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Effect of corpus allatum and ovaries on amount of pupal and adult fat body in the housefly, Musca domestica
J. Imect Physiol., 1969, Vol. 15, pp. 1729 to 1747. PevgamonPress. Printed in Great Britain
EFFECT OF CORPUS ALLATUM AND OVARIES ON AMOUNT OF PUPAL AND ADULT FAT BODY IN THE HOUSEFLY, kWSC.kl DOllhWTIC~ T. S. ADAMS
and D. R. NELSON
Metabolism and Radiation Research Laboratory, Entomology Research Division, Agriculture Research Service, U.S.D.A., Fargo, North Dakota 58102 (Received 14January
1969; revised 2 May 1969)
Abstract-The stage of ovarian development was compared with changes in the volume of abdominal fat body in female houseflies. Pupal fat body volume decreased during each stage of oagenesis and had its maximum rate of volume decreases during the vitellogenic phase of ocgenesis (stages 4 to 9). No pupal fat body remained in flies with mature [stage 10) eggs. Adult fat body showed a volume increase during egg stages 2 to 7 and 9 to 10 but decreased during egg stages 7 to 9. Both ovariectomy and allatectomy resulted in a depressed rate of pupal fat body disappearance. This was attributed to the absence of functional ovaries in the allatectomized and ovariectomized females. It was hypothesized that the developing ovaries may act as a nutritional sump for vitellogenic precursors present in the fat body and/or an ovarian hormone may stimulate cytolysis of the pupal fat body. By so doing the pupal fat body volume would decrease more rapidly when developing ovaries were present. The adult fat body volume was not affected in allatectomized or in allatectomized plus ovariectomized flies, but increased in volume in ovariectomized flies. Topical applications of synthetic juvenile hormone on mature female flies resulted in an increased adult fat body volume. From this it was concluded that ovariectomy removed the inhibition on the corpus allaturn and resulted in an increase in juvenile hormone titre which caused an increase in adult fat body volume. INTRODUCTION
THE ADULTof cyclorraphous Diptera contains two histologically distinct fat body types, the adult fat body and the pupal fat body (HARLOW, 1956); pupal fat body has also been termed ‘products of histolysis’ (LLNEVA, 1953) ‘free larval fat cells’ (WEISMANN, 1963), and ‘pupal fat balls’ (ANDERSON,1964). In Musca domestica L. the adult fat body is formed from embryonic rudiments in the posterior portion of the larvae (WEISMANN,1963) ; in Drosophila melanogaster (Meig.) it is believed to arise from cells of the epidermis during metamorphosis (BODENSTEIN,1950). In M. domestica the pupal fat body is derived from larval fat body that is dispersed into the haemolymph as free-floating cells (110 to 115 ,Uwide) at the beginning of the pupal stage-(WEISMANN, 1963). This same process has been reported 1729
1730
T. S. ADAMS ANDD. R. NELSON
for D. melunogaster (BODENSTEIN, 1950). Early in pupal development these dispersed cells in the housefly become filled with large globules of protein and persist throughout the first 3 days after adult emergence (WEISMANN, 1963). Pupal fat body has also been found in the haemolymph of the following newly emerged dipterous insects: CaZZiphoraerythrocephala (Meig.) (PEREZ,1910; THOMSEN, 1952), Phaenicia sericata (Meig.) (EVANS,1935), 1M.domestica vi&a Macq. (CWILICHand MER, 1954), Protophormia terrenovae (R.-D.) (HARLOW,1956), Fan&a cunicularis (L.) and Muscina stubulans (Fall.) (ANDERSON,1964), D. melanogaster (BUTTERWORTH et al., 1965), and Dacus tryoni (Froggatt) (MTJNROand BAILEY, 1965). Histochemical investigations of the pupal fat body in Drosophila showed that these cells contained stores of lipids, proteins, and glycogen, whereas the adult fat body contained only lipid and glycogen stores (BUTTERWORTH et al., 1965); the same pattern was observed in M. domestica (WEISMANN, 1963). WEISMANN (1963) correlated the disappearance of the pupal fat body with the development of the ovaries and assumed that the pupal fat body was a nutritive reserve for ovarian maturation and that the adult fat body was used for general metabolic maintenance. Several papers support the hypothesis that pupal fat body disappearance is correlated with ovarian development. The pupal fat body of P. sericata disappeared more rapidly in females on a diet containing protein that promoted ovarian maturation than in those maintained on sugar and water only, which prevented ovarian development (EVANS,1935). This same pattern was observed by other investigatorsfor the following insects fed only sugar and water: C. erythrocephalu (THOMSEN, 1952), P. terrenovae (HARLOW,1956), and F. canicuZaris (ANDERSON, 1964). Also, irradiated D. tryoni females with stunted ovaries used pupal fat body at a slower rate than the controls (MUNROand BAILEY,1965). Hypertrophy of the fat body was demonstrated in ovariectomized Bombyx mori (L.) (HAMASAKI,1932), MeZanopZns dzjkrentialis (Thomas) (PFEIFFER,1945), Drosophila virilis Sturt. (BODENSTEIN, 1947), Oncopeltus fasciatus (Dallas) (JOHANSSON, 1958), D. melanogaster (DOANE,1961; BUTTERWORTH and BODENSTEIN, 1968), but only a slight increase of the fat body was observed in C. erythrocephala (THOMSEN and HAMBURGER, 1955). Allatectomy resultedin hypertrophied fat bodies in C. erythrocephalu (THOMSEN, 1942), M. differential& (PFEIFFER, 1945), Drosophila hydei (Sturtevant) (VOGT,1949), Peripluneta americana (L.) (BODENSTEIN, 1953), 0. fasciatus (JOHANSSON, 1958), Phormia regina Meig. (ORR, 1964), and Locusta migratoria migratoriodes (R. & F.) (MINKS, 1967). No hypertrophy was observed in allatectomized Leucophaea maderae (F.) (SCHARRER, 1955). The Drosophila mutant, adipose, has aberrant ovaries and a hypertrophied fat body, but when wild-type ovaries were implanted into these mutant females, the fat body did not hypertrophy and the implanted ovaries developed (DOANE,1961). Subsequently, BUTTERWORTH and BODENSTEIN (1967) found that the ovaries in Drosophila had a role in protein deposition, growth, and histolysis when larval fat bodies were transplanted into adults.
CORPUS ALLATUM
AND OVARIALEFFECTON
HOUSEFLYFATBODY
1731
Thus, both the corpus allatum and the ovary have a rale in the function of the fat body in insects. This study was conducted to examine more closely the relationship between the corpus allaturn, ovary, and the pupal and adult fat bodies in the abdomen of the housefly, Musca domestica, during the first gonotropic cycle. MATERIALS
AND METHODS
Rearing Flies of the FW, strain were reared on CSMA medium (ANON., 1956) that was fortified with 0.015 g of brewer’s yeast/g of CSMA. Adults were given water and powdered food consisting of dried non-fat milk, sugar, and whole powdered egg at a ratio by weight of 6 : 6 : 1. Virgin female flies used in these studies were from a 4-hr emergence period All flies were held in plastic cages and were sexed under CO, narcosis. (17.8 x 26.7 x 34.4 cm) that were covered at one end by a Tubegauze sleeve. The rearing temperature was 25.6 + 1°C at 40 to 50 per cent r.h. with a 12-hr photoperiod. Egg development scores Egg development was scored on the basis of ten stages which were adapted from those described for the eye gnat, IA&elates collusor (Townsend) (ADAMS and MULLA, 1967). In this scoring technique, stage I is the germarium, stage 4 is the beginning of vitdlogenesis, and stage 10 is the mature egg. Surgical procedures and fat body removal techniques Flies were allatectomized, ovariectomized, and sham operated by previously described techniques (ADAMS and HINTZ, 1969). Removal of the ring gland will be referred to hereafter as allatectomy. The fat body was removed from the fly by first removing the abdomens and placing them ventral side up in a drop of 1% saline solution. Then the abdomen was slit longitudinally and the viscera removed. The pupal fat body was scraped from the abdomen and removed from the gut by dipping it in saline solution. Adult fat body was picked from the abdomen with microforceps. Both the adult and pupal fat bodies were then taken up in a separate microhematocrit tube in a drop of saline solution and the tubes were sealed with a torch and centrifuged for 1 min in a microhematocrit centrifuge. The amount of fat body in the tube was measured in a dissection microscope at 40 x with an ocular micrometer and then converted to mm3 of fat body. Statistical treatment of the data Simple analysis of variance and standard deviations were conducted on nontransformed data by the methods described in SNEDECOR (1956), and the means were separated by Duncan’s multiple range test (LITTLE, 1963). Regression analysis and the covariance analyses were conducted on mm3 of fat body vs. log, of age by the North Dakota State University computing centre at Fargo.
1732
T. S. ADAMS AND D. R. NELSON
Relationship of ovarian development to fat body volume From a group of females held at 22.2 _+l”C, samples of 10 flies were taken periodically and frozen. Ovarian development was scored and the fat body volume was determined for each female. The mean volume of fat body was then calculated for each of the stages of obgenesis in the first gonotropic cycle. ESfect of allatectomy, allatectomy plus ovariectomy, and ovariectomy on fat body volume The females were allatectomized at a mean age of 6 hr after emergence and were ovariectomized 24 hr after emergence. They were held at 25.6 ? 1°C until 7 days after emergence. At this time the flies were frozen and the fat body volume was determined. The four groups of 40 flies each were allatectomized, allatectomized plus ovariectomized, ovariectomized, and control flies. The fat body was measured in 10 flies from each group. This experiment was replicated four times, Effect of allatectomy on fat body volume in$ies of different ages About 150 flies were allatectomized at a mean age of 12 hr after emergence and were held at 22.2 i 1°C and another 150 were control operated. In the first study, samples were removed from the allatectomized and control flies at 12-hr intervals 12 to 96 hr after emergence. This experiment was repeated and samples were taken at 12, 48, 96, 144, and 192 hr after emergence. Each sample consisted of 10 flies which were frozen, the fat body then measured, and ovarian development scored. Effect of ovariectomy on fat body volume in flies of different ages About 150 flies were ovariectomized 24 hr after emergence and a comparable number were control operated. All flies were held at 22.2 + 1°C and during the first study they were sampled at 24-hr intervals from 24 to 144 hr after emergence. The experiment was repeated a second time under the same conditions and sampled at 24, 72, 120, 168, and 216 hr. Each sample consisted of 10 flies that were frozen before the fat body volume was determined. Effect of synthetic juvenile hormone on the volume of adult fat body Synthetic juvenile hormone (trans,trans-lO,ll-epoxy farnesenic acid methyl ester) was topically applied in mineral oil (1~1) to 200 virgin females 6 days after emergence at a concentration of 25 pg/,ul. Control females received 1~1 of mineral oil. Seven days after treatment the females were frozen and fat body volume was determined. RESULTS
Relationship of Ovarian Development to Fat Body Volume Correlation of ovarian development with pupal and adult fat body volume The total abdominal fat body volume (pupal fat body plus adult fat body volumes) decreased with ovarian maturation (Table 1). Newly emerged females with stage 2 ovaries contained 1.82 mm3 of total fat body; this volume decreased
1733
CORPUS ALLATUM AND OVARIAL EFFECTON HOUSEFLY FAT BODY
44 per cent to l-01 mm3 in females with mature eggs. The total fat body volume varied between l-63 and l-93 mm3 in females with ovaries in stages 2 to 6, but decreased in volume when the ovaries were in stages 6 to 9. No further changes occurred between stages 9 and 10. TABLE I-RELATIONSHIP OF OVARIAN DEVELOPMENTTO VOLUMES OF PUPAL AND ADULT FAT BODY IN THE HOUSEFLY Tissue vol. (mm3) . , Ovarian stage
No. flies
Pupal fat body ( + S.D.)
