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Distention-mediated egg maturation in the mosquito, Aedes aegypti
J. Insect Physiol. Vol. 33, ICo. 2. pp. 83-87, 1987 Printed
in Great
Britain.
Copyright
All rights reserved
DISTENTION-MEDIATED THE MOSQUITO, MARC
Department
of Plant.
Soil and Entomological (Received 12 May
J.
0@22-191Oj87 $3.00 + 0.00 1987 Pergamon Journals Ltd
0
EGG MATURATION AEDES AEGYPTI
IN
KLOWDEN
Sciences,
University
of Idaho,
Moscow,
ID 83843, U.S.A.
1986; revised 16 July 1986)
Abstract-Abdominal distention accelerates the release of a factor from the head of blood-fed Aedes aeg.vpti mosquitoes. The critical period during which the head is required for oijgenesis following blood ingestion is approx 6 h with a 5 ~1 meal, but small blood meals of 1~1 require the head to be present for significantly longer. Increasing the abdominal distention by supplementing the 1 ~1 meal with saline results in a critical period similar to that with 5 ~1 of blood. The information from the distended abdomen appears to travel via the ventral nerve cord. Transection of the ventral nerve cord prevents oiigenesis from occurring after small blood meals. but not with larger blood volumes. Topical application of 100 pg of juvenile hormone III can substitute for the distention message. Key Word In&x:
Aedes aegypti, &genesis,
abdominal
INTRODUCTION
distention,
juvenile
hormone
a unifying hypothesis that accounts for previous reports demonstrating either neural or humoral involvement in release of egg-development neurosecretory hormone.
In anautogenous Aedes aegypti mosquitoes, postemergence follicular growth proceeds to the so-called resting stage under the influence of juvenile hormone (Gwadz and Spielman, 1973), but its development advances no further until a blood meal is ingested. The blood not only provides precursors for vitellogenesis but also serves as a trigger for the subsequent endocrine events that are necessary for the production and uptake of vitellogenin. Experiments in which mosquitoes were decapitated at intervals following a blood meal demonstrated that one of these endocrine events included the release of a factor from the head that was essential in order for egg maturation to occur (Clements, 1956; Gillett, 1956). Lea named this factor egg-development neurosecretory hormone. which was produced in the medial neurosecretory cells of the brain and released from the corpus cardiacum in response to blood ingestion (Lea, 1967, 1972). Although a blood meal is necessary for this hormone release, the mechanism by which this release occurs has not yet been determined. Lea (1972) ruled out nervous involvement in its release and demonstrated that the release signal was transported by the haemolymph. This has been confirmed in several studies (Bellamy and Bracken, 1971; Chang and Judson, 1977; Van Handel and Lea, 1984). However, conflicting with this hypothesis of a humoral release of the egg-development neurosecretory hormone are experiments that indicate abdominal distention is involved in the initiation of oiigenesis (Gillett, 1957; Larsen and Bodenstein, 1959; Gillett ef al., 1975; Spielman and Wong, 1974). In this report, further evidence is presented that demonstrates that nol: only is a component of the blood meal necessary in order for a factor stimulating oiigenesis to be released from the head, but that this release is modulated b;! the nervous system as a result of abdominal distention. These data may provide
MATERIALS
AND METHODS
Aedes aegypti (L.) larvae and adults were reared at 27°C under a 14 h light: 10 h dark photoperiodic regime. Adults were maintained at 70% r.h., with 1, 10 or 20% sucrose solution continuously available from cotton wicks. Mosquitoes used in these experiments were 4- to 5-days after emergence. Blood from a laboratory rat, obtained by heart puncture, and saline (Ephrussi and Beadle, 1936) were administered to the midguts of mosquitoes in measured amounts by enema (Briegel and Lea, 1975). In some experiments, the ventral nerve cord was transected anterior to the second abdominal ganglion; in sham-operated controls the arthrodial membrane was opened, but the ventral nerve cord was not disturbed. Decapitation removed the medial neurosecretory cells and corpus cardiacum. All wounds from ventral nerve cord transection and decapitation were sealed with paraffin wax. Juvenile hormone III (Sigma, St Louis, MO.) was dissolved in acetone and topically applied to the abdomens; controls received acetone alone. Oljcytes were considered to be developing if they contained more than 200 pm of yolk at 48 h after the blood meal. A one-way analysis of variance and an arcsine test for the equality of 2 percentages (Sokal and Rohlf, 1969) were used to statistically compare responses. RESULTS
Egg development When
number volume 83
resulting from
small blood meals
scale, the mean of eggs maturing was directly related to the of blood administered if the volumes exexpressed
on a logarithmic
MARC J. KLOWDEN
84
Table
140 D-a
120 0 f!
%
1%
I. Effects of
#%,,,a 10% ‘_ 20%
swrose sucrose
Group
100
1
0
3.0
0 (46)
ND
0. I
0
2 (90)
ND
C
0.1 0.1 0.1
0.9 1.9 3.9
I8 (67) 75 (40) 80 (35)
Il.2 19.8 21.5
0.05 0.05
0 3.0
ow
ND ND
0.1 0.1
I.9 I .9*
71 (61)
ND ND
*Saline containing 8 peg of soybean ND = not determined.
