- Email: [email protected]
Oöstatic hormone production in houseflies, Musca domestica, with developing ovaries
J. Insect Physiol., 1968, Vol. 14, pp. 983 to 993. PergamonPress. Printed in Great Britain
OijSTATIC HORMONE PRODUCTION IN HOUSEFLIES, MUSCA DOMESTK’A, WITH DEVELOPING OVARIES T.
S. ADAMS,
A. M. HINT&
and
J. G. POMONIS
Metabolism and Radiation Research Laboratory, Entomology Research Division, Agricultural Research Service, United States Department of Agriculture, Fargo, North Dakota (Received 2 February 1968)
Abstract-An oijstatic hormone is produced by house@ with eggs in stages 4 to 10. This hormone suppresses the development rate of stages 2 to 4 eggs. Complete inhibition of the second gonotropic cycle at stage 4 occurs when mature eggs from the first cycle are retained. Extracts from mature female flies inhibited ovarian development when injected into 24-hr-old flies with eggs in stage 2 of development. The oijstatic hormone is soluble in 95% ethanol, chloroform-methanol (2 : l), 20% glycerol, and water. The oijstatic hormone maintains the cycle of egg production in the housefly.
INTRODUCTION
OVARIAN-INHIBITING hormones have been reported from various Arthropoda. In decapod crustaceans the eye stalk produces an ovarian inhibitor that is stored in the sinus gland (CARLISLE and KNOWLES, 1959). This hormone is reported for the following decapods: Leander serratus (PANOUSE, 1943), Lysmata sexicaudata (CARLISLE, 1.953), Car&u maenas (DEMEUSY, 1962). CARLISLE and BUTLER (1956) suggested a steroid nature for this decapod ovarian inhibitor and demonstrated that injections of ‘queen bee’ substance inhibited ovarian development in the prawn, Leander serratus; extracts of prawn eye stalks inhibited ovarian development when fed to worker bees. The decapod ovarian inhibitor was extractable in alcohol. IVANOV and MESCHERSKAYA (1935) showed that the o&heca-bearing cockroaches, Blattella germanica and Blatta orientalis, produced an ovarian inhibiting hormone from the corpora lutea in the proximal part of the ovariole. This hormone decreased the permeability of the oijcyte and was soluble in water. Studies by ADAMS and MULLA (1967) on the eye gnat, Hippelates collusor, showed that oi5cytes in the second gonotropic cycle did not proceed past stage 4 when mature eggs from the first cycle were retained. The present study was undertaken to find whether an oiistatic hormone was produced by houseflies with developing ovaries, whether it was extractable, and to develop a bioassay system for this hormone. 983
984
T. S. ADAMS,A. M. HINTZ,ANDJ. G. POMONIS MATERIALS
AND METHODS
Rearing All flies (h&sea domestica) for this study were of the FW, strain. Larvae were reared on CSMA medium (ANONYMOUS, 1956) that was fortified with 0*015 g brewer’s yeast/g CSMA. Adults were given water and powdered food consisting of dried non-fat milk, sugar, and whole powdered egg at a ratio of 6 : 6 : 1 by weight. The flies were allowed to emerge for a 4 hr period before sexing. Virgin females were used in these studies unless otherwise stated. Flies were held in plastic cages 7 x 10.5 x 13.5 in. that were covered with Tubegauz sleeves at one end. The rearing temperature was 78 + 2°F.
Scoring Egg development was scored on a basis of ten stages which-were adapted from those described for H. coZZusor(ADAMSand MULLA, 1967). In this scheme, stage 1 is the germarium, stage 4 is the beginning of vitellogenesis, and stage 10 is the mature egg.
Simultaneous ovarian growth Females were held at 78 + 2”F, and samples of twenty females were removed twice daily for 6 days; another sample of about two hundred females was taken on the seventh day. Ovarian development was scored for the first and second gonotropic cycles in each female examined. The data were compiled, and the percentage of the follicles in each ovarian stage of the second gonotropic cycle at each ovarian stage in the first cycle was calculated.
Egg retention studies Two groups of five hundred females/group were mated on the third day after emergence with an excess number of males of the same age. After 4 hr from introducing the males, an ovipositional substrate of used larval rearing medium covered with a moist black cloth was given to one group of flies. This substrate was renewed daily for the remainder of the study. The other group was encouraged to retain their eggs by withholding the ovipositional substrate. Samples of ten females were removed from each cage twice daily, both before and after mating. The egg scores were determined for the first and all observable secondary gonotropic cycles. This study was conducted at a test temperature of 80 f 2”F, and it was repeated using virgin females at a test temperature of 72 f 2°F. The means of the egg stages were determined for all gonotropic cycles and plotted against time. More accurate measurements were made from graphs plotted on log-log paper. Extracts The first series of extracts were prepared in 95% ethanol from excised mature ovaries and female abdomens containing mature eggs. The abdomens were depressed with a wooden dowel to extrude the eggs. The tissues were then
OijSTATIC
HORMONE PRODUCTION
985
IN HOUSEFLIES
rinsed three times with 95% ethanol? and the three rinses were combined, filtered, and taken to dryness on a flash evaporator. Prior to injection, distilled water was added to give the desired concentration. In one experiment, whole female flies were ground in a blender with 95% ethanol and then processed as described. Another experiment used flies which were stored in 95% ethanol. The ethanol was discarded, and the flies were ground in a blender with chloroform-methanol (2 : 1). The ground bodies were filtered and rinsed three times with chloroformmethanol. The rinses were combined and taken to dryness in a flash evaporator. The dried residue was extracted with 20% glycerol to give a solution concentration of 3 equiv./& Extracts of mature (3-day-old) flies and immature (4-hr-old) females were prepared in the same manner. All extracts and dried residues were stored under N, in a freezer at 10°F until the time of injection. Bioassay Newly ernerged virgin female flies were held at 72 & 2°F for 24 hr before injection. (Ovarian development was stage 2 at the time of injection.) The flies were narcotized with CO,, and the solutions were injected through the cervical region into the thorax with a 50 ~1 syringe on a micro-injector. Flies received from O-5 to 1.5 ~1 of solution, depending on the experiment. Controls were injected with either distilled water, 20% glycerol, or immature female extract in 20% glycerol. The flies were held for 48 hr at 78 & 2°F or for 5 days at 72 + 2°F before ovarian development was scored. The percentage inhibition was calculated from the following formula : Mean ovarian stage of control - mean ovarian stage of extract Mean ovarian stage of control
x 100 per cent.
