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Alterations in fatty acid composition during the metamorphosis of Hyalophora cecropia: Correlations with juvenile hormone titre
J. Insect Physiol., 1970, Vol. 16, pp. 851to 864. PergamonPress. Printed in Great Britain
ALTERATIONS IN FATTY ACID COMPOSITION DURING THE METAMORPHOSIS OF HYALOPHORA CIX’ROPIA: CORRELATIONS WITH JUVENILE HORMONE TITRE* WILLIAM Department
F.
STEPHEN
Jr.t
of Biological Sciences,
and
LAWRENCE
Northwestern
I. GILBERT
University,
Evanston,
Illinois 60201
(Received 31 October 1969) Abstract-Gas chromatograpbic analyses of the total fatty acids of whole animals during development revealed that palmitic and stearic acids are always present in lower concentrations than their unsaturated homologues while both oleic and linolenic acid concentrations exceed that of linoleic acid. This also holds true for the neutral lipid fraction but the phospholipids possess a higher concentration of stearic acid. During late pharate adult development there are increases in the relative quantities of saturated and monounsaturated fatty acids and a decrease in polyunsaturated fatty acids. The triglyceride fraction of the fat body reflects the changes observed in the total and neutral lipid fraction of whole animals. During the last three days of pharate adult life the amount of palmitoleic acid doubles in the fat body neutral lipid. The composition of the haemolymph neutral lipid differs from that of the fat body and changes during development. Similarities in fatty acid composition between haemolymph and flight muscle lipid suggest that the developing flight muscle derives most of its fatty acids from haemolymph neutral lipid. The correlation between haemolymph and ovarian neutral and phospholipid fatty acids suggests that the haemolymph lipids are incorporated into the developing oijcytes. When the data are compared with that pertaining to juvenile hormone titre, it appears that juvenile hormone may control aspects of fatty acid synthesis and lipid transport and suggests a r61e for the high concentration of juvenile hormone in the male moth. INTRODUCTION
WE HAVE recently chain
saturated
shown and
monounsaturated
GILBERT, 1969). This dehydrogenate chain
lengthening
therefore
analyzed
morphosis place.
both
The
of
that Hyalophora cecropia can synthesize
insect to the
can lengthen
acid composition
data, when
whether
correlated
from
acetate
acid to stearic
monoenes.
are functions
H. cecropia to determine
present
acids
palmitic
corresponding
and desaturation the fatty
fatty
Our
data
of developmental
of several
tissues
developmental
with the juvenile
only
straight
(STEPHEN and acid and can suggested
that
stage and we
during
the meta-
alterations hormone
do take
content
of
* Supported by grant AM-02818 from the National Institutes of Health. t Former National Institutes of Health pre-doctoral fellow. Supported in part by NIH training grant GM-903. Present address : Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas. 851
8.52
WILLIAMF. STEPHEN JR. ANDLAWR~CE I. GILBERT
H. cecropia, furnish a reasonable explanation for the high concentration of juvenile hormone in the male moth. MATERIALS
AND METHODS
Notes on the handling of animals as well as methodology involved in extracting lipid, column chromatographic separation into discrete lipid classes, and gas chromatographic analyses of the methylated fatty acids have been detailed in the preceding paper (STEPHENand GILBERT, 1969). RESULTS
Total lipid Extraction of 68 animals revealed that: fifth instar larvae yielded an average of 54.6 mg lipid (144% of fresh weight) ; pharate pupae yielded an average of 174.0 mg (5.61% of fresh weight); whereas pupae contained an average of 360.1 mg of lipid (8.09% of fresh weight). Neutral lipid represented only 66.2 per cent of the total lipid in fifth instar larvae, but rose to 88.3 per cent in pharate pupae and 91.0 per cent in newly ecdysed pupae. The extreme difference in phospholipid content between a fifth instar larva and a pupa was only 26.5 mg while the least difference in neutral lipid content between these stages was 270.5 mg. We detected no significant changes in the absolute lipid content of male pharate adults although the average percent lipid of fresh weight rose from 9.39 per cent in early pharate adult development (0 to 5 days) to 11.73 per cent in late development (18 to 20 days). In view of the large increase in neutral lipid during pupation a sample of pupal fat body was extracted for further investigation. After removing contaminating phospholipids, the neutral lipid was further fractionated and the triglycerides were found to comprise 91.5 per cent of the total neutral lipid while other fractions made up only between O-6 and 2.4 per cent.
Fatty acid composition of whole animals during development Initially, fatty acid analyses were carried out on the methyl esters of whole chilled pupae and pharate adults. The average values for each fatty acid are found at the bottom of Table 1 and express the general pattern quite well. Palmitic and stearic acids are always present in lower concentrations than their unsaturated homologues and linoleic acid is present in lesser quantities than oleic and linolenic acids. In general, the quantity of palmitic acid slightly exceeds that of stearic acid while the percentage of oleic acid always exceeds that of palmitoleic acid. Trace peaks are not considered in these analyses because of their sporadic appearance and quantitative insignificance. Changes noted in the fatty acid composition between early pharate adult development (0 to 5 days) and late development (18 to 20 days) are: an increase in total saturated fatty acids from 1 to 3 per cent to 4 to 5 per cent; an increase in monounsaturated fatty acids ; a decrease in polyunsaturated acids.
FATTY ACID COMPOSITIONOF HYAL.OPHORA
853
CECROPIA
The fatty acid composition of a single fifth instar larva was determined and striking differences between it and pupae and pharate adults were observed. Not only are palmitic and stearic acids present in much higher amounts in the larva (12.31% and 4.03% respectively), but the quantity of palmitoleic acid (6.08%) is only half that of palmitic acid. There is also a 4 : 3 ratio of linolenic acid (42%0/b) to oleic acid (29.82%) as compared to the average 8 : 7 ratio of oleic acid to linolenic acid in pupae and pharate adults. Linolenic acid comprised 4.6 per cent of the total. TABLET-RELATIVEPERCENT FATTYACIDCOMPOSITION Day of pharate adult development O(3) l(4) 3(7) 5(l) 18(2) 19(l) 20(l) Average
OF LIPIDFROMMALEPHARATEADULTS
Fatty acid composition Cl6 :0 0.84 I.18 I.48 1.90 3.31 2.92 2.40 1.64
Cl6:l 7.52 16.28 15.37 6.40 13.76 14.17 13.33 13.51
Cl8:O
Cl8:l
Cl8:2
Cl8:3
0.65 0.72 0.95 0.80 1.69 2.05 1.56 1 a06
38.29 44.87 43.57 3140 41.38 41.05 43.12 41.99
9.60 2.97 4.48 11.60 3.07 3.65 2.76 5.06
43.08 33.59 34.09 47.90 36.91 36.09 36.75 36.68
Number in parentheses denotes number of determinations. a separate animal.
Each determination was on
Since both the ratios of neutral lipid to phospholipid and the fatty acid compositions of these fractions varied between larvae and pupae, the lipids of a number of animals were separated into neutral lipid and phospholipid fractions and the fatty acid compositions analyzed. Fatty
acid composition
of neutral lipids and phospholipids
of whole animals
The comments about pupal and pharate adult total lipid apply without exception to the neutral lipid of chilled pupae (Table 2). However, the phospholipids are characterized by a higher concentration of stearic acid (11 to 12%) than is found in the neutral or total lipids. The ratio of palmitoleic acid to palmitic acid is slightly more than 2 : 1 as opposed to the 4 : 1 to 20 : 1 ratios observed in the total and neutral lipids. The amount of linolenic acid is also at the lower limit of the range observed in the neutral and total lipid samples. Our data suggest that individual variation in fatty acid composition is less for phospholipids than neutral lipids. The fatty acid pattern of the neutral lipids extracted from 100 to 200 nonfeeding first instar larvae was different from those described in Table 2. Palmitic acid comprises 17 per cent of the total fatty acid complement and is more than three times the concentration of palmitoleic acid. The stearic acid content is high
854
WILLIAM F. STEPHEN JR. ANDLAWRENCEI. GILBERT
(8-l per cent) and the linolenic acid content lower (26 per cent) than in any of the neutral lipid or total lipid extracts alluded to above. This stage is characterized by a relatively high concentration of saturated fatty acids. The phospholipid fatty acids of first instar larvae are similar to those of the pupa except for the large quantity of palmitic acid in the larvae. The concentration of Cl8 acids corresponds well to that found in the pupal phospholipids. TABLE
~-FATTY
ACID
COMPOSITION
OF NEUTRAL
LIPID AND
PHOSPHOLIPID
OF WHOLE
H. cecropia First instar larvae*
Chilled pupae Fatty acids
Neutral lipid
Phospholipid
Neutral lipid
Phospholipid
C16:O C16:l C18:O C18:l C18:2 C18:3 c2o:o
0.76 11.51 0.35 44.57 3.71 38.98 -
2.95 5.28 11.60 37.73 12.60 29.73 -
17.01 5.03 8.13 39.19 4.58 26.03 -
8.13 1.18 13.38 35.31 9.63 30.82 1.61
* 100 to 200 larvae, 1 to 2 days after emergence were pooled for this experiment.