8 (48)
2 (54) trypsin inhibitor.
increases m saline (Table 1C). Eggs produced by these females hatched normally and larvae were successfully reared to the adult stage. When the volume of blood was reduced to 0.05 ~1, none of the mosquitoes
20
0
Mean no. eggs/female
B
E
40
% with eggs (N)
A
80
60
Saline volume (/Ll)
Blood volume (jll)
D 8 f
saline supplements on the ability of small blood meals to initiate oikenesis
SWrosB
1
0.1
I
I
03
l111111
05
Blood
I
1.0
III
3.0
5.0
developed
eggs, and only a small proportion
under-
volume +I)
Fig. 1. The number of eggs developing in mosquitoes maintained on 1, 10 or 20% sucrose as a function of the log,, of the blood volume.
ceeded 0.3 ~1 (Fig. 1). The slopes, representing the rate at which blood was converted into eggs, were not significantly different for mosquitoes maintained on 1% [Y = 56.0 + 86.4 (log,,)X], 10% [Y = 60.0 + 89.7 (log,,,) X] or 20% sucrose [Y = 62.9 + 89.4 (log,,) X]. However, mosquitoes maintained on 20% sucrose that received less than 0.3 ~1 of blood no longer showed a relationship between blood volume and number of eggs; approx 20 eggs matured regardless of the volume administered between 0.1 and 0.3 ~1. Mosquitoes maintained on 1 or 10% sucrose failed to develop eggs at blood volumes below 0.3 ~1 (Fig. 1). Although the number ofeggs that matured was not dependent on the concentration of the sucrose diet, the percentage that matured any eggs at all increased with increasing sucrose concentration. The abdomens of mosquitoes maintained on 20% sucrose were also significantly more distended than those of mosquitoes on either 1 or 10% sucrose (unpublished data). For these reasons, abdominal distention was considered as a factor responsible for the greater tendency of mosquitoes that were maintained on 20% sucrose to develop eggs at small blood volumes. This ability of distention to supplement the blood meal was examined in another series of experiments. If 3 ~1 of saline alone were administered to mosquitoes, none devel-
)-
)-
) -
, -
)-
(B) )-
1
I
I
I
,-
oped eggs (Table 1A) and 0.1 ~1 of blood alone initiated egg maturation in only a small number of
/
,
,
females (Table 1B). However, when the 0.1 11 of blood was administered simultaneously with saline in order to increase abdominal distention, a larger percentage of mosquitoes developed eggs when the volume of saline was increased up to a total volume of 2 ~1. With a total volume of 4 /*I (0.1 ~1 of blood plus 3.9 ~1 of saline), the percentage with eggs was not increased significantly compared to the 2 ~1 total volume. The mean number of eggs in mosquitoes that did reproduce was not significantly increased by these
0.5 25
1.0 2.0
2.0 10
Volume
admlnlstered
‘A; 3.0 0
Blood Salhe
(pl)
Fig. 2. influence of ventral nerve cord on distentionmediated oOgenesis in A. czegypti. A: Percentage of mosquitoes maturing eggs after the administration of 3 /II of blood and saline in varying proportions. B: Number of eggs maturing per female. (Dashed lines: VNC Transected; solid lines: sham-operated controls; vertical lines: standard errors).
Abdominal distention
5ji
bloqd
,
I
85
different from controls (Fig. 2A). The numbers of eggs which did mature in controls and experimentals given comparable blood/saline meals did not differ significantly (Fig. 2B).
I
80
Critical periods and abdominal distention
20
0
Time
of decopitatlon
(h pbm)
Fig. 3. Critical periods for the presence of the head following a 1~1 blood meal (triangles), a 5~1 blood meal (open circles). or a 1 ~1 blood meal supplemented with 4~1 of saline (closed circles). Vertical lines: standard errors. In intact controls, 82 and 89% developed eggs with 1 and 5 ~1 of blood, respectively.
went oijgenesis when 3 ~1 of saline were simultaneously administered [Table 1D). Although abdominal distention was indeed able to influence egg development, it appea.red that a certain component in the blood was also necessary in some minimal quantity. In order to determine whether the blood must be digested in order to release this component, 0.1 p 1 of blood was administered simultaneously with 1.9 ~1 of saline that also contained 8 pg of soybean trypsin inhibitor. Few of the mosquitoes receiving this solution matured eggs (Table IE). Infruence of the ventral nerue cord Transecting the ventral nerve cord of mosquitoes prior to a meal of Cl.1~1 of blood plus 2.9 ~1 of saline abolished the distention-mediated effect on oiigenesis, but the distention signal became progressively less important as the proportion of blood in the total volume of 3 ~1 was increased relative to the saline. With 3 ~1 of blood and no saline, the proportion of nerve cord-transected females that developed eggs was not significantly Table 2. Decapitation
following
Treatment 5 jI I sahne; I ~1 blood 6-7 b later
The interval during which the head was necessary for egg development was determined for blood meals of 1 and 5 ~1. With 5 ~1 of blood, there was a sharp increase in the percentage with eggs when decapitation occurred after 6 h (Fig. 3). In mosquitoes receiving 1 ~1 of blood, however, there was no clearly defined critical period. The percentage with eggs increased slowly with time of decapitation, and by 12 h had finally achieved a level comparable with intact controls. In contrast, when the 1 ~1 of blood was administered simultaneously with 4~1 of saline in order to increase abdominal distention. the critical period was characteristic of mosquitoes receiving 5 ~1 of blood (Fig. 3). To investigate whether abdominal distention alone could release egg-development neurosecretory hormone, an assay similar to that of Chang and Judson (1977) was employed. Mosquitoes were given 5 ~1 of saline, and 67 h later, 1~1 of blood. Immediately following this blood, the insects were decapitated and examined 48 h later. Controls were treated similarly, but not decapitated. It was anticipated that if the saline triggered the hormone’s release, the 1 ~1 of blood, administered after the critical period, would provide precursors for oiigenesis even in the subsequent absence of the head. As shown in Table 2, the blood, introduced after the presumed critical period for distention-induced release of the hormone, failed to support oiigenesis, demonstrating that eggdevelopment neurosecretory hormone was not in circulation at the time of the blood meal and that the previous distention with saline was insufficient to cause the hormone’s release. This confirmed that a component in the blood meal must first be present before abdominal distention can modulate its effects.
