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Utilization of ╬Ф5 7 - and ╬Ф8-sterols by larvae of Heliothis zea.

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Archives of insect Biochemistry and Physiology 3:349-362 (1986)
Utilization of A5,'- and A8-Sterols by Larvae of
Heliothis zea
Karla S . Ritter
Department of Biological Sciences, Drexel University, Philadelphia, Pennsylvania
Larvae from two populations of Heliothis zea were reared on artificial diets
containing various sterols, which supported suboptimal growth, and their
tissue sterols were characterized in order to determine how these dietary
sterols are utilized by this insect. The sterols studied included A5r7-sterols(7dehydrocholesterol or ergosterol), A8-sterols (lanosterol andlor 2Cdihydrolanosterol), and a A5-sterol (4,4-dimethylcholesterol). Although larvae did not
develop on 4,4dimethylcholesterol, those fed primarily A8-4,4,14trimethylsterols developed to the third instar. When the latter sterols were spared with
cholesterol, the larvae reached the sixth instar and contained 4,4,14trimethylsterols as well as cholesterol in their tissues. When larvae were fed 7dehydrocholesterol, < 1% of the larvae from one population developed to the
sixth instar and these larvae contained 7-dehydrocholesterol as their principal
sterol. The other larvae successfully completed their larval stage when they
were transferred from the diet containing 7-dehydrocholesterol (or no sterol)
to a diet containing cholesterol within at least 9 days. The sterol composition of
larvae transferred from a diet containing cholesterol t o a diet containing 7dehydrocholesterol, after they had reached 60% of their final weight, was 54%
cholesterol and 46% 7-dehydrocholesterol. The major sterol isolated from the
tissues of the larvae fed ergosterol was also 7-dehydrocholesterol. Therefore,
although the larva of H. zea can dealkylate and saturate the side chain of the
A5,7,22-24fhnethylsteroI,it carries out little metabolism of the B ring of the
nucleus. These studies demonstrate that, when A5r7- or A8-sterols are
the principal sterols in the diet of H. zea, they are absorbed and incorporated
into i t s tissues, although they slow the rate of growth and may prevent complete
development of the larva.
Key words: Heliothis zed, cholesterol, 7-dehydrocholesterol, 24-dihydrolanosterol, 4,4dimethylcholesterol, ergosterol, lanosterol
Acknowlegments: I thank Mrs. J.R. Landrey for obtaining the MS and 'H-NMR sDectra and
Dr. W.D. Rosenzweig for reading the manuscript. This stidy was partially supporied by NSF
grant PCM-8206541.
Received August 30,1985; accepted November 13,1985.
Address reprint requests to K.S. Ritter, Department of Biological Sciences, Drexel University,
Philadelphia, PA 19104.
0 1986 Alan R.
Liss, inc.
350
Ritter
INTRODUCTION
Unlike many organisms, insects require an exogenous source of sterols in
order to complete their growth and maturation. As in other animals, insects
use the sterols as structural components of membranes and as precursors for
hormones (eg, ecdysteroids) [l].Different species of insects, however, have
different structural requirements for their sterols. For example, Heliofhis zeu
can develop to the adult stage using a variety of A'-, A5-, and A7-sterols as
dietary sterols, although the A7-sterols significantly retard the rate of growth
of the larva [2,3]. In contrast, the fruit fly Drosophilu pucheu requires A7-sterols
as dietary sterols and cannot use A5- or A'-sterols at all [4].
Although the larva of H. zed can mature on a variety of dietary sterols, it
does not always metabolize them to the same products. For example, after
dealkylation of A'-, A5-, and A7-24-alkylsterols,it carries out little metabolism
of the B ring of the resulting 24-desalkylsterols [5]. Therefore, the principal
type of sterol in the tissues of H. zed can be altered by varying the type of
sterol fed to the larva. That is, depending on whether the larva is fed A5-,
A'-, or A7-sterols, the larva will contain primarily cholesterol, cholestanol, or
lathosterol, respectively.
Since the larva apparently can use a variety of sterols as structural components of membranes, it is of interest to explore why some other sterols
(eg, A5, 7-sterols)support only minimal or no growth of larvae [3]. Therefore,
the purpose of this investigation was to study the sterol composition of
larvae, which had been reared on diets containing various sterols that do not
support normal growth (ie, A5T7- and As-sterols) in order to compare and
contrast the metabolic fates of these different dietary sterols in this insect.
MATERIALS AND METHODS
H. zea
Neonate larvae of H. zed were reared individually on artificial diets, which
lacked supplementation with plant materials but were supplemented with
various sterols (15 mgllOO ml diet except where noted), as described previously [3]. In some experiments, the growth of larvae and the metabolites of
different dietary sterols were compared in two different populations of H.
zeu. Larvae representing one of these populations (A) were kindly supplied
by Dr. Wayne Brooks (North Carolina State University, Raleigh) and the
larvae in the other population (B) by Dr. Norman Leppla (USDA, ARS,
Gainesville, Florida); each group of stock insects was maintained through at
least 20 generations.
