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The medial neurosecretory cells and egg maturation in mosquitoes
J. Insect Physiol., 1967, Vol. 13, pp. 419 to 429. Pwgmwn
Press Ltd.
Printed in Great Britain
THE MEDIAL NEUROSECRETORY CELLS AND EGG MATURATION IN MOSQUITOES ARDEN
0. LEA
Florida State Board of Health, Entomological Research Center, Vero Beach, Florida
(Receiked 21 July 1966) Abstract-When the medial neurosecretory cells (mnc) were surgically removed from aedine mosquitoes immediately after emergence, egg maturation was suppressed. In older females mnc-ablation had little effect. Ablation did not interfere with ingestion or absorption of a blood’meal. In blood-fed Aedes tueniorhynchus,arrest of egg development by mnc-ablation was reversed by transplantation of 1 pair of mnc from an anaesthetized donor. In sugar-fed A. tueniorhynchus, 4 pairs of mnc were required to restore autogenous egg maturation. In blood-fed hosts, male or female Aedes or female Culex were equally effective as donors. If the donor was killed, rather than anaesthetized, 4 pairs of mnc were needed to restore egg development. Both the mnc and the corpora allata are essential for egg maturation in mosquitoes. However, transplanted mnc could not restore egg development arrested by allatectomy, and transplanted corpora sllata could not restore egg Consequently, the corpora allata and development arrested by mnc-ablation. the mnc regulate different processes in egg maturation. INTRODUCTION
THOMSEN (1942, 1952) has d emonstrated that both the corpora allata and the medial neurosecretory cells are required for egg maturation in Calliphora erythrocephala. This has been confirmed in Schistocerca spp. by HIGHNAM(1962), HIGHNAMet al. (1963), and STRONG (1965) and in Tenebrio m&or by MQRDUE (1965). In Cdiphora, egg development, which had been arrested by allatectomy, could be restored by transplantation of corpora allata, whereas, after ablation of the .medial neurosecretory cells (mnc), only a few females matured eggs when mnc or corpora cardiaca-allata were implanted (THOMSEN, 1942, 1952). In Schistocerca, implantation of corpora allata after allatectomy, or of brain tissue after mnccauterization, resulted in increased yolk deposition, but not in mature eggs (HIGHNAM, 1962, 1963). In aedine mosquitoes the corpora allata are essential for egg development, and egg maturation, suppressed by allatectomy, can be restored by transplantation of corpora allata (LEA, 1963). The present report demonstrates that in mosquitoes: (1) the medial neurosecretory cells are as essential as the corpora allata, but they regulate different processes in egg development; (2) ablation of the mnc does not interfere with absorption of protein from a blood meal ; and (3) the arrest of egg maturation by mnc-ablation can be completely reversed by implantation of mnc. 419
420
AFCDEN 0. LEA METHODS
Ablation
of medial neurosecretory
cells
Surgical techniques were modelled on those described by THOMSEN (1952) for Calliphoru erythrocephala. A mosquito was anaesthetized with nitrogen and imbedded almost vertically in modelling clay. The head was bent forward and held down by a strip of clay. Once the scales on the occiput had been removed, the entire preparation was submerged in Ringer’s solution (EPHRUSSIand BEADLE, 1936). Two converging incisions were made in the occiput. Starting posteriorly, these incisions joined just behind the compound eyes forming a triangular flap (Fig. 1). Th is fl ap was held closed by the paired dorsal dilator muscles, which have one insertion on the occiput and the other on the dorsal plate of the oesophageal sucking pump. The brain was exposed by pulling back the triangular flap in the occiput and stretching the dorsal dilators. Two groups of neurosecretory cells, 10 or more in each, lie in the pars intercerebralis on either side of the midline and were distinguishable by their bluish appearance (Fig. 1). The mnc were removed with forceps, taking as little surrounding tissue as possible. The two groups of lateral neurosecretory cells remained intact. The flap was then closed and the saline removed. On drying, the cut edges of the occiput sealed themselves. The drying time was reduced to a few seconds by directing a stream of air at the wound. This operation removed the mnc perikarya but left the mnc axons in place. Autopsy and paraldehyde-fuchsin staining of sectioned heads established that all the mnc could be removed by this technique. The controls for mnc-ablation were females which had their head capsule opened briefly under saline. Transplantation
of medial neurosecretory
cells
Donors of mnc to be transplanted were anaesthetized for 30 set with nitrogen. A large piece of the brain was placed in saline, and nearly all the tissue surrounding the mnc was teased away. The mnc were then picked up in a glass needle and injected into the abdomen of a host along with a small quantity of saline. The mnc of the host were ablated at emergence, and transplantation of mnc was made the following day. The day after implantation the host was fed on blood. In one experiment, donors of mnc were killed by exposure to chloroform vapour for 5-10 min. Controls for the transplantation experiments were mnc-ablated hosts implanted with a piece of salivary gland. The technique for ablation and transplantation of the paired corpora allata has been described (LEA, 1963). Triglycerides were determined in individual mosquitoes according to VAN HANDEL (1961). Experimental (2)
animals
The following taeniorhynchus,
species of Aedes were employed: (1) aegypti, Miami North Key Largo strain; (3) triseriatus, Savannah
strain; strain,
Frc;. I, Occiput opened exposing the protocerebrum with the paired groups of bluish medial nt’urosecretorv cells.