Adult fat body (+ SD.)
Total fat body (rf: S.D.)
2 3 4 5 6 7 8 9 10
29 28 21 8 9 10 4 4 41
l-38 + 0.11 1.15 c 0.11 o-99 k o-09 0.65 kO.15 O-69 + 0.07 o-21 i- 0.10 0.17 F 0.10 O-08 F 0.08 O-00 i o-00
044 * 0.08
1.82 + 0.07 1.93 kO.11 I.91 + 0.09 1.63 z!z0.20 I.73 * 0.09 1.56kO.18 1.24 + 0.20 0.98 + 0.23 I.01 + o-04
0.78 + 0.06 0.92 + 0.06 0,98 * 0.10 1-04+0~10 I.35 + o-07 1.07 + o-20 0.89 + 0.16 1*01+ o-04
Adult fat body (%) 24 40 50 60 60 86 86 91 100
The volume of pupal fat body decreased with ovarian maturation from l-38 mm3 in females with stage 2 ovaries to O-00 mm3 in females with stage 10 ovaries, whereas the adult fat body increased from O-44 mm3 in females with stage 2 ovaries to a maximum volume of l-35 mm3 in females with stage 7 ovaries and then decreased to 1.01 mm3 in mature females. When adult fat body was expressed as the percentage of the total fat body, we found that females with stage 2 ovaries contained 24 per cent adult fat body and this percentage increased steadily with ovarian maturation to 100 per cent in females with mature eggs (Table 1). Correlation of ovarian development with volzcme increase and decrease of the adult and pupal fat body The volume of the pupal fat body decreased in all ovarian stage intervals except 5 to 6 (Fig. 1). The maximum pupal fat body volume decrease of 0.48 mm3 occurred during the ovarian stage interval of 6 to 7. During the ovarian stage intervals of 2 to 3, 3 to 4, and 4 to 5, the volume of pupal fat body decreased 0.25, O-17, and O-34 mm3, respectively. A small decrease in pupal fat body volume of O-04,0-09, and O-OSmm3 occurred during ovarian stage intervals of 7 to 8, 8 to 9, and 9 to 10, respectively. Adult fat body volume increased during the ovarian stage intervals of 2 to 3, through 6 to 7, and in 9 to 10 but decreased during the ovarian stage intervals of 7 to 8 and 8 to 9. A maximal volume increase of O-45 mm3 of adult fat body
1734
T. S. ADAMSANDD. R. NELSON
occurred during the ovarian stage interval of 2 to 3, and during ovarian stage intervals of 3 to 4, 4 to 5, 5 to 6, and 6 to 7 adult fat body volume increased 0.15, 0.04, O-06, and 0.31 mms, respectively. During the ovarian stage intervals of 7 to 8 and 8 to 9 the adult fat body volume decreased 0.28 and 0.18 mm3, respectively, but in the egg stage interval of 9 to 10 the volume of adult fat body increased 0.11 111111~. % E .6 g .5 2 .J > >3 0” m .z 2 .’ _I 0 9 5 -_I x-2 S’ g -3 g -9 f -5 5 &.-.6 %I
ADULT F/iiBODY PUP& WrBODY
5- 6 ,,,,LL&,, OVARIAN
STAGE INTERVALS
7- *
* - “f ~‘p;siY WrELLOOENlC
FIG. 1. Changes in the volumes of adult and pupal fat body in the housefly during different ovarian stage intervals.
A clearer pattern of fat body volume increase and decrease emerged when ovarian development was broken into previtellogenic (stages 2 to 4), vitellogenic (stages 4 to 9), and postvitellogenic (stages 9 to 10) phases lasting 49, 74 and 47 hr, respectively (Table 2). In the previtellogenic phase of ovarian development the volume of adult fat body increased 0.48 mm3 (9.7 x 10m3mm3/hr) and the pupal fat body volume decreased 0.39 mm3 (7.9 x 1OL3mm3/hr) for a net volume increase in total fat body of 0.09 mm3 (1.8 x lO-3 mm3/hr). During vitellogenesis both the pupal fat body and the adult fat body decreased 0.91 mm3 (12.2 x 10-s mms/hr) and 0.03 mms (0.4 x 10e3 mm/hr), respectively, for a net volume loss of 0.94 mms The remainder of the pupal fat body, 0.08 mms (12.7 x 1O-3 mm3/hr). (1.7 x 10-s mm3/hr) disappeared during the post-vitellogenic period, but the adult fat body volume increased 0.12 mm3 (2-5 x 10~~ mm3/hr) for a net volume increase of 0.04 mm3 (0.8 x 1O-3 mm3/hr). Thus, there was a net volume increase in total fat body during the pre- and post-vitellogenic periods because of an increase in adult fat body, and a net volume loss during the vitellogenic period because of a decrease in volume of both the pupal and adult fat bodies. Effect of ovariectomy, fat body
allatectomy,
and ovariectomy
plus allatectomy
on abdominal
Allatectomized, allatectomized plus ovariectomized, and ovariectomized females had total fat body volumes of 1.64, 1.83, and 1.73 mm3, respectively, which were 34, 44, and 37 per cent greater than in the control flies (1.26 mm3) (Table 3).
- I.38
180
Total
-21.4
-7.9 - 12.2 -1.7 +0.57
+ 0.48 - 0.03 +0*12
AV
+ 12.5
+9*7 -0.4 +2*5
AV/hr x IO3
Adult fat body
* AV calculated from means presented in Table 1. t Calculated from graph in Fig. 3. $ Includes total time from stage 9 to the end of the experiment at 180 hr.
-0.39 -0.91 - 0.08
49 74 47:
Pre-vitellogenic (2-4) Vitellogenic (4-9) Post-vitellogenic (9-10)
AV/hr x IO3
Pupal fat body
-0.81
+ 0.09 - 0.94 + 0.04
AV
- 8.9
+I*8 -12.7 +0*8
AV/hr x 10S
Fat body net change
Volumetric (mm”) changes in abdominal fat body*
PHASES TO VOLUME CHANGES IN THE PUPAL AND ADULT FAT BODY OF THE HOUSEFLY
AV
OF VITELLOGENIC
Otigenetic phase
&-&LATIONSHIP
Duration (hr) t
TABLE
1736
T. S. ADAMSAND D. R. NELSON
Analysis of variance showed that the treatments had a significant effect (0.1 confidence level), and the mean separation is shown in Table 3. No pupal fat body remained in the control flies 7 days after emergence whereas 61 to 100 per cent of the operated flies contained pupal fat body. Allatectomized, allatectomized plus ovariectomized, and ovariectomized flies contained O-46, O-55, and 0.19 mm3 of pupal fat body, respectively, and these volumes represented 11 to 30 per cent of the total fat body in the operated flies. The treatments had a significant effect on pupal fat body volume (O-01 confidence level); the mean separation is shown in Table 3. Adult fat body volume varied from 1.18 to 1.54 mm3 in all groups, and no significant difference was observed between the means. However, the ovariectomized flies showed a 21 per cent increase in adult fat body volume over the controls. Both ovariectomy and allatectomy resulted in the retention of pupal fat body. Allatectomy had no effect on the adult fat body volume, but ovariectomy resulted in an increase in volume of adult fat body. Therefore, the increase in total fat body volume in the treated flies was due primarily to pupal fat body retention. Eflect of allatectomy on abdominal fat body volume in flies of dz@erent ages Totalfat body. In all age groups examined (except flies 24 hr after emergence) the allatectomized flies contained more total fat body than the controls (Fig. 2A) and the analysis of covariance showed that allatectomy and age had a significant effect (0.10 confidence level) on the total fat body volume. In flies 24 to 36 hr after emergence the volume of total fat body in both the control and the allatectomized flies increased over the 12-hr volume (184 mm3), then in the control flies it decreased gradually to 1.28 mm3 in 192~hr flies for a net loss of 30 per cent. In contrast, the 192-hr allatectomized flies contained 180 mm3 of total fat body, about the same volume as at 12 hr after emergence. Regression analysis of the data, mm3 total abdominal fat vs. log, age, gave a correlation coefficient (r) of O-056 (insignificant) for the allatectomized females and -0.657 (significant at the 0.05 confidence level) for the controls. Thus, total fat body volume and age did not correlate in the allatectomized flies but the volume of the total fat body of the control flies correlated negatively with age. Pupal fat body. Pupal fat body (1.63 mm3 in flies 12 hr after emergence) disappeared by 144 hr in the control females (Fig. 2B), but allatectomized flies 192 hr after emergence contained 0.45 mm3 of pupal fat body. Also, all allatectomized females at all ages contained some pupal fat body, but the number of control females that contained pupal fat body decreased in the samples taken after 72 hr. The covariance analysis showed that allatectomized flies contained significantly (0.01 confidence level) more pupal fat body than the controls and that the volume was significantly affected by age. Regression analysis of the data, mm3 pupal fat body vs. log, age, gave a correlation coefficient of -0.789 for the allatectomized females and - 0.933 for the controls (significant at the 0.01 confidence level).