Dietary Sterols
The dietary sterols used in these experiments were: cholesterol* (J.T.Baker
Chemical Co, Phillipsburg, NJ); 7-dehydrocholesterol (Sigma Chemical Co,
*The systematic names for the sterols used in this study are as follows: agnosterol, 4,4,14atrimethyl-5a-cholesta-7,9(11),24-trien-3~-0l; cholesterol, cholest-5-en-3P-ol; 7-dehydrocholesterol, cholesta-5,7-dien-3P-ol; 24-dihydroagnosterol, 4,4,14a-trimethyl-5a-cholesta-7,9(ll)-dien3P-01; 24dihydrolanosterol, lanost-8(9)-en-3P-ol; 4,4-dimethylcholesterol, 4a,4P-dimethylcholest-5-en-3P-01; ergosterol, ergosta-5,7,22-trien-3P-ol; lanosterol, lanosta-8(9),24-dien-3P-ol.
Utilization of Sterols by H. zea
351
St. Louis, MO), ergosterol (ICN Pharmaceuticals, Inc., Cleveland, OH), 4,4dimethylcholesterol (a gift from the late Dr. H.W. Kircher, University of
Arizona), "lanosterol" and 24-dihydrolanosterol(Mann Research Labs, New
York, NY). Each sterol was recrystallized as necessary from ethanol, examined by GLC, ? RPLC, U V S , MS, and 'H-NMR and shown to be at least 98%
pure with the exception of the "lanosterol." This latter sterol was found to
be a mixture of lanosterol, 24-dihydrolanosterol, agnosterol, and 24-dihydroagnosterol, as reported by others [6]. In GLC analysis, the 24-dihydrolanosterol plus 24-dihydroagnosterol represented 39% of the sample, and
lanosterol plus agnosterol represented 61% of the sample; in RPLC analysis
at 205 nm, agnosterol and dihydroagnosterol represented < 8% of the sample
(ie, approximately 92% of the sterol was As-sterol). The spectra for lanosterol
and 24-dihydrolanosterol from MS and 'H-NMR agreed with those reported
by others [7l.
Isolation of Sterols
Late sixth-instar larvae were collected and extracted in order to determine
the sterol composition of their tissues after they had consumed different
dietary sterols. After the intestine of each larva was removed (in order to
remove any unabsorbed dietary sterol), the cadavers were stored at -20°C.
The various groups of cadavers were homogenized in acetone using a Sorvall
Omnimixer and the sterols obtained by continuous extraction with acetone
in a Soxhlet extractor for 24 h. The acetone extracts were saponified over
night at 60°C in 5% KOH in 90% ETOH and the 4,4,14-trimethylsterols andl
or 4-desmethysterols separated from the other neutral lipids by TLC on Silica
Gel G using a solvent system of benzene and ethyl acetate (9:l). The different
fractions were collected and the compounds eluted with anhydrous ether.
These procedures were carried out under darkened conditions and the recoveries made as rapidly as possible to prevent degradation of the sterols.
Similar extraction and purification techniques were used to isolate sterols
from the diet and frass of the larvae.
Identification of Sterols
The sterols isolated from the larvae, diet, and frass were characterized by:
GLC between 225°C and 235°C on 3% QF-1, 0.75% SE-30, andlor 1%XE-60
columns, using a Perkin-Elmer Sigma 3B gas chromatograph; analytical RPLC
at 45°C on a Zorbax ODS (CIS)column with a mobile phase of acetonitrileisopropanol (80:20) and a flow rate of 2 mllmin, using a Perkin-Elmer Series
3B liquid chromatograph equipped with a variable wavelength detector; U V S
by stopping the flow of mobile phase during RPLC and scanning the individ-
+Abbreviations: ethanol = ETOH; gas-liquid chromatography = GLC; V, - V o N o(where V, is
the retention volume of the sterol and Vo is the void volume) = k'; k' for test sterol/k' for
cholesterol = aC; proton nuclear magnetic resonance spectroscopy = 'H-NMR; mass spectrometry = MS; retention time relative to cholesterol = RRT; reversed-plase liquid chromatography = RPLC; side chain = SC; singlet = s; doublet = d; thin-layer chromatography =
TLC; ultraviolet spectroscopy = UVS.
352
Ritter
ual peaks between 190 and 300 nm; MS via direct probe on a Model 4000
Finnigan instrument with electron impact ionization at 70 eV; andlor ‘HNMR at 360 MHz at ambient temperature on a Bruker instrument, model
WH360, in CDC13 with Si(CH3)4as the usual internal standard. Separation
of individual sterols in mixtures was carried out by preparative RPLC at 30°C
on a Perkin-Elmer preparative C18 column, with a mobile phase of acetonitrile
and a flow rate of 10 mllmin, using a Perkin-Elmer Series 1 liquid
chromatograph.
For all experiments, when f values are indicated, they represent the actual
range of the results.
RESULTS
Growth on ”Lanosterol”
”Lanosterol” was fed to the two populations of larvae in order to determine whether this mixture of 4,4,14-trimethylsterols would support the development of the larvae. After 14 days, one population (A) had completed 13
-t 2% of the 295 molts that were possible (five per larva), and the other
population (B) had completed 39-t 5% of its 185 possible molts. The maximum instar reached by either group was the third (Table 1).