MEDIAL NRUROSECRRTORY CELLS AND EGG MATURATION
IN MOSQUITOES
421
originally colonized by HAYES and MORLAN (1957) at Savannah, Georgia;* and (4) sollicitans, eggs from wild females were collected periodically, since this species has not been colonized. The North Key Largo strain of taeniorhynchus is highly autogenous (i.e. matures an initial batch of eggs without a blood meal), but expressivity depends on the larval diet (LEA, 1964); the other species require a blood meal to mature their initial batch of eggs (LER, 1963). All species were reared at 27°C 75 larvae per rearing container. The larval diets for aegypti, triseriatus, and sollicitans were the same as those reported by LEA (1963), except that ground Purina Lab Chow was substituted for the rabbit food or dog food previously specified. The ‘High-larval-diet’ (LEA, 1963) was used for sollicitans. Larvae of taeniorhynchus, used for mnc-ablation, were reared on a minimum quantity of food. The diet consisted of powdered brewer’s yeast (Y), finely ground Purina Lab Chow (LC), and non-nutritive cellulose (C), which was fed on the following schedule: day of hatch, 25 mg Y; second and third day, 25 mg LC, 50 mg C; fourth day, 50 mg LC, 50 mg C; fifth day, 25 mg LC, 50 mg C. To maintain uniformity among the adults, only females of the first day’s emergence, from containers which yielded at least 70 adults from 75 larvae, were used. Since this diet reduced the expression of autogeny, these females were always blood-fed as adults. All taeniorhynchus used as donors were reared on the ‘High-larval-diet’ of LEA (1964) and were fed only on sugar. All females to be used for mnc-ablation were collected within 5 min of emergence. To have maximum biting by females operated at 1 hr after emergence, taeniorhynchus and sollicitans were starved l-2 days before blood-feeding, while aegypti and triseriatus were starved 2-4 days. When ablation was performed l-3 days after emergence, all species were blood-fed 24 hr after the operation. All experimental females were examined 5 days after the blood meal. Ovarian development was recorded as the length of the yolk mass along the long axis of the largest follicle. However, all data are reported here as the number of females which matured eggs ( ~450 ,u of yolk), because, if eggs did not mature, the yolk length generally did not exceed 68 p [the approximate dividing line between Stage II and III of CHRISTOPHERS(191111. RESULTS Ablation of the medial neurosemetmy cells at emergence Suppressed egg maturation. To ascertain whether the medial neurosecretory cells were essential for egg maturation, the mnc perikarya were ablated within 1 hr of emergence. Egg development after a blood meal was suppressed in the four * In biseriatus,
contrast
to the results
allatectomy
strain (4 out of 17 allatectomized controls).
of allatectomy
suppressed
egg
females
reported
maturation matured
by LEA (1963) after
with another
blood-feeding
eggs compared
in the
strain of Savannah
to 23 out of 23 unoperated
422
ARDEN
0. LEA
species of Aedes tested (Table 1). Although these females were unmated, insemination did not alter the results in aegypt& since 20 females, inseminated after mnc-ablation but before blood-feeding, did not deposit more yolk than mncablated virgins. TABLE~-SUPPRESSIONOF EGGMATURATION BY ABLATION OFMEDIAL NEUROSECRETORY CELLS IN Aedes FEDONBLOOD* rnnc-Ablated Species
A. A. A. A.
aegypti taeniorhynchus triseriatus sollicitans
No.
86 140 29 3.5
With mature eggs O(O%) 6 (4%) 12 (41%) 19 (54%)
Sham-operated No.
With mature eggs
82
75 (91%)
46 31 36
42 (91%) 31 (100%) 36 (100%)
* Operated 1 hr after emergence. In all cases, the percentage difference between rnnc-ablated and sham-operated females was >3 times the standard error of the per cent difference. No effect on blood-meal size. In mosquitoes, the quantity of blood ingested in a single meal must exceed a certain minimum before egg development is initiated. The mnc-ablated taeniorhynchus, sollicitans, and triseriatus took at least as much blood as their controls. This was established by weighing before and after bloodfeeding. Only mnc-ablated aegypti frequently took smaller meals than their controls, However, the difference was not enough to account for the suppression of egg development, because the controls still matured eggs even if limited to smaller meals (Z = 143mg) than the mnc-ablated females (5 = 2.6 mg). Furthermore, mnc-ablated aegypti, which were refed 3 days after the first blood meal, did not produce eggs. No efSect on absorption of blood meal. Mosquitoes synthesize a considerable amount of fat from the protein in a blood meal (VANHANDEL, 1965). To determine whether mnc influence digestion or absorption, the mnc were ablated from newly emerged taeniorhynchus, aegypti, and sollicitans. After 2 days’ starvation, mncablated females and unoperated controls were given blood meals of equal weight and, thereafter, maintained on water. Five animals of each treatment were analysed individually for triglycerides 1, 24, 48, and 72 hr after feeding. The triglycerides found at 1 hr represented the amount in the ingested blood and in the mosquito Net synthesis was calculated as the difference at the start of the experiment. between the amount of triglycerides after 1 hr and the amount at each of the other intervals. While only the results at 48 hr are reported in Table 2, all mnc-ablated females synthesized at least as much fat as the controls from blood meals of equal
MEDIALNEUROSRCRRTORY CELLSANDEGGMATURATION IN MOSQUITOES
423
size.* Since the accumulation of triglycerides depended on absorption of protein from the blood meal, ablation of the mnc perikarya does not suppress egg development in mosquitoes by interference with protein digestion. TABLE 2-SYNTI-IESIS OP TRIGLYCERIDESFROM A BLOOD MRAL BY ~XK-ABLATED AND UNOPERATRD MOSQUITOES
Species A. taeniorhynchus A. sollicitans A. aegypti
Treatment
Mean wt. of blood meal (mg)
Net synthesis triglyceridest +g + S.E.)
Unoperated mnc-Ablated Unoperated mnc-Ablated Unoperated mnc-Ablated
3.2 3.3 8.5 8.9 4.5 2.9
52+5 4959 177 z!z21 189 rf:28 109 zk10 76+17
t Five females hilled 48 hr after blood-feeding, analysed individually.
Delayed ablation of medial neurosecretmy cells If the time between emergence and mnc-ablation was not critical, operating would be facilitated by using older females. However, in taeniorhynchus, triseriatus, and sollicitans, the longer ablation was delayed the more the percentage of females which matured eggs approached that of the controls (Fig. 2). In aegypti, removal of the mnc on the seventh day prevented egg maturation as effectively (0 out of 25 matured eggs) as operating at emergence.
FIG. 2. Egg maturation increased as mnc-ablation
was delayed after emergence.
Transplantation of medial newosecretory cells Donor anaesthetixed. (a) Into mnc-ablated host fed on blood. To test the ability of transplanted mnc to resore egg development, taeniorhynchus females had their mnc ablated at emergence, and the following day were implanted with mnc * mnc-Ablation limits the amount of triglycerides synthesized from sugar (VAN HANDEL and LEA, 1965) but not from blood.
ARIXN 0. LEA
424
from another animal. One day after implantation, the hosts were blood-fed. Donors were A. taeniorhynchus, sollicitans, trimiatus, and Culex nigripalpm, anaesthetized briefly with nitrogen. With all donors tested, 1 pair of mnc restored egg development (Table 3) ; in one test Q pair of mnc was effective (Table 5). Restoration depended neither on the sex or genus of the donor, nor on the age or previous diet (sugar or blood) of the donor. TABLE ~---RE~~~RATI~N OF EGG MATURATION BYTRANSPLANTATION OF LIvE MEDIAL NEUROSECRETORY CELLS INTO m?lC-ABLATED HOST FED ON BLOOD Donor
Species
Host *
Sex
Diet and age
With mature
NO.
eggs
A. taeniorhynchus
? 9 6
?
