clarence G. Thompson and

Linda J. Peterson^

Introduction

In 1974 the U.S. Department of

Agriculture initiated the Combined

Forest Pest Research and Development Program, an interagency effort

that concentrated on the Douglas-fir

tussock moth in the West, on the

southern pine beetle in the South, and

on the gypsy moth in the Northeast.

The work reported in this publication

was funded in whole or in part by the

program. This manual is one in a

series on the Douglas-fir tussock

moth.

This leaflet provides laboratory rearing

procedures for the Douglas-fir tussock

moth, Orgyiapseudotsugata(McDunnough). These procedures have been

developed over the past 14 years of

continuous rearing of the tussock

moth at the Forest Service's Forestry

Sciences Laboratory in Corvallis,

Oregon. The artificial diet was

developed over a period of several

years with advice and suggestions from

many sources. Similar diets have been

described elsewhere (Lyon and Flake

1966); however, the recipe given here

has been used in the Corvallis

laboratory and by our cooperators

with success for a number of years.

The techniques can be used for continuous rearings or for one-generation

rearings of field-collected eggs or

larvae in order to assess levels of

disease, parasitism, and other aspects

of population quality.

Research entomologist and formerly laboratory

technician, respectively. Forest Service, Pacific

Northwest Forest and Range Experiment Station,

Forestry Sciences Laboratory, Corvalllis, Oreg.
The Artificial Diet

Following are instructions for preparing Douglas-fir tussock moth diet

65-W-PEN (the 65th modification in

development ofthe diet). We find it

convenient to package ingredients for

several batches of diet at one time (fig.

1). The recipe makes one batch—four

liters (5 pans)—of diet.

Figure 1.—Components of the

tussock moth artificial diet prepackaged for convenient use.

Equipment

5 flat-bottom enamel or stainless steel

pans, (25 x 40 cm)

1 stainless steel beaker

(4-liter capacity)

1 graduated cylinder (100 ml)

1 graduated cylinder (1000 ml)

1 glass stirring rod

Disposable 10-ml pipettes

1 high-speed commercial blender

(4-liter capacity)
Ingredients (Diet No. 65-W-PEN):

Package A Agar, granulated 64.0 g

Package B

Liquids:

Casein, technical

Sugar, granulated

Wesson's salts **w*'

Alphacel

210.0 g

210.0 g

60.0 g

30.0 g

Package C Wheat germ 180.0 g

Package D Ascorbic acid

Chlorotetracycline

24.0 g

280.0 mg

Package E Penicillin G, K salt 800,000 units

SorbicAcid(10%in95o/o 32.0 ml

ETOH. Mix 10 gm/100 ml

or multiples of this ratio)

Methyl parahydroxybenzoate 40.0 ml

(15%in95<í7oETOH. Mix

15 gm in 100 ml or multiples

of this ratio)

Vitamin solution^ 10.0 ml

Choline chloride (lO^i^o in H2O) 60.0 ml

Potassium hydroxide (4M) 30.0 ml

Wheat germ oil, cold pressed 20.0 ml

Distilled water 3000.0 ml

' Prepare vitamin stock solution in 100-ml

quantities. The solution should be refrigerated.

Ingredients include:

Niacin 600 mg

Calcium pantothenate 600 mg

Riboflavin 300 mg

Thiamin hydrochloride 150 mg

Pyridoxine hydrochloride 150 mg

Folie acid 150 mg

Biotin 12 mg

Vitamin B,2 1.2 mg

Sterile distilled water 100 ml
Rearing Methods

Procedure

1. Place 1400 ml distilled water in a

4-liter stainless steel beaker. Stir in

the agar(package A), and cover

with aluminum foil. Autoclave

along with the 4-liter blender and 5

pans for 20 minutes at 15 pounds

pressure and slow exhaust.