Fatty acid composition of fat body neutral lipid fractions Since the fatty acid compositions of neutral lipids and phospholipids were different, we determined the composition of the individual neutral lipid fractions to find out whether any particular fraction was responsible for the differences. Fat body lipid was chosen for these analyses because of the central metabolic role of the fat body and any differences in fatty acid composition of the neutral lipid classes might reflect the utilization of specific fatty acids. The data reveal that the composition of the triglyceride fraction is very similar to that of the fat body total lipid, and this in turn reflects the composition of the neutral or total lipids of whole chilled pupae. The partial glyceride fractions contain greater amounts of saturated acids (including arachidic acid) and lesser concentrations of linolenic acid. The sterol ester fraction is characterized by higher concentrations of monoenoic acids and the least amount of linolenic acid.
Fatty acid composition of whole animal phospholipids during adult development and life Although the fatty acid composition of the total lipid of H. cecropia changes little during adult development, changes in the phospholipid fatty acid composition could be masked by the large amount of neutral lipid present. Therefore, the phospholipid fatty acid compositions of pupae, pharate adults, and adults were determined (Table 3). Two developmental changes were observed. The first is a decrease in the concentrations of both palmitoleic and oleic acids as the animals develop and age. The second change is a corresponding increase in the amount of
FATTY ACID COMPOSITION OF HYALOPHORA
linolenic acid. less regular.
CECROPlA
855
Both males and females show this pattern although the females are
TABLE ~-RELATIVE
PER CENT FATTY ACID COMPOSITION OF WHOLE ANIMAL PHOSPHOLIPIDS DURING ADULT DEVELOPMENT* Developmental
stage
--
Fatty acid
pupae
15 to 19 day pharate adults
Male
C16:O C16:l C18:O C18:l C18:2 C18:3
6.38 9.16 9.16 45.91 10.09 19.30
6.85 6.32 8.20 41.53 11.42 25.67
5.13 3.96 11.12 39.36 9.20 31.23
5.28 2.11 9.19 38.01 9.08 36.32
Female
C16:O C16:l C18:O C18:l C18:2 Cl8:3
8.68 8.68 9.03 50.12 10.30 13.19
5.08 3.60 8.96 50.97 10.06 21.33
6.20 3.96 11.74 37.99 11.74 28.36
4.88 2.71 12.10 41.19 8.58 30.50
Chilled Sex
1 day adults
2 to 5 day adults -
* Each developmental stage represents the pooled extract of 8 to 12 animals. The females contained eggs and these contribute to the results obtained with female extract.
In view of these results, a further study of alterations in fatty acid composition during development was undertaken. The neutral and phospholipids of fat body, haemolymph, ovaries, and flight muscle were analyzed at various times during adult development and adult life.
Fatty acid composition of neutral lipids and phospholipids of selected tissues during adult development As noted above, the fatty acid composition of the fat body neutral lipid is similar to that of the fat body total lipid. Table 4 shows a twofold increase in the amount of palmitoleic acid in the fat body neutral lipid during the last 3 days of pharate adult life. The linoleic acid concentration decreases through adult development while the quantity of linolenic acid decreases from day 13 of pharate adult life to adult ecdysis. Fat body phospholipids are relatively high in palmitic acid (10 to 12 per cent), stearic acid (11 to 12 per cent), and linoleic acid (12 to 27 per cent). The amount of stearic acid rises during the first 13 days of pharate adult life. The palmitoleic acid content remains low and constant at about 2-S per cent. The oleic acid values (27 to 35 per cent) and the linolenic acid values (17 to 26 per cent) are lower than those of the neutral lipid.
856 TABLE
WILLIAM ~-RELATIVE
F. STEPHFX JR. ANDLAWRENCEI. GILBERT
PER CENT FATTY ACID COMPOSITION DEVELOPMENT*
OF FAT BODY
LIPIDS DURING
ADULT
Developmental stage
Fatty acid
Chilled pupae
10 day pharate adults
13 day pharate adults
18 day pharate adults
1 day adults
Neutral lipid
C16:O C16:l C18:O C18:l C18:2 C18:3
0.76 440 0.46 35.46 9.87 49.50
4.17 8.11 0.93 45.43 5.80 35.58
2.51 7.41 0.86 38.62 6.36 44.25
4.01 8.03 1.28 42.96 5.55 38.17
4.48 15.30 0.65 45.48 2.52 31.57
Phospholipid
C16:O C16:l C18:O C18:l C18:2 C18:3
9.73 2.46 10.90 35.30 18.67 22.92
12.46 2.41 13.85 26.82 27.19 17.27
10.64 2.46 20.57 28.67 11.52 26.13
-
-
Lipid fraction
-
* Each developmental stage represents the extract of pooled tissue from 2 to 5 female insects.
Haemolymph neutral lipids have an unusual composition and exhibit developmental change (Table 5). Palmitic acid is prominent and is much more abundant than palmitoleic acid during the first 13 days of pharate adult life. However, by the time of adult ecdysis, palmitoleic acid is in 2 : 1 excess, although palmitic acid is still present in greater amounts than are usually found in H. cecropiu extracts. During the first 13 days of pharate adult development, the concentration of stearic acid declines by about one-half and that of linoleic acid by about two-thirds. The concentration of both these Cl8 fatty acids decrease even more by the time of adult ecdysis. The linolenic acid content rises during the first 13 days of pharate adult life while the oleic acid concentration remains almost constant during that period. However, at ecdysis, the adults have 9 per cent more oleic acid and 10 per cent less linoleic acid than they did as 13 day pharate adults. The haemolymph phospholipids have 16 to 17 per cent stearic acid as their most unusual feature while the concentration of palmitic acid is three times greater than that of palmitoleic acid. The oleic acid concentration drops 6 per cent between 13 and 18 days of pharate adult development while the concentration of linolenic acid rises 14 per cent during the same period. The ovarian neutral lipids exhibit several unusual features (Table 6). The amount of palmitic acid rises dramatically during the first half of pharate adult development and then declines gradually until adult ecdysis. The palmitoleic acid concentration is only 4 to 5 per cent in pharate adults but is two to three times greater in the adult. The quantity of stearic acid rises modestly during pharate
OF HYALOPHORA
FATTY ACID COMPOSITION TABLE
S---RELATIVE PER CENT
FATTY ACID COMPOSITION ADULT DEVELOPMENT*
OF HAEMOLYMPH
Developmental
857
CECROPIA
LIPIDS DURING
stage
-
Fatty acid
Chilled pupae
10 day pharate adults
Neutral lipid
C16:O C16:l Cl8:O C18:l C18:2 C18:3
IO.82 5.59 4.66 34.60 11.39 32.95
15.86 3.85 3.23 30.82 9.95 36.29
12-18 4.81 2.19 33.38 4.47 39.96
-
7.84 13.84 1.66 42.40 4.16 30.40
Phospholipid
C16:O C16:l C18:O Cl8:l C18:2 C18:3
-
-
11.53 3.51 15.68 29.43 11.90 27.95
5.88 2.34 16.95 23.10 9.43 42.30
-
Lipid fraction
* Each insects.
TABLE
developmental
6--RELATIVEPER
stage
represents
CENTFATTY
13 day pharate adults
18 day pharate adults
1 day adults
the extract
ACID COMPOSITION ADULT DEVELOPMENT*
of pooled
tissue
of 2 to 5 female
OF OVARIAN AND EGG LIPIDS DURING -
Developmental
pupae
10 day pharate adults
18 day pharate adults
1 day adults
3 day adults
Cl6:O C16:l C18:O C18:l C18:2 C18:3
2.81 3.71 2.30 35-17 12.83 43.19
13.00 5.26 3.32 29-87 10.37 38.17
11.50 5.21 4.20 37.62 6.63 34.84
8.78 14.08 2.41 47.54 2.31 25.88
8.55 7.74 2.61 46.74 3.18 31.19
C16:O C16:l C18:O C18:l C18:2 C18:3
10.83 2.27 11.17 33.59 19.98 23.17
12.58 I.54 18.19 25.61 13.58 28.51
9.03 2.29 17.52 32.61 10.51 28.03
9.35 13.55 3.27 50.00 2.80 21.03
8.64 13.64 5.91 48.64 3.64 19.55
Fatty acid
Chilled
Neutral lipid
Phospholipid
Lipid fraction
* Each
stage
developmental
stage represents
the extract
of pooled
tissue of 2 to 5 animals.