%-%FFECT
OVAR~ECTOMY,
+ 33.9 i-43.8 -I-36.6 -
1.73 & 0.208 1.26 ?z0.14b
Change ?I (%)$
1.64 k 0*21&lb 1.83 4,0*21’
Volume (mm”) (+S.D.)i
Total fat body
OF ALLATECTOMY,
AND
With pupal fat body (%) 92 100 61 00
Volume @mS) ( ?I S.D.) t 0.46 zk0.20& 0.55 + 0.04s 0.19 f O*ll@ o*oo * O*OOb
Pupal fat body
ALLATECTOMY PLUS OVAR~ECTOMY IN THE HOUSEFLY
10.8 0.0
27.5 30.4
Fat body (%)
ON THE VOLUME
PAT BODY
+21.6 -
1.26 + 0~14~
-6.6 +0*4
Change + (%)5
1~54~0~15~
1.18 1 O-20& I.28 rt o-17*
Volume (mm3) ( ?I S.D.)t
Adult fat body
OP ABDOMINAL
* Flies were allatectomized at 6 hr after emergence, ovariectomized at 24 hr, and then held for 6 days at 25.6 zk1°C. t Those values not followed by the same letter are significantly different from each other at the 5 per cent level as determined by Duncan’s multiple-range test. Fat body volume of treated-fat body volume of control X 100 per cent. $ Percentage change in fat body volume = Fat body volume control
Allatectomy Allatectomy .plus ovariectomy Ovariectomy Controls
Treatment *
TABLE
g %
2
2 “m p ++
m
g
E 4 # 2
g
2
3 5;
$ !z
2
T. S. ADAMS AND D. R. NELSON
1738
Adult fat body. Adult fat body increased with age in both the allatectomized and control flies (Fig. ZC). Females 12 hr after emergence contained 0.21 mm3 of adult fat body. This volume increased to l-28 mm3 in the controls and to l-39 mm3 in allatectomized flies 192 hr after emergence. Analysis of covariance showed no significant effect for allatectomy on adult fat body volume, but age had a significant effect (O-10 confidence level). o-;----a -
?“-A.
CONTRCL &LIITECTOMY
2.3. 2.1 1.9 17. 15. w.
0 AGE (HR
1
FIG. 2. Effect of allatectomy on the volumes of total, pupal, and adult fat body in houseflies of different ages after emergence. A, volume of total fat body, B, volume of pupal fat body, and C, volume of adult fat body.
Regression analysis of the data, mm3 adult fat body vs. log, age, gave significant correlation coefficients (0.01 level) of O-939 for the controls and O-907 for the allatectomized flies. Correlation
of fat body volume with ovarian stages
Since 180 hr after emergence the ovaries in the allatectomized flies had not proceeded beyond stage 4 but the controls had reached stage 10 (Fig. 3), the data were rearranged from their chronological treatment (as in Fig. 2) to give volume changes within the various ovarian stage intervals (a physi,ological adjustment). This was accomplished by calculating the mean volume of adult and pupal fat bodies for each stage of oiigenesis in allatectomized flies as was previously done for the controls (Table 1). The volume change (AV) from one ovarian stage to another was determined and the rate of change (AV/h r ) was calculated by dividing the volume change by the duration (hr) of the ovarian stage intervals (determined from Fig. 3). These data are summarized in Table 4.
CORPUS
ALLATUM
AND
OVARIAL
EFFECT
ON
HOUSEFLY
FAT
1739
BODY
The decrease in the volume of pupal fat body during the ovarian stage interval of 2 to 3 was the same in the control (O-23 mma) and allatectomized flies (O-26 mm3). However, the rate of change was twice as great in the controls (13.5 x lo3 mm3/hr) as in the allatectomized flies (7.4 x 10-s mma/hr). During the ovarian stage interval of 3 to 4 the pupal fat body decreased 0.16 mm3 (5.0 x 10-3 mma/hr) in the controls and O-29 mm3 (2.0 x 10-a mm3jhr) in the allatectomized flies. The volume decrease IOLS9i&i a 8-
/I o------o
/
CONTROL ALLATECTOMY
e--.-.4
I’
s w 7-
. I’
zl 0 6-
I/.
%
f’
5 ti5%4-
Q __A__$
$/.
f
,--;
_p--~_.~.‘.‘~$‘-
%3Y g p(?J-----vt IO
/-_y--’ I 20
o I
I 40
I
II,,,
AGE (i-ii FIG.
3
I’
80
100
I 200
1 300
.)
3. The effect of allatectomy on ovarian maturation in the housefly. Ages are hours after emergence.
during the ovarian stage interval of 2 to 4 was 0.39 mm3 (870 x 10e3 mm3/hr) for the controls and 0.55 mm3 (3.0 x 10-a mm3/hr) for the allatectomized flies. When the decrease in pupal fat body was considered for the total elapsed time (180 hr) the change in the allatectomized flies was the same as during stages 2 to 4,0*55 mm3 (3.0 x 1O-3 mm3/hr), whereas the change was twice as great in the controls, 1.38 mm3 (7.7 x 10-s mm3/hr). The volume changes observed during the ovarian stage intervals of 2 to 3 and 3 to 4 did not differ appreciably between the control and allatectomized flies (although the rates did). This demonstrates that the developing ovaries in the controls (stages 4 to 10) had an effect on the pupal fat body volume decrease which was not observed in the allatectomized flies because the ovaries did not develop beyond stage 4. The change in adult fat body was the same in the control and allatectomized flies during the ovarian stage interval of 2 to 3 and was twice as great in the allatectomized flies as in the controls during stage interval 3 to 4. However, when the stage duration was considered the rate of increase in adult fat body was approximately twice as great in the control as in the allatectomized flies (stages 2 to 3 : 20.0 x 10-Z vs. 9.4 x 10-s mms/hr, respectively, and stage 3 to 4: 4.4 x 10~~ vs. 2.5 x 1O-3 mm3/hr, respectively), but when the total elapsed time for the experiment was considered (180 hr) no appreciable difference was observed between the increase in adult fat body in the controls and in the allatectomized flies (O-69 and
Control total (all stage intervals 2-10)
-7.7
-8.0
-5.0
-13.5
-3.0
-2.0 -7.4
AVjhr x 1Oa
* Calculated from Fig. 3. t Included total time from start of stage 3 to end of experiment, 180 hr. 1 Calculated from Table 1.
Mean change/hr
-1.38$
-0.16: -0*39x
32
-0.23:
-0.55
-- 0.29 0.26
49
49
17
180
145 35 t
AV
Pupal fat body
180
3-4
Control
58
56 61
No.
Interval duration (hr)*
Total Mean change/hr
2-3
34 2-3
Control
Total Mean change/hr
Allatectomy Allatectomy
Treatment
Ovarian stage interval
to.55
-I-0.48
+0*14
+ 0.34
+ 0.69
+ 0.36 +0*33
AV
+3.2
+9*7
+4.4
+ 20.0
+3.8
+2*5 + 9.4
AV/hr x lOa
Adult fat body
-0.83
+ 0.09
- 0.02
to.11
+ 0.14
+ + 0.07 0.07
AV
-4.5
+1.8
-0*4
+ 7.3
1-O-8
+0*5 +2*0
AV/hr x lOa
Total fat body
Fat body volume (mm”) change during indicated stage intervals
TABLE ~---COMPARISONOF PUPALAND ADULT FAT BODY VOLUMECHANGESWITHIN OVARIANSTAGEINTERVALSAND WITH TIME IN ALLATECTOMIZED AND CONTROLHOUSEFLIES
3 $ Z
w
:
:
1
9 .m
CORPUS ALLATUM
AND OVARIAL EFFECT ON HOUSEFLY
FAT BODY
1741
0.55 mm3, respectively) and in the rates of increase (3.8 and 3.2 x 10-s mm3/hr, respectively). Thus, the increase in adult fat body volume appears to be independent of ovarian maturation and the corpus allatum (although a stimulator-y effect of juvenile hormone may be cancelled out by the nutritional demands of the developing ovary). The allatectomized flies’ total fat body volume increased 0.14 mm3: (0.8 x 1O-3 mms/hr) during the ovarian stage interval of 2 to 4 whereas the control flies showed a volume increase of 0*09 mm3 (1.8 x 1O-3 mm3/hr). When the total elapsed time was considered, the volume of total fat body increased 0.14 mm3 (0.8 x 1O--smm3/hr) in the allatectomized flies and decreased 0.83 mm3’ (4.5 x 10-s mms/hr) in the control flies. ESfect of ovariectomy
on abdominal fat body volume in flies of dz@er-ent ages
Total fat body. Ovariectomized females showed a steady increase with age in the total volume of fat body from 1.09 mm3 24 hr after emergence to 1.83 mm3 at 216 hr (Fig. 4). This represented a total increase of 68 per cent in total fat body
FIG. 4. Effect of ovariectomy on the volumes of total, pupal, and adult fat body in houseflies of different ages after emergence. A, volume of total fat body, B, volume of pupal fat body, and C, volume of adult fat body.
volume over the 24-hr value and 110 per cent more total fat body than the 216-hr control. The total fat body of the control operated flies increased from 1.09 mm3 at 24 hr to 1.32 mms at 96 hr, and then it decreased to 0.91 mm3 at 216 hr for a 16 per cent loss in volume over the 24-hr samples. The ovariectomized females contained significantly more (t-test, 0.01 confidence level) fat body than the controls in samples taken at 144, 168, and 216 hr.
1742
T. S. ADAMS
AND D.R.
NELSON
Regression analysis of the data, mm3 total fat body vs. log, of the age, gave a correlation coefficient (r) of -0.519 for the controls and 0.564 for the ovariectomized flies. Thus, the volume change gave a poor fit for a linear model, but the fat body volume increased in the ovariectomized flies and decreased in the controls with age. Pupal fat body. In both the ovariectomized flies and the control operated ones, -the volume of pupal fat body decreased with age from its 24-hr volume of 0.86 mm3 (Fig. 4B). At 144 hr the pupal fat body had completely disappeared from the .control females, but 100 per cent of the ovariectomized flies contained pupal fat body (mean volume of 0.46 mm3). At 168 hr, 80 per cent of the ovariectomized flies had pupal fat body (mean volume of 0.33 mm3), and at 216 hr, 33 per cent of the flies had pupal fat body (mean volume of 0.20 mm3). Analysis of covariance showed that ovariectomized flies contained significantly (O-10 confidence level) more pupal fat body than the controls and that the volume was affected significantly (0.10 confidence level) by age. The ovariectomized flies at 144, 168, and 216 hr contained significantly (t test, 0.01 confidence level) more pupal fat body than the controls. Regression analysis of the data, mm3 pupal fat body vs. log, age, gave a correlation coefficient (r) of -0.920 for the controls and -0.947 for the ovariectomized flies. Thus, the change of pupal fat body volume was negatively correlated with age and was linear in the transformed data for both the control and the ,ovariectomized flies. Adult fat body. Adult fat body volume increased with age in both the control operated flies and in the ovariectomized ones (Fig. 4C). The volume in the controls at 24 hr was 0.23 mm3 and at 216 hr it had increased to 0.91 mm3. At 216 hr the adult fat body volume was 1.63 mm3 in ovariectomized flies. Thus, the ovariec-tomized flies had 79 per cent more fat body than the controls. The analysis of covariance gave no significant effect for ovariectomy on the -volume of adult fat body but showed that age had a significant effect (0.1 level). However, when a t-test was applied to the data from individual flies, the 216-hr .age group had significantly (0.01 confidence level) more fat body than the controls; .a11other differences were insignificant. Regression analysis of the data, mm3 adult fat body vs. log, age, gave a correlation coefficient (r) of O-695 for the control operated flies and O-838 for the ovariectomized flies. Both values were significant and showed that adult fat body volume -increased with female age in both the ovariectomized flies and the controls. Comparison of the effect of allatectomy .adult fat bodies
and ovariectomy
on the volumes of pupal and
Analysis of covariance applied to the data on the pupal fat body volume between -the allatectomy and ovariectomy control groups showed that the allatectomy ,controls contained a significantly greater volume of pupal fat body than the ovariectomy controls, but the volumes of the adult fat bodies did not differ. This meant that direct quantitative comparisons could not be made between the
CORPUS ALLATUM
AND OVARIAL EFFECT ON HOUSEFLY FAT BODY
1743
effects of allatectomy and ovariectomy. However, the data were adjusted computing the percentage of adult fat body for each age group. Percentage
of adult fat body =
by
Adult fat body mm3 x 100 Total fat body mm3
*
Covariance analysis showed that there was no significant difference between the control groups when the data were adjusted in this manner, and the combined values had a correlation coefficient (r) of 0.912. Up to 72 hr after emergence the percentage of adult fat body increased at identical rates in the controls, allatectomized, and ovariectomized flies, but after 72 hr the allatectomized and ovariectomized flies showed a slower rate of increase (Fig. 5). This change in
80_ -
O----Q ----.a
CONTROL OVARIECTOMY ALLATECTOMY
2 a cn 40a
FIG.