Growth on ”Lanosterol” Spared With Cholesterol
Diets containing different quantities of cholesterol, in addition to lanosterol,” were fed to larvae (A) to ascertain whether the 4-desmethylsterol
would spare the 4,4,14-trimethylsterols.Larvae fed diet containing cholesterol (2.5 mgllOO ml diet) in addition to ”lanosterol” (15 mgllOO ml diet)
reached a maximum of the fourth instar in 14 days; in contrast, control larvae
fed a diet containing cholesterol alone (2.5 mgllOO ml diet) reached only the
second instar. Larvae fed “lanosterol” diet, which contained twice as much
cholesterol (5 mgllOO ml diet), molted as many as five times, reaching the
sixth instar in 14 days; in contrast, control larvae fed a diet containing
cholesterol alone (5 mgllOO ml diet), molted up to three times, reaching only
the fourth instar in 14 days. When the amount of cholesterol in the ”lanosterol” diet was increased to 10 or 15 mgllOO ml of diet, a greater percentage
of larvae reached the sixth instar in 14 days; similarly, control larvae fed a
diet containing only cholesterol (10 or 15 mgllOO ml diet) reached the sixth
instar in 14 days but in fewer numbers (Table 1).
Utilization of ”Lanosterol” Spared With Cholesterol
Studies were carried out to investigate whether larvae can metabolize
4,4,14-trimethylsterols, because they developed as far as the third instar
when fed these sterols in the absence of other sterols. Since the intestine of
the sixth-instar larva can be removed more easily than that of the third instar
larva, in these studies the larvae (A as well as B) were fed ”lanosterol” diet
spared with cholesterol (5 mgllOO ml diet) to allow them to reach the sixth
instar (Table 1).Then the sterols in the diet, frass, and tissues were identified
and the relative amounts of the different sterols in each sample determined.
Utilization of Sterols by H. zea
353
TABLE 1. Effect of Various Sterols on the Development of Larvae of H. zeu
Dietary sterol
None
None
"Lanosterol"
"Lanosterol"
"Lanosterol"
and cholesterol
Cholesterol
"Lanosterol"
and cholesterol
"Lanosterol"
and cholesterol
Cholesterol
"Lanosterol"
and cholesterol
Cholesterol
"Lanosterol"
and cholesterol
Cholesterol
24-Dihydrolanosterol
4,4-Dimethylcholesterol
~
Amount of
sterol in diet
(mg1100 ml)
Population
Total no.
of molts
possible"
Average YO
of molts
completed
by day 14b
Maximum
instar
reached
by day 14
A
B
A
B
205
260
295
185
0
0
13 f 2
39 5
1
1
3
3
A
A
170
195
30 f 4
2*2
4
A
1,095
B
A
1,045
255
A
A
155
305
94 5
64 f 15
A
A
A
135
710
165
94
76
15
A
115
0
0
0
15
15
15
2.5
2.5
15
5
15
5
5
15
10
10
15
15
15
15
15
*
*3
60 * 8
14 & 7
67
*
*3
+ 13
"Five molts per larva.
bWhen error values are listed, each number is the average of two experiments
range of the results.
2
6
6
4
6
6
6
6
3
1
the actual
Sterol content of the "lanosterol"-cholesterol diet. One hundred grams of
this diet were removed from unused rearing vials, the sterols reisolated, and
five peaks detected by RPLC. The crcs of 0.65 0.02, 0.82 k 0.02, 0.93 k
0.02, 1.00 0.02, and 1.18 k 0.02 indicated that agnosterol, lanosterol, 24dihydroagnosterol, cholesterol, and 24-dihydrolanosterol were present, respectively. The relative areas of these peaks at 205 nm were 3, 58, 1, 16, and
22%, respectively. (Note: because of differences in extinction coefficients, the
areas from RPLC do not necessarily reflect the true relative concentrations of
the sterols in the mixture.) The UV spectra of agnosterol and 24-dihydroagnosterol revealed that the
,,A
of both these sterols were 237, 243, and 252
nm, confirming the presence of heterocyclic conjugated double bonds,
whereas the spectra from lanosterol, cholesterol, and dihydrolanosterol were
characteristic of end absorption indicating an absence of conjugated double
bonds. Three peaks were observed by GLC on XE-60 and SE-30. The RRTs
of these peaks indicated that 24% of the reisolated dietary sterol was cholesterol (RRT of 1.00 on XE-60 and SE-30), 29% 24-dihydrolanosterol and 24dihydroagnosterol (RRT of 1.29 0.01 on XE-60; 1.51 on SE-30), and 47%
lanosterol and agnosterol (RRT of 1.53 k 0.02 on XE-60; 1.65 on SE-30; Table 2).
*
*
*
354
Ritter
TABLE 2. Percentage of Sterols, by GLC, in Diet, Frass, and Larvae of Two Populations (A
and B) of H. zed fed "Lanosterol" Plus Cholesterol (15 mg 5 mgll00 ml Diet,
Respectively)
+
Source of
sterols
Diet
Frass (A)
Frass (B)
Larvae (A)
Larvae (B)
Weight of
sample (8)
100
100
20
42
10
Percentage of sterol isolated from sample
24-Dihydrolanosterol
Lanosterol
Cholesand
and
terola
24-dih ydroagno~terol~
agnosterol'
24
27
25
59
64
29
28
29
22
24
47
45
46
19
12
aRRT of cholesterol: XE 60, 1-00; SE-30, 1.00.
bRRT of 24-dihydrolanosterol and 24-dihydroagnosterol: XE 60, 1.29; SE-30, 1.51.