8.58
WILLIAM F. STEPHENJR. ANDLAWRENCEI. GILBERT
adult life but drops to the pupal level at adult ecdysis. The concentrations of linoleic and linolenic acid decrease constantly from chilled pupa to adult while the oleic acid concentration rises during late pharate adult life. The ovarian phospholipids provide an interesting developmental picture. Although the amount of palmitic acid varies between 9 to 13 per cent, the palmitoleic acid content is only 2 per cent during pharate adult development but rises dramatically to 14 per cent at adult ecdysis. The stearic acid concentration is 18 per cent during the latter part of the pharate adult life but is only 3 to 6 per cent in the adult while the quantity of oleic acid almost doubles during the same developmental period. The linoleic acid content decreases dramatically (from 20 to 3 per cent) during adult development while the linolenic acid concentration drops only slightly. The data collected on flight muscle neutral lipids were not sufficient to show developmental trends. However, palmitic acid is moderately prominent, being present in equal or greater concentrations than palmitoleic acid. In other respects, these samples have typical neutral lipid fatty acid patterns. The phospholipid fatty acids show essentially no change from late pharate adult life to adult ecdysis with the exception of the decrease in linoleic acid concentration and a corresponding increase in the quantity of oleic acid. TABLE
~----RELATI~E PER CENT
FATTY
ACID
COMPOSITION
OF FLIGHT
MUSCLE
LIPIDS DURING
LATEADULTDEVELOPMENT * Developmental stage
Lipid fraction Neutral lipid
Phospholipid
Fatty acid
18 day pharate adults
C16:O C16:l C18:O C18:l C18:2 C18:3
8.36 5.78 3.01 31.00 11.56 40.28
C16:O C16:l C18:O C18:l C18:2 C18:3
8.15 3.49 5.20 33.33 14.65 35.18
1 day adults
7.55 4.41 4.80 37.06 7.95 38.24
3 day adults 4.23 4.23 0.98 37.81 7.58 45.18 6.14 2.68 4.22 42.86 7.67 36.43
* Each developmental stage represents the extract of pooled tissue of 2 to 5 female insects. DISCUSSION
The pattern of total lipid change that can be deduced from these studies is that lipid (almost entirely neutral lipid) is accumulated during the latter portion of the fifth instar and that a further increase occurs during spinning. Then, a small
FAlTY ACID COMPOSITION
OF HYALOPHORA
CECROPIA
859
amount is lost as the pupae enter diapause. When adult development commences, males conserve lipid while females utilize it. Key points, documented by DOMROE~Eand GILBERT (1964) are that H. cecropia derives its energy for flight almost solely from the oxidation of fatty acids and that the male conserves its lipid during pharate adult life since it is the more active flier. The qualitative nature and biosynthesis of the fatty acids of H. cecropia have been discussed previously (STEPHENand GILBERT, 1969). Here, our interest lies in the quantitative relations of these fatty acids and the various parameters which affect these relations. Fatty acid composition of whole animals through development Although most insects have a high proportion of unsaturated fatty acids (FAST, 1964), H. cecropia is extreme in this regard. Over 97 per cent of the fatty acids of pupae and early pharate adults are unsaturated as are more than 90 per cent of the fatty acids of adults (YOUNG, 1967). Larvae have a greater proportion of saturated fatty acids than other developmental stages. The fatty acid pattern most characteristic of insect lipid is large amounts of palmitic and oleic acids and a concentration of linoleic acid exceeding that of linolenic acid. Most Diptera have large amounts of palmitoleic acid and most Lepidoptera more linolenic than linoleic acid (FAST, 1964; GILBERT, 1967a). The fatty acid composition of H. cecropia is intermediate between that of the majority of Lepidoptera and that of Diptera. Linolenic and oleic acids are the most abundant fatty acids, being found in concentrations far exceeding that of linoleic acid while palmitic acid is the major saturated fatty acid. It is the relatively large quantity of palmitoleic acid that gives H. cecropiu lipid its somewhat dipteran character. The newly emerged first instar larva has about 22 to 23 per cent saturated acids and a relatively small amount of palmitoleic acid. By the fifth instar, the relative quantity of saturated fatty acids has fallen to 16 per cent. According to HILDITCH and WILLIAMS (1964) the major fatty acids of leaves are palmitic, oleic, linoleic, and linolenic acids with linolenic acid being most prominent, followed by linoleic, palmitic and oleic acids. About equal amounts of palmitic and palmitoleic acids are formed from acetate by the fifth instar larvae (STEPHENand GILBERT, 1969). Th us, the prominence of palmitic acid at this stage must be due to a slow rate of desaturation of dietary palmitic acid. The changes we observe in the fatty acid composition of whole pharate adults are probably due to the oxidation of fatty acids to acetyl-CoA followed by resynthesis of the fatty acid chains. The fact that we observed this phenomenon directly by the use of r4C-labelled fatty acids in H. cecropia (STEPHEN and GILBERT, 1969) and that males conserve lipid during adult development support this conclusion. Preferential oxidation of polyunsaturated fatty acids is not a factor since it has been shown that if any oxidative preference exists, it favours saturated and monounsaturated fatty acids (STEPHENand GILBERT, 1969; BEENAKKERS and GILBERT, 1968). In first instar larvae, the concentrations of both juvenile hormone (GILBERT and SCHNEIDERMAN, 1961) and saturated fatty acids are high. In fifth instar larvae,
860
WILLIAMF. STEPHENJR. ANDLAWRENCE I. GILBERT
the concentration of juvenile hormone is much lower and the concentration of saturated fatty acids also drops significantly. In chilled pupae and early pharate adults there is no juvenile hormone and saturated fatty acids represent at most 3 per cent of the total fatty acids. However post-17 day pharate adults show increases in the concentration of both saturated fatty acids and juvenile hormone. In adults, where the juvenile hormone concentration is quite high, YOUNG (1967) reports that saturated fatty acids comprise 10 per cent of the total fatty acids. The means by which juvenile hormone may act to affect the concentration of saturated fatty acids and the relation of this increased concentration of saturated fatty acids to the developmental metabolism of H. cecropiu will be discussed subsequently. Although phospholipids comprise less than 10 per cent of the pupal lipids, they account for over 60 per cent of the total saturated fatty acids. Stearic acid content is high and relatively constant (9 to 13 per cent). In chilled pupae, the concentration of phospholipid palmitic acid is about four times that of the neutral lipid palmitic acid. However, first instar larval neutral lipids have twice the palmitic acid of larval phospholipids. Two major changes in phospholipid fatty acid composition occur during development. They are a regular decline in the concentration of monoenoic acids and a corresponding increase in that of linolenic acid. THOMAS and GILBERT (1967) have shown that the amount of cardiolipin in H. cecropiu flight muscle increases markedly as the pharate adults develop. Since 77 per cent of the fatty acids of H. cecropiu cardiolipin is linolenic acid, perhaps the increase in the amount of this phospholipid with its specific requirement for linolenic acid accounts for the relative rise in linolenic acid. Fatty acid composition changes in neutral lipid and phospholipid of selected tissues The fatty acid composition of specific insect organs and tissues during development has been the subject of few studies. BEENAKKER~and GILBERT (1968) analyzed the fatty acid composition of lipid fractions from male H. cecropia fat body and haemolymph in both chilled pupae and pharate adults just prior to adult ecdysis. Their data are comparable with the present results on total neutral lipid because triglyceride represents such a large proportion of fat body neutral lipids and diglyceride is almost the sole constituent of haemolymph neutral lipids. Changes in the fat body fatty acid composition show the conversion of polyunsaturated fatty acids into saturated and monounsaturated fatty acids, because the alternative mechanisms of preferential fatty acid oxidation and release would produce a change in the opposite direction. In the haemolymph of females, we note a decrease in the concentrations of saturated fatty acids and linolenic acid during the latter part of adult development. BEENAKKER~and GILBERT (1968) reported significant increases in the concentrations of saturated fatty acids and a much lower concentration of linolenic acid in male H. cecropiu. This sexual dichotomy may be explained if we consider that the amount of lipid in the haemolymph is small and fatty acid concentrations are the result of the dynamic processes of release and absorption.
FATTY
ACID
COMPOSITION
OF
HYALOPHORA
CECROPIA
861
To analyze these processes we must first consider the contributions of other organs. In males, the major developing structure is the flight muscle while in females both flight muscle and ovaries must be taken into account. Although containing somewhat more polyunsaturated fatty acids than either haemolymph or fat body, both the neutral lipid and phospholipid of flight muscle are similar in general pattern to haemolymph and fat body neutral lipid. This suggests that the developing flight muscle derives most of its fatty acids from haemolymph neutral lipid and that the phospholipids are then synthesized in situ. The fatty acid compositions of ovarian neutral lipid and phospholipid are very similar to the compositions of their respective haemolymph fractions at the same stage. This suggests that haemolymph neutral lipid and phospholipid are incorporated as such into the developing oiicyte. TELFER (1961) has shown that specific haemolymph proteins are incorporated into the developing oijcytes of Saturniid moths and THOMAS and GILBERT (1969) have demonstrated that they are lipoproteins. Thus, it is probable that the haemolymph lipids are incorporated into the developing oocytes as lipoprotein complexes. The reduction in polyunsaturated fatty acid content of ovarian lipids after the eighteenth day of pharate adult life (chorion already layed down) reflects the conversion of polyunsaturated fatty acids into saturated and monounsaturated fatty acids via acetyl-CoA. The dramatic increase in monoenoic fatty acids results from either a rapid turnover or synthesis accompanied by the incorporation of recently synthesized monounsaturated fatty acids. Two explanations are possible for the sexual dimorphism in the fatty acid composition of haemolymph neutral lipid. The first is based on the suggestions given above that in males the major path of fatty acids is to flight muscle via haemolymph neutral lipid while in females much of the fatty acids are transported to the ovaries via phospholipid. If the recently suggested hypothesis (BEENAKKERS and GILBERT, 1968) on lipid release in insects can be extended to include phospholipids, we would expect a depletion of the polyunsaturated fatty acids in the of males while females would present a more balanced ‘active compartment’ picture. The fact that flight muscle lipids have somewhat more polyunsaturated fatty acids than haemolymph suggests that at least part of the depletion of haemolymph neutral lipid polyunsaturates is due to preferential absorption. The second explanation is related to the possible control of lipid release by juvenile hormone.
The possible control of lipid synthesis and release by juvenile hormone Not only does the fatty acid composition of the total lipid, neutral lipid, and phospholipid of whole animals and specific organs vary with developmental stage, but the rates at which fatty acids are synthesized and oxidized also vary in Lipids are synthesized more rapidly by pharate pupae meaningful patterns. than by early or mid-fifth instar larvae. This is in accord with the observation that the amount of fat body increases dramatically just prior to spinning.