enemas of saline and blood
Efects
of juvenile hormone
The possible role of juvenile hormone in distention-mediated oijgenesis was examined using mosquitoes which had their ventral nerve cords transected prior to receiving 0.1 ~1 of blood and 1.9 ~1 of saline. Immediately after the blood-saline enema, 100 pg of juvenile hormone III were topically applied. A small percentage of controls which received acetone alone developed eggs, but significantly more females developed eggs after the juvenile hormone treatment (Table 3).
% with eggs (N) Decapitated immediately after blood meal Non-decapitated controls
Table
3. Topical
treatment
Tr:atment Ventral nerve cord transected; 0.5 PI blood plus 2.5 ~1 saline -
DlSCUSSION Blood ingestion triggers the release of a reproductive factor from the head of A. aegypti females, and this release can be accelerated by abdominal
0 (30) 77 (26)
of ventral nerve cord hormone III
transected
females
with juvenile
% with eggs (N) 100 pg juvenile Acetone
controls
hormone
50 (26) 9(21)
86
MARC J. KLOWDEN
distention. This is evidenced by a change in the critical period for the presence of the head following a blood meal when a small volume of blood is supplemented with saline, compared with the effect of the blood alone (Fig. 3). As previously demonstrated by Spielman and Wong (1974), mosquitoes were more likely to produce eggs from a small blood meal when the midgut distention was increased (Table lC), but without some critical component from the blood meal, this distention alone was insufficient (Tables ID and 2). The data agree with previous studies concluding that release of egg-development neurosecretory hormone is triggered by components resulting from the initial products of blood digestion (Chang and Judson, 1977; Van Handel and Lea, 1984). The information relating the degree of abdominal distention reaches the head via the ventral nerve cord. Transection of the nerve cord anterior to the second abdominal ganglion before a meal of 0.5 ~1 of blood and 2.5 ~1 of saline was introduced all but abolished the effect distention had on oogenesis. As the proportion of the blood in the meal increased, however, the distention message became less important, and was no longer necessary when the blood comprised all of the 3 ~1 that was administered (Fig. 2A). This may explain why Lea (1972) was unable to show any involvement of the nervous system in release of egg-development neurosecretory hormone. Because the mosquitoes in his experiments were fed on blood to repletion (Lea, personal communication), the possible contribution of the nervous system may have been overridden by the large meals that were ingested. Spielman and abdominal distention a mosquitoes in their experiment to take small meals. Midgut endocrine cells are present in adult A. aegypti (Brown et al., 1985), which could respond to midgut distention, but these cells are probably not involved in distention-mediated oogenesis because their activity would not depend upon intact nervous connections to the head. The possibility that abdominal distention could affect the induction of digestive enzymes in the midgut, and thus indirectly reproduction, was considered. However, Briegel and Lea (1975) using blood volumes as low as 0.5 ~1, demonstrated that this induction was the result of a secretogogue stimulus and not abdominal distention. Although an intact ventral nerve cord influenced whether or not eggs developed at small blood volumes, the numbers of eggs maturing in mosquitoes that received comparable amounts of blood was not affected (Table 1. Fig. 2B). This suggested that the utilization of the blood is neither dependent upon distention nor occurs more readily when diluted with saline. Although females maintained on 20% sucrose are more likely to develop eggs from blood volumes of less than 0.3 ~1. no relationship exists between the amount of blood at these volumes and the number of eggs that develop (Fig. 1). Even when additional distention was created with saline, the proportion of insects developing eggs increased without a significant increase in the mean number of eggs per female (Table 1). This suggested that at these small blood volumes, the role of the blood meal as a releaser of a factor from the head had been separated