'RRT of lanosterol and agnosterol: XE 60, 1.53; SE-30, 1.65.
Sterol content of "lanosterol"-cholesterol frass. One hundred grams of frass
from the larvae A were collected from the rearing vials and the sterols
isolated and examined by RPLC. Five peaks with acs of 0.66, 0.83, 0.95, 1.00,
and 1.19 indicated that agnosterol, lanosterol, 24-dihydroagnosterol, cholesterol, and 24-dihydrolanosterol were present, respectively; the relative areas
of these peaks at 205 nm were 3, 57, 1, 20, and 19%, respectively. The UV
spectra of agnosterol and 24-dihydroagnosterol revealed that the A,
of both
of these sterols were 237, 243, and 252 nm, confirming the presence of
heterocyclic conjugated double bonds, whereas the spectra from lanosterol,
cholesterol, and 24-dihydrolanosterol were characteristic of end absorption
indicating an absence of conjugated double bonds. Three peaks were observed by GLC on XE-60 and SE-30. The relative areas and RRTs of the three
peaks observed indicated that 27% of the sterol was cholesterol (RRT of 1.00
on XE-60 and SE-30), 28% 24-dihydrolanosterol andlor 24-dihydroagnosterol
(RRT of 1.28 0.02 on XE-60; 1.50 on SE-30), and 45% lanosterol andlor
agnosterol (RRT of 1.54
0.01 on XE-60; 1.64 on SE-30). Similar relative
amounts of these sterols were isolated from the frass of the other larvae (BTable 2).
Sterol content of "lanosterol"-cholesterol larvae. The sterols extracted from
the tissues (minus the intestine) of 42 g larvae A as well as 10 g of larvae B
produced five peaks by RPLC with acs of 0.65, 0.83, 0.94, 1.00, and 1.18,
which indicated that agnosterol, lanosterol, 24-dihydroagnosterol, cholesterol, and 24-dihydrolanosterol were present, respectively. The relative areas
of these peaks from sterols isolated from larvae A at 205 nm were 1, 31,1,49,
and 17%, respectively. The corresponding relative areas of peaks from sterols
isolated from larvae B were 1, 25, 1, 49, and 24%, respectively. The UV
spectra of agnosterol and 24-dihydroagnosterol revealed that the A,,
of both
of these sterols were 237, 243, and 252 nm, confirming the presence of
heterocyclic conjugated double bonds, whereas the spectra from lanosterol,
cholesterol, and 24-dihydrolanosterol were characteristic of end absorption
indicating an absence of conjugated double bonds. Three peaks were observed for both samples by GLC on XE-60 and SE-30. The RRTs of these
peaks for sterols from larvae A indicated that 59% was cholesterol (RRT of
Utilization of Sterols by H. zea
355
1.01 f 0.01 on XE-60 and SE-30), 22% 24-dihydrolanosterol andlor 24-dihydroagnosterol (RRT of 1.31 k 0.02 on XE-60; 1.50 on SE-30), and 19% lanosterol andlor agnosterol (RRT of 1.56 f 0.02 on XE-60; 1.64 on SE-30). Three
similar peaks were observed for larvae B, and the relative percentages of
these peaks were 64,24, and 12%, respectively (Table 2).
Growth on 24-Dihydrolanosterol
24-Dihydrolanosterol was fed to larvae A to determine how this 4,4,14trimethylsterol, alone, affected the development of the larvae. The results
were similar to those obtained for "lanosterol"; that is, after 14 days, the
maximum instar reached was the third (Table 1).
Growth on 4,4-Dirnethylcholesterol
4,4-Dimethylcholestero1 was fed to larvae A to determine whether the
absence of the 14-methyl group in this sterol, and the presence of a double
bond at the 5 position instead of the 8 position, would affect the development
of larvae fed this sterol. No larvae molted to the second instar, although they
ate the diet, produced frass, and lived for as long as 13 days (Table 1).
Growth on 7-Dehydrocholesterol
When larvae A were fed 7-dehydrocholesterol, no larvae molted, although
they ate the diet, produced frass, and lived for as long as 15 days. In contrast,
when larvae from the second population of H. zed (B) were fed 7-dehydrocholesterol, they completed 24% of their larval molts in 14 days; however, less
than 1%of the larvae reached the sixth instar in this time interval (Table 3).
Utilization of 7-Dehydrocholesterol
Sterols were isolated from 1.9 g of larvae B, which eventually reached the
late sixth instar after feeding on diet containing 7-dehydrocholesterol, and
were identified by comparing their RRTs (on QF-l), acs, and UV spectra to
those of standards. 7-Dehydrocholesterol represented 76% (by GLC) of the
tissue sterols (RRT of 1.11k 0.02; ac of 0.75 0.01; ,A,
271, 282, and 293
nm) and cholesterol represented the remaining 24% (RRT of 1.00 f 0.01; ac
of 1.00 0.02; UV spectrum characteristic of end absorption; Table 4).
