PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 15, 158-165 (1981)

Aldrin Epoxidase Activity and Cytochrome P-450 Content of

Microsomes Prepared from Alfalfa and Cabbage Looper
Larvae Fed Various Plant Diets’

D. E. FARNSWORTH, R. E. BERRY, S. J. Yu, AND L. C. TERRIERE

Depurtment of Entomology* Oregon Stute University, Corvallis, Oregon 97331

Received November 16, 1980; accepted February II, 1981

The alfalfa looper (Culifornico uutogruphicu) and the cabbage looper (Trichoplusiu ni) were reared
through the fourth instar on a semidefined artificial diet and for the first 60 hr of the last instar on
fresh leaves of one of their host plants. Microsomes preparedfrom midguts and a carcass segment
which included the fat body were then assayed for aldrin epoxidase activity and cytochrome P-450
content. Epoxidation by midgut microsomes of alfalfa loopers fed peppermint leaves was at a rate
up to eight times that of larvae fed alfalfa, snap beans, or broccoli. The epoxidase activity of the
carcass microsomes was induced about fourfold by the peppermint diet. Midgut microsomes pre-
pared from cabbage looper larvae reared on peppermint leaves were also more active (about four
times) than those reared on alfalfa, broccoli, or cabbage. Although the epoxidase activity of the
carcass microsomes of the cabbage loopers was low, about one-eighth that of the midgut micro-
somes, this enzyme was greatly induced by peppermint leaves. In neither species did the peppermint
leaf diet cause a corresponding increase in the cytochrome P-450 content of the microsomes, the
maximum difference between peppermint and the other plants being about twofold. Bioassays of
cabbage looper larvae reared on broccoli or peppermint and of alfalfa loopers reared on alfalfa or
peppermint indicated that the stimulation of microsomal oxidase activity by the peppermint con-
stituents provided increased tolerance for carbaryl and methomyl but not acephate.