from its role as a source of metabolic precursors. Although release may have been triggered by some component in the blood, endogenous reserves, and not the blood, may have been the major source of yolk proteins. As additional blood was ingested above 0.3 ~1. it began to be incorporated into yolk, allowing more eggs to be produced. Spielman et al. (1971) treating non-blood fed A. aegypti with 20-hydroxyecdysone. demonstrated that there are sufficient endogenous reserves in the mosquito to produce a small batch of eggs. There is some evidence that juvenile hormone may play a role in oijgenesis not only before the blood meal (Lea, 1963; Gwadz and Spielman, 1973). but after it as well (Borovsky, 1981; Kelly et al.. 198 1; Borovsky et al., 1985; Guilvard et al.. 1984). When mosquitoes with their ventral nerve cords transected received 0.1 ~1 of blood simultaneously with 1.9 ~1 of saline, topical application of physiological levels of juvenile hormone III stimulated oogenesis (Table 3). This suggested that exogenous juvenile hormone may be able to substitute for the distention message. It may be that one of the ways that distention modulates vitellogenesis is by the regulation of juvenile hormone synthesis following a blood meal. The possible reasons for the presence of a dual mechanism for hormone release can only be speculated upon. In some hemipterans, larvae rely on a nervous message from the distended abdomen to initiate the moulting cycle (Wigglesworth, 1934) and adult females require an intact ventral nerve cord in order for mating to activate the corpus allatum (Mundall and Engelmann, 1977). In the adult mosquito, aerodynamic considerations may have resulted in a selective pressure to rapidly excrete the excess water in the blood meal. This rapid diuresis (Nijhout and Carrow. 1978) may have necessitated a move away from a sole reliance on prolonged abdominal distention as an indication that precursors for vitellogenesis were present. Acknowledgements--I thank Drs E. Van Handel and A. 0. Lea for their critical reading of the manuscript. This research was supported by grant AI- 19004 from the National Institutes of Health. Contribution No. 86711 from the Idaho Agricultural Experiment Station, REFERENCES Bellamy R. E. and Bracken G. K. (1971) Quantitative aspects of ovarian development in mosquitoes. Can. Ent. 103, 763-773. Borovsky D. (1981) In vitro stimulation of vitellogenesis in Aedes aegypfi with juvenile hormone, juvenile-hormone analogue (ZR515) and 20-hvdroxvecdvsone. J. Insecf . . . Phy.&l. 27, 371-378. Borovsky D.. Thomas B. R., Carlson D. A., Whisenton L. R. and Fuchs M. S. (1985) Juvenile hormone and 20-hydroxyecdysone as primary and secondary stimuli of vitellogenesis in Aedes aegypti. Archs Insect Biochem. Physiol. 2, 75-90. Briegel H. and Lea A. 0. (1975) Relationship between protein and proteolytic activity in the midgut of mosquitoes. J. Insect Physiol. 21, 1597-1604. Brown M. R., Raikhel A. S. and Lea A. 0. (1985) Ultrastructure of midgut endocrine cells in the adult mosquito, Aedes aegypti. Tiss. Cell. 17, 709-721. Chang Y. H. and Judson C. L. (1977) Peptides as stimulators of egg development neurosecretory hormone re-
Abdominal lease in the mosquito Aedes aegypti. Comp. Biochem. Physiol. SIC, 147-l 5 1. Clements A. N. (1956) Hormonal control of ovary development in mosquitoes. J. exp. Biol. 33, 211-223. Ephrussi B. and Beadle G. W. (1936) A technique of transplantation for Drosophila. Am. Nat. 70, 218225. Gillett J. D. (1956) Initiation and promotion of ovarian development in the mosquito Aedes (Stegomyia) uegypti (Linnaeus). Ann. Fop. Med. Purasit. 50, 375-380. Gillett J. D. (1957) Variation in the time of release of the ovarian development hormone in Aedes aegypti. Nafure 180, 656657. Gillett J. D., Cole S. J. and Reeves D. (1975) The influence of the brain hormone on retention of blood in the mid-gut of the mosquito Aedts uegypti (L.). Proc. R. Sot. Lond. B 190, 3599367. Guilvard E.. DeReggi h4. and Rioux J. A. (1984) Changes in ecdysteroid and juvenile hormone titers correlated to the initiation of vitellogenesis in two Aedes species (Diptera, Culicidae). Gen. camp. Endocr. 53, 218223. Gwadz R. W. and A. Spielman (1973) Corpus allatum control of ovarian development in Aedes aegypti. J. Insect PhTsiol. 19, 144-1448. Kelly T. J.. Fuchs M. S. and Kang S. H. (1982) Induction of ovarian development in autogenous Aedes atropalpus by juvenile hormone and 20-hydroxyecdysone. Int. J. Inoerr. Reprod. 3, 101-112. Larsen J. R. and Bodenatein D. (1959) The humoral control of egg maturation in the mosquito. J. exp. Zool. 140, 343-38 I.