TABLE 3. Effect of A5r7-Sterolson the Development of Larvae of H. zeu
Dietary
sterol
7-Dehydrocholesterol
7-Dehydrocholesterol
Ergosterol
Ergostero1
Amount
of sterol
in diet
(mg1100 ml)
Population
15
15
15
15
A
B
A
B
Total
no. of
molts
possiblea
360
1,290
165
150
Average
% of molts
completed
by day 14b
Maximum
instar
reached
by day 14
o+o
1
6
4
5
24 f 3
38 & 5
49 f 11
aFive molts per larva.
bEach value is the average of two experiments f the actual range of the results.
356
Ritter
TABLE 4. Percentage of Sterols, by GLC, in Diet, Frass, and Larvae of Two Populations of
H . zea Fed A5,'-Sterols
Exogenous
sterol
7-Dehydrocholesterol
7-Dehydrocholesterol
Cholesterol
and then 7dehydrocholesterold
Ergosterol
Ergosterol
Ergosterol
Ergosterol
Ergosterol
Ergosterol
Type of
sample
Diet
Weight of
sample (g)
50
Percentage of sterol isolated from sample
7-DehydroCholesterola
cholesterolb
Ergosterol'
0
100
0
Larvae (B)
1.9
24
76
0
Larvae (B)
6.9
54
46
0
252
100
2.7
8.1
18.7
5.1
0
0
17
15
16
13
0
0
73
61
54
60
100
Diet
Frass (A)
Larvae (A)
Larvae (8)
Larvae (BY
Larvae (B)f
100
10
23
30
27
aRRT of cholesterol: XE-60, 1.00 f 0.01; QF-1, 1.00 f 0.01.
bRRT of 7-dehydrocholesterol: XE-60, 1.22 0.02; QF-1, 1.13 0.01.
'RRT of ergosterol: XE-60, 1.31 f 0.02, QF-1, 1.23 f 0.02.
dThe larvae were fed diet containing cholesterol until they molted into the fifth instar (ie, had
reached 60% of their final weight), and then they were fed diet containing 7-dehydrocholesterol
until the late sixth instar.
'20 f 1days old.
*40 f 1days old.
+
+
Growth on Diet Containing Cholesterol Before and After Incubation on Diet
Containing 7-Dehydrocholesterol
Larvae (A and B) were allowed to develop to the early second, third,
fourth, and fifth instars on diet containing cholesterol and then were transferred to diet containing only 7-dehydrocholesterol in order to determine
whether the poor growth of neonate larvae (both A and B) on 7-dehydrocholesterol was characteristic only of the first instar or whether the growth of all
instars would be retarded after they were exposed to this sterol. Also, the
reverse experiments were carried out using first-instar larvae that had been
maintained on 7-dehydrocholesterol for various amounts of time (ie, 3-9
days, which corresponded to the chronological age of the cholesterol larvae
transferred to 7-dehydrocholesteral in the second through fifth instars) in
order to determine whether normal growth would resume after they were
transferred to diet containing cholesterol. Similar studies were carried out
using larvae incubated on diets that lacked sterol instead of containing 7dehydrocholesterol.
The results after 9 days of incubation on the final diets are summarized in
Table 5. The growth of both populations of larvae slowed when the larvae
were transferred from the cholesterol diet to 7-dehydrocholesteral diet, especially as newly molted second, third, and fourth instars; similar results
were obtained when the larvae were transferred to diet containing no sterol
during these instars. Interestingly, those first-instar larvae of corresponding
Utilization of Sterols by H. zed
357
TABLE 5. Effect of Dietary Cholesterol on Growth of Two Populations (A and B) of Larvae
Before and After Incubation on Diet Containing 7-Dehydrocholesterolor No Sterol
Sterol (W mgilOO ml diet) in
Average instar of larvae 9 days after transfer
from initial diet to final diet
(instar transferred to final diet)
_2nd_ _3rd~ _4th _ _5th
A
B
A
B
A
B
A
B
Initial diet
Final diet
Cholesterol
Cholesterol
Cholesterol
7-Dehydrocholesterol
None
7-Dehydrocholesterol
Cholesterol
3.0 2.9 3.8 4.0 5.1 4.9 5.4 5.9
lsta _
lsta_
~ lsta
_ lsta _
1.0 2.3 1.0 1.5 1.0 1.0 1.0 1.4
4.7 4.9 3.3 4.6 4.3 4.0 3.4 3.8
None
Cholesterol
1.0
4.9
Cholesterol
7-Dehydrocholesterol
7-Dehydrocholesterol
None
None
6.0
2.7
5.1
3.6
1.0
4.4
5.8
3.7
1.0
3.3
6.0
4.1
1.0
4.4
6.0
5.1
1.0
3.0
6.0
5.5
1.0
3.2
6.0
5.9
1.0
3.0
6.0
6.0
1.0
2.5
aAlthough these larvae were in the first instar, they were the same age as those in the columns
above them.
chronological age, incubated on diets containing 7-dehydrocholesterol or
lacking sterol for 3-9 days (ie, the chronological age of normal larvae chosen
in the second through fifth instars), began to develop when transferred to
diet containing cholesterol; individuals in these diferent groups subsequently
reached the pupal stage after 16 or more days of incubation on the cholesterol
diet (control larvae reared continuously on cholesteral began to pupate in 15
days).