862
WILLIAMF. STEPHENJR. ANDLAWRENCE I. GILBERT
We can estimate from the data of GILBERT and SCHNEIDERMAN(1961) that juvenile hormone concentration is high in eggs and first instar larvae. It is lower but still significant during the fifth instar, being highest at the beginning, reaching a minimum just prior to spinning and then rising during spinning and pupation. Our data (STEPHEN and GILBERT, 1969) on the incorporation of acetate into lipid during the last larval instar can be inversely correlated with the juvenile hormone concentration. Animals injected on the seventeenth day of pharate adult life and sacrificed on the eighteenth, nineteenth, and twentieth days show much slower lipid synthesis than chilled pupae or four-day pharate adults. GILBERT and SCHNEIDERMAN(1961) have shown that no juvenile hormone is present during early pharate adult life but that it accumulates very rapidly starting on or about the seventeenth day. Our data on the synthesis of unsaturated fatty acids from acetate and palmitate (STEPHEN and GILBERT, 1969) show a similar inverse correlation with juvenile hormone concentration. Juvenile hormone has been shown to affect lipid metabolism, both in Vito (VROMAN et uZ., 1965) and in vitro (GILBERT, 1967b). Working with female Periplaneta americana, VROMAN et al. (1965) sh owed that allatectomy resulted in increased lipid content and increased synthesis of fatty acids (particularly unsaturated fatty acids) from acetate. GILBERT (1967b) demonstrated that incubation of Leucophaea maderae ovaries with active corpora allata stimulated the uptake of 14C-palmitate from the medium into the ovarian lipids. However juvenile hormone inhibited the uptake of 14C-palmitate by fat body lipid. These studies together with our data suggest that juvenile hormone is responsible for changes in fatty acid metabolism and also affects lipid transport. By utilizing our previous data (STEPHEN and GILBERT, 1969) and that of CHINO and GILBERT (1965a) we were able to conduct a detailed analysis of the kinetics of fatty acid synthesis from acetate in H. cecropia. We corrected the radioactive acetate pool for reduction due to oxidation to CO, and incorporation into lipid, and found that injection of acetate into chilled pupae resulted in the rate of synthesis of lipid being no less than 15 per cent greater during the first 24 hr following injection than during the second 24 hr. However, when we injected acetate into 17-day pharate adults, the rate of synthesis is at least 25 per cent greater during the second 24 hr period than during the first. Since four-day pharate adults synthesize lipid at a greater rate than chilled pupae (indicating increasing synthetic capacity), the decline in the rate of lipid synthesis may be due to the incorporation of acetate into protein. This is supported by the facts that SEDEE (1961) demonstrated that acetate is incorporated into amino acids by Calliphora erythrocephala and CHINO and GILBERT (1965a) have shown that acetate is not incorporated into glycogen in H. cecropia while the acetylation of glucosamine is highly unlikely at this stage of development. Two hypotheses can be invoked to explain the increasing rate of lipid synthesis late in development. First, the activity of fatty acid synthesizing enzymes increases during this period. This is unattractive in view of the demonstration of VROMAN et al. (1965) that active corpora allata inhibit fatty acid synthesis and
FATTYACIDCOMPOSITION OF H YALOPHORA
CECROPIA
863
that the titre of juvenile hormone in H. cecropiu rises very rapidly at this time (GILBERT and SCHNEIDERMAN,1961). Alternatively, the acetate could move from a pool that is unavailable for lipid synthesis to one that is. BEENAKKERSand GILBERT (1968) have presented evidence for the existence of inactive and active compartments within the insect fat body which function in lipid transport. If these components and the pools postulated above are identical, some important relationships are suggested. H. cecropia releases high proportions of saturated fatty acids into the haemolymph, yet the concentration of saturated fatty acids increases in whole males and the fat body of males during adult development. Females do not show this phenomenon. If synthesis of saturated fatty acids from non-lipid reserves were involved, one would expect females to show the greatest increase since they have more glycogen at all stages of adult development than males (DOMROESE and GILBERT, 1964). We have already explained why preferential oxidation of polyunsaturated fatty acids is not involved. Let us now consider the following hypothesis. Fat body triglycerider are hydrolyzed and the free fatty acids and monoglycerides are transported to an active compartment. In this compartment, saturated fatty acids are preferentially synthesized into diglycerides which are released into the haemolymph in the form of lipoproteins (CHINO and GILBERT, 1965b; BEENAKKERSand GILBERT, 1968). The polyenoic fatty acids remaining in the active compartment are oxidized to acetyl CoA which is resynthesized into saturated fatty acids which can then be released or transported back into the storage (inactive) compartment. Juvenile hormone acts at the point of resynthesis to block desaturation of palmitic and stearic acids. The higher concentrations of available saturated fatty acids would then stimulate lipid release. As seen above, acetate was actively synthesized into fatty acids in a relatively inaccessible compartment in 17-day pharate adults. Thus, the changes in saturated fatty acid concentration observed in male fat body and whole male pharate adults are explained. The fact that an increase in saturated fatty acid concentration is not observed in female fat body also fits this hypothesis. Females have much less juvenile hormone than males and the effect of juvenile hormone on the concentration of saturated fatty acids in females would therefore be expected to be much less than in males. This hypothesis also suggests a less involved explanation for the sexual difference observed in the haemolymph neutral lipid fatty acid composition late in development. The more intense and more rapid buildup of juvenile hormone in males would result in the release of more saturated fatty acids into the haemolymph in males than in females. A summary of the effects of juvenile hormone on lipid metabolism during the life history of H. cecropia as they have been suggested by this and other studies is now in order. During most of larval life, juvenile hormone concentrations are relatively high and the synthesis of fatty acids, especially unsaturated fatty acids, is low. Late in the fifth instar the juvenile hormone concentration drops to a low level
864
WILLIAMF. STEPHENJR. AND LAWRENCEI. GILBERT
and lipid is rapidly accumulated. As the concentration of juvenile hormone rises during spinning and pupation, the rate of lipid synthesis decreases. Chilled pupae and pharate adults, up to the seventeenth day, synthesize lipid readily, but after 17 days fatty acid synthesis is reduced while the relative rate of saturated fatty acid synthesis increases. Very high titres of juvenile hormone are found in adult males which require very large amounts of lipid for their mating flight. It has been shown that the overall metabolism of male H. cecropia is directed towards its mating flight (GILBERT and SCHNEIDERMAN,1961; DOMROESEand GILBERT, 1964). Perhaps the effects of juvenile hormone on lipid metabolism and release postulated above explain the previously unexplained high concentrations of juvenile hormone in the abdomens of male Saturniid moths. REFERENCES BEENAKKERS A. M. T. and GILBERTL. I. (1968) The fatty acid composition of fat body and haemolymph lipids in Hyalophora cecropiu and its relation to lipid release. J. Insect Physiol. 14, 481-494. CHINO H. and GILBERTL. I. (1965a) Studies on the interconversion of carbohydrate and fatty acid in Hyalophora cecropiu. J. Insect Physiol. 11, 287-295. CHINO H. and GILBERT L. I. (1965b) Lipid release and transport in insects. Biochim. biophys. Actu 98, 94-110. DOMROESE K. A. and GILBERTL. I. (1964) The role of lipid in adult development and flight muscle metabolism in Hyalophora cecropia. J. exp. Biol. 41, 573-590. FAST P. G. (1964) Insect lipids: A review. Mem. ent. Sot. Can. 37, S-50. GILBERT L. I. (1967a) Lipid metabolism and function in insects. Adw. Insect PhysioE. 4, 69-211. GILBERTL. I. (1967b) Changes in lipid content during the reproductive cycle of Leucophaea maderae and effects of the juvenile hormone on lipid metabolism in vitro. Comp. Biochem. Physiol. 21, 237-257. GILBERTL. I. and SCHNEIDERMAN H. A. (1961) The content of juvenile hormone and lipid in Lepidoptera: Sexual differences and developmental changes. Gen. camp. Endocr. 1, 453471. HILDITCH T. P. and WILLIAMS P. N. (1964) The Chemical Constitution of Natural Fats, 4th ed. Chapman & Hall, London. SEDEEP. D. J. W. (1961) Intermediary metabolism in aseptically reared blowfly larvae, Calliphova erythrocephala (Meig.)-II. Biosynthesis of fatty acids and amino acids. Arch. int. Physiol. Biochim. 69, 295-308. STEPHENW. F. JR. and GILBERT L. I. (1969) Fatty acid biosynthesis in the silkmoth, Hyalophora cecropia. J. Insect Physiol. 15, 1833-1854. TELFERW. H. (1961) The route of entry and localization of blood proteins in the oacytes of saturniid moths. 3’. biophys. biochem. Cytol. 9, 747-759. THOMAS K. K. and GILBERT L. I. (1967) Phospholipid synthesis during flight muscle development in the American silkworm, Hyalophora cecropia. Comp. Biochem. Physiol. 21, 279-290. THOMAS K. K. and GILBERT L. I. (1969) The haemolymph lipoproteins of the silkmoth Hyalophora gloveri: Studies on lipid composition, origin and function. Physiol. Chem. Physics 1, 293-3 11. VROMANH. E., KAPLANISJ. N., and ROBBINSW. E. (1965) Effect of allatectomy on lipid biosynthesis and turnover in the female American cockroach PeripZaneta americana (L.). g. Insect Physiol. 11, 897-904. YOUNG R. G. (1967) Fatty acids of some arthropods. Mem. Cornell Exp. St. 401, 2-14.
