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The relationships between corpora allata and fat body and haemolymph lipids in the adult female desert locust
J. Insect Physiol., 1974, Vol. 20, pp. 2143 to 2156. Pergmon
Press. Printed in Great Britain
THE RELATIONSHIPS BETWEEN CORPORA ALLATA AND FAT BODY AND H~MOLYMPH LIPIDS IN THE ADULT FEMALE DESERT LOCUST L. HILL
and M. E. G. IZATT
Department of Zoology, The University, Sheffield SlO 2TN, England
(Received 29 March 1974) Abstract-Allatectomy of l-day-old female desert locusts resulted in an accumulation of lipid in the fat body. This accumulation of lipid was due to the continuation of lipid deposition in the fat body after the period of somatic growth. Somatic growth and feeding activity were unaffected by allatectomy, and so could not be indirect causes of fat body lipid accumulation. Lipid accumulation in allatectomized locusts is more likely to be related directly to a lack of juvenile hormone. Implantation of active corpora allata into l-day-old adult female locusts resulted in a premature development of oiicytes and a
decrease in fat body lipid accumulation; somatic growth was not inhibited. Im~l~tat~on of active corpora allata into old allatectomized locusts resulted in a decrease in the fat body lipid content and the onset of obcyte development. The
lipid synthetic activity of the fat body, measured by the incorporation of r4Cacetate into total fat body lipid, was greatly increased in allatectomized locusts after the period of somatic growth. The protein synthetic activity of the fat body, measured by the incorporation of VI-leucine into total fat body protein, remained low after the period of somatic growth in allatectomized insects. Juvenile hormone might thus have a dual effect on fat body metabolism, that is suppressing lipid synthesis and stimulating vitellogenic protein synthesis. Increased synthesis of lipid by the fat body would then account for the accumulation of lipid in the fat body after allatectomy. Inhibition of release of lipid from the fat body is unlikely to play a part in the accumulation as allatectomy had no effect on haemolymph lipid concentrations. INTRODUCTION
ALTHOUGH abnormal accumulation of lipid in the fat body following allatectomy has been reported in a variety of insects (WEED-PFEIFFER,1945; VOGT, 1949; THOMS~, 1952; BODENSTHN, 1953; ODHIAMBO, 1966; STRONG, 1968a; EL-IBRASHY and BOCTOR,1970), there is no generally accepted explanation for this phenomenon. In ~@~n~~ ~~~~~i~~ (WED-P~IFFER, 1945) and ~~~~~~a algae (VROMANet al., 1965) it was suggested that the corpora allata regulate fat body lipid content by a control of the mechanisms of lipid utilization, but in Leucophaea maderae the suppression of fat body lipogenesis in vitro by the addition of juvenile hormone suggested a direct effect of the hormone on fat body lipid synthesis (GILBERT, 1967). In ~c~~~~~~ff gregaria various indirect effects of allatectomy on fat body lipids have been proposed. For example, that allatectomy resulted in a decrease 2143
2144
L. HILL ANDM. E. G. IZATT
in locomotor activity and thus an accumulation of the fat body lipid reserves (ODHIAMBO, 1966), or that after allatectomy food consumption and growth were increased and the increase of lipid in the fat body was only one aspect of this general effect (WALKER and BAILEY, 1971a-c). In a recent review WYATT (1972) suggested that the available evidence pointed to a direct effect of juvenile hormone on fat body lipid metabolism. In this paper data are presented on the lipids of the fat body and haemolymph of the female desert locust, related to growth and feeding activity, which support this view and help resolve some of the contradictions in previous work on locusts.
MATERIALS
AND METHODS
The adult female desert locusts, Schistocerca gregaria, used in this study were taken from a colony reared in Sheffield at a temperature of 30°C with a constant photoperiod of 12 hr. The insects were reared under crowded conditions and fed a diet of bran and freshly cut barley. One experimental group of animals was fed a diet of bran and fresh lettuce. The methods for the measurement of growth, feeding activity, and fat body and haemolymph lipid content have been previously described (HILL et uZ., 1968, 1972). Allatectomy was performed by the method of STRONG (1963), except that the insects were not covered with saline. Active corpora allata for implantation were taken from mature adult male Locusta migrutoriu (JOHNSONand HILL, 1973) and immediately implanted into adult female desert locusts through a small slit in the side of the abdomen which was then sealed with paraffin wax. Ovariectomy was performed as described by HILL (1962). The DNA content of whole fat bodies was measured with the diphenylamine reagent (SCHNEIDER, 1957). The ability of the fat body to synthesize lipid and protein was measured by the incorporation of labelled precursors into total fat body lipid and total fat body protein. The whole abdominal fat body was dissected from each locust and divided longitudinally into two equal parts. Each part was weighed and placed in 2 ml of incubation medium (WALKER and BAILEY, 1970). 14C-sodium acetate (0.5 &i) (R ad’ioc h emical Centre, Amersham) was added to one part of the fat body and 5 ,&i 3H-leucine (Radiochemical Centre, Amersham) was added to the other part. The fat body portions were incubated for 1 hr at 30°C and then the incubation stopped by the addition of 0.5 ml of 25% trichloroacetic acid. The total lipid content was extracted from the acetate incubation, purified (WALKER and BAILEY, 1970), and dissolved in 10 ml scintillation medium (PPO, 7 g; POPOP, 0.6 g; ethoxyethanol, 500 ml; toluene, 1000 ml). The total protein content of the leucine incubation was purified (HILL, 1965), the solid protein dissolved in O-2 ml 1 N NaOH, and 10 ml scintillation medium added. Samples were counted in a Packard Tricarb scintillation counter. All counts were corrected for background and expressed as dis/min for the lipid or protein fraction of the whole fat body.
2145
RELATIONSHIPS BEZWEEN LOCUST CORPORA ALLATA, FAT BODY AND LIPIDS
RESULTS Somatic gr~th
ati feeding activity
AZZatectomy and growth.
The effect of allatectomy of l-day-old adult female desert locusts on growth is shown in Table 1. The somatic dry weight of the locust is the total dry weight minus the weight of the ovaries and fat body. The pattern of growth was essentially the same whether the somatic dry weight, the dry weight of the flight muscles, or the dry weight of a cuticle sample was measured. There were no consistent differences in growth pattern between normal and allatectomized adult female locusts, and allatectomized locusts did not grow significantly more than control locusts.
I
2
3
4
5
6
Days FIG.
1. The
total
dry
weight l-day-old
*-*
Allatectomized
o-0
Control
7
6
after
of food
IO
9
I,
12
13
14
15
16 17
I6
IS
locust
after
allatectomy
operation
consumed
aduit female
desert
per
of
locusts.
~rn~~a~tat~~ of active corpora allata. The implantation of four active corpora aliata into adult female desert locusts 1 day after the final ecdysis resulted in the premature deposition of yolk in the proximal o8cytes. Five days after the implantation the mean length of the proximal oiicytes was 54i mm. Ten days after implantation the mean length of the proximal oijcytes was 73 mm, that is almost fully developed, whilst in control animals of this age yolk deposition was just starting in the proximal oiicytes. There was no inhibition of growth associated with the premature oijcyte development which resulted from the impl~tation of active corpora allata (Table 2). The pattern of growth was, however, changed. Somatic growth appeared to be compressed into the first 5 days after the implantation, and between the fifth and tenth days after the implantation, when the metabolic demands of the developing oiicytes would be greatest (HILL et al., 1968), little further somatic growth occurred. Allatectomy and food consumption. Fig. 1 shows the total food intake of adult female desert locusts ~latectomized 1 day after the final ecdysis and of control animals. Although the period of intense feeding associated with somatic growth
538 + 602 f 7.59 + 754 f 794 + 791 f
27 37 36 32 23 39
Allatectomized
69.5 90.1 112.8 97.2 76.6 103.8
* 6.0 + 8.1 f. 13.0 + 5~5 + 10.9 + 11.5
30.3 f 3.9
Controls
50.2 79.8 121.9 124.0 129.3 llS*l
zk2.4 zk3.6 + 70 f 9-O + 6.3 f 13.1
-
Allatectomized
Flight muscle weight (mg f SE.)
5.8 6.4 8.0 6-l 9.1 11.5
z!z0.6 + 0.3 c 0.6 f 0.2 k 0.8 +0*4
3.0 f 0.2
Controls
4.0 4.5 6.5 7.2 7.5 8.6
+ 0.4 f 0.2 f 0.4 + 0.5 * 0.3 + 0.6
-
Allatectomized
Tergites’ weight (mg 4 SE.)