Utilization of 7-DehydrocholesterolAfter Incubation on Cholesterol
Larvae B were reared on diet containing cholesterol until apolysis at the
end of the fifth instar (at which time they weighed an average of 0.19 k 0.02
g or 60% of their final weight) and then were transferred to diet containing
7-dehydrocholesterol.This preincubation of the larvae on cholesterol allowed
the larvae to develop to the late sixth instar, which was easily dissected.
Identification of the tissue sterols from 6.9 g of these larvae was made by
comparing the RRTs, acs, and UV spectra of the sterols to those of standards.
The relative amounts of the sterols in the tissues were determined by GLC
analysis. Fifty-four percent of the sterol of these larvae was cholesterol (RRT
of 1.00 +_ 0.01; cyc of 0.99 f O.Ol), and 46% was 7-dehydrocholesterol (RRT of
1.13 k 0.02; a, of 0.76 +_ 0.01; A,,
of 271, 282, 293 nm; Table 4).
Growth on Ergosterol
Larvae A that were fed diet containing ergosterol completed 38% of their
165 total larval molts and developed as far as the fourth instar after 14 days,
and the other larvae (B) completed 49% of their 150 molts after 14 days and
developed as far as the fifth instar (Table 3).
~
358
Ritter
Utilization of Ergosterol
Sterol content of ergosterol diet. When the sterol was isolated from 252 g of
ergosterol diet and its RRT (on QF-1), a,, and UV spectrum compared to
those of standards, ergosterol was the only sterol detected (RRT on QF-1 of
1.23 f 0.02; a, of 0.70 k 0.02; A,,
of 271, 282, and 293 nm; Table 4).
Sterol content of ergosterol frass. When the sterols were isolated from 100
g of frass collected from the larvae A reared on ergosterol diet, ergosterol
(RRT of 1.23 0.01 on QF-1; a, of 0.70 f 0.01; A,,
of 271, 282, and 293 nm)
was the only sterol detected (Table 4). The fragmentation pattern of this
sterol in MS was consistent with that of a standard of ergosterol: M+ = mle
396, 42%; M+-H20-CH3 = 363, 50%; Mf-H20-SC = 253, 83%; M+-H20SC-C3H6 = 211, 100%.
Sterol content of ergosterol larvae. The metabolites (>3%) of dietary ergosterol were characterized from both populations of larvae (A and B) as well as
from those specific individuals in B who reached the sixth instar in 20 f 1
days and those who reached the sixth instar in 40 f 1 days in order to
determine whether there were differences in degree of metabolism of ergosterol in larvae requiring different lengths of time to reach the sixth instar
(Table 4). The relative amounts of the sterols in the tissues were determined
by GLC analysis. The sterol composition of 2.7 g of larvae A fed ergosterol
was: 17% cholesterol (RRT of 1.01 f 0.01 on XE-60 and QF-1; a, of 0.99 f
O.Ol), 73% 7-dehydrocholesterol (RRT of 1.22 0.02 on XE-60 and 1.13 f
0.01 on QF-1; a, of 0.76 f O.Ol), and 10% ergosterol (RRT of 1.31 f 0.02 on
XE-60 and 1.23 0.01 on QF-1; a, of 0.70 f 0.01). The character of UV
absorption of each of the latter two peaks indicated that both sterols had a
conjugated double bond system and so could be A537-sterols(A,
271, 282,
and 293 nm), whereas the UV spectrum for the first sterol was characteristic
of end absorption, indicating an absence of conjugated double bonds. Similar
evidence from GLC, RPLC, and UV analyses indicated that the sterol composition of 8.1 g of larvae B fed ergosterol was 15% cholesterol, 61% 7dehydrocholesterol, and 23% ergosterol.
Likewise, the sterol composition of those larvae B (18.7 g) fed ergosterol
and reaching the late sixth instar in 20 days was 16% cholesterol, 54% 7dehydrocholesterol, and 30% ergosterol. When the individual sterols, from
these mixtures of sterols, were separated using preparative RPLC, not only
did GLC analysis of the individual peaks confirm the above identifications
but the M+ and fragmentation pattern of each sterol in MS was consistent
with the presence of cholesterol (M+ = mle 386, 100%; M+-85 = 301, 75%;
Mf-C7H11 = 275, 63%; M+-SC = 273, 50%; M+-SC-H20 = 255, 63%), 7dehydrocholesterol (M+ = 384, 100%; Mf-CH3 = 369,l8%; M+-H20-CH3=
351, 88%; M+-H,O-C,H, = 325, 75%; Mf-H20-SC = 253, 69%; M+-C3H*-SC
= 227, 44%), and ergosterol (M+ = 396) [8]. The presence of these three
sterols was confirmed by 'H-NMR. The chemical shift of 'H in ppm from
Si(CH& at various carbons in cholesterol was C-18,0.68 (s);C-26 and 27,036
and 0.87 (d, J =6 Hz); C-21, 0.91 (d, J = 6 Hz); C-19, 1.01 (s). The 'H-NMR
spectrum for the 7-dehydrocholesterol peak was C-18, 0.62 (s); C-26 and 27,
0.86 and 0.87 (d, J = 6 Hz); C-21, 0.94 (d, J = 6 Hz); C-19, 0.95 (s). The 'H-
Utilization of Sterols by H. zea
359
NMR spectrum for the ergosterol peak was C-18, 0.62 (s); C-26 and 27, 0.82
and 0.85 (d, J = 6 Hz); C-21, 1.03 (d, J = 6 Hz); C-19, 0.94 (s). Similarly the
sterol composition of those larvae B (5.1 g) fed ergosterol and reaching the
late sixth instar in 40 days was 13% cholesterol, 60% 7-dehydrocholesterol,
and 27% ergosterol (Table 4).