5.
Percentage
of the total abdominal
ovariectomized,
allatectomized,
fat body and control
as adult
fat body
in
houseflies.
rate occurred at the time vitellogenesis started in the control fly population. The major difference between the allatectomized, ovariectomized, and control flies was the presence of vitellogenesis in the controls and its absence in the treated flies. The effects of allatectomy and ovariectomy on pupal and adult fat body volume changes were compared further by calculating the net change in volume over time in the pupal and adult fat bodies. The net change in the treatment was compared to the net change in the respective control, and a percentage of increase or decrease over the respective control was calculated. When these percentages were compared, the allatectomized females had 33 per cent of their pupal fat body remaining and the ovariectomized females had 49 per cent remaining. Although there was little difference in the decrease in pupal fat body volume between ovariectomized and allatectomized flies, the volume of adult fat body increased 15 per cent (insignificant)
1744
T. S. ADAMSAND
D.R. NELSON
in the allatectomized flies and 107 per cent in the ovariectomized flies. Thus, the increase in adult fat body volume in ovariectomized flies compared with essentially no change in allatectomized flies indicates a possible stimulation of adult fat body by the corpus allatum. EfSect of synthetic juvenile hormone on the adult fat body Adult female flies with mature ovaries and without pupal fat body were treated with 25 pg of synthetic juvenile hormone and the volume of adult fat body was measured 7 days later. The treated flies contained 1.28 mm3 of adult fat body compared with 1.08 mm3 in the controls. The increase in adult fat body volume was significant at the 0.01 confidence level (determined by a t-test). This proves that juvenile hormone stimulates an increase in adult fat body volume as suggested by our previous experiments. DISCUSSION
AND
CONCLUSIONS
Our studies showed that the pupal fat body volume decreased at its greatest rate lduring the vitellogenic phase of oijgenesis and that no pupal fat body remained after the ovaries had reached maturity (stage 10). A relationship between the disappearance of pupal fat body and the development of the ovaries is indicated by the retention of pupal fat body in Diptera with stunted ovaries: P. sericata (EVANS, 1935), C. erythrocephala (THOMSEN, 1952), P. terranovae (HARLOW, 1956), M. domestica (WEISMANN, 1963), F. canicularis (ANDERSON, 1964), D. tryoni (MUNRO and BAILEY, 1965). The correlation between ovarian maturation and pupal fat body cytolysis is further demonstrated in our study by the retarded rate of pupal fat body loss in houseflies without ovaries or in those with non-developing ovaries because of allatectomy. Since the pupal fat body is retained in allatectomized females (no juvenile hormone present) and in ovariectomized females (in which the corpus allatum is continuously active) (Adams, unpublished observations), the retention of the pupal fat body is not due to the presence or absence of the corpus allatum and juvenile hormone, but is dependent upon the functionality of the ovary. Thus, the maturing ovary may increase the rate of disappearance of pupal fat body by acting as a nutrient sump, as suggested for 0. fasciatus (JOHANSSON,1958), M. domestica (WEISMANN,1963), and D. tryoni (MUNRO and BAILEY, 1965), and/or by a direct stimulation of pupal fat body cytolysis. DOANE (1961), from studies on the mutant female sterile (2) adipose of D. melanogaster, proposed the existence of a hormone from the ovaries which regulated the utilization of stored lipid in the fat body. An oiistatic hormone has been found in M. domestica (ADAMS et al., 1968) which is produced by the developing ovary and which maintains the cyclicity of ovarian development by its action on the corpus allatum (Adams, unpublished observations). This hormone also may be responsible for the effect of the ovaries on pupal fat body cytolysis and on the decrease in adult fat body volume during vitellogenesis. The adult fat body in the housefly showed a volume increase with age. When this was correlated with ovarian development, adult fat body volume increased
CORPUSALLATUMAND OVARIALEFFECT ONHOUSEFLYFATBODY
174.5
during the pre-vitellogenic and post-vitellogenic phases and decreased during the latter part of the vitellogenic phase. Thus, both the adult fat body and the pupal fat body decreased during vitellogenesis. This also has been observed in P. tewanovae (HARLOW, 1956). Allatectomy did not result in an increase in adult fat body when compared to the control group. In this respect, the housefly resembles L. maderae (SCHAR~R, 1955). Allatectomy has been reported to result in an increase in fat body in a number of other insects: C. erythocephala (THOMSEN, 1942),M. d$erentiaZis (PFEIFFER,1945), D. hydei (VOGT, 1949), P. americana (BODENSTEIN, 1953), 0. fascia&s (JOHANSSON, 1958),P. regina (ORR, 1964), and L. m. migratoriodes (MINKS, 1967).
FIG.6. Summary of the interrelationships between the ovaries, corpus allaturn, pupal fat body, and adult fat body in the housefly.
Ovariectomized houseflies had a greater volume of adult fat body than the controls. Similar results have been reported for B. mori (HAMA~AKI, 1932), M. diflerentialis (PFEIFFER,1945), D. virilis (BODENSTEIN, 1947), 0. fasciatus (JOHANSSON, 1958), and D. melanogaster (DOANE, 1961;.BUTTERWORTH and BODENSTEIN, 1968),but not for P. regina (ORR, 1964). The corpus allatum is active in ovariectomized houseflies (Adams, unpublished observations) and it was assumed that these flies contained more juvenile hormone than the controls. This excess of juvenile hormone in ovariectomized females resulted in an increase of adult fat body volume; this was substantiated by the increase of adult fat body in flies topically treated with synthetic juvenile hormone. Our data may be summarized by presenting the following hypothesis, which is diagrammed in Fig. 6. The corpus allatum produces juvenile hormone which
1746
T. S. ADAMSANDD. R. NELSON
stimulates the onset of vitellogenesis in the ovaries of the housefly (ADAMS and The developing ovaries draw on reserve substances in the pupal fat body first and then on the substances in the adult fat body. Secondly, the maturing ovaries produce an oijstatic hormone that inhibits the release of juvenile hormone (ADAMSet al., 1968); this hormone may stimulate cytolysis of the pupal fat body. Both the pupal and adult fat body are drawn upon for general metabolic maintenance. Ovariectomy removed the inhibition on the corpus allatum and juvenile hormone is released continuously. The increased titre of juvenile hormone and the absence of the nutritive sump effect of the ovaries resulted in an increase volume of adult fat body. The stimulator-y effect of juvenile hormone on adult fat body volume is demonstrated in both our ovariectomy experiments and in the synthetic juvenile hormone experiments. Ovariectomized females that were allatectomized did not show hypertrophy of the adult fat body, but ovariectomized females had hypertrophied adult fat bodies; this shows that hypertrophy is not an ovarian effect but one of the corpus allatum. HINTZ, 1969).
Acknowledgements-The authors express their thanks to Dr. BERTA SCHAFZRER of the Yeshiva University and to Dr. F. M. BUTTERWORTHof the Oakland University for their well-founded comments and criticisms of the manuscript. The authors also thank Dr. WM. BOWERSof the U.S.D.A.‘s Pioneering Insect Physiology Laboratory for supplying the synthetic juvenile hormone used in these studies.
REFERENCES ADAMST. S. and HINTZ A. M. (1969) Relationship of age, ovarian development, and the corpus allatum to mating in the housefly, Musca domestica. J. Insect Physiol. 15, 201-215. ADAMST. S., HINTZ A. M., and POMONISJ. G. (1968) O&static hormone production in houseflies, Musca domestica, with developing ovaries. J. Insect Physiol. 14, 983-993. ADAMST. S. and MULLA M. S. (1967) The reproductive biology of Hi&elates coZZusor--II. Gametogenesis. Ann. ent. Sot. Am. 61, 368-372. ANDERSONJ. R. (1964) Methods for distinguishing nulhparous from parous flies and for estimating the ages of Fannia canicularis and some other cyclorraphous diptera. Ann. ent. Sot. Am. 57, 226-236. ANON. (1956) The Peet-Grady method. Soap them. Spec. 32, 243-244. BODENSTEIND. (1947) Investigations on the reproductive system of Drosophila. r. exp. zooz. 104, 101-151. BODENSTEIND. (1950) The Post Embryonic Development of Drosophila (Ed. by DEMERECM.), Chapter 4. John Wiley, New York. BODENSTEIN D. (1953) Studies on the humoral mechanisms in growth and metabolism of the cockroach, Periplaneta americana. BioZ. Bull., Woods Hole 124, 105-116. BUTTERWORTH F. M. and BODENSTEIND. (1967) Adipose tissue of Drosophila melanogasterII. The effect of the adult internal environment histolysis in the larval fat body. J. exp. ZooZ. 164,
on growth, 251-263.
protein
deposition,
and
BUTTERWORTH F. M. and BODENSTEIND. (1968) Adipose tissue of DrosophiZamelanogasterIII. The effect of the ovary on cell growth adult tissue. 3. exp. ZooZ. 167, 207-218.
and the storage
of lipid and glycogen
in the
BUTTERWORTH F. M., BODENSTEIND., and KING R. C. (1965) Adipose tissue of Drosophila melanogaster--I. An experimental study of the larval fat body. J. exp. ZooZ. 158,141-l 54.