A. sollicitans A. triseriatus
: Culex nigripalpus Piece of salivary gland (control) * A. taeniorhynchus: mnc-ablated anaesthetized donor, blood-fed.
22 23 12 10 13 11 73
Emerged 5 min Sugar, l-8 days Sugar, 5 days Sugar, 7; blood, 1 day Sugar, 1 day Sugar, 1 day
at emergence,
implanted
19 (86%) 19 (83%) 4 (33%) 6 (60%) 11(85%) 8 (73%) 2 (3%)
with 1 pair of mnc from
(b) Into mnc-ablated host fed on sugar. When taeniorhynchus were reared on the ‘High-larval-diet’ and were fed only on sugar as adults, 90 per cent were autogenous (i.e. matured an initial batch of eggs without a blood meal) (LEA, 1964). TABLE
~-RESTORATION OFAUTOGENOUS EGG MATURATION NEUROSECRBTORYCELLS INTO mnC-ABLATED
BY TRANSPLANTATION HOST FED ONSUGAR
OF MEDIAL
Host:: Pairsmnc implanted
Donor?
No.
With mature eggs
4
A. taeniorhynchus’ A. sollicitansb
10 13
9 (90%) 8 (62%)
2 2
A. sollicitansb A. sollicitansc
10 13
3 (30%) 103%)
1 1
A. taeniorhynchus” A. sollicitansb
20 17
2 (10%) 16%)
91
0 (0%)
4
0
t Donor’s diet and age: * sugar, 1 day; ’ sugar, 7; blood, 1 day; ’ sugar, 7 days. f A. taeniorhynchus: reared on ‘High-larval-diet’ (LEA, 1964), mnc-ablated at emergence, implanted with mnc from anaesthetized donor, sugar-fed.
MEDIAL NEUROSRCRETORYCELLS AND EGG MATURATION IN MOSQUITOES
425
Removal of the mnc at emergence prevented autogenous egg maturation in 91 out Therefore, these mnc-ablated of 91 sugar-fed females from the ‘High-larval-diet’. taeniorhynchus could be used as hosts to test the ability of transplanted mnc to restore autogenous egg development. If 4 pairs of mnc from anaesthetized taeniorhynchus or sollicitans donors were implanted, egg development was resumed without a blood meal (Table 4). The number of eggs matured was within the range of autogenous egg production by unoperated sugar-fed taeniorhynchus. Transplantation of 1 or 2 pairs of mnc was ineffective. Thus, autogenous egg maturation could be restored, but implanted mnc were less efficient in sugar-fed than in blood-fed taeniorhynchus. Donor killed. Transplantation of 1 pair of mnc from an anaesthetized donor restored egg development (Table 5), whereas transplantation of 1 pair of mnc from a killed donor (exposed to chloroform for 5-10 min) failed to restore egg maturation in the 80 hosts which were tested with a variety of donors. However, if the number of pairs of mnc from killed donors was increased to 2 to 4 pairs, the mnc-ablated hosts did mature eggs (Table 5). Thus, mnc transplanted from anaesthetized donors were more efficient than mnc from killed donors. TABLE S--RESTORATION OF EGG MATURATION BY TRANSPLANTATION OF DEAD MEDIAL NRUROSECRRTORYCELLS INTO mnC-ABLATED HOST FED ON BLOOD
Pairs of mnc implanted
Donor species diet and age
1
A. taeniorhynchus
Donor killed (with chloroform)
Donor anaesthetized (with nitrogen)
No. of With mature hosts * eggs
No. of With mature hosts * eggs
45
38 (84%)
13
9 (69%)
7 (54%)
10 12
6 (60%) 12 (100%)
40
28 (70%)
34
1(3%)
13 13
1 (8%)
Sugar, O-8 days
B
A. sollicitans Sugar, 7; blood, l-2
A. sollicitans Sugar, l-10
days
* A. taeniorhynchw:
days
1 2
1
30
2 (7%)
2 4
25 12
g (32%) 9 (75%)
mnc-ablated
at emergence, implanted with mnc, blood-fed.
The non-interchangeability of the mnc and corpora allata Egg maturation in Aedes is suppressed by allatectomy but can be restored by transplantation of corpora allata (LEA, 1953). Since mnc-ablation had the same effect, it was possible that the sole function of one of these tissues was to stimulate the production of hormone by the other. If this were true, implantation of extra corpora allata into a mnc-ablated host, or implantation of extra mnc into an allatectomized host, would restore egg development.
ARDEN0. LEA
426
1. Corpora allata fail to replace mnc. mnc-Ablated taeniorhynchus, operated at emergence, were implanted the following day either with 1 pair of corpora allata from a sugar-fed taeniorhynchzls or 2 pairs from blood-fed sollicitans donors. The host was blood-fed 1 day afeer implantation. The transplantation of extra corpora allata into a mnc-ablated host failed to restore egg maturation (Table 6a, d). TABLE
SUBSTITUTRFOR mnc AM) OF INRESTORINGEGGMATURATION
6-INABILITYOFCORPORAALLATATO SUBSTITUTEFOR CORPORAALLATA
mnc TO
Host * Treatment Experiment
Donor? Ablated
a b
A. tatviiorhynchus
C
d
A. sollicitans
Implanted (No. of pairs)
g h
A. taeniorhynchus A. sollicitans
With mature egga
mnc 1 corpora aIIata mnc lmnc Corpora allata 1 corpora allata
28
2 corpora alIata 2mnc Corpora allata 2 corpora alIata
20 8 15
2 (10%) 8 (100%) 12 (80%)
Corpora aIlata 1 mnc Corpora alIata 2 mnc
11 10
0 (0%) 0 (0%)
mnc IlXlC
;
No. 39 18
3 (8%) (83%) 18 (64%)
15
* A. taeniorhynchus : mnc or corpora a.lIataablated at emergence, implanted with corpora allata or mnc from anaesthetizeddonor, blood-fed. t A. taeniorhynchus, sugar, l-4 days; A. sollicitans, sugar, 7; blood, 1 day. The taeraiorhynchus host was appropriate for this test, because it retained the ability to mature eggs after mnc-ablation, provided mnc were implanted prior to blood-feeding (Table 6b, e). The donor’s corpora allata were ‘active’, because they restored egg maturation in allatectomized hosts fed on blood (Table 6c, f). 2. mnc Fail to replace corpora allata. Allatectomized taeniorhynchus, operated at emergence, were implanted the following day either with 1 pair of mnc from sugar-fed taeniorhynchus or 2 pairs from blood-fed sollicitans donors. The host was blood-fed 1 day after implantation. The transplantation of extra mnc into an allatectomized host failed to restore egg maturation (Table 6g, h). Even 4 extra pairs of mnc from the taerziorhynchus donors did not initiate egg development in the 17 hosts tested. The activity of the donor’s mnc and the appropriateness of &is allatectomized host as a test animal have been demonstrated (Table 6b, e and c, f). Since the mnc and corpora allata were not interchangeable, different processes, each essential for egg maturation, must be influenced by the hormones of these tissues.