2. Place 1600 ml sterile distilled water

in the blender. Add the preweighed

packaged ingredients (packages B,

C, D, E). Add the liquid ingredients

in their proper amounts. Blend on

low for 4 minutes. The temperature

of this mixture after blending

should be about 38° C.

3. AUow the autoclaved hot agar to

cool to 55° C with intermittent

stirring. It takes 30-40 minutes for

the agar solution to cool to 55° C.

4. Add the 55° C agar solution to the

mixture in the blender and blend

for 4 minutes on low. The temperature of this mixture should be

about 52° C.

5. Immediately pour equal amounts of

diet into each pan to a depth of

about 1 cm.

6. Allow the pans to remain

uncovered for about 10 minutes;

then cover loosely with aluminum

foil. After 10-20 minutes, cover

tightly, label, and refrigerate (4° C).

Eggstage

In nature, a diapause takes place in the

overwintering eggs. Atleast three and

preferably four months exposure to

cool temperatures is necessary to break

the diapause. Usually eggs collected in

the field in January or later have completed their cold exposure requirement

and will hatch after about 14 days incubation at a constant temperature of

21-23° C. Eggs collected in the field

in the fall or produced in laboratory

rearings should be stored for 3-4

months at 2-5° C before attempting to

hatch them.

Following cold treatment, the eggs are

surface sterilized to remove microbial

contaminants such as nucleopolyhedrosis virus (NPV), unless the purpose of the rearing is to measure field

incidence of NPV. The technique for

surface sterilization is as follows:

1. Remove the eggs from the cocoon

(figs. 2 and 3).

2. Gently break up the egg mass

without damaging the eggs (fig. 3).

Figure 2.—Field-collected egg masses

of the tussock moth.

Figure 3.—The egg masses are

stripped from the maternal cocoon

and gently broken up.


Place the eggs in a flask or beaker

containing 0.1 percent sodium

hypochlorite (this is roughly equivalent to a 2 percent solution in water

of common household bleaches

such as Clorox" or Purex".)

Briskly agitate the sodium

hypochlorite solution containing

the egg-mass fragments, preferably

with a laboratory shaker or stirrer.

If it is not necessary to sterilize each

egg mass separately, up to 10 egg

masses can be sterilized in one

600-ml beaker or flask with a lab

stirrer (fig. 4). If it is necessary to

keep each egg mass separate, a laboratory shaker capable of handling

a number of flasks is preferred (fig.

5).

Figure 4.—Eggs from up to 10 masses

can be surface sterilized in 0.1 percent

sodium hypochlorite using a simple

lab stirrer.

Figure 5.—Where egg masses must be

treated individually or where very

large numbers of eggs must be surfaced sterilized, a laboratory shaker

with multiple receptacles is used.
After 15 minutes agitation, replace

the spent sodium hypochlorite solution with a fresh solution and

agitate for 15 minutes more.

Remove the tussock moth eggs by

pouring the sodium hypochlorite

solution through cheesecloth (fig. 6)

where the eggs are retained.

Thoroughly rinse the eggs with

water, and place them in a flask or

beaker with fresh water. Agitate for

5 minutes to remove the last traces

of the disinfectant.

Again remove the eggs from the

rinse water by pouring through

cheesecloth and gently blot to

remove excess water (fig. 7).

Hold the eggs (fig. 8) at 25° C,

preferably at 45 percent RH and

with alternating 12-hour periods of

light and dark. Hatch usually occurs in 12-14 days.

Figure 6.—Sterilized eggs are

removed from the sodium hypochlorite solution by straining with

cheesecloth.

Figure?.—After washing the eggs,

excess moisture is removed by blotting

with paper towels.

Figure 8.—Following surface sterilization, the eggs are set up in desired

numbers for hatching.

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11
Larval stage

1. Gently transfer newly hatched

larvae with sterile toothpicks or

camel's hair brushes to rearing containers(disposable plasticl 5 X 100

mm petri cüshes are recommended)

containing a block of artificial diet

(fig. 9). For stock cultures, put 10

larvae in each dish. For virus incidence determination (from unsterilized eggs) put 25 larvae in each

dish and maintain these dishes for

14 days only (fig. 10).