CORPUSALLATUMANDOVARIAL EFFECTON HOUSEFLYFAT BODY
1747
CWILICH R. and MER G. G. (1954) Determination of the age of the housefly, Musca domestica vicina, by the persistence of larval fat body cells in the imago. Riv. Parassit. 15, 357-359. DOANE W. W. (1961) Developmental physiology of the mutant female sterile(s) adipose of Dvosophilu meZunogaster--III. Corpus allatum complex and ovarian transplantations. r. exp. Zool. 146, 275-298. EVANS A. C. (1935) Some notes on the biology and physiology of the sheep blow fly, Lucilia sericata. Bull. ent. Res. 26, 115-125. HAMA~AIUS. (1932) On the effect of castration in the silkworm, Bombyx mori. Proc. imp. Acad. Japan 8, 267-270. HARLOWP. M. (1956) A study of ovarial development and its relation to adult nutrition in the blow fly, Protophormia terranovae. J. exp. Biol. 33, 777-797. JOHANSSONA. (1958) Relation of nutrition to endocrine-reproductive functions in the milkweed bug, Oncopeltus fasciatzls. Nytt. Mag. Zool. 7, I-132. LINEVA V. A. (1953) Physiological age of females of Musca domestica. l?nt. Obozr. 33, 161-173. LITTLE T. M. (1963) Correlation and regression. A supplement to Experimental Methods for Extension Workers. Univ. Calif. Agric. Ext. Serv. Publ. MINKS A. K. (1967) Biochemical aspects of juvenile hormone action in the adult Locusta migratoria. Archs nee& 2001. 17, 175-258. MUNROJ. and BAILEY P. T. (1965) I n fluence of radiation on ovarian maturation and histolysis of pupal fat body in Diptera. Nature, Lond. 207, 437-438. ORR C. W. M. (1964) The influence of nutritional and hormonal factors on the chemistry of the fat body, blood, and ovaries of the blowfly, Phormia regina. J. Insect Physiol. 10, 103-119. PEREZ C. (1910) Recherches histologiques sur la metamorphose des muscides Calliphora erythrocephala (Mg.). Arch. Zool. exp. g&n. 5, l-24. PFEIFFERI. W. (1945) Effect of the corpora allata on the metabolism of adult female grasshoppers. 3. exp. Zool. 99, 183-233. SCHARRERB. (1955) Hormones in invertebrates. The Hormones 3, 57-95. SNEDECORG. W. (1956) Statistical Methods applied to Experiments in Agriculture and Biology, 5th ed. Iowa State University Press, Ames. THOMSEN E. (1942) An experimental and anatomical study on the corpus allatum in the Vidensk. Medd. Dansk Nut. Fore-n. 106, 320-405. blow fly, Calliphora erythrocephula. THOMSEN E. (1952) Functional significance of the neurosecretory brain cells and the corpus cardiacum in the female blow fly, Calliphora erythrocephala. J. exp. Biol. 29, 137-172. THOMSEN E. and HAMEKJRGER K. (1955) Oxygen consumption of castrated females of the blowfly, Calliphora erythrocephala. J. exp. Biol. 32, 692-699. VOGT 34. (1949) Fettkorper und Oenocyten der Drosophila nach Extirpation der adulten Bingdruse. Z. Zellforsch. 34, 160-164. WEISMANN R. (1963) Untersuchungen i.iber den larvalen und imaginalen Fettkorper der Imago von Musca domestica. Mitt. Schweiz. ent. Ges. 35, 185-210.
EFFECT OF CORPUS ALLATUM AND OVARIES ON AMOUNT OF PUPAL AND ADULT FAT BODY IN THE HOUSEFLY, kWSC.kl DOllhWTIC~ T. S. ADAMS
and D. R. NELSON
Metabolism and Radiation Research Laboratory, Entomology Research Division, Agriculture Research Service, U.S.D.A., Fargo, North Dakota 58102 (Received 14January
1969; revised 2 May 1969)
Abstract-The stage of ovarian development was compared with changes in the volume of abdominal fat body in female houseflies. Pupal fat body volume decreased during each stage of oagenesis and had its maximum rate of volume decreases during the vitellogenic phase of ocgenesis (stages 4 to 9). No pupal fat body remained in flies with mature [stage 10) eggs. Adult fat body showed a volume increase during egg stages 2 to 7 and 9 to 10 but decreased during egg stages 7 to 9. Both ovariectomy and allatectomy resulted in a depressed rate of pupal fat body disappearance. This was attributed to the absence of functional ovaries in the allatectomized and ovariectomized females. It was hypothesized that the developing ovaries may act as a nutritional sump for vitellogenic precursors present in the fat body and/or an ovarian hormone may stimulate cytolysis of the pupal fat body. By so doing the pupal fat body volume would decrease more rapidly when developing ovaries were present. The adult fat body volume was not affected in allatectomized or in allatectomized plus ovariectomized flies, but increased in volume in ovariectomized flies. Topical applications of synthetic juvenile hormone on mature female flies resulted in an increased adult fat body volume. From this it was concluded that ovariectomy removed the inhibition on the corpus allaturn and resulted in an increase in juvenile hormone titre which caused an increase in adult fat body volume. INTRODUCTION
THE ADULTof cyclorraphous Diptera contains two histologically distinct fat body types, the adult fat body and the pupal fat body (HARLOW, 1956); pupal fat body has also been termed ‘products of histolysis’ (LLNEVA, 1953) ‘free larval fat cells’ (WEISMANN, 1963), and ‘pupal fat balls’ (ANDERSON,1964). In Musca domestica L. the adult fat body is formed from embryonic rudiments in the posterior portion of the larvae (WEISMANN,1963) ; in Drosophila melanogaster (Meig.) it is believed to arise from cells of the epidermis during metamorphosis (BODENSTEIN,1950). In M. domestica the pupal fat body is derived from larval fat body that is dispersed into the haemolymph as free-floating cells (110 to 115 ,Uwide) at the beginning of the pupal stage-(WEISMANN, 1963). This same process has been reported 1729
1730
T. S. ADAMS ANDD. R. NELSON
for D. melunogaster (BODENSTEIN, 1950). Early in pupal development these dispersed cells in the housefly become filled with large globules of protein and persist throughout the first 3 days after adult emergence (WEISMANN, 1963). Pupal fat body has also been found in the haemolymph of the following newly emerged dipterous insects: CaZZiphoraerythrocephala (Meig.) (PEREZ,1910; THOMSEN, 1952), Phaenicia sericata (Meig.) (EVANS,1935), 1M.domestica vi&a Macq. (CWILICHand MER, 1954), Protophormia terrenovae (R.-D.) (HARLOW,1956), Fan&a cunicularis (L.) and Muscina stubulans (Fall.) (ANDERSON,1964), D. melanogaster (BUTTERWORTH et al., 1965), and Dacus tryoni (Froggatt) (MTJNROand BAILEY, 1965). Histochemical investigations of the pupal fat body in Drosophila showed that these cells contained stores of lipids, proteins, and glycogen, whereas the adult fat body contained only lipid and glycogen stores (BUTTERWORTH et al., 1965); the same pattern was observed in M. domestica (WEISMANN, 1963). WEISMANN (1963) correlated the disappearance of the pupal fat body with the development of the ovaries and assumed that the pupal fat body was a nutritive reserve for ovarian maturation and that the adult fat body was used for general metabolic maintenance. Several papers support the hypothesis that pupal fat body disappearance is correlated with ovarian development. The pupal fat body of P. sericata disappeared more rapidly in females on a diet containing protein that promoted ovarian maturation than in those maintained on sugar and water only, which prevented ovarian development (EVANS,1935). This same pattern was observed by other investigatorsfor the following insects fed only sugar and water: C. erythrocephalu (THOMSEN, 1952), P. terrenovae (HARLOW,1956), and F. canicuZaris (ANDERSON, 1964). Also, irradiated D. tryoni females with stunted ovaries used pupal fat body at a slower rate than the controls (MUNROand BAILEY,1965). Hypertrophy of the fat body was demonstrated in ovariectomized Bombyx mori (L.) (HAMASAKI,1932), MeZanopZns dzjkrentialis (Thomas) (PFEIFFER,1945), Drosophila virilis Sturt. (BODENSTEIN, 1947), Oncopeltus fasciatus (Dallas) (JOHANSSON, 1958), D. melanogaster (DOANE,1961; BUTTERWORTH and BODENSTEIN, 1968), but only a slight increase of the fat body was observed in C. erythrocephala (THOMSEN and HAMBURGER, 1955). Allatectomy resultedin hypertrophied fat bodies in C. erythrocephalu (THOMSEN, 1942), M. differential& (PFEIFFER, 1945), Drosophila hydei (Sturtevant) (VOGT,1949), Peripluneta americana (L.) (BODENSTEIN, 1953), 0. fasciatus (JOHANSSON, 1958), Phormia regina Meig. (ORR, 1964), and Locusta migratoria migratoriodes (R. & F.) (MINKS, 1967). No hypertrophy was observed in allatectomized Leucophaea maderae (F.) (SCHARRER, 1955). The Drosophila mutant, adipose, has aberrant ovaries and a hypertrophied fat body, but when wild-type ovaries were implanted into these mutant females, the fat body did not hypertrophy and the implanted ovaries developed (DOANE,1961). Subsequently, BUTTERWORTH and BODENSTEIN (1967) found that the ovaries in Drosophila had a role in protein deposition, growth, and histolysis when larval fat bodies were transplanted into adults.
CORPUS ALLATUM
AND OVARIALEFFECTON
HOUSEFLYFATBODY
1731
Thus, both the corpus allatum and the ovary have a rale in the function of the fat body in insects. This study was conducted to examine more closely the relationship between the corpus allaturn, ovary, and the pupal and adult fat bodies in the abdomen of the housefly, Musca domestica, during the first gonotropic cycle. MATERIALS
AND METHODS
Rearing Flies of the FW, strain were reared on CSMA medium (ANON., 1956) that was fortified with 0.015 g of brewer’s yeast/g of CSMA. Adults were given water and powdered food consisting of dried non-fat milk, sugar, and whole powdered egg at a ratio by weight of 6 : 6 : 1. Virgin female flies used in these studies were from a 4-hr emergence period All flies were held in plastic cages and were sexed under CO, narcosis. (17.8 x 26.7 x 34.4 cm) that were covered at one end by a Tubegauze sleeve. The rearing temperature was 25.6 + 1°C at 40 to 50 per cent r.h. with a 12-hr photoperiod. Egg development scores Egg development was scored on the basis of ten stages which were adapted from those described for the eye gnat, IA&elates collusor (Townsend) (ADAMS and MULLA, 1967). In this scoring technique, stage I is the germarium, stage 4 is the beginning of vitdlogenesis, and stage 10 is the mature egg. Surgical procedures and fat body removal techniques Flies were allatectomized, ovariectomized, and sham operated by previously described techniques (ADAMS and HINTZ, 1969). Removal of the ring gland will be referred to hereafter as allatectomy. The fat body was removed from the fly by first removing the abdomens and placing them ventral side up in a drop of 1% saline solution. Then the abdomen was slit longitudinally and the viscera removed. The pupal fat body was scraped from the abdomen and removed from the gut by dipping it in saline solution. Adult fat body was picked from the abdomen with microforceps. Both the adult and pupal fat bodies were then taken up in a separate microhematocrit tube in a drop of saline solution and the tubes were sealed with a torch and centrifuged for 1 min in a microhematocrit centrifuge. The amount of fat body in the tube was measured in a dissection microscope at 40 x with an ocular micrometer and then converted to mm3 of fat body. Statistical treatment of the data Simple analysis of variance and standard deviations were conducted on nontransformed data by the methods described in SNEDECOR (1956), and the means were separated by Duncan’s multiple range test (LITTLE, 1963). Regression analysis and the covariance analyses were conducted on mm3 of fat body vs. log, of age by the North Dakota State University computing centre at Fargo.