The maximal inhibition obtainable by using the above formula is 60 per cent. RESULTS
Simultaneous growth of the Jirst and second gonotropic cycle Simultaneous growth occurred in the first and second gonotropic cycles but stopped at stage 4 (in most cases) in the second cycle when mature eggs were retained (Table 1). When eggs in the first cycle were at stage 2, those in the second cycle were always in stage 1. When eggs in the first cycle were at stage 3, 85 per cent of those in the second cycle were at stage 1, and the remainder were at stage 2. Eggs at stages 4 to 7 in the first gonotropic cycle were followed by stage 2 eggs in the second cycle in most cases (656-96.4 per cent) with the remainder being in stages 3 and 4. Stages 8 and 9 of the first cycle were usually accompanied by stage 3 eggs in the second gonotropic cycle. Mature eggs in the first cycle were accompanied by stage 4 eggs in the second cycle in 48.8 per cent of the females examined, with the remaining percentages as follows: 20.3 with stage 2 eggs, 29.9 with stage 3 eggs, 4.0 with stage 5 eggs, and 3.0 with stage 6 eggs. From this, we found that the second gonotropic cycle starts development when the first cycle is at an egg stage of 3.
986
T. S. ADAMS, A. M. HINTZ, AND J. G. POMONIS
Egg retention and deposition The development of the first and all secondary gonotropic cycles in oviposited and gravid mated females is presented in Fig. l(A) and l(B), respectively. Females that were allowed to deposit eggs experienced three complete gonotropic TABLE ~-SIMULTANEOUS GROWTH OF EGGS IN THE FIRST AND SECONDGONOTROPICCYCLE OF HOUSEFLIES Percentage eggs in second cycle at indicated stage Stages in fkst cycle 2 3 4 5 6 7 8 9 10
No.
50 49 38 28 97 29 26 52 551
1 100.0
83.6 10.5 -
3
2
4
5
6
-
-
-
-
16.4 86.9 96.4
_ 2.6 3.6
_ _ _
_ _ _
_ _ _
89.7 65.6 23.1 23-l 20.3
6.2 31.0 76.9 53.8 23.9
4.1 3.4 23.1 48.8
4.0
3-o
-
cycles in 160 hr. The females that retained mature eggs for 130 hr completed the first gonotropic cycle, but the second cycle was inhibited at stage 4. After 130 hr, the females deposited many eggs, and development in the second cycle progressed beyond stage 4. Both the virgin female populations refused to oviposit and retained mature eggs from the first cycle for 130 hr. The second cycle was completely suppressed at stage 4. No graph is presented because the trend is duplicated in Fig. l(B). _. In both oviposited and gravid mated females, 68 and 70 hr respectively were required to complete the first gonotropic cycle (Table 2). Mated and oviposited females took 82 and 92 hr for the completion of the second and third gonotropic This represents an increase of 14 and 24 hr respectively cycles, respectively. over the first cycle. This increase is due to a prolongation of stages 2 to 4. It required 45 and 55 hr to complete stages 2 to 4 in the second and third gonotropic cycles in the mated and oviposited females, respectively, and 50 hr for the second cycle in the mated and gravid females. This contrasts with the 30 hr each taken to complete stages 2 to 4 in the first cycle for both the mated oviposited and mated gravid flies. Stages 4 to 10 took 37 and 38 hr to complete the first to third gonotropic cycles in the mated and oviposited females and 40 hr to complete stages 4 to 10 in the first cycle mated gravid flies. There was no difference between the first and subsequent gonotropic cycles in the time required to complete stages 4 to 10. The first gonotropic cycle in both populations of virgin gravid females took 100 hr for completion, and the second cycle was inhibited at stage 4 (Table 2).
OijSTATIC HORMONE
PRODUCTION
987
IN HOUSEFLIES
A
20
,
*
.
*
40
60
60
IO0
AGE
(Hours
, 120 at
60.
140
160
160
E)
FIG. 1. The effect of egg retention and deposition on the development of secondary gonotropic cycles in the housefly. (A) Mated and oviposited females, (B) mated and gravid females. TABLE
2-DURATION
OF OVARIAN
STAGESEQUENCES
INOVIPOSITRD
AND
GRAVID
POPULATIONS
OF HOUSEFLIES
Duration (hr) of Population Mated-viposited
Mated-gravid
*
*
Virgin-gravid.?
* Held at 80 + 2°F. t Held at 72 + 2°F.
Gonotropic cycle
Stages 2-4
Stages 4-10
Complete cycle
1 2 3 1 2 1 2 1 2
30 45 55 30 50 32 66 36 60
38 37 37 40 Inhibited at stage 4 68 Inhibited at stage 4 64 Inhibited at stage 4
68 82 92 70 100 100 -
988
T. S. ADAMS,A. M. HINTZ, AND
J. G. POMONIS
The duration of stages 2 to 4 in the first cycles was 32 and 36 hr and 66 and 60 hr for the second gonotropic cycles. Again, a prolongation of the duration of stages 2 to 4 in the second cycle occurred.