657 + 30 631+43 608+12
-
Implants
Each figure is the mean of five observations.
328 f 30
450 + 23 642 + 30 626 + 24
5 10 30
Controls
0
Days after implantation
Somatic weight (mg f. SE.)
69.5 -I 6-O 90.1 f a.1 97.2 f 5.5
30‘3 k 3.9
Control5
al.6 i 8.7 90.5 + 5.5
83-o+ 3.1
-
Implants
Flight muscle weight (mg + SE)
5.8 + 0.6 6.4 f 0.3 6.1 rt 0.2
3-o It 0.2
Controls
8.2 & 0.5 6.9 f 0.6 9.1 f 0.4
-
Implants
Tergites’ weight (mg f S.E.)
TA~LE~--THEEFFECTSOFTHEIMPLANTATIONOFFOURACTIVECORPORAALLATAINTO l-day-old ADULTFEMALEDESERTLOCUSTS ONSOMATICDRYWEIGHT,DRYUTEIGHTOFTHEFLIGHTMUSCLES,ANDDRYWEIGHTOFTHESECOND,THIRD,ANDFOURTHABDOMINAL TERGITRS
Each figure is the mean of five observations.
23 30 36 24 42 41
328 + 30
450 f 642 f 686 + 626 f 632 f 7.52 +
0
5 10 20 30 40 50
Controls
Days after operation
Somatic weight (mg f S.E.)
TABLE I-THEEFFECTSOFALLATECTOMYOF~ -day-old ADULTFEMALEDESERTLOCUSTSONSOMATICDRY WEIGHT, DRYWEIGHTOF THEFLIGHT MUSCLES, ANDDRYWEIGHT OF THESECOND,THIRD,ANDFOuRTHABDOMINALTERGITES
RELATIONSHIPSBETWEEN LOCUST CORPORAALLATA, FAT BODY AND LIPIDS
2147
was reduced in allatectomized animals, both groups consumed about the same total quantity of food during the 19-day period; 5.01 g by control animals and 4.83 g by allatectomized animals. Fat body DNA content. The total fat body DNA content of adult female desert locusts allatectomized 1 day after the final ecdysis is shown in Table 3. There were no differences between allatectomized and control animals. TABLE ~---THE EFFECT OF ALLATECTOMY OF ~-DAY-OLD ADULT FEMALE DESERT LOCUSTS ON THE TOTAL DNA CONTENT OF THE FAT BODY Total fat body DNA
content
@g rt S.E.) Days after operation 0 5 10 20 30 40
Controls 56+9 87~10 102 + 10 145+5 169440 133It13
Allatectomized 106+17 131-118 173 -1:21 193 + 21 129 4 37
Each figure is the mean of five observations.
Fat body lipid content Eflect of diet. Fig. 2 shows the total lipid content of the fat body during somatic growth and the first gonotrophic cycle of adult female desert locusts fed on bran and fresh barley or bran and fresh lettuce. The pattern of lipid accumulation was the same in both cases, but the total quantity of lipid stored in the fat body was greater in the barley fed animals. Effects of allatectomy. Fig. 3 shows the total fat body lipid content after allatectomy of l-day-old adult female locusts. During the period of somatic growth, that is the first 10 days of adult life, there were no differences between allatectomized and control animals, in both cases there was an accumulation of lipid. After this period the fat body lipid content of control animals decreased, but continued to increase in allatectomized animals for a further 30 days. If adult female desert locusts were allatectomized 10 or 20 days after the final ecdysis there was no excessive accumulation of lipid in the fat body 40 days after the operation (Table 4). Eflects of omriectonty. Fig. 3 shows the total fat body lipid content after ovariectomy of l-day-old adult female desert locusts. Ovariectomy did not result in an accumulation of lipid in the fat body. ~mp~ntut~~ of active corpora aZZutu. The effect on total fat body lipid content of the implantation of four active corpora allata into adult female desert locusts 1 day after the final ecdysis is shown in Table 5. After 5 days there was no significant difference between implanted animals and control animals, but after 10 days
L. HILL AND M. E. G. IZATT
2148 200
E”
r
06orley l Lettuce
0
5
Age,
IO
fed fed
2.0 to
2.6 to
3.6 to
4.6 to
5.6 to
6.6 to
1”
2.4
3.5
4.5
5.5
6.5
6.0
oviduct
Oocyte
days
length,
mm
FIG. 2. The effect of diet on the total lipid content of the fat body of the adult female desert locust during somatic growth and the first gonotrophic cycle. The diet consisted of either fresh barley and bran or fresh lettuce and bran.
E” 400
Allatectamized
d._ P ._
Ovariectamized
ST i
200
+ ,o 0 2 0
5
20
IO
Days
after
30
40
operation
FIG. 3. The effect of allatectomy or ovariectomy of l-day-old
adult female desert locusts on the total lipid content of the fat body.
TABLE
~-THE
TOTAL FAT BODY LIPID CONTENT 40days AFTER ALLATECTOMY ADULT FEMALE DESERT LOCUSTS OF DIFFERENT AGES
Age when allatectomized (days) 1 10 20
Fat body lipid content (mg k SE.) Controls 89+25 155*41 151 k46
Allatectomized 367 f 21 49+11 175 * 22
OF
RELATIONSHIPSBETWBJZNLOCUST.CORPORAALLATA, FAT BODY AND LIPIDS
2149
the lipid content of the fat body of implanted animals was significantly lower (PC 0.001) than in control animals. The effect on total fat body lipid content of the implantation of four active corpora allata into 35-day-old adult female desert locusts which had been allatectomized 1 day after the final ecdysis is shown in Table 6. After 5 days there was a significant reduction (P = 0.5-0.02) in the lipid content of the fat body and yolk deposition in the proximal oiicytes had just begun. Ten days after implantation there was a further significant reduction (P = 0.05) in the lipid content of the fat body and the mean length of the proximal oijcytes was 5.0 mm. TABLE S-THE EFFECTOF THE IMPLANTATION OF FOUR ACTIVE CORPORAALLATA INTO l-day-old ADULT FEMALE DESERT LOCUSTS ON TOTAL FAT BODY LIPID CONTENT Fat body lipid content (mg 5 S.E.) Days after implantation
Implants
Controls
0
-
10+4
5 10 30
93*12 42+10 2257
77+16 142 f 16 72518
Each figure is the mean of five observations. TABLE ~-THE EFFECT OF THE IMPLANTATION OF FOUR ACTIVE CORPORA ALLATA INTO ADULT FEMALE DESERTLOCUSTS ALLATECTOMIZEDFOR 35 days ON TOTAL FAT BODY
LIPID
CONTENT
Fat body lipid content (mg Z!Z S.E.) Days after implantation
0 5 10
Allatectomized controls 255 * 30 283 + 59 324 + 29
Implants
Controls
109f22 46+22
49f18 120f31 88f25
Each figure is the mean of five observations.
Failure of ovarian development in normal animals. A small proportion of normal of lipid accumulated in the fat locusts failed to develop oijcytes. Large quantities bodies of these animals which was not significantly different from that of allatectomized animals of the same age. Haemolymph lipid concentration Normal animals. The total haemolymph lipid concentration desert locust during somatic growth and the first gonotrophic
of the adult female cycle is shown in
L. HILL AND M. E. G. IZATT
2150
Table 7. The concentration of lipid increased significantly (P-C O-001) during somatic growth and fell significantly (P = O-02) at the end of the gonotrophic cycle. TABLE~--THETOTALHABMOLYMPHLIPIDCONCENTRATIONDURINGSOMATICGROU~TH ANDTHEFIRSTGONOTROPHICCYCLEOFTHEADULT FEMALEDESERT
(days)
Haemolymph lipid concentration (mg/ml k SE.)
0 5 10
8.7 + 0.5 (5) 11.3 f0.5 (10) 15.3 kO.9 (15)
Age
Length of proximal oijcytes (mm) 2.0-2.5 2.6-3.5 3.6-4.5 4.6-5-S S-6-6.5 6.6-8-O In oviduct
17.0 18.7 14.4 18.6 165 14.8 12.7
+ 2.0 f 3.8 + 0.8 + 2.4 + 1.3 + 0.6 rt 0.7
LOCUST
(9) (6) (9) (10) (10) (8) (19)
Figures in parentheses refer to the number of observations in each group.
