An improved method for the laboratory rearing of the corn earworm. Heliothis zea, described. The rearing medium is a modification of the commonly used wheat germ An oviposition chamber, a feeder for adults, and a simple and inexpensive contrnlled humidity chamber are described

Cohen, R. W

Friedman, S.

Waldbauer, G. P

English

ValpoScholar

The Great Lakes Entomologist 
Volume 17 
Number 2 - Summer 1984 Number 2 - Summer 
1984 
Article 10 
June 1984 
An Improved Procedure for Laboratory Rearing of the Corn 
Earworm, Heliothis Zea (Lepidoptera: Noctuidae) 
G. P. Waldbauer 
University of Illinois 
R. W. Cohen 
University of Illinois 
S. Friedman 
University of Illinois 
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Recommended Citation 
Waldbauer, G. P.; Cohen, R. W.; and Friedman, S. 1984. "An Improved Procedure for Laboratory Rearing of 
the Corn Earworm, Heliothis Zea (Lepidoptera: Noctuidae)," The Great Lakes Entomologist, vol 17 (2) 
Available at: https://scholar.valpo.edu/tgle/vol17/iss2/10 
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THE GREAT LAKES ENTOMOLOGIST 1984 113 
AN IMPROVED PROCEDURE FOR LABORATORY REARING OF 

THE 
CORN EARWORM, 
HELlOTHIS ZEA 

(LEPIDOPTERA: NOCTUIDAE) 