Extract studies When 24-hr-old females were injected with extracts from mature female flies, the mean ovarian inhibition was 29.8 per cent or 9.6 per cent inhibition/female equivalent injected (Table 3). Females that were injected on 2 consecutive days with extracts from mature female abdomens had a greater percentage of ovarian inhibition than those injected once; 40 and 36 per cent inhibition compared with 28 and 26 per cent inhibition, respectively. An extract prepared from mature excised ovaries had oiistatic activity (23 per cent inhibition) when injected into female flies. Extracts from whole ground female flies were more active than comparable concentrations of extracts from unground female abdomens (11.6 per cent inhibition/female equivalent compared with 7.9 per cent inhibition/female equivalent). Injections of mature female extracts inhibited ovarian development in all nine experiments, and the mean differences between egg stages in water-injected females and extract-injected flies were statistically significant from 0 at the 0.01 level. All the extracts were toxic when injected into females and induced a mean mortality of 37.9 per cent (Table 3). Distilled water-injected flies had a mean was lowest in flies injected with mortality of 10.0 per cent. Female mortality 1~1 of extract at a concentration of 1.5 equiv./$ and highest in females receiving 1.5 ~1 of either the extract or distilled water. Ovarian development was not inhibited in females injected with extracts from whole immature females in 20% glycerol (Table 4). The mean percentage inhibition for the five replicates was 1.4 per cent or O-5 per cent inhibition/female equivalent. The mean differences between egg stages in the 20% glycerol-injected females and extract-injected flies were not statistically significant from 0. Mortality was higher in extract-injected females than in 20% glycerol-injected ones (19.2 per cent mortality compared with 9.2 per cent). This indicates that ovarian inhibition is not correlated with mortality. Extracts from mature females inhibited ovarian development in injected flies when compared with extracts from immature females (control extract) (Table 5). Ovarian inhibition ranged from 6.7 to 20.6 per cent with a mean inhibition of 13.4 per cent or 3.7 per cent/equivalent injected. The mean difference between ovarian stages in females injected with mature female extract and control extract was significantly different from 0 at the 0.05 level. The mortality in females injected with mature female extract was 18 per cent and in immature females, 21 per cent. Storage of the prepared extract solution for 7 days at 10°F reduced the o&static activity by approximately 50 per cent in injected flies (Table 5). Activity was 6.9 per cent/female equivalent in a fresh extract preparation, and the activity decreased
3.3
3.3 3.3 3*0 3.0 3.0
1-S 1.5
Experiment
1
23* 4* St
8: 9:
219 -
39 38
19 17 6 44 22 21
13
37.9
4.80 13.60
46.70 32.00 88.00 12.00 53.00 55.00
36.00
No. Mortality scored (%)
Mean stage
5.82
-
6.20 f 1.26 6*08+ 1.40
5.63 kO.89 5*60& 1.58 5.33 + 1.30 7.60 -I 1.35 5.27k 1.18 4.62f0.20
6.07 k 1.76
females
OF AN O&TATIC
EXTRACTED
FROM
MATURE
305 -
37 39
22 12 51 49 51
22
10.00
5.10 4.80
12.00 16.00 38.00 0.00 2.00 0.00
12.00
No. Mortality scored (%)
+ * 2.34 1.86 f 0.98 f 0.99 f 1.06 +_1.41
8.19
-
7.51 f 0.73 7.46 f 1.17
7.60 9.40 8.33 9.94 7.48 7.51
8.50 zk1.95
Mean stage
Water-injected females (control)
SUBSTANCE
HOUSEFLIES
2.37 5
1.31 1.38
1.97 3.80 3.00 2.34 2.21 2.89
2.93
Stage difference
29.78
1744 18.50
25.92 40.42 36.01 23.14 29.54 38.48
28.58
Inhibition 1%)
9.57 * 0.30
11.62 12.33
7.85 8.08 7.20 7.77 9.85 12.83
8.67
Per cent inhibition/ female equiv.
Ovarian inhibition (control-extract)
FEMALE
* Injected with 1 ~1 of extract when 24 hr old and again with 0.5 ,~l at 48 hr. All others injected with 1~1. t Excised mature ovaries used for extract. $ Injected with extract prepared from ground whole females held for 2 days after injection at 78 & 2°F. Others injected with ethanol rinse of mature female abdomens and held for 5 days at 72 zk2°F. 8 Difference is significantly different from 0 at the 0.01 level as determined by t-tests.
Total Mean
7:
-
Concentration (female equiv./pl)
6:
3-ACTIVITY
Extract-injected
TABLE
k
s 4
5 9
z 2
0
3
B
8 g
8 z 5 ij
-19.20
10~00 4.00 38.00 20.00 24.00
46 48 27 38 39
198 -
(%)
scored
Mortality
female
7.43
8.78 7.68 7.76 6.68 6.51 -
z!I1 a39 k 1.62 k 1 a85 ?I 0.66 + 1.25
Ovarian mean stage
with stage 2 (immature) extract t
No.
injected
* All flies injected with 1 ~1 of solution. t Injected at a concentration of 3.0 per cent female
Total Mean
2 3 4 5
1
Replicate
Females
cquiv./$.
227
48 48 47 46 38
scored
No.
Females
9.20
4.00 2.00 8.00 16.00 16.00
(%)
Mortality
glycerol
7.58
8.35 8.06 7.70 6.98 6.76 -
+ k + f k
l-43 1.25 1.80 1.11 0.68
Ovarian mean stage
injected with 20% (control)
0.09
+ 0.25
- 0.06 + 0.30
- 0.43 + 0.38
Mean stage difference
l-37
-5.14 $4.71 - 0.78 l-4.35 +3.69
(%)
Inhibition
Ovarian inhibition (glycerol-stage 2 extract)
TABLE 4-O~STATIC ACTIVITYOFEXTRACTS FROM IMMATURE FEMALE HOUSEFLIES*
4 ;
LI
.J 5 u
s x 5
x+
; g
9
230
46 27 27 43 44 43
21.00
-
8.00 38+0 38.00 12.00 12.00 18.00
Mortality (%)
8.14
8.78 7.76 7.68 8.54 7.02 9.05 -
f 1.39 + 1.85 + 1.62 &-1.37 + 1.07 + 1.13
Mean stage
-
236
47 39 24 37 44 45
No. scored
18.00
-
6.00 16.00 SO*00 20.00 8.00 8.00
Mortality (%)
7.04
6.97 6.36 6.54 7.46 6.50 844 -
-I 1.43 + 1.29 z?z 1.53 f 2.12 + 1.25 _+I.30
Mean stage
l*lOS
-
1.81 1.46 1.14 1.08 0.52 0.61
13.38
-
20.62 18.04 14.84 12.64 740 6.74
Inhibition (%)
3.73 f 0.69
-
6.87 3.60 2.97 4.21 246 2.25
Per cent inhibition/ female equiv.
Stage 2-stage 10
ON OVARIAN DEVELOPMENT
Stage difference
FEMALEEXTRACTS
* Flies were injected with extract at a concentration of 3 female equiv./pl. t Injected with 1.5 ~1 of extract held for 1 week in freezer in solution. All others injected with 1.0 ~1. $ Injected with extract held for 30 days in freezer in dried form before adding 20% glycerol. $ This difference is significant at the 0.05 level as determined by a t-test.