distention
87
Lea A. 0. (1963) Some relationships between environment, corpora allata, and egg maturation in aedine mosquitoes. J. § Physiol. 9, 793-809. Lea A. 0. (1967) The medial neurosecretorv cells and eee maturation in ‘mosquitoes. J. Insecf Physiol. 13,419--4F< Lea A. 0. (1972) Regulation of egg maturation in the mosquito by the neurosecretory system: the role of the corpus cardiacum. Gen. camp. Endocr. (Suppl.) 3, 602608. Mundall E. and Engelmann F. (1977) Endocrine control of vitellogenin synthesis in Triufomu protracta. J. Insecf Physiol. 23, 825-836. Nijhout H. F. and Carrow G. M. (1978) Diuresis after a bloodmeal in female Anophelesfieeborni. J. insect Physiol. 24, 293-298. Sokal R. R. and Rohlf F. J. (1969) Biometry, W.H. Freeman. San Francisco. Spielman A. and Wong J. (1974) Dietary factors stimulating oogenesis in Aedes aegypti. Biol. Bull. mar. biol. Lab., Woods Hole 147, 433442. Spielman A., Gwadz R. W. and Anderson W. A. (1971) Ecdysone-initiated ovarian development in mosquitoes. J. Insect Physiol. 17, 1807-1814. Van Handel E. and Lea A. 0. (1984) Vitellogenin synthesis in blood-fed Aedes uegypti in the absence of the head, thorax and ovaries. J. Insect Physiol. 30, 871-875. Wigglesworth V. B. (1934) The physiology of ecdysis in Rhodnius urolixus (Hemiptera). II. Factors controlline moulting and ‘metamorphosis’. Q. J/ Microsc. Si. 77: 191-223.
in Great
Britain.
Copyright
All rights reserved
DISTENTION-MEDIATED THE MOSQUITO, MARC
Department
of Plant.
Soil and Entomological (Received 12 May
J.
0@22-191Oj87 $3.00 + 0.00 1987 Pergamon Journals Ltd
0
EGG MATURATION AEDES AEGYPTI
IN
KLOWDEN
Sciences,
University
of Idaho,
Moscow,
ID 83843, U.S.A.
1986; revised 16 July 1986)
Abstract-Abdominal distention accelerates the release of a factor from the head of blood-fed Aedes aeg.vpti mosquitoes. The critical period during which the head is required for oijgenesis following blood ingestion is approx 6 h with a 5 ~1 meal, but small blood meals of 1~1 require the head to be present for significantly longer. Increasing the abdominal distention by supplementing the 1 ~1 meal with saline results in a critical period similar to that with 5 ~1 of blood. The information from the distended abdomen appears to travel via the ventral nerve cord. Transection of the ventral nerve cord prevents oiigenesis from occurring after small blood meals. but not with larger blood volumes. Topical application of 100 pg of juvenile hormone III can substitute for the distention message. Key Word In&x:
Aedes aegypti, &genesis,
abdominal
INTRODUCTION
distention,
juvenile
hormone
a unifying hypothesis that accounts for previous reports demonstrating either neural or humoral involvement in release of egg-development neurosecretory hormone.
In anautogenous Aedes aegypti mosquitoes, postemergence follicular growth proceeds to the so-called resting stage under the influence of juvenile hormone (Gwadz and Spielman, 1973), but its development advances no further until a blood meal is ingested. The blood not only provides precursors for vitellogenesis but also serves as a trigger for the subsequent endocrine events that are necessary for the production and uptake of vitellogenin. Experiments in which mosquitoes were decapitated at intervals following a blood meal demonstrated that one of these endocrine events included the release of a factor from the head that was essential in order for egg maturation to occur (Clements, 1956; Gillett, 1956). Lea named this factor egg-development neurosecretory hormone. which was produced in the medial neurosecretory cells of the brain and released from the corpus cardiacum in response to blood ingestion (Lea, 1967, 1972). Although a blood meal is necessary for this hormone release, the mechanism by which this release occurs has not yet been determined. Lea (1972) ruled out nervous involvement in its release and demonstrated that the release signal was transported by the haemolymph. This has been confirmed in several studies (Bellamy and Bracken, 1971; Chang and Judson, 1977; Van Handel and Lea, 1984). However, conflicting with this hypothesis of a humoral release of the egg-development neurosecretory hormone are experiments that indicate abdominal distention is involved in the initiation of oiigenesis (Gillett, 1957; Larsen and Bodenstein, 1959; Gillett ef al., 1975; Spielman and Wong, 1974). In this report, further evidence is presented that demonstrates that nol: only is a component of the blood meal necessary in order for a factor stimulating oiigenesis to be released from the head, but that this release is modulated b;! the nervous system as a result of abdominal distention. These data may provide
MATERIALS
AND METHODS
Aedes aegypti (L.) larvae and adults were reared at 27°C under a 14 h light: 10 h dark photoperiodic regime. Adults were maintained at 70% r.h., with 1, 10 or 20% sucrose solution continuously available from cotton wicks. Mosquitoes used in these experiments were 4- to 5-days after emergence. Blood from a laboratory rat, obtained by heart puncture, and saline (Ephrussi and Beadle, 1936) were administered to the midguts of mosquitoes in measured amounts by enema (Briegel and Lea, 1975). In some experiments, the ventral nerve cord was transected anterior to the second abdominal ganglion; in sham-operated controls the arthrodial membrane was opened, but the ventral nerve cord was not disturbed. Decapitation removed the medial neurosecretory cells and corpus cardiacum. All wounds from ventral nerve cord transection and decapitation were sealed with paraffin wax. Juvenile hormone III (Sigma, St Louis, MO.) was dissolved in acetone and topically applied to the abdomens; controls received acetone alone. Oljcytes were considered to be developing if they contained more than 200 pm of yolk at 48 h after the blood meal. A one-way analysis of variance and an arcsine test for the equality of 2 percentages (Sokal and Rohlf, 1969) were used to statistically compare responses. RESULTS
Egg development When
number volume 83
resulting from
small blood meals
scale, the mean of eggs maturing was directly related to the of blood administered if the volumes exexpressed
on a logarithmic
MARC J. KLOWDEN
84
Table
140 D-a
120 0 f!