MEDIAL NEUROSECRETORY
CELLS AND EGG MATURATION
IN MOSQUITOES
427
DISCUSSION
The ablation experiments established that the medial neurosecretory cells are as essential for egg maturation in blood-fed mosquitoes as for autogenous egg maturation in sugar-fed females which derive their yolk protein entirely from protein reserves carried over from the larval stage (Tables 1 and 3). Egg development depends on the secretory function of the mnc rather than on a nervous function, because : (1) if ablation was delayed several days after emergence, females could mature eggs despite the removal of their mnc perikarya (Fig. 2) ; and (2) the mnc perikarya of dead animals, severed from their axons in the brain and implanted into the abdomen of another mosquito, stimulated egg development (Table 5). Therefore, the mnc exert their influence through a hormone which is ,produced by both sexes and is not species specific. In blood-fed mosquitoes, mnc-ablation did not interfere with digestion or absorption (Table Z), nor did the operation reduce the quantity of blood ingested by sollicitans, taeniorhynchus, or triseriatus. THOMSEN and MILLER (1963) have shown that the protease level in the midgut of meat-fed Calliphora was suppressed after mnc ,removal, and STRANG~AYS~DIXON(1961) 0bserved.a reduced intake of protein solution by mnc-ablated blowflies. Thus, the effect of mnc-ablation on protein ingestion and digestion in Calliphora appears to differ from that in mosquitoes. The timing of mtic-ablation was critical, except in aegypti, because delay after emergence resulted in a higher percentage of both sugar-fed (taeniorhynchus) and blood-fed females with mature eggs. At the time of ablation in older animals, hormone must have been released or have been stored in a site unaffected by ablation of the perikarya from the protocerebrum. This probably also accounts for the small percentage of so&itans and triseriatus which matured eggs despite the removal of their mnc 1 hr after emergence. The choice of a host for transplanted mnc depended on a low percentage of egg maturation after mnc-ablation and on the resumption of egg development after mnc implantation. Blood-fed taeniorhymhus, which had been reared on the larval diet described under Methods, was the host which best met these requirements. A. tueniorhynchus reared on the ‘High-larval-diet’ (LEA, 1964) were not used, because, if they were blood-fed after mnc-ablation, 30 per cent matured eggs, or, if they were sugar-fed after mnc-ablation, implantation of 4 pairs of mnc was needed to restore (autogenous) egg development (Table 4). Whereas transplantation of 1 pair of live mnc did restore egg development in a mnc-ablated tueniorhynchus fed on blood, 1 pair of dead mnc had no effect (Table 5). Obviously, the mnc perikarya of one donor did not contain enough hormone to complete egg development (assuming no inactivation of hormone due to, the chloroform treatment). Therefore, it is likely that perikarya from an anaesthetized donor survive and function in the abdomen of the host. Furthermore, where 1 pair of live mnc restored egg maturation in a blood-fed host, 1 pair of live mnc had no effect in a sugar-fed host; ,4 pairs were required (Table 4). Therefore, the bloodfed host itself may stimulate secretion of hormone by live mnc. As a consequence
428
ARDEN0. LEA
of the probable survival and activation of the mnc in the host, the degree of activity of the mnc in the donor prior to transplantation could not be assayed by tests of live mnc. In either sugar-fed autogenous taeniorhynchus or blood-fed Aedes spp., egg development is arrested by allatectomy but can be restored by transplantation of corpora allata (LEA, 1963). Thus, both a hormone from the corpora allata and a hormone from the mnc are required for egg maturation in mosquitoes. In other insects, it has been postulated that the mnc regulate the production of hormone by the corpora allata (for review see HIGHNAM, 1964), and that the corpora allata influence synthesis of hormone by the mnc (LEA and THOMSEN, 1962). Thus, the arrest of egg maturation, by allatectomy for example, might be due to the failure of the corpora allata to stimulate the mnc. Because the effect of both allatectomy and mnc-ablation was reversible in taeniorhynchus, this mosquito could be used to determine whether the corpora allata and the mnc also have independent functions in egg development. Table 6 showed that the corpora allata and the mnc were not interchangeable, and therefore, irrespective of their possible influence on each other, they regulate different processes essential for egg development. Acknowledgements-The assistance of Mr. GORDONEVANSwith adult feeding and ovarian examination is acknowledged with thanks. This work was supported by Public Health Service Research Grant No. AI-06587. REFERENCES CHRISTOPHER~ S. R. (1911) Development of the egg follicle in anophelines. P&d&m 2, 73-88. EPHRUSSIB. and BEADLEG. W. (1936) A technique of transplantation for Drosophila. Am. Nat. 70, 218-225. HAYES R. 0. and MORLAN H. B. (1957) Notes on Aedes triseriutus egg incubation and colonization. Mosquito News 17, 33-36. HIGHNAM K. C. (1962) Neurosecretory control of ovarian development in Schistocercu gregaria. Quart. J. micr. Sci. 103, 57-72. HIGHNAMK. C. (1964) Endocrine relationships in insect reproduction. Insect Reproduction. Symp. R. ent. Sot. Lond. 2, 26-42. HIGHNAMK. C., LUSISO., and HILL L. (1963) The role of the corpora allata during oijcyte growth in the desert locust, Schistocerca gregaria Forsk. .J. Insect Physiol. 9, 587-596. LEA A. 0. (1963) Some relationships between environment, corpora allata, and egg maturation in aedine mosquitoes. J. Insect Physiol. 9, 793-809. LEA A. 0. (1964) Studies on the dietary and endocrine regulation of autogenous reproduction in Aedes taeniorhynchus @Vied.). J. med. Ent. 1, 4044. LEA A. 0. and THOMSENE. (1962) Cycles in the synthetic activity of the medial neurosecretory cells of Calliphora erythrocephala and their regulation. Mem. sot. Endow. 12, 345-347. MORDUE W. (1965) The neuro-endocrine control of oijcyte development in Tenebrio molitor L. J. Insect Physiol. 11, 505-511. STRANGWAYS-DIXON J. (1961) The relationship between nutrition, hormones, and reproduction in the blowfly Calliphora erythrocephala (Meig.)-II. The effect of removing the ovaries, the corpus allatum and the median neurosecretory cells upon selective feeding, and the demonstration of the corpus allatum cycle. 3. exp. Biol. 38, 637-646.