Effects of allatectomy. The total haemolymph lipid concentration of adult female desert locusts allatectomized 1 day after the final ecdysis was not significantly different from that in control animals (Table 8). Implantation of active corpora allata. The implantation of four active corpora allata into adult female desert locusts 1 day after the final ecdysis had no effect on the haemolymph lipid concentration. The implantation of four active corpora TABLE
~-THE
EFFECT OF ALLATECTOMY OF l-day-old ADULT FEMALE LOCUSTS ONTOTAL HARMOLYMPH LIPID CONCENTRATION
DESERT
Haemolymph lipid concentration (mg/mlk S.E.) Days after operation 0
5 10 20 30 40 50
Control
8.7 11.3 15.3 13.3 15.8 17.5 14.7
+ 0.5 f0.5 rf:0.9 k 0.5 + 0.9 + 1.4 f 0.9
(5) (10) (15) (25) (20) (23) (18)
Allatectomized
7.1 + 0.7 12.8 f 0.8 15.4f 1.3 15.1 + 1.4 15.8 + 1.4 15.0_+ 1.2
(5) (5) (5) (9) (5) (10)
Figures in parentheses refer to the number of observations in each group.
RELATIONSHIPSBETWEENLOCUST
CORPORAALLATA,FAT
BODY AND LIPIDS
2151
alfata into 35-day-old adult female desert locusts which had been allatectomized 1 day after the final ecdysis resulted in a transient but signitkant (P = O.Ol~,~l) increase in the haemolymph lipid concentration after 5 days. Ten days after the implantation the haemolymph lipid concentration had returned to normal levels (Table 9). TABLE %--THE EFFECT ON TOTAL HAEMOLYMPH LIPID CONCENTRATION IMPLANTATION OF FOUR ACTIVE CORPORA ALLATA INTO 35day-old ADULT D~ERTLOCUS~ALLATE~TOMIZ~ lday AFTERTHE FINAL ECDYSIS
OF THE FEMALE
lipid concentration (mg/mI+S.E.)
Haemolymph
Days after implantation
Allatectomized controls
Implants
Controls
0 5 10
18.7 + 0.3 12.2 + 1.5 13*0+1*0
22.0 f 1.3 13.9+ l-6
15.8 + 0.9 13.3 -f 0.5 14.6 of:0.9
Each figure is the mean of five observations.
Fat body lipid and protein synthesis Fig. 4 shows the effects of allatectomy on the incorporation of 14C-acetate into the lipids of the whole fat body of the adult female desert locust. During somatic growth there was no difference in incorporation between allatectomized and control “0
x
to-
E
-
c
2 a
L :, ‘Z 0, :: 5j s .L cp 2 a
_
5-
0
5
Days
IO
after
IS
20
30
operation
FIG. 4. The lipid synthetic activityof the fat body of adult female desert locusts allatectomized 1 day after the finalecdysis measured bythelevelof~co~oration ofl*C-acetate into totalfat body lipid.
locusts. After this period, however, the incorporation in control locusts decreased whilst in allatectomized locusts it continued to increase for a further 10 days before decreasing to a lower level. The implantation of four active corpora allata into
2152
L. HILL ANDi%%.E. G.
IZATT
adult female desert locusts 1 day after the final ecdysis had no effect on acetate incorporation 5 days after the implantation. Fig. 5 shows the effects of allatectomy on the incorporation of 8H-1eucine into the proteins of the whole fat body of the adult female desert locust. In both allatectomized and control animals the incorporation was low during the first part of somatic growth. Incorporation remained very low in allatectomized animals, but increased greatly in control animals towards the end of somatic growth. Implantation of four active corpora allata into adult female desert locusts 1 day after the final ecdysis resulted in an increase in incorporation 5 days after the implantation.
LI,
1 Days
after
20
30
operation
FIG. 5. The protein synthetic activity of the fat body of adult female desert locusts allatectomized 1 day after the fmal ecdysis measured by the level of incorporation of 8H-leucine into total fat body protein. Four active corpora allata were implanted into 1 -day-old adult female desert locusts and the activity of the fat body measured after 5 days (implant). DISCUSSION
It is now well established that a hypertrophy of the fat body caused by an excessive accumulation of lipid follows allatectomy of young adult insects. Allatectomy also has effects on other processes in the adult insect including egg development, sexual behaviour, life span, and flight activity (ENGEL~N, 1970; PENER, 1972). It is known that juvenile hormone acts directly on the fat body to control the synthesis of vitellogenic protein (PAN et al., 1969; PAN and WYATT, 1971), but it is not known whether juvenile hormone is directly involved in the control of other processes. The first observation of lipid accumulation following allatectomy assumed a relationship between ovarian development and fat body hypertrophy. WEEDPFE~FFER(1945) concluded that the corpora allata in MeZunopEusreleased a hormone with a primary function in the adult female ‘to facilitate the metabolism or production of materials necessary for egg growth’. VROMANet al. (1965) similarly
RELATIONSHIPS
BETWEEN LOCUST CORPORA ALLATA,
FAT BODY AND LIPIDS
2153
suggested that the greater accumulation of triglycerides in the fat body of allatectomized Periplaneta was related to the failure of ovarian development. Although there are cyclic changes in the fat body lipid content of Schistocerca which can be correlated with gonotrophic cycles (HILL et al., 1968), the fact that lipid accumulates in the fat body of male insects after allatectomy must discredit any hypothesis which seeks to involve a relationship between ovarian development and fat body hypertrophy. Further, if Schistocerca is ovariectomized immediately after the final ecdysis, there is no hypertrophy of the fat body in spite of the non-utilization of lipid for ovarian development. In Locusta the juvenile hormone titre in the haemolymph was normal after ovariectomy (JOHNSON and HILL, unpublished observations), and so lipid accumulation in the fat body is more likely to be directly associated with a lack of juvenile hormone than with a failure of reproductive development. The suggestion that allatectomy influences fat body lipid content indirectly through effects on growth and feeding (WALKER and BAILEY, 1971a, b) is not supported by our data. Allatectomy of adult female Schistocerca immediately after the final ecdysis did not prolong the period of somatic growth and the allatectomized insects did not grow more than controls. The DNA content of the fat body was also unaltered by allatectomy. The amount of lipid that accumulates in the fat body is closely related to nutritional factors. Locusts fed on different diets showed the same pattern of fat body lipid accumulation, but the quantities accumulated differed. However, there was no difference in total food consumption between allatectomized and control locusts, so neither qualitative nor quantitative differences in diet can play a part in the accumulation of lipid in the fat body of allatectomized Schistocerca. An accumulation of lipid in the fat body could be due to an increased synthesis of lipid by the fat body, a decrease in lipid utilization, an inhibition of release of lipid from the fat body, or a combination of these factors. What evidence is there for the involvement of the corpora allata in any of these processes ? During the period of somatic growth lipid accumulated in the fat bodies of allatectomized and control locusts at the same rate. As STRONG(1968b) pointed out, allatectomy of Locusta did not initiate the process of lipid accumulation but prevented a process which began in early life from being terminated. Lipid accumulation in allatectomized Schistocerca did not continue indefinitely, but slowed down and ceased after 40 days. Although lipid accumulated in the fat body of Schistocerca allatectomized early in adult life, no lipid accumulated if allatectomy was performed after the period of somatic growth. The corpora allata of locusts become active towards the end of the period of somatic growth (JOHNSONand HILL, 1973), and so a correlation can be drawn between the appearance of juvenile hormone in the haemolymph and the cessation of the accumulation of lipid in the fat body. The fact that lipid did not accumulate in the fat body of locusts allatectomized after the period of somatic growth might be explained by a combination of factors such as the previous suppression of lipid accumulation by juvenile hormone and the lower feeding activity of older animals.