G. P. Waldbauer, R. W. Cohen and S. Friedmanl 
ABSTRACT 
An improved method for the laboratory rearing of the corn earworm. Heli this IS 
described. The rearing medium is a modification of the commonly used wheat germ 
An oviposition chamber, a feeder for adults, and a simple and inexpensive contrnlled 
humidity chamber are described. 
The corn earworm, H liothis zea (Boddie), is often used as a laboratory animal and is 
one f the most important insect pests in North America. The rearing procedure outlined 
below is based in part on the techniques of Berger (1963), Ignoffo (1965). and Young et 
al. (1976). The artificial diet used here is a lineal descendant of the wheat germ medium 
first developed by Adkisson et al. (1960) for rearing the pink bollworm, Pectinophora 
gossypiella (Saunders). This medium was subsequently modified for use with Heliothis 
zea by Vanderzant et al. (1962), Berger (1963), and Ignoffo (1966). We have made a few 
additional modifications, largely following Kogan and Parra's (1981) soybean looper diet. 
Up to now we have reared 34 sueeessive generations of H. zea using this procedure. 
PHYSICAL CONDITIONS DURING REARING 
The larvae are held in an environment chamber at 2SoC and a 16:8 light:dark 
photoperiod to prevent the induction of diapause. There is no need to control humidity 
externally because the larvae, confined in closed cups on a moist diet, are already in a 
sufficiently humid microenvironment. 
pupae, and adults are held in controlled humidity chambers (Fig. 1) in a rearing 
room at 2SoC and a 16:8 Jight:dark photoperiod. The relative humidity (Lh.) in the 
chamber is kept at about 70%. Maintenancc of a high r.h. is mportant becausc the insects 
otherwise desiccate and die prematurely. Adults in dry conditions, for example, often die 
without laying eggs. This problem is especially acute in northern climates where the r.h. 
may be as low as 1~20% in heated buildings in the winter. 
The humidity chamber (Fig. I) is 1.35 m tall by 1.47 m long by 0.54 m wide. It 
consists of a welded 3 cm angle-iron frame covered top and sides with transparent sheet 
plastic held on with masking tape. The underside of the chamber fits flush against the 
floor. The entire expanse of the front opens through two hinged doors. Three shelves of 
expanded metal, held by adjustable shelf brackets, permit the passage of air. Moisture is 
provided by a 38 by 49-cm plastic pan of deionized water in the bottom f the chamber 
with a small blower directed at its surface. The blower is clamped to one of two 1.3-cm 
diameter steel rods that extend from top to bottom in the middle of the ehamber. The 
percent r.h. is controlled by covering o  uncovering two rows of 18 I.S-cm wide circular 
holes on each end of the plastic top. These holes are easily made with a heated cork borer. 
During the scotophase an "artificial moon" must be provided because the moths will 
neither mate nor lay eggs in complete darkness. The "moon" is a IS W light bulb in a 
IDepartment of Entomology, University of Illinois, Urbana, IL 61801.
1
Waldbauer et al.: An Improved Procedure for Laboratory Rearing of the Corn Earworm,
Published by ValpoScholar, 1984
114 THE GREAT LAKES Er-.'TOMOLOGIST VoL 17, No.2 
/;
If 
Fig. I. Frontal view of the controlled humidity chamber with one door open. Note the pall 0! '" a.ler 
(A), the blower (B), and the holes that can be covered and uncovered to control reiati\(: hUIT'.idit... ~. ­
gooseneck lamp placed about 2.5 m from the humidity control chamber. The hemi~pheri­
cal metal shade of the lamp is placed with its open side almost flush against the wall. 50 
that only a small amount of light escapes. 
THE EGGS 
The oviposition chamber (Fig. 2) is made from a cylindrical, one-gallon. cardboard ice 
cream container (Standard Packaging Corp., Union, NJ) with the insert pushed out of the 
rim of the lid. Therim is then used to hold a cheesecloth cover in place on the container. 
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The Great Lakes Entomologist, Vol. 17, No. 2 [1984], Art. 10
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1984 THE GREAT LAKES ENTOMOLOGIST 115 
About 80% or more of the eggs are laid on the che secloth that covers the top of the 
oviposition chamber. The cheesecloth, with the adhering eggs, is removed daily and 
replaced with a fresh cloth. The cloth is easily changed, without moths escaping, by 
removing the rim from the chamber, placing a fresh cloth over the old one, and then 
gently pulling the old cloth out from beneath. The eggs are surface sterilized by placing 
the cloth in a 10% formalin (3.7% formaldehyde) solution for 3 min followed by a 6-min 
rinse in tepid running tap water. (Sterilization with formaldehyde, unlike sterilization with 
sodium hypochlorite, does not cause the eggs to detach from the cloth.) The cloths are 
dried on a blotter and then placed individually in 8-oz paper food cups with a transparent 
plastic lid (Dixie Cup, American Can Co., Easton, PA). During peak egg laying each 
oviposition chamber produces several thousand eggs per day. Developing eggs tum dark 
on the second day and hatch on the third day. 
THE LARVAE 
Larvae are reared on the artificial medium whose ingredients are listed in Table I. The 
medium is prepared as follows: The group A ingredients, water and agar, are poured into a 
large flask and placed in an autoclave where the agar dissolves within 15 min. The flask is 
then allowed to cool for 10 min. Meanwhile, the wheat germ and about one-half of the 
water (group B) are placed in a small blender and blended to a fine pabulum. The rest of 
the group B ingredients are then poured into a large blender. The wheat germ pabulum is 
rinsed into the large blender with the remaining water, and the mixture is blended. The 
agar solution is then added and the mixture is again blended slowly until it cools to below 
60° C. Finally, the group C ingredients are added, and the diet is blended for 3 or 4 min. 
The diet is immediately poured into l-oz transparent plastic condiment cups (Fill-Rite, 
Inc., Newark, NJ) using large beakers that have been preheated in a 60° C oven. The cups 
are approximately half filled. The diet cups may be capped after one-half hour. Six or 
seven pin holes are made in the center of each opaque plastic cap (Bio-Serv, Inc., 
Frenchtown, NJ) to allow for air circulation. 
Fig. 2. The oviposition chamber. Note the stoppered hole for the insertion of moths (A), the 
hatchway for insertion of the feeder (B), and the feeder, shown without cotton wool (e). 
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Waldbauer et al.: An Improved Procedure for Laboratory Rearing of the Corn Earworm,
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116 THE GREAT LAKES ENTOMOLOGIST Vol. 17. No. 2 
Table I. Ingredients for a  amount of Heliothis zea medium sufficient to rear 
about 250 larvae. 
Group A: 

Group B: 

Group C: 
Agar 
Water 
Vitamin-free easein 
Alfalfa meal 
Sucrose 
Wheat germ 
Wesson's salt mixture 
Alphacel
4M 
KOH 
Water 
Wheat germ oil 
Vanderzant modification vitamin mixture 
(Nutritional Biochemicals) 
Sorbic acid 
Methyl-p-hydroxybenzoate 
Ascorbic acid 
Streptomycin 

10% formaldehyde (27.0% formalin) 

75 g 
2200 ml 
126 \2 
54 g 
96 g 
108 g 
36 " 

18 '" g 

18 in! 