1732
T. S. ADAMS AND D. R. NELSON
Relationship of ovarian development to fat body volume From a group of females held at 22.2 _+l”C, samples of 10 flies were taken periodically and frozen. Ovarian development was scored and the fat body volume was determined for each female. The mean volume of fat body was then calculated for each of the stages of obgenesis in the first gonotropic cycle. ESfect of allatectomy, allatectomy plus ovariectomy, and ovariectomy on fat body volume The females were allatectomized at a mean age of 6 hr after emergence and were ovariectomized 24 hr after emergence. They were held at 25.6 ? 1°C until 7 days after emergence. At this time the flies were frozen and the fat body volume was determined. The four groups of 40 flies each were allatectomized, allatectomized plus ovariectomized, ovariectomized, and control flies. The fat body was measured in 10 flies from each group. This experiment was replicated four times, Effect of allatectomy on fat body volume in$ies of different ages About 150 flies were allatectomized at a mean age of 12 hr after emergence and were held at 22.2 i 1°C and another 150 were control operated. In the first study, samples were removed from the allatectomized and control flies at 12-hr intervals 12 to 96 hr after emergence. This experiment was repeated and samples were taken at 12, 48, 96, 144, and 192 hr after emergence. Each sample consisted of 10 flies which were frozen, the fat body then measured, and ovarian development scored. Effect of ovariectomy on fat body volume in flies of different ages About 150 flies were ovariectomized 24 hr after emergence and a comparable number were control operated. All flies were held at 22.2 + 1°C and during the first study they were sampled at 24-hr intervals from 24 to 144 hr after emergence. The experiment was repeated a second time under the same conditions and sampled at 24, 72, 120, 168, and 216 hr. Each sample consisted of 10 flies that were frozen before the fat body volume was determined. Effect of synthetic juvenile hormone on the volume of adult fat body Synthetic juvenile hormone (trans,trans-lO,ll-epoxy farnesenic acid methyl ester) was topically applied in mineral oil (1~1) to 200 virgin females 6 days after emergence at a concentration of 25 pg/,ul. Control females received 1~1 of mineral oil. Seven days after treatment the females were frozen and fat body volume was determined. RESULTS
Relationship of Ovarian Development to Fat Body Volume Correlation of ovarian development with pupal and adult fat body volume The total abdominal fat body volume (pupal fat body plus adult fat body volumes) decreased with ovarian maturation (Table 1). Newly emerged females with stage 2 ovaries contained 1.82 mm3 of total fat body; this volume decreased
1733
CORPUS ALLATUM AND OVARIAL EFFECTON HOUSEFLY FAT BODY
44 per cent to l-01 mm3 in females with mature eggs. The total fat body volume varied between l-63 and l-93 mm3 in females with ovaries in stages 2 to 6, but decreased in volume when the ovaries were in stages 6 to 9. No further changes occurred between stages 9 and 10. TABLE I-RELATIONSHIP OF OVARIAN DEVELOPMENTTO VOLUMES OF PUPAL AND ADULT FAT BODY IN THE HOUSEFLY Tissue vol. (mm3) . , Ovarian stage
No. flies
Pupal fat body ( + S.D.)
Adult fat body (+ SD.)
Total fat body (rf: S.D.)
2 3 4 5 6 7 8 9 10
29 28 21 8 9 10 4 4 41
l-38 + 0.11 1.15 c 0.11 o-99 k o-09 0.65 kO.15 O-69 + 0.07 o-21 i- 0.10 0.17 F 0.10 O-08 F 0.08 O-00 i o-00
044 * 0.08
1.82 + 0.07 1.93 kO.11 I.91 + 0.09 1.63 z!z0.20 I.73 * 0.09 1.56kO.18 1.24 + 0.20 0.98 + 0.23 I.01 + o-04
0.78 + 0.06 0.92 + 0.06 0,98 * 0.10 1-04+0~10 I.35 + o-07 1.07 + o-20 0.89 + 0.16 1*01+ o-04
Adult fat body (%) 24 40 50 60 60 86 86 91 100
The volume of pupal fat body decreased with ovarian maturation from l-38 mm3 in females with stage 2 ovaries to O-00 mm3 in females with stage 10 ovaries, whereas the adult fat body increased from O-44 mm3 in females with stage 2 ovaries to a maximum volume of l-35 mm3 in females with stage 7 ovaries and then decreased to 1.01 mm3 in mature females. When adult fat body was expressed as the percentage of the total fat body, we found that females with stage 2 ovaries contained 24 per cent adult fat body and this percentage increased steadily with ovarian maturation to 100 per cent in females with mature eggs (Table 1). Correlation of ovarian development with volzcme increase and decrease of the adult and pupal fat body The volume of the pupal fat body decreased in all ovarian stage intervals except 5 to 6 (Fig. 1). The maximum pupal fat body volume decrease of 0.48 mm3 occurred during the ovarian stage interval of 6 to 7. During the ovarian stage intervals of 2 to 3, 3 to 4, and 4 to 5, the volume of pupal fat body decreased 0.25, O-17, and O-34 mm3, respectively. A small decrease in pupal fat body volume of O-04,0-09, and O-OSmm3 occurred during ovarian stage intervals of 7 to 8, 8 to 9, and 9 to 10, respectively. Adult fat body volume increased during the ovarian stage intervals of 2 to 3, through 6 to 7, and in 9 to 10 but decreased during the ovarian stage intervals of 7 to 8 and 8 to 9. A maximal volume increase of O-45 mm3 of adult fat body
1734
T. S. ADAMSANDD. R. NELSON
occurred during the ovarian stage interval of 2 to 3, and during ovarian stage intervals of 3 to 4, 4 to 5, 5 to 6, and 6 to 7 adult fat body volume increased 0.15, 0.04, O-06, and 0.31 mms, respectively. During the ovarian stage intervals of 7 to 8 and 8 to 9 the adult fat body volume decreased 0.28 and 0.18 mm3, respectively, but in the egg stage interval of 9 to 10 the volume of adult fat body increased 0.11 111111~. % E .6 g .5 2 .J > >3 0” m .z 2 .’ _I 0 9 5 -_I x-2 S’ g -3 g -9 f -5 5 &.-.6 %I
ADULT F/iiBODY PUP& WrBODY
5- 6 ,,,,LL&,, OVARIAN
STAGE INTERVALS
7- *
* - “f ~‘p;siY WrELLOOENlC
FIG. 1. Changes in the volumes of adult and pupal fat body in the housefly during different ovarian stage intervals.
A clearer pattern of fat body volume increase and decrease emerged when ovarian development was broken into previtellogenic (stages 2 to 4), vitellogenic (stages 4 to 9), and postvitellogenic (stages 9 to 10) phases lasting 49, 74 and 47 hr, respectively (Table 2). In the previtellogenic phase of ovarian development the volume of adult fat body increased 0.48 mm3 (9.7 x 10m3mm3/hr) and the pupal fat body volume decreased 0.39 mm3 (7.9 x 1OL3mm3/hr) for a net volume increase in total fat body of 0.09 mm3 (1.8 x lO-3 mm3/hr). During vitellogenesis both the pupal fat body and the adult fat body decreased 0.91 mm3 (12.2 x 10-s mms/hr) and 0.03 mms (0.4 x 10e3 mm/hr), respectively, for a net volume loss of 0.94 mms The remainder of the pupal fat body, 0.08 mms (12.7 x 1O-3 mm3/hr). (1.7 x 10-s mm3/hr) disappeared during the post-vitellogenic period, but the adult fat body volume increased 0.12 mm3 (2-5 x 10~~ mm3/hr) for a net volume increase of 0.04 mm3 (0.8 x 1O-3 mm3/hr). Thus, there was a net volume increase in total fat body during the pre- and post-vitellogenic periods because of an increase in adult fat body, and a net volume loss during the vitellogenic period because of a decrease in volume of both the pupal and adult fat bodies. Effect of ovariectomy, fat body
allatectomy,
and ovariectomy
plus allatectomy
on abdominal
Allatectomized, allatectomized plus ovariectomized, and ovariectomized females had total fat body volumes of 1.64, 1.83, and 1.73 mm3, respectively, which were 34, 44, and 37 per cent greater than in the control flies (1.26 mm3) (Table 3).
- I.38
180
Total
-21.4
-7.9 - 12.2 -1.7 +0.57
+ 0.48 - 0.03 +0*12
AV
+ 12.5
+9*7 -0.4 +2*5
AV/hr x IO3
Adult fat body
* AV calculated from means presented in Table 1. t Calculated from graph in Fig. 3. $ Includes total time from stage 9 to the end of the experiment at 180 hr.
-0.39 -0.91 - 0.08
49 74 47:
Pre-vitellogenic (2-4) Vitellogenic (4-9) Post-vitellogenic (9-10)
AV/hr x IO3
Pupal fat body
-0.81
+ 0.09 - 0.94 + 0.04
AV
- 8.9
+I*8 -12.7 +0*8
AV/hr x 10S
Fat body net change
Volumetric (mm”) changes in abdominal fat body*
PHASES TO VOLUME CHANGES IN THE PUPAL AND ADULT FAT BODY OF THE HOUSEFLY
AV
OF VITELLOGENIC
Otigenetic phase
&-&LATIONSHIP
Duration (hr) t
TABLE
1736
T. S. ADAMSAND D. R. NELSON
Analysis of variance showed that the treatments had a significant effect (0.1 confidence level), and the mean separation is shown in Table 3. No pupal fat body remained in the control flies 7 days after emergence whereas 61 to 100 per cent of the operated flies contained pupal fat body. Allatectomized, allatectomized plus ovariectomized, and ovariectomized flies contained O-46, O-55, and 0.19 mm3 of pupal fat body, respectively, and these volumes represented 11 to 30 per cent of the total fat body in the operated flies. The treatments had a significant effect on pupal fat body volume (O-01 confidence level); the mean separation is shown in Table 3. Adult fat body volume varied from 1.18 to 1.54 mm3 in all groups, and no significant difference was observed between the means. However, the ovariectomized flies showed a 21 per cent increase in adult fat body volume over the controls. Both ovariectomy and allatectomy resulted in the retention of pupal fat body. Allatectomy had no effect on the adult fat body volume, but ovariectomy resulted in an increase in volume of adult fat body. Therefore, the increase in total fat body volume in the treated flies was due primarily to pupal fat body retention. Eflect of allatectomy on abdominal fat body volume in flies of dz@erent ages Totalfat body. In all age groups examined (except flies 24 hr after emergence) the allatectomized flies contained more total fat body than the controls (Fig. 2A) and the analysis of covariance showed that allatectomy and age had a significant effect (0.10 confidence level) on the total fat body volume. In flies 24 to 36 hr after emergence the volume of total fat body in both the control and the allatectomized flies increased over the 12-hr volume (184 mm3), then in the control flies it decreased gradually to 1.28 mm3 in 192~hr flies for a net loss of 30 per cent. In contrast, the 192-hr allatectomized flies contained 180 mm3 of total fat body, about the same volume as at 12 hr after emergence. Regression analysis of the data, mm3 total abdominal fat vs. log, age, gave a correlation coefficient (r) of O-056 (insignificant) for the allatectomized females and -0.657 (significant at the 0.05 confidence level) for the controls. Thus, total fat body volume and age did not correlate in the allatectomized flies but the volume of the total fat body of the control flies correlated negatively with age. Pupal fat body. Pupal fat body (1.63 mm3 in flies 12 hr after emergence) disappeared by 144 hr in the control females (Fig. 2B), but allatectomized flies 192 hr after emergence contained 0.45 mm3 of pupal fat body. Also, all allatectomized females at all ages contained some pupal fat body, but the number of control females that contained pupal fat body decreased in the samples taken after 72 hr. The covariance analysis showed that allatectomized flies contained significantly (0.01 confidence level) more pupal fat body than the controls and that the volume was significantly affected by age. Regression analysis of the data, mm3 pupal fat body vs. log, age, gave a correlation coefficient of -0.789 for the allatectomized females and - 0.933 for the controls (significant at the 0.01 confidence level).