MEDIALNEUROSECRRTORY CELLSAND EGGMATURATION IN MOSQUITOES
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STRONG L. (1965) The relationships between the brain, corpora allata, and oocyte growth in the Central American locust, Schistocercu sp.-I. The cerebral neurosecretory system, the corpora allata, and oijcyte growth. J. Insect Physiol. 11, 135-146. THOMSRNE. (1942) An experimental and anatomical study of the corpus allatum in the blow-fly, Calliphora erythrocephala Meig. V&n&. Me&r dansk naturh. Foren. 106, 320-405. THOMSENE. (1952) Ftmctional significance of the neurosecretory brain cells and the corpus cardiacurn in the female blow-fly, Calliphora erythrocephala Meig. J. exp. Biol. 29, 137-172. THOMSENE. and MP)LLRRI. (1963) Influence of neurosecretory cells and of corpus allatum on intestinal protease activity in the adult Calliphora erythrocephala Meig. J. exp. Biol. 40, 301-321. VAN HANDELE. (1961) Suggested modifications of the micro determination of triglycerides. Clin. Chem. 7, 249-25 1. VAN HANDELE. (1965) The obese mosquito. J. Physiol. 181,478-486. VAN HANDELE. and LRA A. 0. (1965) Medial neurosecretory cells as regulators of glycogen and triglyceride synthesis. Science, N. Y. 149,298-300.
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THE MEDIAL NEUROSECRETORY CELLS AND EGG MATURATION IN MOSQUITOES ARDEN
0. LEA
Florida State Board of Health, Entomological Research Center, Vero Beach, Florida
(Receiked 21 July 1966) Abstract-When the medial neurosecretory cells (mnc) were surgically removed from aedine mosquitoes immediately after emergence, egg maturation was suppressed. In older females mnc-ablation had little effect. Ablation did not interfere with ingestion or absorption of a blood’meal. In blood-fed Aedes tueniorhynchus,arrest of egg development by mnc-ablation was reversed by transplantation of 1 pair of mnc from an anaesthetized donor. In sugar-fed A. tueniorhynchus, 4 pairs of mnc were required to restore autogenous egg maturation. In blood-fed hosts, male or female Aedes or female Culex were equally effective as donors. If the donor was killed, rather than anaesthetized, 4 pairs of mnc were needed to restore egg development. Both the mnc and the corpora allata are essential for egg maturation in mosquitoes. However, transplanted mnc could not restore egg development arrested by allatectomy, and transplanted corpora sllata could not restore egg Consequently, the corpora allata and development arrested by mnc-ablation. the mnc regulate different processes in egg maturation. INTRODUCTION
THOMSEN (1942, 1952) has d emonstrated that both the corpora allata and the medial neurosecretory cells are required for egg maturation in Calliphora erythrocephala. This has been confirmed in Schistocerca spp. by HIGHNAM(1962), HIGHNAMet al. (1963), and STRONG (1965) and in Tenebrio m&or by MQRDUE (1965). In Cdiphora, egg development, which had been arrested by allatectomy, could be restored by transplantation of corpora allata, whereas, after ablation of the .medial neurosecretory cells (mnc), only a few females matured eggs when mnc or corpora cardiaca-allata were implanted (THOMSEN, 1942, 1952). In Schistocerca, implantation of corpora allata after allatectomy, or of brain tissue after mnccauterization, resulted in increased yolk deposition, but not in mature eggs (HIGHNAM, 1962, 1963). In aedine mosquitoes the corpora allata are essential for egg development, and egg maturation, suppressed by allatectomy, can be restored by transplantation of corpora allata (LEA, 1963). The present report demonstrates that in mosquitoes: (1) the medial neurosecretory cells are as essential as the corpora allata, but they regulate different processes in egg development; (2) ablation of the mnc does not interfere with absorption of protein from a blood meal ; and (3) the arrest of egg maturation by mnc-ablation can be completely reversed by implantation of mnc. 419
420
AFCDEN 0. LEA METHODS
Ablation
of medial neurosecretory
cells
Surgical techniques were modelled on those described by THOMSEN (1952) for Calliphoru erythrocephala. A mosquito was anaesthetized with nitrogen and imbedded almost vertically in modelling clay. The head was bent forward and held down by a strip of clay. Once the scales on the occiput had been removed, the entire preparation was submerged in Ringer’s solution (EPHRUSSIand BEADLE, 1936). Two converging incisions were made in the occiput. Starting posteriorly, these incisions joined just behind the compound eyes forming a triangular flap (Fig. 1). Th is fl ap was held closed by the paired dorsal dilator muscles, which have one insertion on the occiput and the other on the dorsal plate of the oesophageal sucking pump. The brain was exposed by pulling back the triangular flap in the occiput and stretching the dorsal dilators. Two groups of neurosecretory cells, 10 or more in each, lie in the pars intercerebralis on either side of the midline and were distinguishable by their bluish appearance (Fig. 1). The mnc were removed with forceps, taking as little surrounding tissue as possible. The two groups of lateral neurosecretory cells remained intact. The flap was then closed and the saline removed. On drying, the cut edges of the occiput sealed themselves. The drying time was reduced to a few seconds by directing a stream of air at the wound. This operation removed the mnc perikarya but left the mnc axons in place. Autopsy and paraldehyde-fuchsin staining of sectioned heads established that all the mnc could be removed by this technique. The controls for mnc-ablation were females which had their head capsule opened briefly under saline. Transplantation
of medial neurosecretory
cells
Donors of mnc to be transplanted were anaesthetized for 30 set with nitrogen. A large piece of the brain was placed in saline, and nearly all the tissue surrounding the mnc was teased away. The mnc were then picked up in a glass needle and injected into the abdomen of a host along with a small quantity of saline. The mnc of the host were ablated at emergence, and transplantation of mnc was made the following day. The day after implantation the host was fed on blood. In one experiment, donors of mnc were killed by exposure to chloroform vapour for 5-10 min. Controls for the transplantation experiments were mnc-ablated hosts implanted with a piece of salivary gland. The technique for ablation and transplantation of the paired corpora allata has been described (LEA, 1963). Triglycerides were determined in individual mosquitoes according to VAN HANDEL (1961). Experimental (2)
animals
The following taeniorhynchus,
species of Aedes were employed: (1) aegypti, Miami North Key Largo strain; (3) triseriatus, Savannah
strain; strain,
Frc;. I, Occiput opened exposing the protocerebrum with the paired groups of bluish medial nt’urosecretorv cells.