INTRODUCTION gated cutworm (Peridroma sauci) which

The microsomal monooxygenase system
performs an important protective function
in insects, as in higher animals, metaboliz-
ing a wide variety of xenobiotics which the
insect may encounter. Among these chemi-
cals are insecticides and insect growth reg-
ulators used in insect control, and allelo-
chemicals present in the host plant (l-5).
There is considerable variation in the activ-
ity of these enzymes in the larval forms of
lepidopterous species (6-8) possibly due to
the evolutionary history of the species (9).
In addition, there is growing evidence that
this metabolic system can be stimulated to
even greater activity by the allelochemicals
present in the host plant (1, 3-5).
We have reported that larvae of varie-
’ Oregon Agricultural Experiment
paper No. 5693.
0048-3575/81/020158-08$02.00/O
Copyright @ 1981 by Academic Press, Inc.
All rights of reproduction in any form reserved.
have been fed for 60 hr on fresh leaves of
peppermint are more tolerant of insecti-
cides such as carbaryl and methomyl than
those given fresh leaves of snap beans (3,
5). This difference in susceptibility was at-
tributed to increased microsomal oxidase
activity as indicated by a nearly IO-fold in-
crease in aldrin epoxidation by the midgut
microsomes of mint-fed larvae. Among the
plant species examined so far, snap beans,
alfalfa, garden beets, curled dock, and
peppermint, the latter species was by far
the most active in stimulating the micro-
somal enzymes. This is apparently due to
the high concentrations of certain monoter-
penes in peppermint (3). In a similar study
of the southern armyworm (Prodenia
eridaniu), Brattsten (4) found several plants
Station technical to be stimulatory of aldrin epoxidase and
p-chloro-N-methylaniline N-demethylase
158
 ENZYME INDUCTION IN ALFALFA
activity of midgut homogenates. Additional
experiments reported here indicate that this
phenomenon occurs in two other lepidop-
tera.
METHODS
Insects. Cabbage looper and alfalfa
looper larvae were reared from egg to the
completion of the fourth instar on Bio Serv
cabbage looper diet (Frenchtown, N.J.).
The larvae were maintained in environ-
mental control chambers at 25°C with a 168
hr L:D photoperiod. Beginning with the
fifth instar (less than 3 hr after ecdysis) the
insects were continued on the artificial diet
or transferred to fresh leaves of alfalfa,
snap beans, broccoli, or peppermint (alfalfa
looper) and alfalfa, broccoli, cabbage, or
peppermint (cabbage looper). After 60 hr,
larvae were removed from their respective
diets for enzyme assay. Some assays were
also performed on third-, fourth-, and
fifth-instar alfalfa looper larvae coilected
from peppermint fields in August and Sep-
tember 1979.
Enzyme assays. Aldrin epoxidase assays
and cytochrome P-450 measurements were
performed on microsomes prepared from
washed midguts as described elsewhere (3,
10) and on the larval carcass remaining after
removal of the alimentary tract. This frac-
tion included the body segment between
forelegs and prolegs and contained the fat
body. These were washed with isotonic salt
solution prior to homogenization. The mid-
guts or carcasses were homogenized in
batches of 24-40 when both cytochrome
P-450 and aldrin epoxidase assays were to
be performed and in batches of 5- 10 when
only epoxidase assays were scheduled.
Aldrin epoxidase assays were performed
as described elsewhere (10) with the incu-
bation mixture containing 0.1-0.2 mg mi-
crosomal protein (midgut) or 0.2-0.5 mg
(carcass). Cytochrome P-450 was measured
as its carbon monoxide binding complex
( 11) at microsomal protein concentrations
of 0.5-1.0 mg/ml. Spectra were recorded
with an Aminco DW-2a spectrophotometer.
These protein concentrations were doubled
AND CABBAGE LOOPERS 159
when the n-octylamine binding complex of
cytochrome P-450 was measured. Type II
binding spectra were measured by the di-
rect addition of saturating concentrations of
n-octylamine to the sample cuvette. Protein
determinations were by the method of
Bradford (12).
Bioassays. Larval susceptibility to in-
secticides were determined with fifth-instar
larvae reared through the fourth instar on
the artificial diet then divided into two
groups, one reared for 72 hr on fresh alfalfa
leaves (alfalfa looper) or broccoli leaves
(cabbage looper) and the other on fresh
peppermint leaves. Ten larvae from each
group were then fed bean leaf disks (3 cm
diameter) which had been dipped in sus-
pensions of acephate (prepared with 75%
soluble powder), methomyl (90% suspend-
able powder), or carbaryl(80% suspendable
powder). Larval mortalities were evaluated
after 24 and 48 hr. An equal number of lar-
vae were fed untreated bean leaf disks as
controls.
RESULTS
Source of Microsomes
The richest source of microsomal oxidase
activity in lepidopterous larvae appears to
be either the fat body or the midgut, but this
varies with the species (7, 8, 13). Kuhr
(14- 16) reports that fat body microsomes
are considerably more active than gut
microsomes in the cabbage looper. Lack of
time during the experiment prevented our
use of the fat body alone. Instead, we used
the midgut and a carcass segment contain-
ing the fat body for the preparation of mi-
crosomes.
Host Plant Stimulation of Enzyme
Activity
In preliminary experiments with midgut
microsomes from the alfalfa looper it was
determined that the aldrin epoxidase sys-
tem required NADPH and was inhibited
(92%) by carbon monoxide. No experi-
ments of this type were performed with
microsomes from the cabbage looper since
160 FARNSWORTH ET AL.