Mean
Total
3t 4$ 5: 6:
2t
1
Replicate
No. scored
Stage 10 extract (mature) injected females
INJECTIONS OF MATUREAND IMMATURE IN THEHOUSEFLY*
Stage 2 extract (immature) injected females
TABLE S-THEEFFECTOFTHORACIC
E
#
g 9
$ 9
J 0 z 2
m
%
3 4 i;
&
992
T. S. ADAMS,A. M. HINTZ,ANDJ. G. POMONIS
to 3.6 and 3-O per cent/female equivalent in two replications of flies injected with aged extract. Storage of the dried residue in the freezer also reduced activity. Experiments 4, 5, and 6 were conducted with extracts which were stored as a dry residue for approximately 30 days in a freezer. Inhibition in females injected with these extracts varied from 2.2 per cent inhibition/female equivalent to 4.2 per cent inhibition/female equivalent. DISCUSSION The pattern of ovarian growth in the first and second gonorropic cycles for M. domestica is similar to that of H. collusor (ADAMS and MULLA, 1967). Stage 4 in the second cycle of M. domestica first occurred when the first cycle was at stage 6; in H. collusor, stage 4 of the second cycle was not reached until mature eggs were present in the first cycle. Also, in the eye gnat, the-second gonotropic cycle did not proceed past stage 4 as long as mature eggs in the first cycle were retained. In M. domestica, a small number of females were found to have stage 5 or 6 eggs in the second cycle when mature eggs from the first cycle were retained. Simultaneous growth of the first and second gonotropic cycles occurred in both species. Secondary gonotropic cycles in the housefly required more time for completion than the first cycle. This is due to a suppression of the development rate of stages 2 to 4. It is believed that this suppression and subsequent inhibition at stage 4 in the second gonotropic cycle are due to an oostatic hormone. The oiistatic hormone was recovered from extracts prepared from mature females, abdomens with mature eggs, and mature ovaries but not from extracts of immature females. Extracts from mature females inhibited ovarian development in the first gonotropic cycle of injected flies. The housefly oijstatic hormone is soluble in 95% ethanol, chloroform-methanol (2 : l), 20% glycerol, and water. This suggests a lipoidal nature for the oijstatic hormone. Extracts of ground whole flies in ethanol had more oostatic activity than extracts prepared from ethanolic rinses because more hormone was extracted by grinding in ethanol. Chloroform-methanol extract residue taken up in 20% glycerol was less active than the other extracts because the flies were stored in ethanol, and the storage ethanol was discarded. Inhibition of ovarian development in the housefly is not due to nervous inhibition as a result of stretch receptor activation as it is in Nauphoeta cinerea (ROTH, 1964) because extracts inhibited ovarian development when injected into immature flies. ENGELMANN(1964) suggested that the brood sac in Leucophea maderae secreted a substance which acted on neurons in the brain, which in turn inhibited the corpus allatum. Since allatectomy of female flies at an age of 12 hr inhibited ovarian development at stage 4 (ADAMS, 1967), it seems plausible to assume that the o&static hormone in the housefly inhibits the release of juvenile hormone from the corpus allatum, as suggested by ENGELMANNfor the cockroach. IVANOV and MESCHER~KAYA(1935) showed that the hormone produced by the corpus luteum in the cockroaches, Blatta and Blattella, made developing
OilSTATICHORMONE PRODUCTION IN HOUSEFLIES
993
ovaries impermeable to haemoglobin and neutral red; this hormone, then, had a direct effect on the ovaries themselves without corpus allatum involvement. The mode of action for the ovarian inhibition in Blatta and Bkzttella is different from that suggested for the housefly. From the data presented, the authors propose the following hypothesis. An oiistatic hormone is produced by eggs in stages 4 to 10 which inhibits ovarian development in subsequent gonotropic cycles. The presence of this hormone suppresses egg maturation rates in stages 2 to 4, with total inhibition at stage 4. It is likely that the oijstatic hormone prevents the release of juvenile hormone from the corpus allatum and thereby inhibits ovarian development. The o&static hormone maintains the cyclicity of egg maturation in subsequent gonotropic cycles. Furthermore, any dipteran with cyclical egg maturation such as H. coZZusor might have an oijstatic hormone present. Acknowledgements-The authors express their appreciation to Dr. D. R. NELSON and Mr. J. G. JOHNSONfor assistance in extract preparation; to Mrs. GENEVAOLSTADfor assistance in the bioassay; and to the following student assistants from the North Dakota State University: GLEN GUSTAFSON,LAVERN HOLMLUND, and RICHARDURBANIAHfor rearing houseflies ; and to MARGARETJONES, LYNNE PELTIER, MAUREEN SCOTT, SUSAN SCHNEIDERHAN, and MARY WALLACEfor sexing flies. REFERENCES ADAMST. S. (1967) The relationship of ovaries and the corpus allatum to mating in the house fly, Afusca domestica (Summary of submitted paper No. 60). Am. 2001. 7, 724. ADAMS T. S. and MULLA M. S. (1967) The reproductive biology of Hippelates collupor-II. Gametogenesis. Ann. ent. Sot. Am. 60, 1177-1182. ANONYMOUS (1956) The Peet-Grady method. Soap Chem. Specialities 32,243-244. CARLISLED. B. (1953) Studies on Lysmata sexicaudata F&so (Crustacea: Decapoda)-V. The ovarian inhibiting hormone and the hormonal inhibition of sex reversal. Pubbl. Staz. Zool. Napoli’ 24, 355-372. CARLISLED. Et. and BUTLER C. G. (1956) The ‘queen-substance’ of honey bees and the ovary-inhibiting hormone of crustaceans. Nature, Lond. 177, 276-277. CARLISLE D. B. and KNOWLES F. (1959) Endocrine Control in Crustaceans. Cambridge Monographs in Experimental Biology 10, l-120. DEMJXJSYH. (1962) Role de la glande de mue dans l’evolution ovarienne du crabe Carcimcs maenas. Cab. Biol. mar. 3, 37-57. ENGJXMAHNF. (1964) Inhibition of egg maturation in a pregnant viviparous cockroach. Nature, Lond. 202, 724-725. IVAHOVP. P. and MJZSCHER~KAYA K. A. (1935) Die physiologischen Besonderheiten der geschlechtlich unreifen Insektenovarien und die zyklischen Veriinderungen ihrer Eigenschaften. Zooi. Jb. (Physiol.) 55, 281-348. PAHOUSEJ. (1943) Influence de l’ablation du peduncule oculaire sur la croissance de l’ovaire chez la crevette Leander serratus. C. R. Acad. Sci., Paris 217, 553-555. ROTH L. M. (1964) Control of reproduction in female cockroaches with special reference to Nauphoeta ciaerea-II. Gestation and postparturition. Psyche, Camb. 71, 198-244.