ALTERATIONS IN FATTY ACID COMPOSITION DURING THE METAMORPHOSIS OF HYALOPHORA CIX’ROPIA: CORRELATIONS WITH JUVENILE HORMONE TITRE* WILLIAM Department
F.
STEPHEN
Jr.t
of Biological Sciences,
and
LAWRENCE
Northwestern
I. GILBERT
University,
Evanston,
Illinois 60201
(Received 31 October 1969) Abstract-Gas chromatograpbic analyses of the total fatty acids of whole animals during development revealed that palmitic and stearic acids are always present in lower concentrations than their unsaturated homologues while both oleic and linolenic acid concentrations exceed that of linoleic acid. This also holds true for the neutral lipid fraction but the phospholipids possess a higher concentration of stearic acid. During late pharate adult development there are increases in the relative quantities of saturated and monounsaturated fatty acids and a decrease in polyunsaturated fatty acids. The triglyceride fraction of the fat body reflects the changes observed in the total and neutral lipid fraction of whole animals. During the last three days of pharate adult life the amount of palmitoleic acid doubles in the fat body neutral lipid. The composition of the haemolymph neutral lipid differs from that of the fat body and changes during development. Similarities in fatty acid composition between haemolymph and flight muscle lipid suggest that the developing flight muscle derives most of its fatty acids from haemolymph neutral lipid. The correlation between haemolymph and ovarian neutral and phospholipid fatty acids suggests that the haemolymph lipids are incorporated into the developing oijcytes. When the data are compared with that pertaining to juvenile hormone titre, it appears that juvenile hormone may control aspects of fatty acid synthesis and lipid transport and suggests a r61e for the high concentration of juvenile hormone in the male moth. INTRODUCTION
WE HAVE recently chain
saturated
shown and
monounsaturated
GILBERT, 1969). This dehydrogenate chain
lengthening
therefore
analyzed
morphosis place.
both
The
of
that Hyalophora cecropia can synthesize
insect to the
can lengthen
acid composition
data, when
whether
correlated
from
acetate
acid to stearic
monoenes.
are functions
H. cecropia to determine
present
acids
palmitic
corresponding
and desaturation the fatty
fatty
Our
data
of developmental
of several
tissues
developmental
with the juvenile
only
straight
(STEPHEN and acid and can suggested
that
stage and we
during
the meta-
alterations hormone
do take
content
of
* Supported by grant AM-02818 from the National Institutes of Health. t Former National Institutes of Health pre-doctoral fellow. Supported in part by NIH training grant GM-903. Present address : Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas. 851
8.52
WILLIAMF. STEPHEN JR. ANDLAWR~CE I. GILBERT
H. cecropia, furnish a reasonable explanation for the high concentration of juvenile hormone in the male moth. MATERIALS
AND METHODS
Notes on the handling of animals as well as methodology involved in extracting lipid, column chromatographic separation into discrete lipid classes, and gas chromatographic analyses of the methylated fatty acids have been detailed in the preceding paper (STEPHENand GILBERT, 1969). RESULTS
Total lipid Extraction of 68 animals revealed that: fifth instar larvae yielded an average of 54.6 mg lipid (144% of fresh weight) ; pharate pupae yielded an average of 174.0 mg (5.61% of fresh weight); whereas pupae contained an average of 360.1 mg of lipid (8.09% of fresh weight). Neutral lipid represented only 66.2 per cent of the total lipid in fifth instar larvae, but rose to 88.3 per cent in pharate pupae and 91.0 per cent in newly ecdysed pupae. The extreme difference in phospholipid content between a fifth instar larva and a pupa was only 26.5 mg while the least difference in neutral lipid content between these stages was 270.5 mg. We detected no significant changes in the absolute lipid content of male pharate adults although the average percent lipid of fresh weight rose from 9.39 per cent in early pharate adult development (0 to 5 days) to 11.73 per cent in late development (18 to 20 days). In view of the large increase in neutral lipid during pupation a sample of pupal fat body was extracted for further investigation. After removing contaminating phospholipids, the neutral lipid was further fractionated and the triglycerides were found to comprise 91.5 per cent of the total neutral lipid while other fractions made up only between O-6 and 2.4 per cent.
Fatty acid composition of whole animals during development Initially, fatty acid analyses were carried out on the methyl esters of whole chilled pupae and pharate adults. The average values for each fatty acid are found at the bottom of Table 1 and express the general pattern quite well. Palmitic and stearic acids are always present in lower concentrations than their unsaturated homologues and linoleic acid is present in lesser quantities than oleic and linolenic acids. In general, the quantity of palmitic acid slightly exceeds that of stearic acid while the percentage of oleic acid always exceeds that of palmitoleic acid. Trace peaks are not considered in these analyses because of their sporadic appearance and quantitative insignificance. Changes noted in the fatty acid composition between early pharate adult development (0 to 5 days) and late development (18 to 20 days) are: an increase in total saturated fatty acids from 1 to 3 per cent to 4 to 5 per cent; an increase in monounsaturated fatty acids ; a decrease in polyunsaturated acids.
FATTY ACID COMPOSITIONOF HYAL.OPHORA
853
CECROPIA
The fatty acid composition of a single fifth instar larva was determined and striking differences between it and pupae and pharate adults were observed. Not only are palmitic and stearic acids present in much higher amounts in the larva (12.31% and 4.03% respectively), but the quantity of palmitoleic acid (6.08%) is only half that of palmitic acid. There is also a 4 : 3 ratio of linolenic acid (42%0/b) to oleic acid (29.82%) as compared to the average 8 : 7 ratio of oleic acid to linolenic acid in pupae and pharate adults. Linolenic acid comprised 4.6 per cent of the total. TABLET-RELATIVEPERCENT FATTYACIDCOMPOSITION Day of pharate adult development O(3) l(4) 3(7) 5(l) 18(2) 19(l) 20(l) Average
OF LIPIDFROMMALEPHARATEADULTS
Fatty acid composition Cl6 :0 0.84 I.18 I.48 1.90 3.31 2.92 2.40 1.64
Cl6:l 7.52 16.28 15.37 6.40 13.76 14.17 13.33 13.51
Cl8:O
Cl8:l
Cl8:2
Cl8:3
0.65 0.72 0.95 0.80 1.69 2.05 1.56 1 a06
38.29 44.87 43.57 3140 41.38 41.05 43.12 41.99
9.60 2.97 4.48 11.60 3.07 3.65 2.76 5.06
43.08 33.59 34.09 47.90 36.91 36.09 36.75 36.68
Number in parentheses denotes number of determinations. a separate animal.
Each determination was on
Since both the ratios of neutral lipid to phospholipid and the fatty acid compositions of these fractions varied between larvae and pupae, the lipids of a number of animals were separated into neutral lipid and phospholipid fractions and the fatty acid compositions analyzed. Fatty
acid composition
of neutral lipids and phospholipids
of whole animals
The comments about pupal and pharate adult total lipid apply without exception to the neutral lipid of chilled pupae (Table 2). However, the phospholipids are characterized by a higher concentration of stearic acid (11 to 12%) than is found in the neutral or total lipids. The ratio of palmitoleic acid to palmitic acid is slightly more than 2 : 1 as opposed to the 4 : 1 to 20 : 1 ratios observed in the total and neutral lipids. The amount of linolenic acid is also at the lower limit of the range observed in the neutral and total lipid samples. Our data suggest that individual variation in fatty acid composition is less for phospholipids than neutral lipids. The fatty acid pattern of the neutral lipids extracted from 100 to 200 nonfeeding first instar larvae was different from those described in Table 2. Palmitic acid comprises 17 per cent of the total fatty acid complement and is more than three times the concentration of palmitoleic acid. The stearic acid content is high
854
WILLIAM F. STEPHEN JR. ANDLAWRENCEI. GILBERT
(8-l per cent) and the linolenic acid content lower (26 per cent) than in any of the neutral lipid or total lipid extracts alluded to above. This stage is characterized by a relatively high concentration of saturated fatty acids. The phospholipid fatty acids of first instar larvae are similar to those of the pupa except for the large quantity of palmitic acid in the larvae. The concentration of Cl8 acids corresponds well to that found in the pupal phospholipids. TABLE
~-FATTY
ACID
COMPOSITION
OF NEUTRAL
LIPID AND
PHOSPHOLIPID
OF WHOLE
H. cecropia First instar larvae*
Chilled pupae Fatty acids
Neutral lipid
Phospholipid
Neutral lipid
Phospholipid
C16:O C16:l C18:O C18:l C18:2 C18:3 c2o:o
0.76 11.51 0.35 44.57 3.71 38.98 -
2.95 5.28 11.60 37.73 12.60 29.73 -
17.01 5.03 8.13 39.19 4.58 26.03 -
8.13 1.18 13.38 35.31 9.63 30.82 1.61
* 100 to 200 larvae, 1 to 2 days after emergence were pooled for this experiment.