other workers have already established
these facts (15, 16).
The results of the cytochrome P-450 and
aldrin epoxidase assays of fifth-instar lar-
vae of alfalfa looper reared on the various
diets are presented in Table 1. The midgut
epoxidase activity of the peppermint-fed
larvae was five to eight times that of the
larvae receiving artificial diet, alfalfa, snap
bean, or broccoli leaves. The cytochrome
P-450 content did not increase to a similar
extent in the peppermint-fed larvae, the
maximum change being about twofold in
these larvae compared to those fed alfalfa
leaves. It is evident that the carcass micro-
somal epoxidase, though much lower in ac-
tivity than the midgut enzyme, was induced
less, up to fourfold, in the peppermint-fed
larvae while the cytochrome P-450 content
of these microsomes was not affected.
Similar results were obtained in the
studies of the cabbage looper microsomes,
Table 2. Enhancement in midgut epoxidase
activity due to peppermint was approxi-
mately four times that of the other diets and
there was no increase in cytochrome P-450
content. One difference between the two
species was the considerable enhancement
of carcass epoxidase activity, about 20-
fold, in the peppermint-fed larvae com-
pared to those on the other diets. Except
for the mint-fed larvae, these microsomes
were much less active in aldrin epoxidation
than those prepared from the midguts. Due
to an oversight, we did not record the larval
weights in these experiments.
We were able to collect third-, fourth-,
and fifth-instar alfalfa looper larvae from
peppermint plants in the field for a determi-
nation of their aldrin epoxidase activity.
The results of these assays were as follows:
third-instar midgut microsomal epoxidase,
1.6 5 0.13; fourth instar, 2.4 + 0.5; and fifth
instar, 3.0 ? 0.04 nmol dieldrin/min/mg
protein, mean ? SE of two experiments,
each with two or three assays. Comparing
these results with those in Table 1, it ap-
pears that some induction also occurred in
the earlier instars.
Spectral Characteristics of Cytochrome
P-450
Typical spectral difference curves for the
CO-P-450 binding complex of midgut mi-
crosomes from the two species are shown
in Figs. 1 and 2. Only curves from larvae
reared on artificial diet and peppermint are
shown but these did not vary in spectral
characteristics from those obtained with the
other diets. There was little evidence of the
presence of cytochrome P-420, and the ab-
sorption maxima was at 450 nm in all cases.

TABLE 1
Effect of Host Plunt on Microsomal Aldrin Epoxiduse Activity und Cytochrome P-450
Content of Alfulfu Looper Larvue

Midgut Carcass”
nmol dld/min/ nmol P-450/ nmol dldlmini nmol P-4501 Larval wt at assay’
Diet” mg protein* mg protein’ mg protein” mg protein’ (mg)
Artificial diet 0.58 + 0.12 0.23 0.10 2 0.01 0.08 426.7 _t 16.7
Alfalfa 0.38 -r- 0.02 0.16 0.09 t 0.01 0.13 109.0 + 6.3
Snap beans 0.43 + 0.05 0.18 0.08 -r- 0.01 0.11 123.0 ? 5.8
Broccoli 0.49 k 0.06 0.26 0.17 + 0.02 0.06 207.4 + 8.7
Peppermint 2.98 k 0.29 0.36 0.39 + 0.11 0.08 185.0 k 9.5
N Newly molted fifth-instar larvae were fed leaves of host plant or continued on the artificial diet for 60 hr prior
to enzyme assay.
b Mean t SE of four experiments, each in duplicate. did, Dieldrin.
’ Average of two experiments, each with single assay.
” See Methods for description.
(’ Mean + SE of two experiments, each in duplicate.
‘Average of 30-40 larvae.
 ENZYME INDUCTION IN ALFALFA AND CABBAGE LOOPERS 161

TABLE 2
Effect of Host Plunt on Microsomul Aldrin Epoxiduse Activity und Cytochrame P-450
Content of Cubbuge Looper Lurvue

Midgut Carcass”
nmol dldlmini nmol P-4501 nmol dldlmini nmol P-4501
Diet” mg protein” mg protein” mg protein” mg protein’
Artificial diet 0.39 + 0.01 0.29 0.05 t 0.01 0.05
Alfalfa 0.36 t 0.07 0.17 0.08 -t 0.01 0.16
Broccoli 0.42 + 0.02 0.30 0.04 i- 0.01 0.06
Cabbage 0.27 k 0.01 0.25 0.04 k 0.01 0.06
Peppermint 1.58 2 0.10 0.28 0.84 k 0.21 0.14
” Newly molted fifth-instar larvae were fed leaves of host plant or continued on the artificial diet for 60 hr
prior to enzyme assay.
’ Mean t SE of two experiments, each in duplicate. dld, Dieldrin.
’ Average of two experiments, each with single assays.
” See Methods for description.