DISCUSSION
H. zed is similar to other insects [9] in that it cannot complete its development using 4,4,14-trimethylsterols as its sole dietary sterols. However, the
present study indicates that the larva can develop as far as the third instar
not only on "lanosterol" but also on pure 24-dihydrolanosterol. When cholesterol was used to spare the "lanosterol" (in a 1:3 ratio, respectively) the
larvae then reached the sixth instar. When the fate of the dietary "lanosterol"
spared with cholesterol was examined in the metabolic studies, the ratio of
cholesterol to the other sterols in the tissues increased to 1:l.Therefore, H.
zea may be able to convert some of the 4,4,14-trimethylsterols to cholesterol
because 1)the relative percentage of cholesterol in the tissues was at least
double that in the diet; 2) there was little change in the relative percentages
of sterols in the frass, which indicated that cholesterol was not selectively
absorbed from the diet; 3) the relative percentage of lanosterol in the tissues
was reduced by more than twofold, in contrast to the relative percentage of
24-dihydrolanosterol (which was only slightly below that in the diet), indicating that lanosterol can preferentially be converted to cholesterol or that it
can first be converted to 24-dihydrolanosterol which may then be converted
to cholesterol. The ratio of agnosterol to 24-dihydroagnosterol in the tissues
also decreased from that of the diet. Despite this evidence, proof that 4,4,14trimethylsterol can be converted to cholesterol must await future studies
using radioactive sterol.
Since larvae did not develop at all on dietary 4,4-dimethylcholestero1,
apparently the presence of the gem-dimethyl group at C-4 prevents this
sterol from being a suitable component of membranes (andlor hormones) or
a substrate that can be metabolized to other sterols such as cholesterol.
There was some difference in the ability of the sterols to support the
development of the two populations (A and B) of H. zed. Although some
individuals in both groups reached the third instar with "lanosterol" as their
dietary sterol, population B completed more of its total possible molts in 14
days (39% vs 13%). Population B was also able to use A5r7-sterolsmore
efficiently than population A, because, unlike populations tested previously
[3,10], some larvae molted when fed 7-dehydrocholesterol (24% of their total
possible molts were completed in 14 days vs 0% for population A).
Because a few individuals in population B actually reached the sixth instar
when fed 7-dehydrocholesterol, unlike those in population A, it was possible
to examine the sterol content of these individuals to ascertain the metabolic
fate of this sterol. Since 7-dehydrocholesterol was the principal sterol recovered from their tissues, after the sterol was absorbed by the intestine
much of it was probably used directly in the membranes. Therefore, the
uptake and direct utilization of A5,7-desmethylstero1in tissues was similar to
360
Ritter
that of the A0-,A5-, and A7-desmethylsterols, which also became the principal
tissue sterols of larvae despite the differences in their B rings [5]. However,
since the rate of growth of the larva varied when it was fed these different
sterols (A5 > A' > A7 > A5,7, with the rate being exceptionally slow on the
A5,7-desmethylsterol[3], this indicated that the position and degree of unsaturation of the B ring affects the rate of absorption of the dietary sterol andlor
synthesis of membranes using the dietary sterol andlor conversion of the
molecule to other essential molecules (eg, ecdysteroids).
The inability of 7-dehydrocholesterol to support normal growth was not
limited to the first instar; development of second- third-, and fourth-instar
larvae was also slowed when they were transferred from diet that contained
cholesterol to diet that contained 7-dehydrocholesterol. Similar results were
obtained when they were transferred to diet that contained no sterol (Table
5). When larvae reared on cholesterol were transferred to diet containing 7dehydrocholesterol, prior to the onset of feeding in the sixth instar, and the
sterol composition of the tissues was determined at the end of the sixth
instar, the ratio of cholesterol to 7-dehydrocholesterol in the larva (54:46) was
similar to the percent of weight gained by the larva during the time spent on
the two different sterols (60:40, respectively). This confirmed the evidence
presented above that most of the dietary 7-dehydrocholesterol was incorporated directly into the tissues of the larvae.
Although 7-dehydrocholesterol inhibited growth of larvae, the negative
effect of 7-dehydrocholesterol on growth was reversible up to at least 9 days
after incubation on this sterol; larvae that had remained in the first instar for
this period of time finally completed their development and pupated after
they were transferred to diet containing cholesterol. Other first-instar larvae
also completed their development and pupated after they were transferred
from diet containing no sterol to diet containing cholesterol. Therefore, the
poor growth or absence of growth of larvae on diets containing 7-dehydrocholesterol, as well as no sterol, is reversible during at least the first 9 days
of incubation and is probably due to lack of a suitable sterol substrate rather
than a toxic effect of 7-dehydrocholesterol.