2154
L. HILL
AND
M. E. G.
IZATT
The changes in fat body lipid content can thus be best explained by assuming that juvenile hormone suppresses lipid accumulation. More direct evidence to support this view comes from the results of the implantation of active corpora allata into normal and allatectomized Schistocerca, and from the in vitro studies on fat body lipid and protein synthesis. When active corpora allata were implanted into adult female Schistocerca immediately after the final ecdysis, thus providing juvenile hormone early in adult life, significantly less lipid accumulated in the fat body during somatic growth. However, the quantity of lipid involved was about that required by the prematurely developing oijcytes (HILL et al., 1968), and so is not an unequivocal demonstration of an effect of juvenile hormone independent of Similarly the implantation of active corpora allata into old ovarian development. allatectomized animals resulted in a significant decrease in fat body lipid content associated with the initiation of oijcyte growth. In this case the decrease in fat body lipid was considerably more than was required by the developing o6cytes; a decrease in fat body lipid of 200 mg when the requirements of the oijcytes could not have exceeded 40 mg. The incorporation of 14C-acetate into fat body lipids is a more direct measure of the synthetic activity of the fat body. During somatic growth acetate incorporation was high, but decreased at the end of somatic growth when the corpora allata became active. This decrease at the end of somatic growth did not occur in allatectomized animals, and acetate incorporation remained high for some time before decreasing slowly. As the fat bodies of the allatectomized locusts were no larger, in terms of cell numbers (the DNA content being unchanged), than control fat bodies, more lipid must be synthesized by allatectomized animals. This situation contrasts with the protein synthetic ability of the fat body which was low during the period of somatic growth, but increased in control animals when the corpora allata became active, or earlier if active corpora allata were implanted, whilst remaining low in allatectomized locusts. These results suggest that juvenile hormone has a dual effect on fat body metabolism, a stimulation of protein synthesis and a suppression of lipid synthesis. Although accumulation of lipid in the fat body of allatectomized locusts can be explained by an increased lipid synthesis by the fat body, the possibility remains that inhibition of lipid release from the fat body, or a decrease in lipid utilization, might play some part. However, it is unlikely that release of lipid from the fat body is affected by allatectomy. An adipokinetic hormone from the glandular lobes of the corpora cardiaca controls fat body lipid release in locusts (MAYERand CANDY, 1969; GOLDSWORTHYet al., 1972a). Allatectomized locusts respond normally to this hormone (GOLDSWORTHYet al., 1972b) and haemolymph lipid levels are not affected by allatectomy. Further, exercise results in the release of lipids from the fat body of allatectomized locusts (STRONG, 1968a; GOLDSWORTHY et al., 1972b). All these facts point to a normal release of lipid from the fat body of allatectomized locusts. The possibility still remains that the action of juvenile hormone on the excitability and/or responsiveness of the nervous system (PENER, 1972) might result in a decreased utilization of lipid. This might play a part in maintaining the
RELATIONSHIPS BETWEENLOCUSTCORPORA ALLATA,FAT BODYANDLIPIDS
2155
high fat body lipid content in old allatectomized animals when the lipid synthetic activity of the fat body has decreased. If, then, juvenile hormone acts directly on the fat body to suppress lipid synthesis, this action would be in accord with the function of juvenile hormone as a ‘switch’ into reproductive activity, and with the ‘oogenesis-flight syndrome’ (JOHNSON, 1969). The value of this mechanism is clear in the locust where the onset of reproductive development can be delayed for a variable time whilst the locust migrates. During migration lipid is needed as an energy source, but for reproduction protein is needed for developing oijcytes and spermatophores. In non-migratory insects the situation is less clear, but might be seen as a mechanism for the accumulation of energy reserves before reproduction starts. REFERENCES BODENSTEIND. (1953) Studies on the humoral mechanisms in growth and metamorphosis of the cockroach,PeripIaneta urneticanu-HI. Humoral effects on metabolism. J. exp. Zool. 124,105-116. EL-IBRASHY M. T. and BOCTORI. Z. (1970) Effect of allatectomy upon lipid metabolism of the female moth of Spodoptera littorals’s Boisd. 2. vergl, Physiol. 68, 11 l-l 16. ENGELMANNF. (1970) The Physiology of Insect Reproductkm. Pergamon Press, Oxford. GILBERT L. I. (1967) Changes in lipid content during the reproductive cycle of Leucophaea maderue and effects of the juvenile hormone on lipid metabolism in vitro. Camp. Biochem. Physiol. 21, 237-257. GOLDSWORTHYG. J., JOHNSONR. A., and MORDUEW. (1972b) In vivo studies on the release of hormones from the corpora cardiaca of locusts. J. co?np. Phy~o~. 79, 85-96. GOLDSWORTHYG. J., MORDUEW., and GUTHKELCHJ. (1972a) Studies on insect adipokinetic hormone. Gen. camp. h’ndocr. 18, 545-551. HILL L. (1962) Neurosecretory control of haemolymph protein concentration during ovarian development in the desert locust. r. Insect Physiol. 8, 609-619. HILL L. (1965) The incorporation of i4C-glycine into the proteins of the fat body of the desert locust during ovarian development. r. Insect Physiol. 11, 1605-1615. HILL L., IZATTM. E. G., HORNEJ. A., and BAILEY E. (1972) Factors affecting concentrations of acetoacetate and D-3-hydroxybutyrate in haemolymph and tissues of the adult desert locust. J. Insect PhysioZ. 18, 1265-1285. HILL L., LuNrz A. J., and STEELE P. A. (1968) The relationships between somatic growth, ovarian growth, and feeding activity in the adult desert locust. J. Insect Physiol. 14, l-20. JOHNSONC. G. (1969) Migration and Dispersal of Insects by Flight. Methuen, London. JOHNSONR. A. and HILL L. (1973) Q uantitative studies on the activity of the corpora allata in adult male Locust5 and Schistocerca. J. Insect Physiol. 19, 2459-2467. MAYER R. J. and CANDY D. J. (1969) Control of haemolymph lipid concentration during locust flight. An adipokinetic hormone from the corpora cardiaca. J. Insect Physiol. 15, 598-611. ODHIAMBOT. R. (1966) The metabolic effects of the corpus allatum hormone in the male desert locust, Schistocerca gregaria. Lipid metabolism. J. exp. BioE. 45, 45-50. PAN M. L., BELL W. J., and TELFER W. H. (1969) Vitellogenic blood protein synthesised by insect fat body. Science, Wash. 165, 393-394. PAN M. L. and WYATT G. R. (1971) J uvenile hormone induces vitellogenin synthesis in the Monarch butterfly, S&nce, Wash. 174, 503-505. PENER M. P. (1972) The corpus allatum in adult Acridids; the interrelations of its functions and possible correlations to the life cycle. Proc. int. Study Conf. Current Future Problems Acridology, London, 1970, pp. 135-147.
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L. HILL ANDM. E. G. IZATT
SCHNEIDERW. C. (1957) Determination of nucleic acies in tissues by pentose analysis. In Methods in EmymoZogy (Ed. by COLOWICKS. P. and KAPLANN. 0.) 3. Academic Press, New York. STRONGL. (1963) A simple apparatus for use in removing corpora allata from locusts. Bull. ent. Res. 54, 19-21. STRONGL. (1968a) The effect of enforced locomotor activity on lipid content in allatectomized males of Locasta mi~ato~‘a mi~ato~.~*~s. J. exp. Biol. 48, 625-630. STRONGL. (1968b) Locomotor activity, sexual behaviour, and the corpus allaturn hormone in males of Locusta. J. Insect Physiol. 14, 16851692. THOMSENE. (1952) Functional significance of the neurosecretory brain cells and the corpus cardiacum in the female blowfly, Ca~~~hora ~yt~r~~~Za Meig. J. exp. Biol. 29,
137-172. VOGT M. (1949) Fettkarper und ijnocyten der Drosophila nach extirpation der adulten ringdriise. Z. Zellforsch. 34, 160-164. VROMANH. E., KAPLANISJ. N., and ROBBINSW. E. (1965) Effect of allatectomy on lipid biosynthesis and turnover in the female American cockroach, Perkplaneta americana (L.). J. Insect Physiol. 11, 897-904. WALKERP. R. and BAILEY E. (1970) Metabolism of glucose, trehalose, citrate, acetate, and palmitate by the male desert locust during adult development. J. Insect PhysioZ. 16,
499-509. WALKERP. R. and BAILEY E. (1971a) Effect of allatectomy on fat body lipid metabolism of the adult male desert locust during adult development. J. Insect Physiol. 17, 813-821. WALKER P. R. and BAILEY E. (1971b) Effect of allatectomy on the growth of the male desert Iocust during adult development. 4: Insect Physiol. 17, 1125-l 137. WALKER P. R. and BAILEY E. (1971~) Effect of allatectomy on fat body lipogenic enzymes of the male desert locust during adult development. J. Insect Physiol. 17, 1359-1369. WEED-PFEIFFER I. W. (1945) Effects of the corpora allata on the metabolism of adult grasshoppers. J. exp. Zool. 99, 183-233. WYATT G. R. (1972) Insect hormones. In ~~och~ical Actions of ~0~0~s (Ed. by LITWACK G.) 2. Academic Press, New York.