%
1%
I. Effects of
#%,,,a 10% ‘_ 20%
swrose sucrose
Group
100
1
0
3.0
0 (46)
ND
0. I
0
2 (90)
ND
C
0.1 0.1 0.1
0.9 1.9 3.9
I8 (67) 75 (40) 80 (35)
Il.2 19.8 21.5
0.05 0.05
0 3.0
ow
ND ND
0.1 0.1
I.9 I .9*
71 (61)
ND ND
*Saline containing 8 peg of soybean ND = not determined.
8 (48)
2 (54) trypsin inhibitor.
increases m saline (Table 1C). Eggs produced by these females hatched normally and larvae were successfully reared to the adult stage. When the volume of blood was reduced to 0.05 ~1, none of the mosquitoes
20
0
Mean no. eggs/female
B
E
40
% with eggs (N)
A
80
60
Saline volume (/Ll)
Blood volume (jll)
D 8 f
saline supplements on the ability of small blood meals to initiate oikenesis
SWrosB
1
0.1
I
I
03
l111111
05
Blood
I
1.0
III
3.0
5.0
developed
eggs, and only a small proportion
under-
volume +I)
Fig. 1. The number of eggs developing in mosquitoes maintained on 1, 10 or 20% sucrose as a function of the log,, of the blood volume.
ceeded 0.3 ~1 (Fig. 1). The slopes, representing the rate at which blood was converted into eggs, were not significantly different for mosquitoes maintained on 1% [Y = 56.0 + 86.4 (log,,)X], 10% [Y = 60.0 + 89.7 (log,,,) X] or 20% sucrose [Y = 62.9 + 89.4 (log,,) X]. However, mosquitoes maintained on 20% sucrose that received less than 0.3 ~1 of blood no longer showed a relationship between blood volume and number of eggs; approx 20 eggs matured regardless of the volume administered between 0.1 and 0.3 ~1. Mosquitoes maintained on 1 or 10% sucrose failed to develop eggs at blood volumes below 0.3 ~1 (Fig. 1). Although the number ofeggs that matured was not dependent on the concentration of the sucrose diet, the percentage that matured any eggs at all increased with increasing sucrose concentration. The abdomens of mosquitoes maintained on 20% sucrose were also significantly more distended than those of mosquitoes on either 1 or 10% sucrose (unpublished data). For these reasons, abdominal distention was considered as a factor responsible for the greater tendency of mosquitoes that were maintained on 20% sucrose to develop eggs at small blood volumes. This ability of distention to supplement the blood meal was examined in another series of experiments. If 3 ~1 of saline alone were administered to mosquitoes, none devel-
)-
)-
) -
, -
)-
(B) )-
1
I
I
I
,-
oped eggs (Table 1A) and 0.1 ~1 of blood alone initiated egg maturation in only a small number of
/
,
,
females (Table 1B). However, when the 0.1 11 of blood was administered simultaneously with saline in order to increase abdominal distention, a larger percentage of mosquitoes developed eggs when the volume of saline was increased up to a total volume of 2 ~1. With a total volume of 4 /*I (0.1 ~1 of blood plus 3.9 ~1 of saline), the percentage with eggs was not increased significantly compared to the 2 ~1 total volume. The mean number of eggs in mosquitoes that did reproduce was not significantly increased by these
0.5 25
1.0 2.0
2.0 10
Volume
admlnlstered
‘A; 3.0 0
Blood Salhe
(pl)
Fig. 2. influence of ventral nerve cord on distentionmediated oOgenesis in A. czegypti. A: Percentage of mosquitoes maturing eggs after the administration of 3 /II of blood and saline in varying proportions. B: Number of eggs maturing per female. (Dashed lines: VNC Transected; solid lines: sham-operated controls; vertical lines: standard errors).
Abdominal distention
5ji
bloqd
,
I
85
different from controls (Fig. 2A). The numbers of eggs which did mature in controls and experimentals given comparable blood/saline meals did not differ significantly (Fig. 2B).
I
80
Critical periods and abdominal distention
20
0
Time
of decopitatlon
(h pbm)
Fig. 3. Critical periods for the presence of the head following a 1~1 blood meal (triangles), a 5~1 blood meal (open circles). or a 1 ~1 blood meal supplemented with 4~1 of saline (closed circles). Vertical lines: standard errors. In intact controls, 82 and 89% developed eggs with 1 and 5 ~1 of blood, respectively.