Fatty acid composition of fat body neutral lipid fractions Since the fatty acid compositions of neutral lipids and phospholipids were different, we determined the composition of the individual neutral lipid fractions to find out whether any particular fraction was responsible for the differences. Fat body lipid was chosen for these analyses because of the central metabolic role of the fat body and any differences in fatty acid composition of the neutral lipid classes might reflect the utilization of specific fatty acids. The data reveal that the composition of the triglyceride fraction is very similar to that of the fat body total lipid, and this in turn reflects the composition of the neutral or total lipids of whole chilled pupae. The partial glyceride fractions contain greater amounts of saturated acids (including arachidic acid) and lesser concentrations of linolenic acid. The sterol ester fraction is characterized by higher concentrations of monoenoic acids and the least amount of linolenic acid.
Fatty acid composition of whole animal phospholipids during adult development and life Although the fatty acid composition of the total lipid of H. cecropia changes little during adult development, changes in the phospholipid fatty acid composition could be masked by the large amount of neutral lipid present. Therefore, the phospholipid fatty acid compositions of pupae, pharate adults, and adults were determined (Table 3). Two developmental changes were observed. The first is a decrease in the concentrations of both palmitoleic and oleic acids as the animals develop and age. The second change is a corresponding increase in the amount of
FATTY ACID COMPOSITION OF HYALOPHORA
linolenic acid. less regular.
CECROPlA
855
Both males and females show this pattern although the females are
TABLE ~-RELATIVE
PER CENT FATTY ACID COMPOSITION OF WHOLE ANIMAL PHOSPHOLIPIDS DURING ADULT DEVELOPMENT* Developmental
stage
--
Fatty acid
pupae
15 to 19 day pharate adults
Male
C16:O C16:l C18:O C18:l C18:2 C18:3
6.38 9.16 9.16 45.91 10.09 19.30
6.85 6.32 8.20 41.53 11.42 25.67
5.13 3.96 11.12 39.36 9.20 31.23
5.28 2.11 9.19 38.01 9.08 36.32
Female
C16:O C16:l C18:O C18:l C18:2 Cl8:3
8.68 8.68 9.03 50.12 10.30 13.19
5.08 3.60 8.96 50.97 10.06 21.33
6.20 3.96 11.74 37.99 11.74 28.36
4.88 2.71 12.10 41.19 8.58 30.50
Chilled Sex
1 day adults
2 to 5 day adults -
* Each developmental stage represents the pooled extract of 8 to 12 animals. The females contained eggs and these contribute to the results obtained with female extract.
In view of these results, a further study of alterations in fatty acid composition during development was undertaken. The neutral and phospholipids of fat body, haemolymph, ovaries, and flight muscle were analyzed at various times during adult development and adult life.
Fatty acid composition of neutral lipids and phospholipids of selected tissues during adult development As noted above, the fatty acid composition of the fat body neutral lipid is similar to that of the fat body total lipid. Table 4 shows a twofold increase in the amount of palmitoleic acid in the fat body neutral lipid during the last 3 days of pharate adult life. The linoleic acid concentration decreases through adult development while the quantity of linolenic acid decreases from day 13 of pharate adult life to adult ecdysis. Fat body phospholipids are relatively high in palmitic acid (10 to 12 per cent), stearic acid (11 to 12 per cent), and linoleic acid (12 to 27 per cent). The amount of stearic acid rises during the first 13 days of pharate adult life. The palmitoleic acid content remains low and constant at about 2-S per cent. The oleic acid values (27 to 35 per cent) and the linolenic acid values (17 to 26 per cent) are lower than those of the neutral lipid.
856 TABLE
WILLIAM ~-RELATIVE
F. STEPHFX JR. ANDLAWRENCEI. GILBERT
PER CENT FATTY ACID COMPOSITION DEVELOPMENT*
OF FAT BODY
LIPIDS DURING
ADULT
Developmental stage
Fatty acid
Chilled pupae
10 day pharate adults
13 day pharate adults
18 day pharate adults
1 day adults
Neutral lipid
C16:O C16:l C18:O C18:l C18:2 C18:3
0.76 440 0.46 35.46 9.87 49.50
4.17 8.11 0.93 45.43 5.80 35.58
2.51 7.41 0.86 38.62 6.36 44.25
4.01 8.03 1.28 42.96 5.55 38.17
4.48 15.30 0.65 45.48 2.52 31.57
Phospholipid
C16:O C16:l C18:O C18:l C18:2 C18:3
9.73 2.46 10.90 35.30 18.67 22.92
12.46 2.41 13.85 26.82 27.19 17.27
10.64 2.46 20.57 28.67 11.52 26.13
-
-
Lipid fraction
-
* Each developmental stage represents the extract of pooled tissue from 2 to 5 female insects.
Haemolymph neutral lipids have an unusual composition and exhibit developmental change (Table 5). Palmitic acid is prominent and is much more abundant than palmitoleic acid during the first 13 days of pharate adult life. However, by the time of adult ecdysis, palmitoleic acid is in 2 : 1 excess, although palmitic acid is still present in greater amounts than are usually found in H. cecropiu extracts. During the first 13 days of pharate adult development, the concentration of stearic acid declines by about one-half and that of linoleic acid by about two-thirds. The concentration of both these Cl8 fatty acids decrease even more by the time of adult ecdysis. The linolenic acid content rises during the first 13 days of pharate adult life while the oleic acid concentration remains almost constant during that period. However, at ecdysis, the adults have 9 per cent more oleic acid and 10 per cent less linoleic acid than they did as 13 day pharate adults. The haemolymph phospholipids have 16 to 17 per cent stearic acid as their most unusual feature while the concentration of palmitic acid is three times greater than that of palmitoleic acid. The oleic acid concentration drops 6 per cent between 13 and 18 days of pharate adult development while the concentration of linolenic acid rises 14 per cent during the same period. The ovarian neutral lipids exhibit several unusual features (Table 6). The amount of palmitic acid rises dramatically during the first half of pharate adult development and then declines gradually until adult ecdysis. The palmitoleic acid concentration is only 4 to 5 per cent in pharate adults but is two to three times greater in the adult. The quantity of stearic acid rises modestly during pharate
OF HYALOPHORA
FATTY ACID COMPOSITION TABLE
S---RELATIVE PER CENT
FATTY ACID COMPOSITION ADULT DEVELOPMENT*
OF HAEMOLYMPH
Developmental
857
CECROPIA
LIPIDS DURING
stage
-
Fatty acid
Chilled pupae
10 day pharate adults
Neutral lipid
C16:O C16:l Cl8:O C18:l C18:2 C18:3
IO.82 5.59 4.66 34.60 11.39 32.95
15.86 3.85 3.23 30.82 9.95 36.29
12-18 4.81 2.19 33.38 4.47 39.96
-
7.84 13.84 1.66 42.40 4.16 30.40
Phospholipid
C16:O C16:l C18:O Cl8:l C18:2 C18:3
-
-
11.53 3.51 15.68 29.43 11.90 27.95
5.88 2.34 16.95 23.10 9.43 42.30
-
Lipid fraction
* Each insects.
TABLE
developmental
6--RELATIVEPER
stage
represents
CENTFATTY
13 day pharate adults
18 day pharate adults
1 day adults
the extract
ACID COMPOSITION ADULT DEVELOPMENT*
of pooled
tissue
of 2 to 5 female
OF OVARIAN AND EGG LIPIDS DURING -
Developmental
pupae
10 day pharate adults
18 day pharate adults
1 day adults
3 day adults
Cl6:O C16:l C18:O C18:l C18:2 C18:3
2.81 3.71 2.30 35-17 12.83 43.19
13.00 5.26 3.32 29-87 10.37 38.17
11.50 5.21 4.20 37.62 6.63 34.84
8.78 14.08 2.41 47.54 2.31 25.88
8.55 7.74 2.61 46.74 3.18 31.19
C16:O C16:l C18:O C18:l C18:2 C18:3
10.83 2.27 11.17 33.59 19.98 23.17
12.58 I.54 18.19 25.61 13.58 28.51
9.03 2.29 17.52 32.61 10.51 28.03
9.35 13.55 3.27 50.00 2.80 21.03
8.64 13.64 5.91 48.64 3.64 19.55
Fatty acid
Chilled
Neutral lipid
Phospholipid
Lipid fraction
* Each
stage
developmental
stage represents
the extract
of pooled
tissue of 2 to 5 animals.