%-%FFECT
OVAR~ECTOMY,
+ 33.9 i-43.8 -I-36.6 -
1.73 & 0.208 1.26 ?z0.14b
Change ?I (%)$
1.64 k 0*21&lb 1.83 4,0*21’
Volume (mm”) (+S.D.)i
Total fat body
OF ALLATECTOMY,
AND
With pupal fat body (%) 92 100 61 00
Volume @mS) ( ?I S.D.) t 0.46 zk0.20& 0.55 + 0.04s 0.19 f O*ll@ o*oo * O*OOb
Pupal fat body
ALLATECTOMY PLUS OVAR~ECTOMY IN THE HOUSEFLY
10.8 0.0
27.5 30.4
Fat body (%)
ON THE VOLUME
PAT BODY
+21.6 -
1.26 + 0~14~
-6.6 +0*4
Change + (%)5
1~54~0~15~
1.18 1 O-20& I.28 rt o-17*
Volume (mm3) ( ?I S.D.)t
Adult fat body
OP ABDOMINAL
* Flies were allatectomized at 6 hr after emergence, ovariectomized at 24 hr, and then held for 6 days at 25.6 zk1°C. t Those values not followed by the same letter are significantly different from each other at the 5 per cent level as determined by Duncan’s multiple-range test. Fat body volume of treated-fat body volume of control X 100 per cent. $ Percentage change in fat body volume = Fat body volume control
Allatectomy Allatectomy .plus ovariectomy Ovariectomy Controls
Treatment *
TABLE
g %
2
2 “m p ++
m
g
E 4 # 2
g
2
3 5;
$ !z
2
T. S. ADAMS AND D. R. NELSON
1738
Adult fat body. Adult fat body increased with age in both the allatectomized and control flies (Fig. ZC). Females 12 hr after emergence contained 0.21 mm3 of adult fat body. This volume increased to l-28 mm3 in the controls and to l-39 mm3 in allatectomized flies 192 hr after emergence. Analysis of covariance showed no significant effect for allatectomy on adult fat body volume, but age had a significant effect (O-10 confidence level). o-;----a -
?“-A.
CONTRCL &LIITECTOMY
2.3. 2.1 1.9 17. 15. w.
0 AGE (HR
1
FIG. 2. Effect of allatectomy on the volumes of total, pupal, and adult fat body in houseflies of different ages after emergence. A, volume of total fat body, B, volume of pupal fat body, and C, volume of adult fat body.
Regression analysis of the data, mm3 adult fat body vs. log, age, gave significant correlation coefficients (0.01 level) of O-939 for the controls and O-907 for the allatectomized flies. Correlation
of fat body volume with ovarian stages
Since 180 hr after emergence the ovaries in the allatectomized flies had not proceeded beyond stage 4 but the controls had reached stage 10 (Fig. 3), the data were rearranged from their chronological treatment (as in Fig. 2) to give volume changes within the various ovarian stage intervals (a physi,ological adjustment). This was accomplished by calculating the mean volume of adult and pupal fat bodies for each stage of oiigenesis in allatectomized flies as was previously done for the controls (Table 1). The volume change (AV) from one ovarian stage to another was determined and the rate of change (AV/h r ) was calculated by dividing the volume change by the duration (hr) of the ovarian stage intervals (determined from Fig. 3). These data are summarized in Table 4.
CORPUS
ALLATUM
AND
OVARIAL
EFFECT
ON
HOUSEFLY
FAT
1739
BODY
The decrease in the volume of pupal fat body during the ovarian stage interval of 2 to 3 was the same in the control (O-23 mma) and allatectomized flies (O-26 mm3). However, the rate of change was twice as great in the controls (13.5 x lo3 mm3/hr) as in the allatectomized flies (7.4 x 10-s mma/hr). During the ovarian stage interval of 3 to 4 the pupal fat body decreased 0.16 mm3 (5.0 x 10-3 mma/hr) in the controls and O-29 mm3 (2.0 x 10-a mm3jhr) in the allatectomized flies. The volume decrease IOLS9i&i a 8-
/I o------o
/
CONTROL ALLATECTOMY
e--.-.4
I’
s w 7-
. I’
zl 0 6-
I/.
%
f’
5 ti5%4-
Q __A__$
$/.
f
,--;
_p--~_.~.‘.‘~$‘-
%3Y g p(?J-----vt IO
/-_y--’ I 20
o I
I 40
I
II,,,
AGE (i-ii FIG.
3
I’
80
100
I 200
1 300
.)
3. The effect of allatectomy on ovarian maturation in the housefly. Ages are hours after emergence.
during the ovarian stage interval of 2 to 4 was 0.39 mm3 (870 x 10e3 mm3/hr) for the controls and 0.55 mm3 (3.0 x 10-a mm3/hr) for the allatectomized flies. When the decrease in pupal fat body was considered for the total elapsed time (180 hr) the change in the allatectomized flies was the same as during stages 2 to 4,0*55 mm3 (3.0 x 1O-3 mm3/hr), whereas the change was twice as great in the controls, 1.38 mm3 (7.7 x 10-s mm3/hr). The volume changes observed during the ovarian stage intervals of 2 to 3 and 3 to 4 did not differ appreciably between the control and allatectomized flies (although the rates did). This demonstrates that the developing ovaries in the controls (stages 4 to 10) had an effect on the pupal fat body volume decrease which was not observed in the allatectomized flies because the ovaries did not develop beyond stage 4. The change in adult fat body was the same in the control and allatectomized flies during the ovarian stage interval of 2 to 3 and was twice as great in the allatectomized flies as in the controls during stage interval 3 to 4. However, when the stage duration was considered the rate of increase in adult fat body was approximately twice as great in the control as in the allatectomized flies (stages 2 to 3 : 20.0 x 10-Z vs. 9.4 x 10-s mms/hr, respectively, and stage 3 to 4: 4.4 x 10~~ vs. 2.5 x 1O-3 mm3/hr, respectively), but when the total elapsed time for the experiment was considered (180 hr) no appreciable difference was observed between the increase in adult fat body in the controls and in the allatectomized flies (O-69 and
Control total (all stage intervals 2-10)
-7.7
-8.0
-5.0
-13.5
-3.0
-2.0 -7.4
AVjhr x 1Oa
* Calculated from Fig. 3. t Included total time from start of stage 3 to end of experiment, 180 hr. 1 Calculated from Table 1.
Mean change/hr
-1.38$
-0.16: -0*39x
32
-0.23:
-0.55
-- 0.29 0.26
49
49
17
180
145 35 t
AV
Pupal fat body
180
3-4
Control
58
56 61
No.
Interval duration (hr)*
Total Mean change/hr
2-3
34 2-3
Control
Total Mean change/hr
Allatectomy Allatectomy
Treatment
Ovarian stage interval
to.55
-I-0.48
+0*14
+ 0.34
+ 0.69
+ 0.36 +0*33
AV
+3.2
+9*7
+4.4
+ 20.0
+3.8
+2*5 + 9.4
AV/hr x lOa
Adult fat body
-0.83
+ 0.09
- 0.02
to.11
+ 0.14
+ + 0.07 0.07
AV
-4.5
+1.8
-0*4
+ 7.3
1-O-8
+0*5 +2*0
AV/hr x lOa
Total fat body
Fat body volume (mm”) change during indicated stage intervals
TABLE ~---COMPARISONOF PUPALAND ADULT FAT BODY VOLUMECHANGESWITHIN OVARIANSTAGEINTERVALSAND WITH TIME IN ALLATECTOMIZED AND CONTROLHOUSEFLIES
3 $ Z
w
:
:
1
9 .m
CORPUS ALLATUM
AND OVARIAL EFFECT ON HOUSEFLY
FAT BODY
1741
0.55 mm3, respectively) and in the rates of increase (3.8 and 3.2 x 10-s mm3/hr, respectively). Thus, the increase in adult fat body volume appears to be independent of ovarian maturation and the corpus allatum (although a stimulator-y effect of juvenile hormone may be cancelled out by the nutritional demands of the developing ovary). The allatectomized flies’ total fat body volume increased 0.14 mm3: (0.8 x 1O-3 mms/hr) during the ovarian stage interval of 2 to 4 whereas the control flies showed a volume increase of 0*09 mm3 (1.8 x 1O-3 mm3/hr). When the total elapsed time was considered, the volume of total fat body increased 0.14 mm3 (0.8 x 1O--smm3/hr) in the allatectomized flies and decreased 0.83 mm3’ (4.5 x 10-s mms/hr) in the control flies. ESfect of ovariectomy
on abdominal fat body volume in flies of dz@er-ent ages
Total fat body. Ovariectomized females showed a steady increase with age in the total volume of fat body from 1.09 mm3 24 hr after emergence to 1.83 mm3 at 216 hr (Fig. 4). This represented a total increase of 68 per cent in total fat body
FIG. 4. Effect of ovariectomy on the volumes of total, pupal, and adult fat body in houseflies of different ages after emergence. A, volume of total fat body, B, volume of pupal fat body, and C, volume of adult fat body.
volume over the 24-hr value and 110 per cent more total fat body than the 216-hr control. The total fat body of the control operated flies increased from 1.09 mm3 at 24 hr to 1.32 mms at 96 hr, and then it decreased to 0.91 mm3 at 216 hr for a 16 per cent loss in volume over the 24-hr samples. The ovariectomized females contained significantly more (t-test, 0.01 confidence level) fat body than the controls in samples taken at 144, 168, and 216 hr.