Press. Printed in Great Britain
THE RELATIONSHIPS BETWEEN CORPORA ALLATA AND FAT BODY AND H~MOLYMPH LIPIDS IN THE ADULT FEMALE DESERT LOCUST L. HILL
and M. E. G. IZATT
Department of Zoology, The University, Sheffield SlO 2TN, England
(Received 29 March 1974) Abstract-Allatectomy of l-day-old female desert locusts resulted in an accumulation of lipid in the fat body. This accumulation of lipid was due to the continuation of lipid deposition in the fat body after the period of somatic growth. Somatic growth and feeding activity were unaffected by allatectomy, and so could not be indirect causes of fat body lipid accumulation. Lipid accumulation in allatectomized locusts is more likely to be related directly to a lack of juvenile hormone. Implantation of active corpora allata into l-day-old adult female locusts resulted in a premature development of oiicytes and a
decrease in fat body lipid accumulation; somatic growth was not inhibited. Im~l~tat~on of active corpora allata into old allatectomized locusts resulted in a decrease in the fat body lipid content and the onset of obcyte development. The
lipid synthetic activity of the fat body, measured by the incorporation of r4Cacetate into total fat body lipid, was greatly increased in allatectomized locusts after the period of somatic growth. The protein synthetic activity of the fat body, measured by the incorporation of VI-leucine into total fat body protein, remained low after the period of somatic growth in allatectomized insects. Juvenile hormone might thus have a dual effect on fat body metabolism, that is suppressing lipid synthesis and stimulating vitellogenic protein synthesis. Increased synthesis of lipid by the fat body would then account for the accumulation of lipid in the fat body after allatectomy. Inhibition of release of lipid from the fat body is unlikely to play a part in the accumulation as allatectomy had no effect on haemolymph lipid concentrations. INTRODUCTION
ALTHOUGH abnormal accumulation of lipid in the fat body following allatectomy has been reported in a variety of insects (WEED-PFEIFFER,1945; VOGT, 1949; THOMS~, 1952; BODENSTHN, 1953; ODHIAMBO, 1966; STRONG, 1968a; EL-IBRASHY and BOCTOR,1970), there is no generally accepted explanation for this phenomenon. In ~@~n~~ ~~~~~i~~ (WED-P~IFFER, 1945) and ~~~~~~a algae (VROMANet al., 1965) it was suggested that the corpora allata regulate fat body lipid content by a control of the mechanisms of lipid utilization, but in Leucophaea maderae the suppression of fat body lipogenesis in vitro by the addition of juvenile hormone suggested a direct effect of the hormone on fat body lipid synthesis (GILBERT, 1967). In ~c~~~~~~ff gregaria various indirect effects of allatectomy on fat body lipids have been proposed. For example, that allatectomy resulted in a decrease 2143
2144
L. HILL ANDM. E. G. IZATT
in locomotor activity and thus an accumulation of the fat body lipid reserves (ODHIAMBO, 1966), or that after allatectomy food consumption and growth were increased and the increase of lipid in the fat body was only one aspect of this general effect (WALKER and BAILEY, 1971a-c). In a recent review WYATT (1972) suggested that the available evidence pointed to a direct effect of juvenile hormone on fat body lipid metabolism. In this paper data are presented on the lipids of the fat body and haemolymph of the female desert locust, related to growth and feeding activity, which support this view and help resolve some of the contradictions in previous work on locusts.
MATERIALS
AND METHODS
The adult female desert locusts, Schistocerca gregaria, used in this study were taken from a colony reared in Sheffield at a temperature of 30°C with a constant photoperiod of 12 hr. The insects were reared under crowded conditions and fed a diet of bran and freshly cut barley. One experimental group of animals was fed a diet of bran and fresh lettuce. The methods for the measurement of growth, feeding activity, and fat body and haemolymph lipid content have been previously described (HILL et uZ., 1968, 1972). Allatectomy was performed by the method of STRONG (1963), except that the insects were not covered with saline. Active corpora allata for implantation were taken from mature adult male Locusta migrutoriu (JOHNSONand HILL, 1973) and immediately implanted into adult female desert locusts through a small slit in the side of the abdomen which was then sealed with paraffin wax. Ovariectomy was performed as described by HILL (1962). The DNA content of whole fat bodies was measured with the diphenylamine reagent (SCHNEIDER, 1957). The ability of the fat body to synthesize lipid and protein was measured by the incorporation of labelled precursors into total fat body lipid and total fat body protein. The whole abdominal fat body was dissected from each locust and divided longitudinally into two equal parts. Each part was weighed and placed in 2 ml of incubation medium (WALKER and BAILEY, 1970). 14C-sodium acetate (0.5 &i) (R ad’ioc h emical Centre, Amersham) was added to one part of the fat body and 5 ,&i 3H-leucine (Radiochemical Centre, Amersham) was added to the other part. The fat body portions were incubated for 1 hr at 30°C and then the incubation stopped by the addition of 0.5 ml of 25% trichloroacetic acid. The total lipid content was extracted from the acetate incubation, purified (WALKER and BAILEY, 1970), and dissolved in 10 ml scintillation medium (PPO, 7 g; POPOP, 0.6 g; ethoxyethanol, 500 ml; toluene, 1000 ml). The total protein content of the leucine incubation was purified (HILL, 1965), the solid protein dissolved in O-2 ml 1 N NaOH, and 10 ml scintillation medium added. Samples were counted in a Packard Tricarb scintillation counter. All counts were corrected for background and expressed as dis/min for the lipid or protein fraction of the whole fat body.
2145
RELATIONSHIPS BEZWEEN LOCUST CORPORA ALLATA, FAT BODY AND LIPIDS
RESULTS Somatic gr~th
ati feeding activity
AZZatectomy and growth.
The effect of allatectomy of l-day-old adult female desert locusts on growth is shown in Table 1. The somatic dry weight of the locust is the total dry weight minus the weight of the ovaries and fat body. The pattern of growth was essentially the same whether the somatic dry weight, the dry weight of the flight muscles, or the dry weight of a cuticle sample was measured. There were no consistent differences in growth pattern between normal and allatectomized adult female locusts, and allatectomized locusts did not grow significantly more than control locusts.
I
2
3
4
5
6
Days FIG.
1. The
total
dry
weight l-day-old
*-*
Allatectomized
o-0
Control
7
6
after
of food
IO
9
I,
12
13
14
15
16 17
I6
IS
locust
after
allatectomy
operation
consumed
aduit female
desert
per
of
locusts.
~rn~~a~tat~~ of active corpora allata. The implantation of four active corpora aliata into adult female desert locusts 1 day after the final ecdysis resulted in the premature deposition of yolk in the proximal o8cytes. Five days after the implantation the mean length of the proximal oiicytes was 54i mm. Ten days after implantation the mean length of the proximal oijcytes was 73 mm, that is almost fully developed, whilst in control animals of this age yolk deposition was just starting in the proximal oiicytes. There was no inhibition of growth associated with the premature oijcyte development which resulted from the impl~tation of active corpora allata (Table 2). The pattern of growth was, however, changed. Somatic growth appeared to be compressed into the first 5 days after the implantation, and between the fifth and tenth days after the implantation, when the metabolic demands of the developing oiicytes would be greatest (HILL et al., 1968), little further somatic growth occurred. Allatectomy and food consumption. Fig. 1 shows the total food intake of adult female desert locusts ~latectomized 1 day after the final ecdysis and of control animals. Although the period of intense feeding associated with somatic growth
538 + 602 f 7.59 + 754 f 794 + 791 f
27 37 36 32 23 39
Allatectomized
69.5 90.1 112.8 97.2 76.6 103.8
* 6.0 + 8.1 f. 13.0 + 5~5 + 10.9 + 11.5
30.3 f 3.9
Controls
50.2 79.8 121.9 124.0 129.3 llS*l
zk2.4 zk3.6 + 70 f 9-O + 6.3 f 13.1
-
Allatectomized
Flight muscle weight (mg f SE.)
5.8 6.4 8.0 6-l 9.1 11.5
z!z0.6 + 0.3 c 0.6 f 0.2 k 0.8 +0*4
3.0 f 0.2
Controls
4.0 4.5 6.5 7.2 7.5 8.6
+ 0.4 f 0.2 f 0.4 + 0.5 * 0.3 + 0.6
-
Allatectomized
Tergites’ weight (mg 4 SE.)