880 ml 
7 g (ca. 10 mil 
36 \2 
6.8 ~g 
6.8 g 

13 \2 

0.5~£ 

15 ~l 

A small camel's hair brush that has been cut down so that it has only six to eight bristles 
is 
used gently to place one newly hatched larva 
in each cup. After capping. the diet .:ups 
are placed upside down in wooden racks (inside dimensions: 52.5 by 35.5 by 3.0 em I with 
a hardware cloth bottom to allow for air circulation. The racks, designed to hold % cups 
in 12 rows of eight each. are held in an environmental chamber as described aoove. They 
are stacked with 2-cm wide sticks between them for spacing. On the third or fourth ill:;' the 
cups are examined and turned over. (Missing or dead larvae are replaced from eight .:ups 
per rack that were set up with an extra larva in each.) Keeping the cups upside down for 
the first few days reduces mortality at the second instar from about 15% to about S;,<: 
because fewer of the negatively geotactic, first instar larvae starve as a result of wandering 
about on the lids of the cups. Six to eight racks of diet cups are set up for each generation. 
THE PUPAE 
Pupation occurs about 17 days after hatching. Most of the larvae dig down to the 
bottom of the diet cup and form a pupation chamber that is lined with a sparse but strong 
mesh of silk. The pupae are dug out 1 or 2 days after about 90% of the lanae ha\'e 
pupated. thus avoiding injury either to teneral pupae or pharate adults. The pupae are 
surface sterilized by immersing them in 0.2% sodium hypochlorite (one part household 
bleach in 24 parts of water) for 3 min, then in 10% sodium thiosulfate (20 g2 0 mI \\aten 
for 3 min, and finally in tepid running tap water for 5 min. 
The pupae can be sexed by using the characteristics of the pupal,abdomen shown in Fig. 
3. The sex ratio of 1155 pupae was essentially 1: I, with 50.8% males and 49.2'<: females. 
The pupae. segregated by sex, are placed in groups of 50 each in cubical screen edosion 
cages that are 30.5 cm on a side and have a cloth sleeve. If more than 50 pupae are placed 
in 
a cage, the newly eelosed adults injure each other and the pharate adults that are aoout 
to eelose. 
THE ADULTS 
The adults eelose 
about 13 days after pupation and are moved to oviposition chambers 
at that time. (Survival from the first instar to the adult is generally mer 80'i:.' Adults are 
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The Great Lakes Entomologist, Vol. 17, No. 2 [1984], Art. 10
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1984 THE GREAT LAKES ENTOMOLOGIST II7 
Fig. 3. Ventral views of the ends of the abdomens of male and female pupae of Retiothis zea. 
moved from the emergence cages to the oviposition chambers by catching them in a 
condiment cup with a screen bottom; moths that will not fly can be forced into the 
oviposition chamber by blowing through the screen bottom of the condiment cup. Ten to 
12 
moths 
of each sex are placed in each oviposition chamber though a 3.5-cm diameter 
hole in the side that can be closed with a rubber stopper (Fig. 2). A feeder, composed of a 
5-cm petri dish held in the center of a 9-cm petri dish with a bit of Plasticene®, is placed 
on the floor of the viposition chamber (Fig. 2). The inner dish holds a wad of cotton 
soaked with a 10% solution (vol.lvol.) of honey in water. The feeder is moved in and out 
of 
the chamber through a 
3 by II-cm opening cut near the bottom of the chamber. The 
cover of the opening can be pulled open or held shut with a length of tape. The feeders 
must be washed and the honey solution changed daily because the solution begins to 
ferment on the second day. Fermenting honey solution causes the moths to bloat and die 
prematurely, greatly decreasing the yield of eggs. The design of the feeder prevents 
spillage and subsequent fermentation of honey water in the oviposition cage. Six t  10 
chambers are set up for each generation. The females begin laying eggs in large numbers 3 
or 4 days later. 
MINIMIZING INBREEDING 
Inbreeding is minimized by promoting heterogeneous mating within the culture and by 
introducing new insects from the field at the end of each summer. Heterogeneous mating 
is 
promoted by identifying the progeny of the adults in eaeh oviposition cage, and by 
mating females from one group 
of progeny with males from another. The lineages are 