Examples of the spectral difference acephate and methomyl with cabbage loop-
curves obtained with the Type II substrate, ers. Alfalfa loopers were much less suscep-
n-octylamine, and microsomes produced by tible to carbaryl after 3 days rearing on
larvae on two of the diets are exhibited in peppermint leaves than after being reared
Figs. 3 and 4. Those shown are typical of all on alfalfa leaves. It can be estimated that
of the diets, exhibiting a trough in the the difference is at least fivefold. A similar
392-nm region. In the case of cytochrome comparison of mortalities indicates an ad-
P-450 of higher animals this characteristic vantage at the lower dose for peppermint-
is thought to be evidence that the micro- fed loopers exposed to methomyl. There
somal P-450 mixture contains a significant was no difference, however, in susceptibil-
amount of the high-spin form (17, 18). ity to the organophosphate compound,
acephate.
Susceptibility to Insecticides
Tables 3 and 4 summarize the results of 0.03
the bioassays of carbaryl, acephate, and
methomyl with alfalfa loopers and of t -Art. duet
---Mint
0.02
t

0.02 -
-Art. diet
-- HIIll
0.01 -

\ .-' ,I
-0.03 I 1
350 400 450
Wowlwpth (nm) Wovelength (nm)
FIG. 1. Cytochrome P-450 curbon monoxide differ- 2. Cytochrome
FIG. P-450 curbon monoxide di&r-
ewe spectru of midgut microsomes from fifth-instur ence spectru of midgut microsomes ,from fifth-instur
u&@ loopers reured on urtijkiul diet or peppermint cubbuge loopers reared on urtijkiul diet or peppermint
Ieuves. Protein concentrution: urtificiul diet. I mgiml; leuves. Protein concentrutions It’ere I mglml jiw both
peppermint. 0.5 mglml. cwws.
162 FARNSWORTH ET AL.

- Art. diet
- Ati.dW
0.01 - --Mint
0.01 -

400 450
Wavelength (nm) 350 400 450
FIG. 3. Type II difference spectra with n-octylumine Wavelength (nm )
of midgut microsomes from fifth-instur alfulfu loopers FIG. 4. Type [Idifference spectru with n-octylumine
reared on urtificiul diet or peppermint leuves. Protein of midgut microsomes from fifth-instur cabbage loop-
concentrution: urtificiul diet, 2.0 mglml; peppermint, ers reured on urtificiul diet or peppermint leaves.
1.1 mglml. Protein concentrations were I mglml for both curves.

As with the alfalfa loopers, the cabbage armyworm, and suggest that induction of
loopers seemed less susceptible to meth- the microsomal oxidases by plant con-
omyl after feeding on peppermint leaves stituents is a common phenomenon. In the
compared to broccoli leaves. Acephate case of herbivores, especially those feeding
toxicity was not affected by the diets. Ap- on a wide variety of plants, induction may
parently the carbamate insecticides are serve as a further mechanism of adaptation.
metabolized more rapidly in looper larvae Another aspect of this phenomenon re-
feeding on peppermint leaves. quires consideration. It is known that the
microsomal oxidase system has an impor-
DISCUSSION tant physiological role in insect develop-
Our results with these two species of ment, namely in the activation of the molt-
lepidoptera support those already reported ing hormone, ecdysterone (19-21), and in
in our work with the variegated cutworm (3, the biosynthesis of the juvenile hormone
5), and by Brattsten (4) with the southern (22). Thus, it would seem that any substan-

TABLE 3
Effect of Host Plant on the Susceptibility of Alfalfa Looper Lurvue to Insecticides
Percentage mortality after feeding oP
Alfalfa leaves Peppermint leaves
Concentration
Insecticide” (%I 24 hr 48 hr 24 hr 48 hr
Carbaryl Control 0 0 0 0
0.05 20 20 0 10
0.10 40 45 10 35
0.50 45 60 20 45
Acephate Control 0 0 0 0
0.05 90 100 90 95
0.15 100 100 90 95
0.30 100 100 100 100
Methomyl Control 0 0 0 0
0.05 65 85 20 55
0.15 65 85 60 80
0.30 85 90 70 95
u Ten larvae were fed bean leaf disks dipped in the insecticide concentration shown.
b Larvae reared through fourth-instar on artificial diet were transferred to treated leaves for 72 hr prior to
assay. Average of two assays, each with a different population.
 ENZYME INDUCTION IN ALFALFA AND CABBAGE LOOPERS 163

TABLE 4
Effect of Host Plant on the Susceptibility of Cabbuge Looper Lurvoe to Insecticides

Percentage mortality after feeding or?