1742
T. S. ADAMS
AND D.R.
NELSON
Regression analysis of the data, mm3 total fat body vs. log, of the age, gave a correlation coefficient (r) of -0.519 for the controls and 0.564 for the ovariectomized flies. Thus, the volume change gave a poor fit for a linear model, but the fat body volume increased in the ovariectomized flies and decreased in the controls with age. Pupal fat body. In both the ovariectomized flies and the control operated ones, -the volume of pupal fat body decreased with age from its 24-hr volume of 0.86 mm3 (Fig. 4B). At 144 hr the pupal fat body had completely disappeared from the .control females, but 100 per cent of the ovariectomized flies contained pupal fat body (mean volume of 0.46 mm3). At 168 hr, 80 per cent of the ovariectomized flies had pupal fat body (mean volume of 0.33 mm3), and at 216 hr, 33 per cent of the flies had pupal fat body (mean volume of 0.20 mm3). Analysis of covariance showed that ovariectomized flies contained significantly (O-10 confidence level) more pupal fat body than the controls and that the volume was affected significantly (0.10 confidence level) by age. The ovariectomized flies at 144, 168, and 216 hr contained significantly (t test, 0.01 confidence level) more pupal fat body than the controls. Regression analysis of the data, mm3 pupal fat body vs. log, age, gave a correlation coefficient (r) of -0.920 for the controls and -0.947 for the ovariectomized flies. Thus, the change of pupal fat body volume was negatively correlated with age and was linear in the transformed data for both the control and the ,ovariectomized flies. Adult fat body. Adult fat body volume increased with age in both the control operated flies and in the ovariectomized ones (Fig. 4C). The volume in the controls at 24 hr was 0.23 mm3 and at 216 hr it had increased to 0.91 mm3. At 216 hr the adult fat body volume was 1.63 mm3 in ovariectomized flies. Thus, the ovariec-tomized flies had 79 per cent more fat body than the controls. The analysis of covariance gave no significant effect for ovariectomy on the -volume of adult fat body but showed that age had a significant effect (0.1 level). However, when a t-test was applied to the data from individual flies, the 216-hr .age group had significantly (0.01 confidence level) more fat body than the controls; .a11other differences were insignificant. Regression analysis of the data, mm3 adult fat body vs. log, age, gave a correlation coefficient (r) of O-695 for the control operated flies and O-838 for the ovariectomized flies. Both values were significant and showed that adult fat body volume -increased with female age in both the ovariectomized flies and the controls. Comparison of the effect of allatectomy .adult fat bodies
and ovariectomy
on the volumes of pupal and
Analysis of covariance applied to the data on the pupal fat body volume between -the allatectomy and ovariectomy control groups showed that the allatectomy ,controls contained a significantly greater volume of pupal fat body than the ovariectomy controls, but the volumes of the adult fat bodies did not differ. This meant that direct quantitative comparisons could not be made between the
CORPUS ALLATUM
AND OVARIAL EFFECT ON HOUSEFLY FAT BODY
1743
effects of allatectomy and ovariectomy. However, the data were adjusted computing the percentage of adult fat body for each age group. Percentage
of adult fat body =
by
Adult fat body mm3 x 100 Total fat body mm3
*
Covariance analysis showed that there was no significant difference between the control groups when the data were adjusted in this manner, and the combined values had a correlation coefficient (r) of 0.912. Up to 72 hr after emergence the percentage of adult fat body increased at identical rates in the controls, allatectomized, and ovariectomized flies, but after 72 hr the allatectomized and ovariectomized flies showed a slower rate of increase (Fig. 5). This change in
80_ -
O----Q ----.a
CONTROL OVARIECTOMY ALLATECTOMY
2 a cn 40a
FIG.
5.
Percentage
of the total abdominal
ovariectomized,
allatectomized,
fat body and control
as adult
fat body
in
houseflies.
rate occurred at the time vitellogenesis started in the control fly population. The major difference between the allatectomized, ovariectomized, and control flies was the presence of vitellogenesis in the controls and its absence in the treated flies. The effects of allatectomy and ovariectomy on pupal and adult fat body volume changes were compared further by calculating the net change in volume over time in the pupal and adult fat bodies. The net change in the treatment was compared to the net change in the respective control, and a percentage of increase or decrease over the respective control was calculated. When these percentages were compared, the allatectomized females had 33 per cent of their pupal fat body remaining and the ovariectomized females had 49 per cent remaining. Although there was little difference in the decrease in pupal fat body volume between ovariectomized and allatectomized flies, the volume of adult fat body increased 15 per cent (insignificant)
1744
T. S. ADAMSAND
D.R. NELSON
in the allatectomized flies and 107 per cent in the ovariectomized flies. Thus, the increase in adult fat body volume in ovariectomized flies compared with essentially no change in allatectomized flies indicates a possible stimulation of adult fat body by the corpus allatum. EfSect of synthetic juvenile hormone on the adult fat body Adult female flies with mature ovaries and without pupal fat body were treated with 25 pg of synthetic juvenile hormone and the volume of adult fat body was measured 7 days later. The treated flies contained 1.28 mm3 of adult fat body compared with 1.08 mm3 in the controls. The increase in adult fat body volume was significant at the 0.01 confidence level (determined by a t-test). This proves that juvenile hormone stimulates an increase in adult fat body volume as suggested by our previous experiments. DISCUSSION
AND
CONCLUSIONS
Our studies showed that the pupal fat body volume decreased at its greatest rate lduring the vitellogenic phase of oijgenesis and that no pupal fat body remained after the ovaries had reached maturity (stage 10). A relationship between the disappearance of pupal fat body and the development of the ovaries is indicated by the retention of pupal fat body in Diptera with stunted ovaries: P. sericata (EVANS, 1935), C. erythrocephala (THOMSEN, 1952), P. terranovae (HARLOW, 1956), M. domestica (WEISMANN, 1963), F. canicularis (ANDERSON, 1964), D. tryoni (MUNRO and BAILEY, 1965). The correlation between ovarian maturation and pupal fat body cytolysis is further demonstrated in our study by the retarded rate of pupal fat body loss in houseflies without ovaries or in those with non-developing ovaries because of allatectomy. Since the pupal fat body is retained in allatectomized females (no juvenile hormone present) and in ovariectomized females (in which the corpus allatum is continuously active) (Adams, unpublished observations), the retention of the pupal fat body is not due to the presence or absence of the corpus allatum and juvenile hormone, but is dependent upon the functionality of the ovary. Thus, the maturing ovary may increase the rate of disappearance of pupal fat body by acting as a nutrient sump, as suggested for 0. fasciatus (JOHANSSON,1958), M. domestica (WEISMANN,1963), and D. tryoni (MUNRO and BAILEY, 1965), and/or by a direct stimulation of pupal fat body cytolysis. DOANE (1961), from studies on the mutant female sterile (2) adipose of D. melanogaster, proposed the existence of a hormone from the ovaries which regulated the utilization of stored lipid in the fat body. An oiistatic hormone has been found in M. domestica (ADAMS et al., 1968) which is produced by the developing ovary and which maintains the cyclicity of ovarian development by its action on the corpus allatum (Adams, unpublished observations). This hormone also may be responsible for the effect of the ovaries on pupal fat body cytolysis and on the decrease in adult fat body volume during vitellogenesis. The adult fat body in the housefly showed a volume increase with age. When this was correlated with ovarian development, adult fat body volume increased
CORPUSALLATUMAND OVARIALEFFECT ONHOUSEFLYFATBODY
174.5
during the pre-vitellogenic and post-vitellogenic phases and decreased during the latter part of the vitellogenic phase. Thus, both the adult fat body and the pupal fat body decreased during vitellogenesis. This also has been observed in P. tewanovae (HARLOW, 1956). Allatectomy did not result in an increase in adult fat body when compared to the control group. In this respect, the housefly resembles L. maderae (SCHAR~R, 1955). Allatectomy has been reported to result in an increase in fat body in a number of other insects: C. erythocephala (THOMSEN, 1942),M. d$erentiaZis (PFEIFFER,1945), D. hydei (VOGT, 1949), P. americana (BODENSTEIN, 1953), 0. fascia&s (JOHANSSON, 1958),P. regina (ORR, 1964), and L. m. migratoriodes (MINKS, 1967).
FIG.6. Summary of the interrelationships between the ovaries, corpus allaturn, pupal fat body, and adult fat body in the housefly.
Ovariectomized houseflies had a greater volume of adult fat body than the controls. Similar results have been reported for B. mori (HAMA~AKI, 1932), M. diflerentialis (PFEIFFER,1945), D. virilis (BODENSTEIN, 1947), 0. fasciatus (JOHANSSON, 1958), and D. melanogaster (DOANE, 1961;.BUTTERWORTH and BODENSTEIN, 1968),but not for P. regina (ORR, 1964). The corpus allatum is active in ovariectomized houseflies (Adams, unpublished observations) and it was assumed that these flies contained more juvenile hormone than the controls. This excess of juvenile hormone in ovariectomized females resulted in an increase of adult fat body volume; this was substantiated by the increase of adult fat body in flies topically treated with synthetic juvenile hormone. Our data may be summarized by presenting the following hypothesis, which is diagrammed in Fig. 6. The corpus allatum produces juvenile hormone which
1746
T. S. ADAMSANDD. R. NELSON
stimulates the onset of vitellogenesis in the ovaries of the housefly (ADAMS and The developing ovaries draw on reserve substances in the pupal fat body first and then on the substances in the adult fat body. Secondly, the maturing ovaries produce an oijstatic hormone that inhibits the release of juvenile hormone (ADAMSet al., 1968); this hormone may stimulate cytolysis of the pupal fat body. Both the pupal and adult fat body are drawn upon for general metabolic maintenance. Ovariectomy removed the inhibition on the corpus allatum and juvenile hormone is released continuously. The increased titre of juvenile hormone and the absence of the nutritive sump effect of the ovaries resulted in an increase volume of adult fat body. The stimulator-y effect of juvenile hormone on adult fat body volume is demonstrated in both our ovariectomy experiments and in the synthetic juvenile hormone experiments. Ovariectomized females that were allatectomized did not show hypertrophy of the adult fat body, but ovariectomized females had hypertrophied adult fat bodies; this shows that hypertrophy is not an ovarian effect but one of the corpus allatum. HINTZ, 1969).
Acknowledgements-The authors express their thanks to Dr. BERTA SCHAFZRER of the Yeshiva University and to Dr. F. M. BUTTERWORTHof the Oakland University for their well-founded comments and criticisms of the manuscript. The authors also thank Dr. WM. BOWERSof the U.S.D.A.‘s Pioneering Insect Physiology Laboratory for supplying the synthetic juvenile hormone used in these studies.
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