657 + 30 631+43 608+12
-
Implants
Each figure is the mean of five observations.
328 f 30
450 + 23 642 + 30 626 + 24
5 10 30
Controls
0
Days after implantation
Somatic weight (mg f. SE.)
69.5 -I 6-O 90.1 f a.1 97.2 f 5.5
30‘3 k 3.9
Control5
al.6 i 8.7 90.5 + 5.5
83-o+ 3.1
-
Implants
Flight muscle weight (mg + SE)
5.8 + 0.6 6.4 f 0.3 6.1 rt 0.2
3-o It 0.2
Controls
8.2 & 0.5 6.9 f 0.6 9.1 f 0.4
-
Implants
Tergites’ weight (mg f S.E.)
TA~LE~--THEEFFECTSOFTHEIMPLANTATIONOFFOURACTIVECORPORAALLATAINTO l-day-old ADULTFEMALEDESERTLOCUSTS ONSOMATICDRYWEIGHT,DRYUTEIGHTOFTHEFLIGHTMUSCLES,ANDDRYWEIGHTOFTHESECOND,THIRD,ANDFOURTHABDOMINAL TERGITRS
Each figure is the mean of five observations.
23 30 36 24 42 41
328 + 30
450 f 642 f 686 + 626 f 632 f 7.52 +
0
5 10 20 30 40 50
Controls
Days after operation
Somatic weight (mg f S.E.)
TABLE I-THEEFFECTSOFALLATECTOMYOF~ -day-old ADULTFEMALEDESERTLOCUSTSONSOMATICDRY WEIGHT, DRYWEIGHTOF THEFLIGHT MUSCLES, ANDDRYWEIGHT OF THESECOND,THIRD,ANDFOuRTHABDOMINALTERGITES
RELATIONSHIPSBETWEEN LOCUST CORPORAALLATA, FAT BODY AND LIPIDS
2147
was reduced in allatectomized animals, both groups consumed about the same total quantity of food during the 19-day period; 5.01 g by control animals and 4.83 g by allatectomized animals. Fat body DNA content. The total fat body DNA content of adult female desert locusts allatectomized 1 day after the final ecdysis is shown in Table 3. There were no differences between allatectomized and control animals. TABLE ~---THE EFFECT OF ALLATECTOMY OF ~-DAY-OLD ADULT FEMALE DESERT LOCUSTS ON THE TOTAL DNA CONTENT OF THE FAT BODY Total fat body DNA
content
@g rt S.E.) Days after operation 0 5 10 20 30 40
Controls 56+9 87~10 102 + 10 145+5 169440 133It13
Allatectomized 106+17 131-118 173 -1:21 193 + 21 129 4 37
Each figure is the mean of five observations.
Fat body lipid content Eflect of diet. Fig. 2 shows the total lipid content of the fat body during somatic growth and the first gonotrophic cycle of adult female desert locusts fed on bran and fresh barley or bran and fresh lettuce. The pattern of lipid accumulation was the same in both cases, but the total quantity of lipid stored in the fat body was greater in the barley fed animals. Effects of allatectomy. Fig. 3 shows the total fat body lipid content after allatectomy of l-day-old adult female locusts. During the period of somatic growth, that is the first 10 days of adult life, there were no differences between allatectomized and control animals, in both cases there was an accumulation of lipid. After this period the fat body lipid content of control animals decreased, but continued to increase in allatectomized animals for a further 30 days. If adult female desert locusts were allatectomized 10 or 20 days after the final ecdysis there was no excessive accumulation of lipid in the fat body 40 days after the operation (Table 4). Eflects of omriectonty. Fig. 3 shows the total fat body lipid content after ovariectomy of l-day-old adult female desert locusts. Ovariectomy did not result in an accumulation of lipid in the fat body. ~mp~ntut~~ of active corpora aZZutu. The effect on total fat body lipid content of the implantation of four active corpora allata into adult female desert locusts 1 day after the final ecdysis is shown in Table 5. After 5 days there was no significant difference between implanted animals and control animals, but after 10 days
L. HILL AND M. E. G. IZATT
2148 200
E”
r
06orley l Lettuce
0
5
Age,
IO
fed fed
2.0 to
2.6 to
3.6 to
4.6 to
5.6 to
6.6 to
1”
2.4
3.5
4.5
5.5
6.5
6.0
oviduct
Oocyte
days
length,
mm
FIG. 2. The effect of diet on the total lipid content of the fat body of the adult female desert locust during somatic growth and the first gonotrophic cycle. The diet consisted of either fresh barley and bran or fresh lettuce and bran.
E” 400
Allatectamized
d._ P ._
Ovariectamized
ST i
200
+ ,o 0 2 0
5
20
IO
Days
after
30
40
operation
FIG. 3. The effect of allatectomy or ovariectomy of l-day-old
adult female desert locusts on the total lipid content of the fat body.
TABLE
~-THE
TOTAL FAT BODY LIPID CONTENT 40days AFTER ALLATECTOMY ADULT FEMALE DESERT LOCUSTS OF DIFFERENT AGES
Age when allatectomized (days) 1 10 20
Fat body lipid content (mg k SE.) Controls 89+25 155*41 151 k46
Allatectomized 367 f 21 49+11 175 * 22
OF
RELATIONSHIPSBETWBJZNLOCUST.CORPORAALLATA, FAT BODY AND LIPIDS
2149
the lipid content of the fat body of implanted animals was significantly lower (PC 0.001) than in control animals. The effect on total fat body lipid content of the implantation of four active corpora allata into 35-day-old adult female desert locusts which had been allatectomized 1 day after the final ecdysis is shown in Table 6. After 5 days there was a significant reduction (P = 0.5-0.02) in the lipid content of the fat body and yolk deposition in the proximal oiicytes had just begun. Ten days after implantation there was a further significant reduction (P = 0.05) in the lipid content of the fat body and the mean length of the proximal oijcytes was 5.0 mm. TABLE S-THE EFFECTOF THE IMPLANTATION OF FOUR ACTIVE CORPORAALLATA INTO l-day-old ADULT FEMALE DESERT LOCUSTS ON TOTAL FAT BODY LIPID CONTENT Fat body lipid content (mg 5 S.E.) Days after implantation
Implants
Controls
0
-
10+4
5 10 30
93*12 42+10 2257
77+16 142 f 16 72518
Each figure is the mean of five observations. TABLE ~-THE EFFECT OF THE IMPLANTATION OF FOUR ACTIVE CORPORA ALLATA INTO ADULT FEMALE DESERTLOCUSTS ALLATECTOMIZEDFOR 35 days ON TOTAL FAT BODY
LIPID
CONTENT
Fat body lipid content (mg Z!Z S.E.) Days after implantation
0 5 10
Allatectomized controls 255 * 30 283 + 59 324 + 29
Implants
Controls
109f22 46+22
49f18 120f31 88f25
Each figure is the mean of five observations.
Failure of ovarian development in normal animals. A small proportion of normal of lipid accumulated in the fat locusts failed to develop oijcytes. Large quantities bodies of these animals which was not significantly different from that of allatectomized animals of the same age. Haemolymph lipid concentration Normal animals. The total haemolymph lipid concentration desert locust during somatic growth and the first gonotrophic
of the adult female cycle is shown in
L. HILL AND M. E. G. IZATT
2150
Table 7. The concentration of lipid increased significantly (P-C O-001) during somatic growth and fell significantly (P = O-02) at the end of the gonotrophic cycle. TABLE~--THETOTALHABMOLYMPHLIPIDCONCENTRATIONDURINGSOMATICGROU~TH ANDTHEFIRSTGONOTROPHICCYCLEOFTHEADULT FEMALEDESERT
(days)
Haemolymph lipid concentration (mg/ml k SE.)
0 5 10
8.7 + 0.5 (5) 11.3 f0.5 (10) 15.3 kO.9 (15)
Age
Length of proximal oijcytes (mm) 2.0-2.5 2.6-3.5 3.6-4.5 4.6-5-S S-6-6.5 6.6-8-O In oviduct
17.0 18.7 14.4 18.6 165 14.8 12.7
+ 2.0 f 3.8 + 0.8 + 2.4 + 1.3 + 0.6 rt 0.7
LOCUST
(9) (6) (9) (10) (10) (8) (19)
Figures in parentheses refer to the number of observations in each group.