8.58
WILLIAM F. STEPHENJR. ANDLAWRENCEI. GILBERT
adult life but drops to the pupal level at adult ecdysis. The concentrations of linoleic and linolenic acid decrease constantly from chilled pupa to adult while the oleic acid concentration rises during late pharate adult life. The ovarian phospholipids provide an interesting developmental picture. Although the amount of palmitic acid varies between 9 to 13 per cent, the palmitoleic acid content is only 2 per cent during pharate adult development but rises dramatically to 14 per cent at adult ecdysis. The stearic acid concentration is 18 per cent during the latter part of the pharate adult life but is only 3 to 6 per cent in the adult while the quantity of oleic acid almost doubles during the same developmental period. The linoleic acid content decreases dramatically (from 20 to 3 per cent) during adult development while the linolenic acid concentration drops only slightly. The data collected on flight muscle neutral lipids were not sufficient to show developmental trends. However, palmitic acid is moderately prominent, being present in equal or greater concentrations than palmitoleic acid. In other respects, these samples have typical neutral lipid fatty acid patterns. The phospholipid fatty acids show essentially no change from late pharate adult life to adult ecdysis with the exception of the decrease in linoleic acid concentration and a corresponding increase in the quantity of oleic acid. TABLE
~----RELATI~E PER CENT
FATTY
ACID
COMPOSITION
OF FLIGHT
MUSCLE
LIPIDS DURING
LATEADULTDEVELOPMENT * Developmental stage
Lipid fraction Neutral lipid
Phospholipid
Fatty acid
18 day pharate adults
C16:O C16:l C18:O C18:l C18:2 C18:3
8.36 5.78 3.01 31.00 11.56 40.28
C16:O C16:l C18:O C18:l C18:2 C18:3
8.15 3.49 5.20 33.33 14.65 35.18
1 day adults
7.55 4.41 4.80 37.06 7.95 38.24
3 day adults 4.23 4.23 0.98 37.81 7.58 45.18 6.14 2.68 4.22 42.86 7.67 36.43
* Each developmental stage represents the extract of pooled tissue of 2 to 5 female insects. DISCUSSION
The pattern of total lipid change that can be deduced from these studies is that lipid (almost entirely neutral lipid) is accumulated during the latter portion of the fifth instar and that a further increase occurs during spinning. Then, a small
FAlTY ACID COMPOSITION
OF HYALOPHORA
CECROPIA
859
amount is lost as the pupae enter diapause. When adult development commences, males conserve lipid while females utilize it. Key points, documented by DOMROE~Eand GILBERT (1964) are that H. cecropia derives its energy for flight almost solely from the oxidation of fatty acids and that the male conserves its lipid during pharate adult life since it is the more active flier. The qualitative nature and biosynthesis of the fatty acids of H. cecropia have been discussed previously (STEPHENand GILBERT, 1969). Here, our interest lies in the quantitative relations of these fatty acids and the various parameters which affect these relations. Fatty acid composition of whole animals through development Although most insects have a high proportion of unsaturated fatty acids (FAST, 1964), H. cecropia is extreme in this regard. Over 97 per cent of the fatty acids of pupae and early pharate adults are unsaturated as are more than 90 per cent of the fatty acids of adults (YOUNG, 1967). Larvae have a greater proportion of saturated fatty acids than other developmental stages. The fatty acid pattern most characteristic of insect lipid is large amounts of palmitic and oleic acids and a concentration of linoleic acid exceeding that of linolenic acid. Most Diptera have large amounts of palmitoleic acid and most Lepidoptera more linolenic than linoleic acid (FAST, 1964; GILBERT, 1967a). The fatty acid composition of H. cecropia is intermediate between that of the majority of Lepidoptera and that of Diptera. Linolenic and oleic acids are the most abundant fatty acids, being found in concentrations far exceeding that of linoleic acid while palmitic acid is the major saturated fatty acid. It is the relatively large quantity of palmitoleic acid that gives H. cecropiu lipid its somewhat dipteran character. The newly emerged first instar larva has about 22 to 23 per cent saturated acids and a relatively small amount of palmitoleic acid. By the fifth instar, the relative quantity of saturated fatty acids has fallen to 16 per cent. According to HILDITCH and WILLIAMS (1964) the major fatty acids of leaves are palmitic, oleic, linoleic, and linolenic acids with linolenic acid being most prominent, followed by linoleic, palmitic and oleic acids. About equal amounts of palmitic and palmitoleic acids are formed from acetate by the fifth instar larvae (STEPHENand GILBERT, 1969). Th us, the prominence of palmitic acid at this stage must be due to a slow rate of desaturation of dietary palmitic acid. The changes we observe in the fatty acid composition of whole pharate adults are probably due to the oxidation of fatty acids to acetyl-CoA followed by resynthesis of the fatty acid chains. The fact that we observed this phenomenon directly by the use of r4C-labelled fatty acids in H. cecropia (STEPHEN and GILBERT, 1969) and that males conserve lipid during adult development support this conclusion. Preferential oxidation of polyunsaturated fatty acids is not a factor since it has been shown that if any oxidative preference exists, it favours saturated and monounsaturated fatty acids (STEPHENand GILBERT, 1969; BEENAKKERS and GILBERT, 1968). In first instar larvae, the concentrations of both juvenile hormone (GILBERT and SCHNEIDERMAN, 1961) and saturated fatty acids are high. In fifth instar larvae,
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the concentration of juvenile hormone is much lower and the concentration of saturated fatty acids also drops significantly. In chilled pupae and early pharate adults there is no juvenile hormone and saturated fatty acids represent at most 3 per cent of the total fatty acids. However post-17 day pharate adults show increases in the concentration of both saturated fatty acids and juvenile hormone. In adults, where the juvenile hormone concentration is quite high, YOUNG (1967) reports that saturated fatty acids comprise 10 per cent of the total fatty acids. The means by which juvenile hormone may act to affect the concentration of saturated fatty acids and the relation of this increased concentration of saturated fatty acids to the developmental metabolism of H. cecropiu will be discussed subsequently. Although phospholipids comprise less than 10 per cent of the pupal lipids, they account for over 60 per cent of the total saturated fatty acids. Stearic acid content is high and relatively constant (9 to 13 per cent). In chilled pupae, the concentration of phospholipid palmitic acid is about four times that of the neutral lipid palmitic acid. However, first instar larval neutral lipids have twice the palmitic acid of larval phospholipids. Two major changes in phospholipid fatty acid composition occur during development. They are a regular decline in the concentration of monoenoic acids and a corresponding increase in that of linolenic acid. THOMAS and GILBERT (1967) have shown that the amount of cardiolipin in H. cecropiu flight muscle increases markedly as the pharate adults develop. Since 77 per cent of the fatty acids of H. cecropiu cardiolipin is linolenic acid, perhaps the increase in the amount of this phospholipid with its specific requirement for linolenic acid accounts for the relative rise in linolenic acid. Fatty acid composition changes in neutral lipid and phospholipid of selected tissues The fatty acid composition of specific insect organs and tissues during development has been the subject of few studies. BEENAKKER~and GILBERT (1968) analyzed the fatty acid composition of lipid fractions from male H. cecropia fat body and haemolymph in both chilled pupae and pharate adults just prior to adult ecdysis. Their data are comparable with the present results on total neutral lipid because triglyceride represents such a large proportion of fat body neutral lipids and diglyceride is almost the sole constituent of haemolymph neutral lipids. Changes in the fat body fatty acid composition show the conversion of polyunsaturated fatty acids into saturated and monounsaturated fatty acids, because the alternative mechanisms of preferential fatty acid oxidation and release would produce a change in the opposite direction. In the haemolymph of females, we note a decrease in the concentrations of saturated fatty acids and linolenic acid during the latter part of adult development. BEENAKKER~and GILBERT (1968) reported significant increases in the concentrations of saturated fatty acids and a much lower concentration of linolenic acid in male H. cecropiu. This sexual dichotomy may be explained if we consider that the amount of lipid in the haemolymph is small and fatty acid concentrations are the result of the dynamic processes of release and absorption.
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To analyze these processes we must first consider the contributions of other organs. In males, the major developing structure is the flight muscle while in females both flight muscle and ovaries must be taken into account. Although containing somewhat more polyunsaturated fatty acids than either haemolymph or fat body, both the neutral lipid and phospholipid of flight muscle are similar in general pattern to haemolymph and fat body neutral lipid. This suggests that the developing flight muscle derives most of its fatty acids from haemolymph neutral lipid and that the phospholipids are then synthesized in situ. The fatty acid compositions of ovarian neutral lipid and phospholipid are very similar to the compositions of their respective haemolymph fractions at the same stage. This suggests that haemolymph neutral lipid and phospholipid are incorporated as such into the developing oiicyte. TELFER (1961) has shown that specific haemolymph proteins are incorporated into the developing oijcytes of Saturniid moths and THOMAS and GILBERT (1969) have demonstrated that they are lipoproteins. Thus, it is probable that the haemolymph lipids are incorporated into the developing oocytes as lipoprotein complexes. The reduction in polyunsaturated fatty acid content of ovarian lipids after the eighteenth day of pharate adult life (chorion already layed down) reflects the conversion of polyunsaturated fatty acids into saturated and monounsaturated fatty acids via acetyl-CoA. The dramatic increase in monoenoic fatty acids results from either a rapid turnover or synthesis accompanied by the incorporation of recently synthesized monounsaturated fatty acids. Two explanations are possible for the sexual dimorphism in the fatty acid composition of haemolymph neutral lipid. The first is based on the suggestions given above that in males the major path of fatty acids is to flight muscle via haemolymph neutral lipid while in females much of the fatty acids are transported to the ovaries via phospholipid. If the recently suggested hypothesis (BEENAKKERS and GILBERT, 1968) on lipid release in insects can be extended to include phospholipids, we would expect a depletion of the polyunsaturated fatty acids in the of males while females would present a more balanced ‘active compartment’ picture. The fact that flight muscle lipids have somewhat more polyunsaturated fatty acids than haemolymph suggests that at least part of the depletion of haemolymph neutral lipid polyunsaturates is due to preferential absorption. The second explanation is related to the possible control of lipid release by juvenile hormone.
The possible control of lipid synthesis and release by juvenile hormone Not only does the fatty acid composition of the total lipid, neutral lipid, and phospholipid of whole animals and specific organs vary with developmental stage, but the rates at which fatty acids are synthesized and oxidized also vary in Lipids are synthesized more rapidly by pharate pupae meaningful patterns. than by early or mid-fifth instar larvae. This is in accord with the observation that the amount of fat body increases dramatically just prior to spinning.