went oijgenesis when 3 ~1 of saline were simultaneously administered [Table 1D). Although abdominal distention was indeed able to influence egg development, it appea.red that a certain component in the blood was also necessary in some minimal quantity. In order to determine whether the blood must be digested in order to release this component, 0.1 p 1 of blood was administered simultaneously with 1.9 ~1 of saline that also contained 8 pg of soybean trypsin inhibitor. Few of the mosquitoes receiving this solution matured eggs (Table IE). Infruence of the ventral nerue cord Transecting the ventral nerve cord of mosquitoes prior to a meal of Cl.1~1 of blood plus 2.9 ~1 of saline abolished the distention-mediated effect on oiigenesis, but the distention signal became progressively less important as the proportion of blood in the total volume of 3 ~1 was increased relative to the saline. With 3 ~1 of blood and no saline, the proportion of nerve cord-transected females that developed eggs was not significantly Table 2. Decapitation
following
Treatment 5 jI I sahne; I ~1 blood 6-7 b later
The interval during which the head was necessary for egg development was determined for blood meals of 1 and 5 ~1. With 5 ~1 of blood, there was a sharp increase in the percentage with eggs when decapitation occurred after 6 h (Fig. 3). In mosquitoes receiving 1 ~1 of blood, however, there was no clearly defined critical period. The percentage with eggs increased slowly with time of decapitation, and by 12 h had finally achieved a level comparable with intact controls. In contrast, when the 1 ~1 of blood was administered simultaneously with 4~1 of saline in order to increase abdominal distention. the critical period was characteristic of mosquitoes receiving 5 ~1 of blood (Fig. 3). To investigate whether abdominal distention alone could release egg-development neurosecretory hormone, an assay similar to that of Chang and Judson (1977) was employed. Mosquitoes were given 5 ~1 of saline, and 67 h later, 1~1 of blood. Immediately following this blood, the insects were decapitated and examined 48 h later. Controls were treated similarly, but not decapitated. It was anticipated that if the saline triggered the hormone’s release, the 1 ~1 of blood, administered after the critical period, would provide precursors for oiigenesis even in the subsequent absence of the head. As shown in Table 2, the blood, introduced after the presumed critical period for distention-induced release of the hormone, failed to support oiigenesis, demonstrating that eggdevelopment neurosecretory hormone was not in circulation at the time of the blood meal and that the previous distention with saline was insufficient to cause the hormone’s release. This confirmed that a component in the blood meal must first be present before abdominal distention can modulate its effects.
enemas of saline and blood
Efects
of juvenile hormone
The possible role of juvenile hormone in distention-mediated oijgenesis was examined using mosquitoes which had their ventral nerve cords transected prior to receiving 0.1 ~1 of blood and 1.9 ~1 of saline. Immediately after the blood-saline enema, 100 pg of juvenile hormone III were topically applied. A small percentage of controls which received acetone alone developed eggs, but significantly more females developed eggs after the juvenile hormone treatment (Table 3).
% with eggs (N) Decapitated immediately after blood meal Non-decapitated controls
Table
3. Topical
treatment
Tr:atment Ventral nerve cord transected; 0.5 PI blood plus 2.5 ~1 saline -
DlSCUSSION Blood ingestion triggers the release of a reproductive factor from the head of A. aegypti females, and this release can be accelerated by abdominal
0 (30) 77 (26)
of ventral nerve cord hormone III
transected
females
with juvenile
% with eggs (N) 100 pg juvenile Acetone
controls
hormone
50 (26) 9(21)
86
MARC J. KLOWDEN
distention. This is evidenced by a change in the critical period for the presence of the head following a blood meal when a small volume of blood is supplemented with saline, compared with the effect of the blood alone (Fig. 3). As previously demonstrated by Spielman and Wong (1974), mosquitoes were more likely to produce eggs from a small blood meal when the midgut distention was increased (Table lC), but without some critical component from the blood meal, this distention alone was insufficient (Tables ID and 2). The data agree with previous studies concluding that release of egg-development neurosecretory hormone is triggered by components resulting from the initial products of blood digestion (Chang and Judson, 1977; Van Handel and Lea, 1984). The information relating the degree of abdominal distention reaches the head via the ventral nerve cord. Transection of the nerve cord anterior to the second abdominal ganglion before a meal of 0.5 ~1 of blood and 2.5 ~1 of saline was introduced all but abolished the effect distention had on oogenesis. As the proportion of the blood in the meal increased, however, the distention message became less important, and was no longer necessary when the blood comprised all of the 3 ~1 that was administered (Fig. 2A). This may explain why Lea (1972) was unable to show any involvement of the nervous system in release of egg-development neurosecretory hormone. Because the mosquitoes in his experiments were fed on blood to repletion (Lea, personal communication), the possible contribution of the nervous system may have been overridden by the large meals that were ingested. Spielman and abdominal distention a mosquitoes in their experiment to take small meals. Midgut endocrine cells are present in adult A. aegypti (Brown et al., 1985), which could respond to midgut distention, but these cells are probably not involved in distention-mediated oogenesis because their activity would not depend upon intact nervous connections to the head. The possibility