OijSTATIC HORMONE PRODUCTION IN HOUSEFLIES, MUSCA DOMESTK’A, WITH DEVELOPING OVARIES T.
S. ADAMS,
A. M. HINT&
and
J. G. POMONIS
Metabolism and Radiation Research Laboratory, Entomology Research Division, Agricultural Research Service, United States Department of Agriculture, Fargo, North Dakota (Received 2 February 1968)
Abstract-An oijstatic hormone is produced by house@ with eggs in stages 4 to 10. This hormone suppresses the development rate of stages 2 to 4 eggs. Complete inhibition of the second gonotropic cycle at stage 4 occurs when mature eggs from the first cycle are retained. Extracts from mature female flies inhibited ovarian development when injected into 24-hr-old flies with eggs in stage 2 of development. The oijstatic hormone is soluble in 95% ethanol, chloroform-methanol (2 : l), 20% glycerol, and water. The oijstatic hormone maintains the cycle of egg production in the housefly.
INTRODUCTION
OVARIAN-INHIBITING hormones have been reported from various Arthropoda. In decapod crustaceans the eye stalk produces an ovarian inhibitor that is stored in the sinus gland (CARLISLE and KNOWLES, 1959). This hormone is reported for the following decapods: Leander serratus (PANOUSE, 1943), Lysmata sexicaudata (CARLISLE, 1.953), Car&u maenas (DEMEUSY, 1962). CARLISLE and BUTLER (1956) suggested a steroid nature for this decapod ovarian inhibitor and demonstrated that injections of ‘queen bee’ substance inhibited ovarian development in the prawn, Leander serratus; extracts of prawn eye stalks inhibited ovarian development when fed to worker bees. The decapod ovarian inhibitor was extractable in alcohol. IVANOV and MESCHERSKAYA (1935) showed that the o&heca-bearing cockroaches, Blattella germanica and Blatta orientalis, produced an ovarian inhibiting hormone from the corpora lutea in the proximal part of the ovariole. This hormone decreased the permeability of the oijcyte and was soluble in water. Studies by ADAMS and MULLA (1967) on the eye gnat, Hippelates collusor, showed that oi5cytes in the second gonotropic cycle did not proceed past stage 4 when mature eggs from the first cycle were retained. The present study was undertaken to find whether an oiistatic hormone was produced by houseflies with developing ovaries, whether it was extractable, and to develop a bioassay system for this hormone. 983
984
T. S. ADAMS,A. M. HINTZ,ANDJ. G. POMONIS MATERIALS
AND METHODS
Rearing All flies (h&sea domestica) for this study were of the FW, strain. Larvae were reared on CSMA medium (ANONYMOUS, 1956) that was fortified with 0*015 g brewer’s yeast/g CSMA. Adults were given water and powdered food consisting of dried non-fat milk, sugar, and whole powdered egg at a ratio of 6 : 6 : 1 by weight. The flies were allowed to emerge for a 4 hr period before sexing. Virgin females were used in these studies unless otherwise stated. Flies were held in plastic cages 7 x 10.5 x 13.5 in. that were covered with Tubegauz sleeves at one end. The rearing temperature was 78 + 2°F.
Scoring Egg development was scored on a basis of ten stages which-were adapted from those described for H. coZZusor(ADAMSand MULLA, 1967). In this scheme, stage 1 is the germarium, stage 4 is the beginning of vitellogenesis, and stage 10 is the mature egg.
Simultaneous ovarian growth Females were held at 78 + 2”F, and samples of twenty females were removed twice daily for 6 days; another sample of about two hundred females was taken on the seventh day. Ovarian development was scored for the first and second gonotropic cycles in each female examined. The data were compiled, and the percentage of the follicles in each ovarian stage of the second gonotropic cycle at each ovarian stage in the first cycle was calculated.
Egg retention studies Two groups of five hundred females/group were mated on the third day after emergence with an excess number of males of the same age. After 4 hr from introducing the males, an ovipositional substrate of used larval rearing medium covered with a moist black cloth was given to one group of flies. This substrate was renewed daily for the remainder of the study. The other group was encouraged to retain their eggs by withholding the ovipositional substrate. Samples of ten females were removed from each cage twice daily, both before and after mating. The egg scores were determined for the first and all observable secondary gonotropic cycles. This study was conducted at a test temperature of 80 f 2”F, and it was repeated using virgin females at a test temperature of 72 f 2°F. The means of the egg stages were determined for all gonotropic cycles and plotted against time. More accurate measurements were made from graphs plotted on log-log paper. Extracts The first series of extracts were prepared in 95% ethanol from excised mature ovaries and female abdomens containing mature eggs. The abdomens were depressed with a wooden dowel to extrude the eggs. The tissues were then
OijSTATIC
HORMONE PRODUCTION
985
IN HOUSEFLIES
rinsed three times with 95% ethanol? and the three rinses were combined, filtered, and taken to dryness on a flash evaporator. Prior to injection, distilled water was added to give the desired concentration. In one experiment, whole female flies were ground in a blender with 95% ethanol and then processed as described. Another experiment used flies which were stored in 95% ethanol. The ethanol was discarded, and the flies were ground in a blender with chloroform-methanol (2 : 1). The ground bodies were filtered and rinsed three times with chloroformmethanol. The rinses were combined and taken to dryness in a flash evaporator. The dried residue was extracted with 20% glycerol to give a solution concentration of 3 equiv./& Extracts of mature (3-day-old) flies and immature (4-hr-old) females were prepared in the same manner. All extracts and dried residues were stored under N, in a freezer at 10°F until the time of injection. Bioassay Newly ernerged virgin female flies were held at 72 & 2°F for 24 hr before injection. (Ovarian development was stage 2 at the time of injection.) The flies were narcotized with CO,, and the solutions were injected through the cervical region into the thorax with a 50 ~1 syringe on a micro-injector. Flies received from O-5 to 1.5 ~1 of solution, depending on the experiment. Controls were injected with either distilled water, 20% glycerol, or immature female extract in 20% glycerol. The flies were held for 48 hr at 78 & 2°F or for 5 days at 72 + 2°F before ovarian development was scored. The percentage inhibition was calculated from the following formula : Mean ovarian stage of control - mean ovarian stage of extract Mean ovarian stage of control
x 100 per cent.