MEDIAL NRUROSECRRTORY CELLS AND EGG MATURATION
IN MOSQUITOES
421
originally colonized by HAYES and MORLAN (1957) at Savannah, Georgia;* and (4) sollicitans, eggs from wild females were collected periodically, since this species has not been colonized. The North Key Largo strain of taeniorhynchus is highly autogenous (i.e. matures an initial batch of eggs without a blood meal), but expressivity depends on the larval diet (LEA, 1964); the other species require a blood meal to mature their initial batch of eggs (LER, 1963). All species were reared at 27°C 75 larvae per rearing container. The larval diets for aegypti, triseriatus, and sollicitans were the same as those reported by LEA (1963), except that ground Purina Lab Chow was substituted for the rabbit food or dog food previously specified. The ‘High-larval-diet’ (LEA, 1963) was used for sollicitans. Larvae of taeniorhynchus, used for mnc-ablation, were reared on a minimum quantity of food. The diet consisted of powdered brewer’s yeast (Y), finely ground Purina Lab Chow (LC), and non-nutritive cellulose (C), which was fed on the following schedule: day of hatch, 25 mg Y; second and third day, 25 mg LC, 50 mg C; fourth day, 50 mg LC, 50 mg C; fifth day, 25 mg LC, 50 mg C. To maintain uniformity among the adults, only females of the first day’s emergence, from containers which yielded at least 70 adults from 75 larvae, were used. Since this diet reduced the expression of autogeny, these females were always blood-fed as adults. All taeniorhynchus used as donors were reared on the ‘High-larval-diet’ of LEA (1964) and were fed only on sugar. All females to be used for mnc-ablation were collected within 5 min of emergence. To have maximum biting by females operated at 1 hr after emergence, taeniorhynchus and sollicitans were starved l-2 days before blood-feeding, while aegypti and triseriatus were starved 2-4 days. When ablation was performed l-3 days after emergence, all species were blood-fed 24 hr after the operation. All experimental females were examined 5 days after the blood meal. Ovarian development was recorded as the length of the yolk mass along the long axis of the largest follicle. However, all data are reported here as the number of females which matured eggs ( ~450 ,u of yolk), because, if eggs did not mature, the yolk length generally did not exceed 68 p [the approximate dividing line between Stage II and III of CHRISTOPHERS(191111. RESULTS Ablation of the medial neurosemetmy cells at emergence Suppressed egg maturation. To ascertain whether the medial neurosecretory cells were essential for egg maturation, the mnc perikarya were ablated within 1 hr of emergence. Egg development after a blood meal was suppressed in the four * In biseriatus,
contrast
to the results
allatectomy
strain (4 out of 17 allatectomized controls).
of allatectomy
suppressed
egg
females
reported
maturation matured
by LEA (1963) after
with another
blood-feeding
eggs compared
in the
strain of Savannah
to 23 out of 23 unoperated
422
ARDEN
0. LEA
species of Aedes tested (Table 1). Although these females were unmated, insemination did not alter the results in aegypt& since 20 females, inseminated after mnc-ablation but before blood-feeding, did not deposit more yolk than mncablated virgins. TABLE~-SUPPRESSIONOF EGGMATURATION BY ABLATION OFMEDIAL NEUROSECRETORY CELLS IN Aedes FEDONBLOOD* rnnc-Ablated Species
A. A. A. A.
aegypti taeniorhynchus triseriatus sollicitans
No.
86 140 29 3.5
With mature eggs O(O%) 6 (4%) 12 (41%) 19 (54%)
Sham-operated No.
With mature eggs
82
75 (91%)
46 31 36
42 (91%) 31 (100%) 36 (100%)
* Operated 1 hr after emergence. In all cases, the percentage difference between rnnc-ablated and sham-operated females was >3 times the standard error of the per cent difference. No effect on blood-meal size. In mosquitoes, the quantity of blood ingested in a single meal must exceed a certain minimum before egg development is initiated. The mnc-ablated taeniorhynchus, sollicitans, and triseriatus took at least as much blood as their controls. This was established by weighing before and after bloodfeeding. Only mnc-ablated aegypti frequently took smaller meals than their controls, However, the difference was not enough to account for the suppression of egg development, because the controls still matured eggs even if limited to smaller meals (Z = 143mg) than the mnc-ablated females (5 = 2.6 mg). Furthermore, mnc-ablated aegypti, which were refed 3 days after the first blood meal, did not produce eggs. No efSect on absorption of blood meal. Mosquitoes synthesize a considerable amount of fat from the protein in a blood meal (VANHANDEL, 1965). To determine whether mnc influence digestion or absorption, the mnc were ablated from newly emerged taeniorhynchus, aegypti, and sollicitans. After 2 days’ starvation, mncablated females and unoperated controls were given blood meals of equal weight and, thereafter, maintained on water. Five animals of each treatment were analysed individually for triglycerides 1, 24, 48, and 72 hr after feeding. The triglycerides found at 1 hr represented the amount in the ingested blood and in the mosquito Net synthesis was calculated as the difference at the start of the experiment. between the amount of triglycerides after 1 hr and the amount at each of the other intervals. While only the results at 48 hr are reported in Table 2, all mnc-ablated females synthesized at least as much fat as the controls from blood meals of equal
MEDIALNEUROSRCRRTORY CELLSANDEGGMATURATION IN MOSQUITOES
423
size.* Since the accumulation of triglycerides depended on absorption of protein from the blood meal, ablation of the mnc perikarya does not suppress egg development in mosquitoes by interference with protein digestion. TABLE 2-SYNTI-IESIS OP TRIGLYCERIDESFROM A BLOOD MRAL BY ~XK-ABLATED AND UNOPERATRD MOSQUITOES
Species A. taeniorhynchus A. sollicitans A. aegypti
Treatment
Mean wt. of blood meal (mg)
Net synthesis triglyceridest +g + S.E.)