that abdominal distention could affect the induction of digestive enzymes in the midgut, and thus indirectly reproduction, was considered. However, Briegel and Lea (1975) using blood volumes as low as 0.5 ~1, demonstrated that this induction was the result of a secretogogue stimulus and not abdominal distention. Although an intact ventral nerve cord influenced whether or not eggs developed at small blood volumes, the numbers of eggs maturing in mosquitoes that received comparable amounts of blood was not affected (Table 1. Fig. 2B). This suggested that the utilization of the blood is neither dependent upon distention nor occurs more readily when diluted with saline. Although females maintained on 20% sucrose are more likely to develop eggs from blood volumes of less than 0.3 ~1. no relationship exists between the amount of blood at these volumes and the number of eggs that develop (Fig. 1). Even when additional distention was created with saline, the proportion of insects developing eggs increased without a significant increase in the mean number of eggs per female (Table 1). This suggested that at these small blood volumes, the role of the blood meal as a releaser of a factor from the head had been separated
from its role as a source of metabolic precursors. Although release may have been triggered by some component in the blood, endogenous reserves, and not the blood, may have been the major source of yolk proteins. As additional blood was ingested above 0.3 ~1. it began to be incorporated into yolk, allowing more eggs to be produced. Spielman et al. (1971) treating non-blood fed A. aegypti with 20-hydroxyecdysone. demonstrated that there are sufficient endogenous reserves in the mosquito to produce a small batch of eggs. There is some evidence that juvenile hormone may play a role in oijgenesis not only before the blood meal (Lea, 1963; Gwadz and Spielman, 1973). but after it as well (Borovsky, 1981; Kelly et al.. 198 1; Borovsky et al., 1985; Guilvard et al.. 1984). When mosquitoes with their ventral nerve cords transected received 0.1 ~1 of blood simultaneously with 1.9 ~1 of saline, topical application of physiological levels of juvenile hormone III stimulated oogenesis (Table 3). This suggested that exogenous juvenile hormone may be able to substitute for the distention message. It may be that one of the ways that distention modulates vitellogenesis is by the regulation of juvenile hormone synthesis following a blood meal. The possible reasons for the presence of a dual mechanism for hormone release can only be speculated upon. In some hemipterans, larvae rely on a nervous message from the distended abdomen to initiate the moulting cycle (Wigglesworth, 1934) and adult females require an intact ventral nerve cord in order for mating to activate the corpus allatum (Mundall and Engelmann, 1977). In the adult mosquito, aerodynamic considerations may have resulted in a selective pressure to rapidly excrete the excess water in the blood meal. This rapid diuresis (Nijhout and Carrow. 1978) may have necessitated a move away from a sole reliance on prolonged abdominal distention as an indication that precursors for vitellogenesis were present. Acknowledgements--I thank Drs E. Van Handel and A. 0. Lea for their critical reading of the manuscript. This research was supported by grant AI- 19004 from the National Institutes of Health. Contribution No. 86711 from the Idaho Agricultural Experiment Station, REFERENCES Bellamy R. E. and Bracken G. K. (1971) Quantitative aspects of ovarian development in mosquitoes. Can. Ent. 103, 763-773. Borovsky D. (1981) In vitro stimulation of vitellogenesis in Aedes aegypfi with juvenile hormone, juvenile-hormone analogue (ZR515) and 20-hvdroxvecdvsone. J. Insecf . . . Phy.&l. 27, 371-378. Borovsky D.. Thomas B. R., Carlson D. A., Whisenton L. R. and Fuchs M. S. (1985) Juvenile hormone and 20-hydroxyecdysone as primary and secondary stimuli of vitellogenesis in Aedes aegypti. Archs Insect Biochem. Physiol. 2, 75-90. Briegel H. and Lea A. 0. (1975) Relationship between protein and proteolytic activity in the midgut of mosquitoes. J. Insect Physiol. 21, 1597-1604. Brown M. R., Raikhel A. S. and Lea A. 0. (1985) Ultrastructure of midgut endocrine cells in the adult mosquito, Aedes aegypti. Tiss. Cell. 17, 709-721. Chang Y. H. and Judson C. L. (1977) Peptides as stimulators of egg development neurosecretory hormone re-
Abdominal lease in the mosquito Aedes aegypti. Comp. Biochem. Physiol. SIC, 147-l 5 1. Clements A. N. (1956) Hormonal control of ovary development in mosquitoes. J. exp. Biol. 33, 211-223. Ephrussi B. and Beadle G. W. (1936) A technique of transplantation for Drosophila. Am. Nat. 70, 218225. Gillett J. D. (1956) Initiation and promotion of ovarian development in the mosquito Aedes (Stegomyia) uegypti (Linnaeus). Ann. Fop. Med. Purasit. 50, 375-380. Gillett J. D. (1957) Variation in the time of release of the ovarian development hormone in Aedes aegypti. Nafure 180, 656657. Gillett J. D., Cole S. J. and Reeves D. (1975) The influence of the brain hormone on retention of blood in the mid-gut of the mosquito Aedts uegypti (L.). Proc. R. Sot. Lond. B 190, 3599367. Guilvard E.. DeReggi h4. and Rioux J. A. (1984) Changes in ecdysteroid and juvenile hormone titers correlated to the initiation of vitellogenesis in two Aedes species (Diptera, Culicidae). Gen. camp. Endocr. 53, 218223. Gwadz R. W. and A. Spielman (1973) Corpus allatum control of ovarian development in Aedes aegypti. J. Insect PhTsiol. 19, 144-1448. Kelly T. J.. Fuchs M. S. and Kang S. H. (1982) Induction of ovarian development in autogenous Aedes atropalpus by juvenile hormone and 20-hydroxyecdysone. Int. J. Inoerr. Reprod. 3, 101-112. Larsen J. R. and Bodenatein D. (1959) The humoral control of egg maturation in the mosquito. J. exp. Zool. 140, 343-38 I.
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