The maximal inhibition obtainable by using the above formula is 60 per cent. RESULTS
Simultaneous growth of the Jirst and second gonotropic cycle Simultaneous growth occurred in the first and second gonotropic cycles but stopped at stage 4 (in most cases) in the second cycle when mature eggs were retained (Table 1). When eggs in the first cycle were at stage 2, those in the second cycle were always in stage 1. When eggs in the first cycle were at stage 3, 85 per cent of those in the second cycle were at stage 1, and the remainder were at stage 2. Eggs at stages 4 to 7 in the first gonotropic cycle were followed by stage 2 eggs in the second cycle in most cases (656-96.4 per cent) with the remainder being in stages 3 and 4. Stages 8 and 9 of the first cycle were usually accompanied by stage 3 eggs in the second gonotropic cycle. Mature eggs in the first cycle were accompanied by stage 4 eggs in the second cycle in 48.8 per cent of the females examined, with the remaining percentages as follows: 20.3 with stage 2 eggs, 29.9 with stage 3 eggs, 4.0 with stage 5 eggs, and 3.0 with stage 6 eggs. From this, we found that the second gonotropic cycle starts development when the first cycle is at an egg stage of 3.
986
T. S. ADAMS, A. M. HINTZ, AND J. G. POMONIS
Egg retention and deposition The development of the first and all secondary gonotropic cycles in oviposited and gravid mated females is presented in Fig. l(A) and l(B), respectively. Females that were allowed to deposit eggs experienced three complete gonotropic TABLE ~-SIMULTANEOUS GROWTH OF EGGS IN THE FIRST AND SECONDGONOTROPICCYCLE OF HOUSEFLIES Percentage eggs in second cycle at indicated stage Stages in fkst cycle 2 3 4 5 6 7 8 9 10
No.
50 49 38 28 97 29 26 52 551
1 100.0
83.6 10.5 -
3
2
4
5
6
-
-
-
-
16.4 86.9 96.4
_ 2.6 3.6
_ _ _
_ _ _
_ _ _
89.7 65.6 23.1 23-l 20.3
6.2 31.0 76.9 53.8 23.9
4.1 3.4 23.1 48.8
4.0
3-o
-
cycles in 160 hr. The females that retained mature eggs for 130 hr completed the first gonotropic cycle, but the second cycle was inhibited at stage 4. After 130 hr, the females deposited many eggs, and development in the second cycle progressed beyond stage 4. Both the virgin female populations refused to oviposit and retained mature eggs from the first cycle for 130 hr. The second cycle was completely suppressed at stage 4. No graph is presented because the trend is duplicated in Fig. l(B). _. In both oviposited and gravid mated females, 68 and 70 hr respectively were required to complete the first gonotropic cycle (Table 2). Mated and oviposited females took 82 and 92 hr for the completion of the second and third gonotropic This represents an increase of 14 and 24 hr respectively cycles, respectively. over the first cycle. This increase is due to a prolongation of stages 2 to 4. It required 45 and 55 hr to complete stages 2 to 4 in the second and third gonotropic cycles in the mated and oviposited females, respectively, and 50 hr for the second cycle in the mated and gravid females. This contrasts with the 30 hr each taken to complete stages 2 to 4 in the first cycle for both the mated oviposited and mated gravid flies. Stages 4 to 10 took 37 and 38 hr to complete the first to third gonotropic cycles in the mated and oviposited females and 40 hr to complete stages 4 to 10 in the first cycle mated gravid flies. There was no difference between the first and subsequent gonotropic cycles in the time required to complete stages 4 to 10. The first gonotropic cycle in both populations of virgin gravid females took 100 hr for completion, and the second cycle was inhibited at stage 4 (Table 2).
OijSTATIC HORMONE
PRODUCTION
987
IN HOUSEFLIES
A
20
,
*
.
*
40
60
60
IO0
AGE
(Hours
, 120 at
60.
140
160
160
E)
FIG. 1. The effect of egg retention and deposition on the development of secondary gonotropic cycles in the housefly. (A) Mated and oviposited females, (B) mated and gravid females. TABLE
2-DURATION
OF OVARIAN
STAGESEQUENCES
INOVIPOSITRD
AND
GRAVID
POPULATIONS
OF HOUSEFLIES
Duration (hr) of Population Mated-viposited
Mated-gravid
*
*
Virgin-gravid.?
* Held at 80 + 2°F. t Held at 72 + 2°F.
Gonotropic cycle
Stages 2-4
Stages 4-10
Complete cycle
1 2 3 1 2 1 2 1 2
30 45 55 30 50 32 66 36 60
38 37 37 40 Inhibited at stage 4 68 Inhibited at stage 4 64 Inhibited at stage 4
68 82 92 70 100 100 -
988
T. S. ADAMS,A. M. HINTZ, AND
J. G. POMONIS
The duration of stages 2 to 4 in the first cycles was 32 and 36 hr and 66 and 60 hr for the second gonotropic cycles. Again, a prolongation of the duration of stages 2 to 4 in the second cycle occurred.