Broccoli leaves Peppermint leaves
Concentration
Insecticide” (%) 24 hr 48 hr 24 hr 48 hr
Acephate Control 0 0 0 0
0.02 45 65 60 70
0.05 65 70 50 80
0.15 80 85 85 95
0.30 100 100 85 100
Methomyl Control 0 0 0 0
0.02 40 45 0 10
0.05 55 60 30 30
0.15 55 70 65 75
0.30 65 70 90 95
” Ten larvae were fed bean leaf disks dipped in the insecticide concentration shown.
’ Larvae reared through fourth instar on artificial diet were transferred to treated leaves for 72 hr prior to
assay. Average of two assays, each with a different population.

tial stimulation of these enzymes, i.e., the
microsomal oxidases, by dietary sub-
stances would be disadvantageous if it oc-
curred at the wrong time during the insect’s
growth and development. One question,
therefore, is how has the herbivore adapted
to a host plant such as peppermint so as to
avoid any derangement in its physiological
processes brought on by as much as lo-fold
increase in microsomal oxidase activity?
One possibility is that the insect’s enzyme
regulation mechanism does not respond to
the plant stimulus at critical times such as
during hormone biosynthesis or activation.
Another possibility is that the induction by
the allelochemicals is highly specific and
thus without effect on the hormone-
metabolizing system.
The realization that insects, like higher
animals, probably possess multiple forms of
cytochrome P-450 raises a difficult problem
in experimental design for studies of the
type described here. The problem is that no
single, or even a group of substrates, can be
relied upon to measure the total enzyme
activity of the microsomal oxidase system.
Our solution to the problem has been to use
aldrin as a model substrate for the micro-
somal oxidase system and to support these
assays with a measurement of the cyto-
chrome P-450 present in the same system.
The bioassays, Tables 3 and 4, offer a third
measure of the effect of the plant constitu-
ents on the metabolic systems.
Our finding of a larger relative increase in
aldrin epoxidase activity than in the cyto-
chrome P-450 level of the same microsomes
is difficult to explain. This may indicate that
the change in enzyme activity brought on
by the peppermint allelochemicals is due to
a change in the catalytic capacity of the
cytochrome P-450 rather than in the
amount of this important component. Even
if the epoxidase system does not represent
all of the oxidase capacity of the midgut or
carcass microsomes, it is evident, at least in
the case of the cabbage looper (Table 2),
that part of the cytochrome P-450 from the
peppermint-fed loopers must have been ac-
tivated. This would explain the fourfold in-
crease in epoxidase activity while there was
no change in the P-450 level of the midgut
microsomes. There is some support for this
in the Type II binding spectra (Fig. 4). The
392-nm trough is more pronounced with the
microsomes from the peppermint-fed lar-
vae, suggesting that relatively more high-
spin P-450 was produced.
In the case of the alfalfa looper (Table 1)
this argument is not as strong because there
was a moderate increase in P-450 content of
both midgut and carcass microsomes in the
164 FARNSWORTH ET AL.

loopers which had been reared on pepper-
mint leaves. In this case, therefore, a selec-
tive and substantial increase in an aldrin-
specific cytochrome P-450 could explain
the eightfold increase in epoxidase activity
along with the twofold increase in cyto-
chrome P-450.
We considered it possible that methomyl,
which contains the S-methyl group, might
have been converted to the more toxic sul-
foxide by microsomal oxidation. The
stimulation of these enzymes by the pep-
permint constituents might therefore have
resulted in increased, rather than decreased
toxicity in loopers feeding on this plant. As
the bioassay results indicated, however,
this was not the case. This supports the
opinion of Kuhr (16) that the sulfoxide of
methomyl is not a significant end product in
its metabolism by the cabbage looper.
The practical value of these and other
studies of this type in helping to improve
our present methods of insect control is in-
creasingly apparent. When it can be dem-
onstrated that insects feeding on one crop
are more or less susceptible to an insec-
ticide than when feeding on another crop, it
is clear that the pest control strategy on the
two crops could be different, at least in
terms of insecticide dose. If it can also be
shown, as seems likely, that both the in-
sect’s capacity to detoxify insecticides and
its response to plant stimulation of this ca-
pacity differs with the developmental stage,
then an element of timing could be intro-
duced into the pest control strategy. Fi-
nally, the likelihood that the enzyme-
inducing constituents of the plant are
biosynthesized on a growth-dependent
schedule introduces a second timing factor
worth considering.
ACKNOWLEDGMENT
This research was supported by USPHS Grant ES
00362-22.
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