Unoperated mnc-Ablated Unoperated mnc-Ablated Unoperated mnc-Ablated
3.2 3.3 8.5 8.9 4.5 2.9
52+5 4959 177 z!z21 189 rf:28 109 zk10 76+17
t Five females hilled 48 hr after blood-feeding, analysed individually.
Delayed ablation of medial neurosecretmy cells If the time between emergence and mnc-ablation was not critical, operating would be facilitated by using older females. However, in taeniorhynchus, triseriatus, and sollicitans, the longer ablation was delayed the more the percentage of females which matured eggs approached that of the controls (Fig. 2). In aegypti, removal of the mnc on the seventh day prevented egg maturation as effectively (0 out of 25 matured eggs) as operating at emergence.
FIG. 2. Egg maturation increased as mnc-ablation
was delayed after emergence.
Transplantation of medial newosecretory cells Donor anaesthetixed. (a) Into mnc-ablated host fed on blood. To test the ability of transplanted mnc to resore egg development, taeniorhynchus females had their mnc ablated at emergence, and the following day were implanted with mnc * mnc-Ablation limits the amount of triglycerides synthesized from sugar (VAN HANDEL and LEA, 1965) but not from blood.
ARIXN 0. LEA
424
from another animal. One day after implantation, the hosts were blood-fed. Donors were A. taeniorhynchus, sollicitans, trimiatus, and Culex nigripalpm, anaesthetized briefly with nitrogen. With all donors tested, 1 pair of mnc restored egg development (Table 3) ; in one test Q pair of mnc was effective (Table 5). Restoration depended neither on the sex or genus of the donor, nor on the age or previous diet (sugar or blood) of the donor. TABLE ~---RE~~~RATI~N OF EGG MATURATION BYTRANSPLANTATION OF LIvE MEDIAL NEUROSECRETORY CELLS INTO m?lC-ABLATED HOST FED ON BLOOD Donor
Species
Host *
Sex
Diet and age
With mature
NO.
eggs
A. taeniorhynchus
? 9 6
?
A. sollicitans A. triseriatus
: Culex nigripalpus Piece of salivary gland (control) * A. taeniorhynchus: mnc-ablated anaesthetized donor, blood-fed.
22 23 12 10 13 11 73
Emerged 5 min Sugar, l-8 days Sugar, 5 days Sugar, 7; blood, 1 day Sugar, 1 day Sugar, 1 day
at emergence,
implanted
19 (86%) 19 (83%) 4 (33%) 6 (60%) 11(85%) 8 (73%) 2 (3%)
with 1 pair of mnc from
(b) Into mnc-ablated host fed on sugar. When taeniorhynchus were reared on the ‘High-larval-diet’ and were fed only on sugar as adults, 90 per cent were autogenous (i.e. matured an initial batch of eggs without a blood meal) (LEA, 1964). TABLE
~-RESTORATION OFAUTOGENOUS EGG MATURATION NEUROSECRBTORYCELLS INTO mnC-ABLATED
BY TRANSPLANTATION HOST FED ONSUGAR
OF MEDIAL
Host:: Pairsmnc implanted
Donor?
No.
With mature eggs
4
A. taeniorhynchus’ A. sollicitansb
10 13
9 (90%) 8 (62%)
2 2
A. sollicitansb A. sollicitansc
10 13
3 (30%) 103%)
1 1
A. taeniorhynchus” A. sollicitansb
20 17
2 (10%) 16%)
91
0 (0%)
4
0
t Donor’s diet and age: * sugar, 1 day; ’ sugar, 7; blood, 1 day; ’ sugar, 7 days. f A. taeniorhynchus: reared on ‘High-larval-diet’ (LEA, 1964), mnc-ablated at emergence, implanted with mnc from anaesthetized donor, sugar-fed.
MEDIAL NEUROSRCRETORYCELLS AND EGG MATURATION IN MOSQUITOES
425
Removal of the mnc at emergence prevented autogenous egg maturation in 91 out Therefore, these mnc-ablated of 91 sugar-fed females from the ‘High-larval-diet’. taeniorhynchus could be used as hosts to test the ability of transplanted mnc to restore autogenous egg development. If 4 pairs of mnc from anaesthetized taeniorhynchus or sollicitans donors were implanted, egg development was resumed without a blood meal (Table 4). The number of eggs matured was within the range of autogenous egg production by unoperated sugar-fed taeniorhynchus. Transplantation of 1 or 2 pairs of mnc was ineffective. Thus, autogenous egg maturation could be restored, but implanted mnc were less efficient in sugar-fed than in blood-fed taeniorhynchus. Donor killed. Transplantation of 1 pair of mnc from an anaesthetized donor restored egg development (Table 5), whereas transplantation of 1 pair of mnc from a killed donor (exposed to chloroform for 5-10 min) failed to restore egg maturation in the 80 hosts which were tested with a variety of donors. However, if the number of pairs of mnc from killed donors was increased to 2 to 4 pairs, the mnc-ablated hosts did mature eggs (Table 5). Thus, mnc transplanted from anaesthetized donors were more efficient than mnc from killed donors. TABLE S--RESTORATION OF EGG MATURATION BY TRANSPLANTATION OF DEAD MEDIAL NRUROSECRRTORYCELLS INTO mnC-ABLATED HOST FED ON BLOOD
Pairs of mnc implanted
Donor species diet and age
1
A. taeniorhynchus
Donor killed (with chloroform)
Donor anaesthetized (with nitrogen)
No. of With mature hosts * eggs
No. of With mature hosts * eggs
45
38 (84%)
13
9 (69%)
7 (54%)
10 12
6 (60%) 12 (100%)
40
28 (70%)
34
1(3%)
13 13
1 (8%)
Sugar, O-8 days
B
A. sollicitans Sugar, 7; blood, l-2
A. sollicitans Sugar, l-10
days
* A. taeniorhynchw:
days
1 2
1
30
2 (7%)
2 4
25 12
g (32%) 9 (75%)
mnc-ablated
at emergence, implanted with mnc, blood-fed.