Extract studies When 24-hr-old females were injected with extracts from mature female flies, the mean ovarian inhibition was 29.8 per cent or 9.6 per cent inhibition/female equivalent injected (Table 3). Females that were injected on 2 consecutive days with extracts from mature female abdomens had a greater percentage of ovarian inhibition than those injected once; 40 and 36 per cent inhibition compared with 28 and 26 per cent inhibition, respectively. An extract prepared from mature excised ovaries had oiistatic activity (23 per cent inhibition) when injected into female flies. Extracts from whole ground female flies were more active than comparable concentrations of extracts from unground female abdomens (11.6 per cent inhibition/female equivalent compared with 7.9 per cent inhibition/female equivalent). Injections of mature female extracts inhibited ovarian development in all nine experiments, and the mean differences between egg stages in water-injected females and extract-injected flies were statistically significant from 0 at the 0.01 level. All the extracts were toxic when injected into females and induced a mean mortality of 37.9 per cent (Table 3). Distilled water-injected flies had a mean was lowest in flies injected with mortality of 10.0 per cent. Female mortality 1~1 of extract at a concentration of 1.5 equiv./$ and highest in females receiving 1.5 ~1 of either the extract or distilled water. Ovarian development was not inhibited in females injected with extracts from whole immature females in 20% glycerol (Table 4). The mean percentage inhibition for the five replicates was 1.4 per cent or O-5 per cent inhibition/female equivalent. The mean differences between egg stages in the 20% glycerol-injected females and extract-injected flies were not statistically significant from 0. Mortality was higher in extract-injected females than in 20% glycerol-injected ones (19.2 per cent mortality compared with 9.2 per cent). This indicates that ovarian inhibition is not correlated with mortality. Extracts from mature females inhibited ovarian development in injected flies when compared with extracts from immature females (control extract) (Table 5). Ovarian inhibition ranged from 6.7 to 20.6 per cent with a mean inhibition of 13.4 per cent or 3.7 per cent/equivalent injected. The mean difference between ovarian stages in females injected with mature female extract and control extract was significantly different from 0 at the 0.05 level. The mortality in females injected with mature female extract was 18 per cent and in immature females, 21 per cent. Storage of the prepared extract solution for 7 days at 10°F reduced the o&static activity by approximately 50 per cent in injected flies (Table 5). Activity was 6.9 per cent/female equivalent in a fresh extract preparation, and the activity decreased
3.3
3.3 3.3 3*0 3.0 3.0
1-S 1.5
Experiment
1
23* 4* St
8: 9:
219 -
39 38
19 17 6 44 22 21
13
37.9
4.80 13.60
46.70 32.00 88.00 12.00 53.00 55.00
36.00
No. Mortality scored (%)
Mean stage
5.82
-
6.20 f 1.26 6*08+ 1.40
5.63 kO.89 5*60& 1.58 5.33 + 1.30 7.60 -I 1.35 5.27k 1.18 4.62f0.20
6.07 k 1.76
females
OF AN O&TATIC
EXTRACTED
FROM
MATURE
305 -
37 39
22 12 51 49 51
22
10.00
5.10 4.80
12.00 16.00 38.00 0.00 2.00 0.00
12.00
No. Mortality scored (%)
+ * 2.34 1.86 f 0.98 f 0.99 f 1.06 +_1.41
8.19
-
7.51 f 0.73 7.46 f 1.17
7.60 9.40 8.33 9.94 7.48 7.51
8.50 zk1.95
Mean stage
Water-injected females (control)
SUBSTANCE
HOUSEFLIES
2.37 5
1.31 1.38
1.97 3.80 3.00 2.34 2.21 2.89
2.93
Stage difference
29.78
1744 18.50
25.92 40.42 36.01 23.14 29.54 38.48
28.58
Inhibition 1%)
9.57 * 0.30
11.62 12.33
7.85 8.08 7.20 7.77 9.85 12.83
8.67
Per cent inhibition/ female equiv.
Ovarian inhibition (control-extract)
FEMALE
* Injected with 1 ~1 of extract when 24 hr old and again with 0.5 ,~l at 48 hr. All others injected with 1~1. t Excised mature ovaries used for extract. $ Injected with extract prepared from ground whole females held for 2 days after injection at 78 & 2°F. Others injected with ethanol rinse of mature female abdomens and held for 5 days at 72 zk2°F. 8 Difference is significantly different from 0 at the 0.01 level as determined by t-tests.
Total Mean
7:
-
Concentration (female equiv./pl)
6:
3-ACTIVITY
Extract-injected
TABLE
k
s 4
5 9
z 2
0
3
B
8 g
8 z 5 ij
-19.20
10~00 4.00 38.00 20.00 24.00
46 48 27 38 39
198 -
(%)
scored
Mortality
female
7.43
8.78 7.68 7.76 6.68 6.51 -
z!I1 a39 k 1.62 k 1 a85 ?I 0.66 + 1.25
Ovarian mean stage
with stage 2 (immature) extract t
No.
injected
* All flies injected with 1 ~1 of solution. t Injected at a concentration of 3.0 per cent female
Total Mean
2 3 4 5
1
Replicate
Females
cquiv./$.
227
48 48 47 46 38
scored
No.
Females
9.20
4.00 2.00 8.00 16.00 16.00
(%)
Mortality
glycerol
7.58
8.35 8.06 7.70 6.98 6.76 -
+ k + f k
l-43 1.25 1.80 1.11 0.68
Ovarian mean stage
injected with 20% (control)
0.09
+ 0.25
- 0.06 + 0.30
- 0.43 + 0.38
Mean stage difference
l-37
-5.14 $4.71 - 0.78 l-4.35 +3.69
(%)
Inhibition
Ovarian inhibition (glycerol-stage 2 extract)
TABLE 4-O~STATIC ACTIVITYOFEXTRACTS FROM IMMATURE FEMALE HOUSEFLIES*
4 ;
LI
.J 5 u
s x 5
x+
; g
9
230
46 27 27 43 44 43
21.00
-
8.00 38+0 38.00 12.00 12.00 18.00
Mortality (%)
8.14
8.78 7.76 7.68 8.54 7.02 9.05 -
f 1.39 + 1.85 + 1.62 &-1.37 + 1.07 + 1.13
Mean stage
-
236
47 39 24 37 44 45
No. scored
18.00
-
6.00 16.00 SO*00 20.00 8.00 8.00
Mortality (%)
7.04
6.97 6.36 6.54 7.46 6.50 844 -
-I 1.43 + 1.29 z?z 1.53 f 2.12 + 1.25 _+I.30
Mean stage
l*lOS
-
1.81 1.46 1.14 1.08 0.52 0.61
13.38
-
20.62 18.04 14.84 12.64 740 6.74
Inhibition (%)
3.73 f 0.69
-
6.87 3.60 2.97 4.21 246 2.25
Per cent inhibition/ female equiv.
Stage 2-stage 10
ON OVARIAN DEVELOPMENT
Stage difference
FEMALEEXTRACTS
* Flies were injected with extract at a concentration of 3 female equiv./pl. t Injected with 1.5 ~1 of extract held for 1 week in freezer in solution. All others injected with 1.0 ~1. $ Injected with extract held for 30 days in freezer in dried form before adding 20% glycerol. $ This difference is significant at the 0.05 level as determined by a t-test.