Effects of allatectomy. The total haemolymph lipid concentration of adult female desert locusts allatectomized 1 day after the final ecdysis was not significantly different from that in control animals (Table 8). Implantation of active corpora allata. The implantation of four active corpora allata into adult female desert locusts 1 day after the final ecdysis had no effect on the haemolymph lipid concentration. The implantation of four active corpora TABLE
~-THE
EFFECT OF ALLATECTOMY OF l-day-old ADULT FEMALE LOCUSTS ONTOTAL HARMOLYMPH LIPID CONCENTRATION
DESERT
Haemolymph lipid concentration (mg/mlk S.E.) Days after operation 0
5 10 20 30 40 50
Control
8.7 11.3 15.3 13.3 15.8 17.5 14.7
+ 0.5 f0.5 rf:0.9 k 0.5 + 0.9 + 1.4 f 0.9
(5) (10) (15) (25) (20) (23) (18)
Allatectomized
7.1 + 0.7 12.8 f 0.8 15.4f 1.3 15.1 + 1.4 15.8 + 1.4 15.0_+ 1.2
(5) (5) (5) (9) (5) (10)
Figures in parentheses refer to the number of observations in each group.
RELATIONSHIPSBETWEENLOCUST
CORPORAALLATA,FAT
BODY AND LIPIDS
2151
alfata into 35-day-old adult female desert locusts which had been allatectomized 1 day after the final ecdysis resulted in a transient but signitkant (P = O.Ol~,~l) increase in the haemolymph lipid concentration after 5 days. Ten days after the implantation the haemolymph lipid concentration had returned to normal levels (Table 9). TABLE %--THE EFFECT ON TOTAL HAEMOLYMPH LIPID CONCENTRATION IMPLANTATION OF FOUR ACTIVE CORPORA ALLATA INTO 35day-old ADULT D~ERTLOCUS~ALLATE~TOMIZ~ lday AFTERTHE FINAL ECDYSIS
OF THE FEMALE
lipid concentration (mg/mI+S.E.)
Haemolymph
Days after implantation
Allatectomized controls
Implants
Controls
0 5 10
18.7 + 0.3 12.2 + 1.5 13*0+1*0
22.0 f 1.3 13.9+ l-6
15.8 + 0.9 13.3 -f 0.5 14.6 of:0.9
Each figure is the mean of five observations.
Fat body lipid and protein synthesis Fig. 4 shows the effects of allatectomy on the incorporation of 14C-acetate into the lipids of the whole fat body of the adult female desert locust. During somatic growth there was no difference in incorporation between allatectomized and control “0
x
to-
E
-
c
2 a
L :, ‘Z 0, :: 5j s .L cp 2 a
_
5-
0
5
Days
IO
after
IS
20
30
operation
FIG. 4. The lipid synthetic activityof the fat body of adult female desert locusts allatectomized 1 day after the finalecdysis measured bythelevelof~co~oration ofl*C-acetate into totalfat body lipid.
locusts. After this period, however, the incorporation in control locusts decreased whilst in allatectomized locusts it continued to increase for a further 10 days before decreasing to a lower level. The implantation of four active corpora allata into
2152
L. HILL ANDi%%.E. G.
IZATT
adult female desert locusts 1 day after the final ecdysis had no effect on acetate incorporation 5 days after the implantation. Fig. 5 shows the effects of allatectomy on the incorporation of 8H-1eucine into the proteins of the whole fat body of the adult female desert locust. In both allatectomized and control animals the incorporation was low during the first part of somatic growth. Incorporation remained very low in allatectomized animals, but increased greatly in control animals towards the end of somatic growth. Implantation of four active corpora allata into adult female desert locusts 1 day after the final ecdysis resulted in an increase in incorporation 5 days after the implantation.
LI,
1 Days
after
20
30
operation
FIG. 5. The protein synthetic activity of the fat body of adult female desert locusts allatectomized 1 day after the fmal ecdysis measured by the level of incorporation of 8H-leucine into total fat body protein. Four active corpora allata were implanted into 1 -day-old adult female desert locusts and the activity of the fat body measured after 5 days (implant). DISCUSSION
It is now well established that a hypertrophy of the fat body caused by an excessive accumulation of lipid follows allatectomy of young adult insects. Allatectomy also has effects on other processes in the adult insect including egg development, sexual behaviour, life span, and flight activity (ENGEL~N, 1970; PENER, 1972). It is known that juvenile hormone acts directly on the fat body to control the synthesis of vitellogenic protein (PAN et al., 1969; PAN and WYATT, 1971), but it is not known whether juvenile hormone is directly involved in the control of other processes. The first observation of lipid accumulation following allatectomy assumed a relationship between ovarian development and fat body hypertrophy. WEEDPFE~FFER(1945) concluded that the corpora allata in MeZunopEusreleased a hormone with a primary function in the adult female ‘to facilitate the metabolism or production of materials necessary for egg growth’. VROMANet al. (1965) similarly
RELATIONSHIPS
BETWEEN LOCUST CORPORA ALLATA,
FAT BODY AND LIPIDS
2153
suggested that the greater accumulation of triglycerides in the fat body of allatectomized Periplaneta was related to the failure of ovarian development. Although there are cyclic changes in the fat body lipid content of Schistocerca which can be correlated with gonotrophic cycles (HILL et al., 1968), the fact that lipid accumulates in the fat body of male insects after allatectomy must discredit any hypothesis which seeks to involve a relationship between ovarian development and fat body hypertrophy. Further, if Schistocerca is ovariectomized immediately after the final ecdysis, there is no hypertrophy of the fat body in spite of the non-utilization of lipid for ovarian development. In Locusta the juvenile hormone titre in the haemolymph was normal after ovariectomy (JOHNSON and HILL, unpublished observations), and so lipid accumulation in the fat body is more likely to be directly associated with a lack of juvenile hormone than with a failure of reproductive development. The suggestion that allatectomy influences fat body lipid content indirectly through effects on growth and feeding (WALKER and BAILEY, 1971a, b) is not supported by our data. Allatectomy of adult female Schistocerca immediately after the final ecdysis did not prolong the period of somatic growth and the allatectomized insects did not grow more than controls. The DNA content of the fat body was also unaltered by allatectomy. The amount of lipid that accumulates in the fat body is closely related to nutritional factors. Locusts fed on different diets showed the same pattern of fat body lipid accumulation, but the quantities accumulated differed. However, there was no difference in total food consumption between allatectomized and control locusts, so neither qualitative nor quantitative differences in diet can play a part in the accumulation of lipid in the fat body of allatectomized Schistocerca. An accumulation of lipid in the fat body could be due to an increased synthesis of lipid by the fat body, a decrease in lipid utilization, an inhibition of release of lipid from the fat body, or a combination of these factors. What evidence is there for the involvement of the corpora allata in any of these processes ? During the period of somatic growth lipid accumulated in the fat bodies of allatectomized and control locusts at the same rate. As STRONG(1968b) pointed out, allatectomy of Locusta did not initiate the process of lipid accumulation but prevented a process which began in early life from being terminated. Lipid accumulation in allatectomized Schistocerca did not continue indefinitely, but slowed down and ceased after 40 days. Although lipid accumulated in the fat body of Schistocerca allatectomized early in adult life, no lipid accumulated if allatectomy was performed after the period of somatic growth. The corpora allata of locusts become active towards the end of the period of somatic growth (JOHNSONand HILL, 1973), and so a correlation can be drawn between the appearance of juvenile hormone in the haemolymph and the cessation of the accumulation of lipid in the fat body. The fact that lipid did not accumulate in the fat body of locusts allatectomized after the period of somatic growth might be explained by a combination of factors such as the previous suppression of lipid accumulation by juvenile hormone and the lower feeding activity of older animals.