As we have reported earlier, although ergosterol supports more rapid
development of the larvae than 7-dehydrocholesterol, this A5t7f22-24/3-methylsterol supports slower development than the other A'-, A5-, and A7-sterols
of the 24-ethyl, 24-methyl, and 24-desmethyl series that we tested [3]. In the
present study, when the sterol composition of the larvae fed ergosterol was
examined, the ergosterol was metabolized to the same major end product in
both populations (ie, 7-dehydrocholesterol). Also, when the sterol composition of members of population B that reached the sixth instar in 20 days was
compared to that of larvae that required 40 days, their relative sterol compositions were similar. Therefore, the amount of time required to reach the
sixth instar did not seem to affect (or was not the result of) the degree of
metabolism of ergosterol. This is the first report of the conversion of ergosterol to 7-dehydrocholesterol in insects. In contrast, Blotella gemanica converts dietary ergosterol to 22-dehydrocholesterol [ll].
7-Dehydrocholesterol is the principal tissue sterol of those insects, which
consume this sterol directly, as well as those consuming ergosterol; this
Utilization of Sterols by H. zed
361
suggests that the difference in the rates of growth of larvae on these two
sterols involves differences in their rates of absorption, andlor ergosterol
mi ht spare 7-dehydrocholesterol. It is not clear why the accumulation of
A5/ -sterols in the tissues of H.zeu inhibits the development of this organism;
other species of insects (eg, Tribolium confusum [12] and Attu cephulotes isthrnicola [13]) develop normally using A5i7-sterolsas tissue sterols.
It is not known if the cholesterol, which was found in larvae fed ergosterol
or 7-dehydrocholesterol, was a metabolic product of these dietary sterols.
Minor amounts of cholesterol have been found consistently in those larvae
fed A'-, A7-sterols and analyzed in the sixth instar [5]. Ergosterol was the
only sterol recovered from the ergosterol diet as well as from the frass, and
7-dehydrocholesterol was the only sterol isolated from the 7-dehydrocholesterol diet; this indicates that there was no contamination of the diet with
other sterols and that no microorganisms in the digestive tract metabolize
the dietary ergosterol to other sterols.
Ergosterol, lanosterol, and 24-dihydrolanosterol, as well as other dietary
24-alkylsterols [5], can be recovered from the tissues of H. zeu; this indicates
that these alkylated sterols enter the hemocoel of the larva prior to dealkylation. Therefore, differences in the position or number of double bonds (ie,
A'-, A5-, A7-, As-, and A5r7-sterols)andlor the addition of alkyl groups (at C4/14! and C-24) do not prevent the uptake of certain dietary sterols. However,
the slow rate of growth of H. zeu on A5,7- and As-sterols, relative to A5-, A'-,
and A7-sterols, might be due to differences in the rates of uptake of these
diverse sterols andlor the inability of A5r7- and As-sterols to fulfill all the
essential metabolic roles necessary to support maximal growth of larvae of
H. zed.
9
LITERATURE CITED
1. Svoboda JA, Thompson MJ: Steroids. In: Comprehensive Insect Physiology Biochemistry
and Pharmacology: Kerkut GA, Gilbert LI, eds. Pergamon Press, New York, Vol 10, pp
137-175 (1985).
2. Ritter KS, Nes WR: The effects of cholesterol on the development of Heliothis zea. J Insect
Physiol, 27, 175 (1981).
3. Ritter KS, Nes WR: The effects of the structure of sterols on the development of Heliothis
zea. J Insect Physiol, 27, 419 (1981).
4. Goodnight KC, Kircher HW: Metabolism of lathosterol by Drosophila puchea. Lipids, 6, 166
(1971).
5. Ritter KS: Metabolism of A'-, A5-, and A7-sterols by larvae of Heliothis zea. Arch Insect
Biochem Physiol, 1, 281, (1984).
6. Nes WR, McKean ML: Biochemistry of Steroids and Other Isopentenoids. University Park
Press, Baltimore, p 443 (1977).
7. Sekula BC, Nes WR: The identification of cholesterol and other steroids in Euphorbia
pulcherimmu. Phytochemistry, 19, 1509 (1980).
8. Nes WR, Krevitz K, Joseph J, Nes WD, Harris B, Gibbons GF, Patterson GW: The
phylogenetic distribution of sterols in tracheophytes. Lipids, 12, 511 (1977).
9. Kircher HW: Sterols and insects. In: Cholesterol Systems in Insects and Animals. Dupont
J, ed. CRC Press, Inc. Boca Raton, pp 1-50 (1982).
10. Ritter KS: Some unusual aspects of the sterol biochemistry of insects. In: Isopentenoids in
Plants, Biochemistry and Function. Nes WD, Fuller G, Tsai L, eds. Marcel Dekker, Inc.
New York, pp 389-400 (1984).
362
Ritter
11. Clark AJ, Bloch K: Conversion of ergosterol to 22-dehydrocholesterol in Blattella germanica.
J Biol Chem 234, 2589 (1959).
12. Svoboda JA, Robbins WE, Cohen CF, Shortino, TJ: Phytosterol utilization and metabolism
in insects: recent studies with Tribolium confusurn. In: Insect and Mite Nutrition. Rodriguez
JG, ed. North-Holland, Amsterdam, pp 505-516 (1972).
13. Ritter KS, Weiss BA, Norrbom AL, Nes WR: Identification of A5r7-24-methylene-and
methylsterols in the brain and whole body of Atta cepkalotes istkmicola. Comp Biochem
Physiol 7 2 B , 345 (1982).
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