Mean
Total
3t 4$ 5: 6:
2t
1
Replicate
No. scored
Stage 10 extract (mature) injected females
INJECTIONS OF MATUREAND IMMATURE IN THEHOUSEFLY*
Stage 2 extract (immature) injected females
TABLE S-THEEFFECTOFTHORACIC
E
#
g 9
$ 9
J 0 z 2
m
%
3 4 i;
&
992
T. S. ADAMS,A. M. HINTZ,ANDJ. G. POMONIS
to 3.6 and 3-O per cent/female equivalent in two replications of flies injected with aged extract. Storage of the dried residue in the freezer also reduced activity. Experiments 4, 5, and 6 were conducted with extracts which were stored as a dry residue for approximately 30 days in a freezer. Inhibition in females injected with these extracts varied from 2.2 per cent inhibition/female equivalent to 4.2 per cent inhibition/female equivalent. DISCUSSION The pattern of ovarian growth in the first and second gonorropic cycles for M. domestica is similar to that of H. collusor (ADAMS and MULLA, 1967). Stage 4 in the second cycle of M. domestica first occurred when the first cycle was at stage 6; in H. collusor, stage 4 of the second cycle was not reached until mature eggs were present in the first cycle. Also, in the eye gnat, the-second gonotropic cycle did not proceed past stage 4 as long as mature eggs in the first cycle were retained. In M. domestica, a small number of females were found to have stage 5 or 6 eggs in the second cycle when mature eggs from the first cycle were retained. Simultaneous growth of the first and second gonotropic cycles occurred in both species. Secondary gonotropic cycles in the housefly required more time for completion than the first cycle. This is due to a suppression of the development rate of stages 2 to 4. It is believed that this suppression and subsequent inhibition at stage 4 in the second gonotropic cycle are due to an oostatic hormone. The oiistatic hormone was recovered from extracts prepared from mature females, abdomens with mature eggs, and mature ovaries but not from extracts of immature females. Extracts from mature females inhibited ovarian development in the first gonotropic cycle of injected flies. The housefly oijstatic hormone is soluble in 95% ethanol, chloroform-methanol (2 : l), 20% glycerol, and water. This suggests a lipoidal nature for the oijstatic hormone. Extracts of ground whole flies in ethanol had more oostatic activity than extracts prepared from ethanolic rinses because more hormone was extracted by grinding in ethanol. Chloroform-methanol extract residue taken up in 20% glycerol was less active than the other extracts because the flies were stored in ethanol, and the storage ethanol was discarded. Inhibition of ovarian development in the housefly is not due to nervous inhibition as a result of stretch receptor activation as it is in Nauphoeta cinerea (ROTH, 1964) because extracts inhibited ovarian development when injected into immature flies. ENGELMANN(1964) suggested that the brood sac in Leucophea maderae secreted a substance which acted on neurons in the brain, which in turn inhibited the corpus allatum. Since allatectomy of female flies at an age of 12 hr inhibited ovarian development at stage 4 (ADAMS, 1967), it seems plausible to assume that the o&static hormone in the housefly inhibits the release of juvenile hormone from the corpus allatum, as suggested by ENGELMANNfor the cockroach. IVANOV and MESCHER~KAYA(1935) showed that the hormone produced by the corpus luteum in the cockroaches, Blatta and Blattella, made developing
OilSTATICHORMONE PRODUCTION IN HOUSEFLIES
993
ovaries impermeable to haemoglobin and neutral red; this hormone, then, had a direct effect on the ovaries themselves without corpus allatum involvement. The mode of action for the ovarian inhibition in Blatta and Bkzttella is different from that suggested for the housefly. From the data presented, the authors propose the following hypothesis. An oiistatic hormone is produced by eggs in stages 4 to 10 which inhibits ovarian development in subsequent gonotropic cycles. The presence of this hormone suppresses egg maturation rates in stages 2 to 4, with total inhibition at stage 4. It is likely that the oijstatic hormone prevents the release of juvenile hormone from the corpus allatum and thereby inhibits ovarian development. The o&static hormone maintains the cyclicity of egg maturation in subsequent gonotropic cycles. Furthermore, any dipteran with cyclical egg maturation such as H. coZZusor might have an oijstatic hormone present. Acknowledgements-The authors express their appreciation to Dr. D. R. NELSON and Mr. J. G. JOHNSONfor assistance in extract preparation; to Mrs. GENEVAOLSTADfor assistance in the bioassay; and to the following student assistants from the North Dakota State University: GLEN GUSTAFSON,LAVERN HOLMLUND, and RICHARDURBANIAHfor rearing houseflies ; and to MARGARETJONES, LYNNE PELTIER, MAUREEN SCOTT, SUSAN SCHNEIDERHAN, and MARY WALLACEfor sexing flies. REFERENCES ADAMST. S. (1967) The relationship of ovaries and the corpus allatum to mating in the house fly, Afusca domestica (Summary of submitted paper No. 60). Am. 2001. 7, 724. ADAMS T. S. and MULLA M. S. (1967) The reproductive biology of Hippelates collupor-II. Gametogenesis. Ann. ent. Sot. Am. 60, 1177-1182. ANONYMOUS (1956) The Peet-Grady method. Soap Chem. Specialities 32,243-244. CARLISLED. B. (1953) Studies on Lysmata sexicaudata F&so (Crustacea: Decapoda)-V. The ovarian inhibiting hormone and the hormonal inhibition of sex reversal. Pubbl. Staz. Zool. Napoli’ 24, 355-372. CARLISLED. Et. and BUTLER C. G. (1956) The ‘queen-substance’ of honey bees and the ovary-inhibiting hormone of crustaceans. Nature, Lond. 177, 276-277. CARLISLE D. B. and KNOWLES F. (1959) Endocrine Control in Crustaceans. Cambridge Monographs in Experimental Biology 10, l-120. DEMJXJSYH. (1962) Role de la glande de mue dans l’evolution ovarienne du crabe Carcimcs maenas. Cab. Biol. mar. 3, 37-57. ENGJXMAHNF. (1964) Inhibition of egg maturation in a pregnant viviparous cockroach. Nature, Lond. 202, 724-725. IVAHOVP. P. and MJZSCHER~KAYA K. A. (1935) Die physiologischen Besonderheiten der geschlechtlich unreifen Insektenovarien und die zyklischen Veriinderungen ihrer Eigenschaften. Zooi. Jb. (Physiol.) 55, 281-348. PAHOUSEJ. (1943) Influence de l’ablation du peduncule oculaire sur la croissance de l’ovaire chez la crevette Leander serratus. C. R. Acad. Sci., Paris 217, 553-555. ROTH L. M. (1964) Control of reproduction in female cockroaches with special reference to Nauphoeta ciaerea-II. Gestation and postparturition. Psyche, Camb. 71, 198-244.