followed in succeeding generations, and females are never mated with their first cousins. 
The culture is outcrossed each summer by mating laboratory-reared females with males 
reared from larvae collected in the field. Using only males from the wild population 
prevents accidental introduction of the naturally occurring microsporidian Nosema 
heliothidis Lutz and Splendore into the culture. This microsporidian is transovarially 
transmitted by females, but is not venereally transmitted by males (J. V. Maddox, pers. 
comm.). 
STARTING AND MAINTAINING A DISEASE-FREE CULTURE 
A culture free of infection by N. heliothidis can be started by using a variation of the 
Pasteur technique. Field-collected larvae are reared to the adult stage, and groups of these 
5
Waldbauer et al.: An Improved Procedure for Laboratory Rearing of the Corn Earworm,
Published by ValpoScholar, 1984
118 THE GREAT LAKES ENTOMOLOGIST VoL 17. ~o. :! 
adults are then placed in oviposition chambers as described above. After sufficient 
have been produced, the females are killed and examined for N. heliothidis spores. 
spores are found in one or more females of a group, the larval progeny of that group are 
checked for the presence of N. heliothidis by examining fecal smears for spores. Spores 
can be seen under a phase contrast microscope. Infected larvae are discarded. 
It is particularly important to take precautions against the introduction of X. heliothidis 
into the culture. It is the pathogen most likely to be encountered when starting a colony 
from field-collected insects. Furthennore, it causes a chronic disease that ma\ 11:0 
unnoticed until it eventually causes an unacceptable level of mortality. In the meantime: il 
slows the developmental rate and may obscure the effects of an experimental \'ariable 
(J. V. Maddox, pers. comm.). 
The culture is maintained essentially disease free by strict adherence to routine S31litary 
procedures. Spatulas are flamed if they are used to dig in diet cups that contain a larva thaI 
may be diseased. Cages and oviposition chambers are autoclaved between Oi'11r>r:>tli"\m, 
Diet cups are used only once, and all discarded cups are sealed in plastic 
line wastebaskets. Perhaps the most important measure is the routine surface 
of 
eggs and pupae described above. 
ACKNOWLEDGMENTS 
We thank Joseph V. Maddox and May Berenbaum for reading the manuscript 
and Ali.:e 
Prickett for the drawings. This material is based upon work supported by the CSDA 
Competitive Research Grants Program, Office of Grants and Program Systems. S-.::icn..-e 
and Education, under Grant No. 83-CRCR-J-1207. 
LITERATURE CITED 
Adkisson, P. L., E. S. Vanderzant, D. L. Bull, and W. E. Allison. 1960. A wheal £enTI 
medium for rearing the pink bollwonn. J. Econ. Entomo\. 53:759-76::. -
Berger, R. S. 1963. Laboratory teehniques for rearing HeliotMs species on .J.l1:ifkial 
medium. USDA Agric. Res. Servo Ars-33-84, 4 p. 
Ignoffo, C. M. 1965. The nuclear-polyhedrosis virus of Heliothis -::ea I Bod.:iic, and 
Heliothis virescens (Fabricius) II. Biology and propagation of diet-reared HeiiNhis. J. 
Invertbr. Pathol. 7:217-226. 
1966. Insect viruses. p. 501-530 in C. N. Smith (ed.). Insect colonization a d 
mass production. Academic Press, New York. 
Kogan, M. and J. R. P. Parra. 1981. Techniques and applications of mea5uremcnl.S of 
consumption and utilization of food by phytophagous insects. p. 3T-352 in G. 
Bhaskaran, S. Friedman, and J. G. Rodriguez (eds.). Current topics ir: ins..'CI 
endocrinology and nutrition. Plenum Press, New York. 
Vanderzant, E. S., C. D. Richardson, and S. W. Fort, Jr. 1962. Rearing of the roll~onn 
on artificial diet. J. Econ. EntomoL 55:140. 
Young, J. R., J. J. Hamm, R. L. Jones. W. D. Perkins, and R. L. Bunon. 19-::6. 
Development and maintenance of an improved laboratory colony of corn e~orms. 
USDA Agric. Res. Servo S-llO, 4 p. 
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An Improved Procedure for Laboratory Rearing of the Corn Earworm, \u3ci\u3eHeliothis Zea\u3c/i\u3e (Lepidoptera: Noctuidae)

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An Improved Procedure for Laboratory Rearing of the Corn Earworm, \u3ci\u3eHeliothis Zea\u3c/i\u3e (Lepidoptera: Noctuidae)

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