2. Feed the insects every 7 days.

Remove the unused old diet at each

feeding. Where virus incidence rearings are made, use a sterile toothpick to add new diet and to remove

the old from each dish (figs. 11 and

12).

Figure 9.—Following egg hatch, the

larvae are transferred in groups of 1

to 25 larvae, depending on intended

uses, to petri dishes containing blocks

of artificial diet. The diet is changed

at weekly intervals.

Figure 10.—A convenient system of

rearing tussock moth larvae. Each

tray is 60 cm x 60 cm and will hold

up to 36 standard petri dishes.

Figure 11.—The artificial diet is cut in

squares( 1 or 2 cm) with a spatula

(knives may scratch the enamel pan)

and removed with sterile toothpicks.

When feeding, it is best to use a fresh

toothpick for each petri dish to avoid

transferring virus or any other contaminating agent.

Figure 12.—Égg and various larval

stages in laboratory rearing.

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Pupal stage

1. Collect pupae, within their

cocoons, as soon as they form.

2. Sex the pupae by placing ¡ the

cocoons on a photographic lighttable where the much larger female

pupae can be identified inside their

cocoons.

3. Glue cocoons containing female

pupae 20 to a card (approximately

15 cm X 25 cm) (fig. 13) and place

in brown paper bags. Place 25 male

pupae, 3 days older than the

females, loosely in the bag and

staple the bag closed.

Figure 13.—Female pupae within

their cocoons glued to cards in

preparation for emergence, mating,

and oviposition. Twenty-five male

pupae are added to the sack before it

is closed.

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Adult stage

The adults emerge, mate, and oviposit

in the paper bags.

1. Thirty days later, remove the cards

with cocoons—now bearing egg

masses—from the bags (fig. 14) and

remove the egg masses from the

cards.

2. Store the eggs at 2°-5° C for 4

months to complete the cycle.

Figure 14.—Egg masses are removed

from the oviposition cages and stored

at 2°- 5° C for a minimum of4

months to break diapause.

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General Laboratory Procedure

The tussock moth is subject to several

extremely virulent diseases. Very minute amounts of NPV can infect and

kill tussock moth larvae, and once

established, the virus can spread rapidly and wipe out entire cultures unless a

strict regime ofsanitation is maintained. Bacterial, fungal, and protozoan diseases can be nearly as serious.

All work surfaces should be kept clean

and sponged daily with a good disinfectant (a 2 percent solution of

common household bleaches containing 5-6 percent sodium hypochlorite

will do very well). Wherever practical,

disposable rearing containers should

be used and discarded after one use.

Healthy stock cultures should be isolated from any work involving, or

potentially involving, diseased tussock

moth larvae. If at all possible, the individual preparing the diet and maintaining healthy stock cultures should

be prohibited from any contact with

areas where disease experiments are

being conducted. Post this sanitation

information in the work area and

make sure it is rigidly followed. One

slip can wipe out months of work!

Ahnost everyone who has worked extensively with tussock moths has experienced some irritation from the

barbed hairs of the larvae. Some individuals are so allergic that they may

require medical treatment after a brief

exposure. Certain precautions can be

used to reduce the irritation. The dry

cast skins and the cocoons are the

primary sources of irritation. If the

cocoons and egg masses are handled in

an exhaust hood, the problem will be

greatly alleviated. Rubber gloves offer

additional protection. It is wise to take

all possible precautions because it appears that some individuals who are

not initially allergic become sensitized

with continued exposure.

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References

Lyon, R. L., and H. W. Flake, Jr.

1966. Rearing Douglas-fir tussock

moth larvae on synthetic media. J.

Econ. Entomol. 59:696-698.

Photos by Alex Yusha

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