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We can estimate from the data of GILBERT and SCHNEIDERMAN(1961) that juvenile hormone concentration is high in eggs and first instar larvae. It is lower but still significant during the fifth instar, being highest at the beginning, reaching a minimum just prior to spinning and then rising during spinning and pupation. Our data (STEPHEN and GILBERT, 1969) on the incorporation of acetate into lipid during the last larval instar can be inversely correlated with the juvenile hormone concentration. Animals injected on the seventeenth day of pharate adult life and sacrificed on the eighteenth, nineteenth, and twentieth days show much slower lipid synthesis than chilled pupae or four-day pharate adults. GILBERT and SCHNEIDERMAN(1961) have shown that no juvenile hormone is present during early pharate adult life but that it accumulates very rapidly starting on or about the seventeenth day. Our data on the synthesis of unsaturated fatty acids from acetate and palmitate (STEPHEN and GILBERT, 1969) show a similar inverse correlation with juvenile hormone concentration. Juvenile hormone has been shown to affect lipid metabolism, both in Vito (VROMAN et uZ., 1965) and in vitro (GILBERT, 1967b). Working with female Periplaneta americana, VROMAN et al. (1965) sh owed that allatectomy resulted in increased lipid content and increased synthesis of fatty acids (particularly unsaturated fatty acids) from acetate. GILBERT (1967b) demonstrated that incubation of Leucophaea maderae ovaries with active corpora allata stimulated the uptake of 14C-palmitate from the medium into the ovarian lipids. However juvenile hormone inhibited the uptake of 14C-palmitate by fat body lipid. These studies together with our data suggest that juvenile hormone is responsible for changes in fatty acid metabolism and also affects lipid transport. By utilizing our previous data (STEPHEN and GILBERT, 1969) and that of CHINO and GILBERT (1965a) we were able to conduct a detailed analysis of the kinetics of fatty acid synthesis from acetate in H. cecropia. We corrected the radioactive acetate pool for reduction due to oxidation to CO, and incorporation into lipid, and found that injection of acetate into chilled pupae resulted in the rate of synthesis of lipid being no less than 15 per cent greater during the first 24 hr following injection than during the second 24 hr. However, when we injected acetate into 17-day pharate adults, the rate of synthesis is at least 25 per cent greater during the second 24 hr period than during the first. Since four-day pharate adults synthesize lipid at a greater rate than chilled pupae (indicating increasing synthetic capacity), the decline in the rate of lipid synthesis may be due to the incorporation of acetate into protein. This is supported by the facts that SEDEE (1961) demonstrated that acetate is incorporated into amino acids by Calliphora erythrocephala and CHINO and GILBERT (1965a) have shown that acetate is not incorporated into glycogen in H. cecropia while the acetylation of glucosamine is highly unlikely at this stage of development. Two hypotheses can be invoked to explain the increasing rate of lipid synthesis late in development. First, the activity of fatty acid synthesizing enzymes increases during this period. This is unattractive in view of the demonstration of VROMAN et al. (1965) that active corpora allata inhibit fatty acid synthesis and
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that the titre of juvenile hormone in H. cecropiu rises very rapidly at this time (GILBERT and SCHNEIDERMAN,1961). Alternatively, the acetate could move from a pool that is unavailable for lipid synthesis to one that is. BEENAKKERSand GILBERT (1968) have presented evidence for the existence of inactive and active compartments within the insect fat body which function in lipid transport. If these components and the pools postulated above are identical, some important relationships are suggested. H. cecropia releases high proportions of saturated fatty acids into the haemolymph, yet the concentration of saturated fatty acids increases in whole males and the fat body of males during adult development. Females do not show this phenomenon. If synthesis of saturated fatty acids from non-lipid reserves were involved, one would expect females to show the greatest increase since they have more glycogen at all stages of adult development than males (DOMROESE and GILBERT, 1964). We have already explained why preferential oxidation of polyunsaturated fatty acids is not involved. Let us now consider the following hypothesis. Fat body triglycerider are hydrolyzed and the free fatty acids and monoglycerides are transported to an active compartment. In this compartment, saturated fatty acids are preferentially synthesized into diglycerides which are released into the haemolymph in the form of lipoproteins (CHINO and GILBERT, 1965b; BEENAKKERSand GILBERT, 1968). The polyenoic fatty acids remaining in the active compartment are oxidized to acetyl CoA which is resynthesized into saturated fatty acids which can then be released or transported back into the storage (inactive) compartment. Juvenile hormone acts at the point of resynthesis to block desaturation of palmitic and stearic acids. The higher concentrations of available saturated fatty acids would then stimulate lipid release. As seen above, acetate was actively synthesized into fatty acids in a relatively inaccessible compartment in 17-day pharate adults. Thus, the changes in saturated fatty acid concentration observed in male fat body and whole male pharate adults are explained. The fact that an increase in saturated fatty acid concentration is not observed in female fat body also fits this hypothesis. Females have much less juvenile hormone than males and the effect of juvenile hormone on the concentration of saturated fatty acids in females would therefore be expected to be much less than in males. This hypothesis also suggests a less involved explanation for the sexual difference observed in the haemolymph neutral lipid fatty acid composition late in development. The more intense and more rapid buildup of juvenile hormone in males would result in the release of more saturated fatty acids into the haemolymph in males than in females. A summary of the effects of juvenile hormone on lipid metabolism during the life history of H. cecropia as they have been suggested by this and other studies is now in order. During most of larval life, juvenile hormone concentrations are relatively high and the synthesis of fatty acids, especially unsaturated fatty acids, is low. Late in the fifth instar the juvenile hormone concentration drops to a low level
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and lipid is rapidly accumulated. As the concentration of juvenile hormone rises during spinning and pupation, the rate of lipid synthesis decreases. Chilled pupae and pharate adults, up to the seventeenth day, synthesize lipid readily, but after 17 days fatty acid synthesis is reduced while the relative rate of saturated fatty acid synthesis increases. Very high titres of juvenile hormone are found in adult males which require very large amounts of lipid for their mating flight. It has been shown that the overall metabolism of male H. cecropia is directed towards its mating flight (GILBERT and SCHNEIDERMAN,1961; DOMROESEand GILBERT, 1964). Perhaps the effects of juvenile hormone on lipid metabolism and release postulated above explain the previously unexplained high concentrations of juvenile hormone in the abdomens of male Saturniid moths. REFERENCES BEENAKKERS A. M. T. and GILBERTL. I. (1968) The fatty acid composition of fat body and haemolymph lipids in Hyalophora cecropiu and its relation to lipid release. J. Insect Physiol. 14, 481-494. CHINO H. and GILBERTL. I. (1965a) Studies on the interconversion of carbohydrate and fatty acid in Hyalophora cecropiu. J. Insect Physiol. 11, 287-295. CHINO H. and GILBERT L. I. (1965b) Lipid release and transport in insects. Biochim. biophys. Actu 98, 94-110. DOMROESE K. A. and GILBERTL. I. (1964) The role of lipid in adult development and flight muscle metabolism in Hyalophora cecropia. J. exp. Biol. 41, 573-590. FAST P. G. (1964) Insect lipids: A review. Mem. ent. Sot. Can. 37, S-50. GILBERT L. I. (1967a) Lipid metabolism and function in insects. Adw. Insect PhysioE. 4, 69-211. GILBERTL. I. (1967b) Changes in lipid content during the reproductive cycle of Leucophaea maderae and effects of the juvenile hormone on lipid metabolism in vitro. Comp. Biochem. Physiol. 21, 237-257. GILBERTL. I. and SCHNEIDERMAN H. A. (1961) The content of juvenile hormone and lipid in Lepidoptera: Sexual differences and developmental changes. Gen. camp. Endocr. 1, 453471. HILDITCH T. P. and WILLIAMS P. N. (1964) The Chemical Constitution of Natural Fats, 4th ed. Chapman & Hall, London. SEDEEP. D. J. W. (1961) Intermediary metabolism in aseptically reared blowfly larvae, Calliphova erythrocephala (Meig.)-II. Biosynthesis of fatty acids and amino acids. Arch. int. Physiol. Biochim. 69, 295-308. STEPHENW. F. JR. and GILBERT L. I. (1969) Fatty acid biosynthesis in the silkmoth, Hyalophora cecropia. J. Insect Physiol. 15, 1833-1854. TELFERW. H. (1961) The route of entry and localization of blood proteins in the oacytes of saturniid moths. 3’. biophys. biochem. Cytol. 9, 747-759. THOMAS K. K. and GILBERT L. I. (1967) Phospholipid synthesis during flight muscle development in the American silkworm, Hyalophora cecropia. Comp. Biochem. Physiol. 21, 279-290. THOMAS K. K. and GILBERT L. I. (1969) The haemolymph lipoproteins of the silkmoth Hyalophora gloveri: Studies on lipid composition, origin and function. Physiol. Chem. Physics 1, 293-3 11. VROMANH. E., KAPLANISJ. N., and ROBBINSW. E. (1965) Effect of allatectomy on lipid biosynthesis and turnover in the female American cockroach PeripZaneta americana (L.). g. Insect Physiol. 11, 897-904. YOUNG R. G. (1967) Fatty acids of some arthropods. Mem. Cornell Exp. St. 401, 2-14.