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The changes in fat body lipid content can thus be best explained by assuming that juvenile hormone suppresses lipid accumulation. More direct evidence to support this view comes from the results of the implantation of active corpora allata into normal and allatectomized Schistocerca, and from the in vitro studies on fat body lipid and protein synthesis. When active corpora allata were implanted into adult female Schistocerca immediately after the final ecdysis, thus providing juvenile hormone early in adult life, significantly less lipid accumulated in the fat body during somatic growth. However, the quantity of lipid involved was about that required by the prematurely developing oijcytes (HILL et al., 1968), and so is not an unequivocal demonstration of an effect of juvenile hormone independent of Similarly the implantation of active corpora allata into old ovarian development. allatectomized animals resulted in a significant decrease in fat body lipid content associated with the initiation of oijcyte growth. In this case the decrease in fat body lipid was considerably more than was required by the developing o6cytes; a decrease in fat body lipid of 200 mg when the requirements of the oijcytes could not have exceeded 40 mg. The incorporation of 14C-acetate into fat body lipids is a more direct measure of the synthetic activity of the fat body. During somatic growth acetate incorporation was high, but decreased at the end of somatic growth when the corpora allata became active. This decrease at the end of somatic growth did not occur in allatectomized animals, and acetate incorporation remained high for some time before decreasing slowly. As the fat bodies of the allatectomized locusts were no larger, in terms of cell numbers (the DNA content being unchanged), than control fat bodies, more lipid must be synthesized by allatectomized animals. This situation contrasts with the protein synthetic ability of the fat body which was low during the period of somatic growth, but increased in control animals when the corpora allata became active, or earlier if active corpora allata were implanted, whilst remaining low in allatectomized locusts. These results suggest that juvenile hormone has a dual effect on fat body metabolism, a stimulation of protein synthesis and a suppression of lipid synthesis. Although accumulation of lipid in the fat body of allatectomized locusts can be explained by an increased lipid synthesis by the fat body, the possibility remains that inhibition of lipid release from the fat body, or a decrease in lipid utilization, might play some part. However, it is unlikely that release of lipid from the fat body is affected by allatectomy. An adipokinetic hormone from the glandular lobes of the corpora cardiaca controls fat body lipid release in locusts (MAYERand CANDY, 1969; GOLDSWORTHYet al., 1972a). Allatectomized locusts respond normally to this hormone (GOLDSWORTHYet al., 1972b) and haemolymph lipid levels are not affected by allatectomy. Further, exercise results in the release of lipids from the fat body of allatectomized locusts (STRONG, 1968a; GOLDSWORTHY et al., 1972b). All these facts point to a normal release of lipid from the fat body of allatectomized locusts. The possibility still remains that the action of juvenile hormone on the excitability and/or responsiveness of the nervous system (PENER, 1972) might result in a decreased utilization of lipid. This might play a part in maintaining the
RELATIONSHIPS BETWEENLOCUSTCORPORA ALLATA,FAT BODYANDLIPIDS
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high fat body lipid content in old allatectomized animals when the lipid synthetic activity of the fat body has decreased. If, then, juvenile hormone acts directly on the fat body to suppress lipid synthesis, this action would be in accord with the function of juvenile hormone as a ‘switch’ into reproductive activity, and with the ‘oogenesis-flight syndrome’ (JOHNSON, 1969). The value of this mechanism is clear in the locust where the onset of reproductive development can be delayed for a variable time whilst the locust migrates. During migration lipid is needed as an energy source, but for reproduction protein is needed for developing oijcytes and spermatophores. In non-migratory insects the situation is less clear, but might be seen as a mechanism for the accumulation of energy reserves before reproduction starts. REFERENCES BODENSTEIND. (1953) Studies on the humoral mechanisms in growth and metamorphosis of the cockroach,PeripIaneta urneticanu-HI. Humoral effects on metabolism. J. exp. Zool. 124,105-116. EL-IBRASHY M. T. and BOCTORI. Z. (1970) Effect of allatectomy upon lipid metabolism of the female moth of Spodoptera littorals’s Boisd. 2. vergl, Physiol. 68, 11 l-l 16. ENGELMANNF. (1970) The Physiology of Insect Reproductkm. Pergamon Press, Oxford. GILBERT L. I. (1967) Changes in lipid content during the reproductive cycle of Leucophaea maderue and effects of the juvenile hormone on lipid metabolism in vitro. Camp. Biochem. Physiol. 21, 237-257. GOLDSWORTHYG. J., JOHNSONR. A., and MORDUEW. (1972b) In vivo studies on the release of hormones from the corpora cardiaca of locusts. J. co?np. Phy~o~. 79, 85-96. GOLDSWORTHYG. J., MORDUEW., and GUTHKELCHJ. (1972a) Studies on insect adipokinetic hormone. Gen. camp. h’ndocr. 18, 545-551. HILL L. (1962) Neurosecretory control of haemolymph protein concentration during ovarian development in the desert locust. r. Insect Physiol. 8, 609-619. HILL L. (1965) The incorporation of i4C-glycine into the proteins of the fat body of the desert locust during ovarian development. r. Insect Physiol. 11, 1605-1615. HILL L., IZATTM. E. G., HORNEJ. A., and BAILEY E. (1972) Factors affecting concentrations of acetoacetate and D-3-hydroxybutyrate in haemolymph and tissues of the adult desert locust. J. Insect PhysioZ. 18, 1265-1285. HILL L., LuNrz A. J., and STEELE P. A. (1968) The relationships between somatic growth, ovarian growth, and feeding activity in the adult desert locust. J. Insect Physiol. 14, l-20. JOHNSONC. G. (1969) Migration and Dispersal of Insects by Flight. Methuen, London. JOHNSONR. A. and HILL L. (1973) Q uantitative studies on the activity of the corpora allata in adult male Locust5 and Schistocerca. J. Insect Physiol. 19, 2459-2467. MAYER R. J. and CANDY D. J. (1969) Control of haemolymph lipid concentration during locust flight. An adipokinetic hormone from the corpora cardiaca. J. Insect Physiol. 15, 598-611. ODHIAMBOT. R. (1966) The metabolic effects of the corpus allatum hormone in the male desert locust, Schistocerca gregaria. Lipid metabolism. J. exp. BioE. 45, 45-50. PAN M. L., BELL W. J., and TELFER W. H. (1969) Vitellogenic blood protein synthesised by insect fat body. Science, Wash. 165, 393-394. PAN M. L. and WYATT G. R. (1971) J uvenile hormone induces vitellogenin synthesis in the Monarch butterfly, S&nce, Wash. 174, 503-505. PENER M. P. (1972) The corpus allatum in adult Acridids; the interrelations of its functions and possible correlations to the life cycle. Proc. int. Study Conf. Current Future Problems Acridology, London, 1970, pp. 135-147.
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SCHNEIDERW. C. (1957) Determination of nucleic acies in tissues by pentose analysis. In Methods in EmymoZogy (Ed. by COLOWICKS. P. and KAPLANN. 0.) 3. Academic Press, New York. STRONGL. (1963) A simple apparatus for use in removing corpora allata from locusts. Bull. ent. Res. 54, 19-21. STRONGL. (1968a) The effect of enforced locomotor activity on lipid content in allatectomized males of Locasta mi~ato~‘a mi~ato~.~*~s. J. exp. Biol. 48, 625-630. STRONGL. (1968b) Locomotor activity, sexual behaviour, and the corpus allaturn hormone in males of Locusta. J. Insect Physiol. 14, 16851692. THOMSENE. (1952) Functional significance of the neurosecretory brain cells and the corpus cardiacum in the female blowfly, Ca~~~hora ~yt~r~~~Za Meig. J. exp. Biol. 29,
137-172. VOGT M. (1949) Fettkarper und ijnocyten der Drosophila nach extirpation der adulten ringdriise. Z. Zellforsch. 34, 160-164. VROMANH. E., KAPLANISJ. N., and ROBBINSW. E. (1965) Effect of allatectomy on lipid biosynthesis and turnover in the female American cockroach, Perkplaneta americana (L.). J. Insect Physiol. 11, 897-904. WALKERP. R. and BAILEY E. (1970) Metabolism of glucose, trehalose, citrate, acetate, and palmitate by the male desert locust during adult development. J. Insect PhysioZ. 16,
499-509. WALKERP. R. and BAILEY E. (1971a) Effect of allatectomy on fat body lipid metabolism of the adult male desert locust during adult development. J. Insect Physiol. 17, 813-821. WALKER P. R. and BAILEY E. (1971b) Effect of allatectomy on the growth of the male desert Iocust during adult development. 4: Insect Physiol. 17, 1125-l 137. WALKER P. R. and BAILEY E. (1971~) Effect of allatectomy on fat body lipogenic enzymes of the male desert locust during adult development. J. Insect Physiol. 17, 1359-1369. WEED-PFEIFFER I. W. (1945) Effects of the corpora allata on the metabolism of adult grasshoppers. J. exp. Zool. 99, 183-233. WYATT G. R. (1972) Insect hormones. In ~~och~ical Actions of ~0~0~s (Ed. by LITWACK G.) 2. Academic Press, New York.