The non-interchangeability of the mnc and corpora allata Egg maturation in Aedes is suppressed by allatectomy but can be restored by transplantation of corpora allata (LEA, 1953). Since mnc-ablation had the same effect, it was possible that the sole function of one of these tissues was to stimulate the production of hormone by the other. If this were true, implantation of extra corpora allata into a mnc-ablated host, or implantation of extra mnc into an allatectomized host, would restore egg development.
ARDEN0. LEA
426
1. Corpora allata fail to replace mnc. mnc-Ablated taeniorhynchus, operated at emergence, were implanted the following day either with 1 pair of corpora allata from a sugar-fed taeniorhynchzls or 2 pairs from blood-fed sollicitans donors. The host was blood-fed 1 day afeer implantation. The transplantation of extra corpora allata into a mnc-ablated host failed to restore egg maturation (Table 6a, d). TABLE
SUBSTITUTRFOR mnc AM) OF INRESTORINGEGGMATURATION
6-INABILITYOFCORPORAALLATATO SUBSTITUTEFOR CORPORAALLATA
mnc TO
Host * Treatment Experiment
Donor? Ablated
a b
A. tatviiorhynchus
C
d
A. sollicitans
Implanted (No. of pairs)
g h
A. taeniorhynchus A. sollicitans
With mature egga
mnc 1 corpora aIIata mnc lmnc Corpora allata 1 corpora allata
28
2 corpora alIata 2mnc Corpora allata 2 corpora alIata
20 8 15
2 (10%) 8 (100%) 12 (80%)
Corpora aIlata 1 mnc Corpora alIata 2 mnc
11 10
0 (0%) 0 (0%)
mnc IlXlC
;
No. 39 18
3 (8%) (83%) 18 (64%)
15
* A. taeniorhynchus : mnc or corpora a.lIataablated at emergence, implanted with corpora allata or mnc from anaesthetizeddonor, blood-fed. t A. taeniorhynchus, sugar, l-4 days; A. sollicitans, sugar, 7; blood, 1 day. The taeraiorhynchus host was appropriate for this test, because it retained the ability to mature eggs after mnc-ablation, provided mnc were implanted prior to blood-feeding (Table 6b, e). The donor’s corpora allata were ‘active’, because they restored egg maturation in allatectomized hosts fed on blood (Table 6c, f). 2. mnc Fail to replace corpora allata. Allatectomized taeniorhynchus, operated at emergence, were implanted the following day either with 1 pair of mnc from sugar-fed taeniorhynchus or 2 pairs from blood-fed sollicitans donors. The host was blood-fed 1 day after implantation. The transplantation of extra mnc into an allatectomized host failed to restore egg maturation (Table 6g, h). Even 4 extra pairs of mnc from the taerziorhynchus donors did not initiate egg development in the 17 hosts tested. The activity of the donor’s mnc and the appropriateness of &is allatectomized host as a test animal have been demonstrated (Table 6b, e and c, f). Since the mnc and corpora allata were not interchangeable, different processes, each essential for egg maturation, must be influenced by the hormones of these tissues.
MEDIAL NEUROSECRETORY
CELLS AND EGG MATURATION
IN MOSQUITOES
427
DISCUSSION
The ablation experiments established that the medial neurosecretory cells are as essential for egg maturation in blood-fed mosquitoes as for autogenous egg maturation in sugar-fed females which derive their yolk protein entirely from protein reserves carried over from the larval stage (Tables 1 and 3). Egg development depends on the secretory function of the mnc rather than on a nervous function, because : (1) if ablation was delayed several days after emergence, females could mature eggs despite the removal of their mnc perikarya (Fig. 2) ; and (2) the mnc perikarya of dead animals, severed from their axons in the brain and implanted into the abdomen of another mosquito, stimulated egg development (Table 5). Therefore, the mnc exert their influence through a hormone which is ,produced by both sexes and is not species specific. In blood-fed mosquitoes, mnc-ablation did not interfere with digestion or absorption (Table Z), nor did the operation reduce the quantity of blood ingested by sollicitans, taeniorhynchus, or triseriatus. THOMSEN and MILLER (1963) have shown that the protease level in the midgut of meat-fed Calliphora was suppressed after mnc ,removal, and STRANG~AYS~DIXON(1961) 0bserved.a reduced intake of protein solution by mnc-ablated blowflies. Thus, the effect of mnc-ablation on protein ingestion and digestion in Calliphora appears to differ from that in mosquitoes. The timing of mtic-ablation was critical, except in aegypti, because delay after emergence resulted in a higher percentage of both sugar-fed (taeniorhynchus) and blood-fed females with mature eggs. At the time of ablation in older animals, hormone must have been released or have been stored in a site unaffected by ablation of the perikarya from the protocerebrum. This probably also accounts for the small percentage of so&itans and triseriatus which matured eggs despite the removal of their mnc 1 hr after emergence. The choice of a host for transplanted mnc depended on a low percentage of egg maturation after mnc-ablation and on the resumption of egg development after mnc implantation. Blood-fed taeniorhymhus, which had been reared on the larval diet described under Methods, was the host which best met these requirements. A. tueniorhynchus reared on the ‘High-larval-diet’ (LEA, 1964) were not used, because, if they were blood-fed after mnc-ablation, 30 per cent matured eggs, or, if they were sugar-fed after mnc-ablation, implantation of 4 pairs of mnc was needed to restore (autogenous) egg development (Table 4). Whereas transplantation of 1 pair of live mnc did restore egg development in a mnc-ablated tueniorhynchus fed on blood, 1 pair of dead mnc had no effect (Table 5). Obviously, the mnc perikarya of one donor did not contain enough hormone to complete egg development (assuming no inactivation of hormone due to, the chloroform treatment). Therefore, it is likely that perikarya from an anaesthetized donor survive and function in the abdomen of the host. Furthermore, where 1 pair of live mnc restored egg maturation in a blood-fed host, 1 pair of live mnc had no effect in a sugar-fed host; ,4 pairs were required (Table 4). Therefore, the bloodfed host itself may stimulate secretion of hormone by live mnc. As a consequence
428
ARDEN0. LEA
of the probable survival and activation of the mnc in the host, the degree of activity of the mnc in the donor prior to transplantation could not be assayed by tests of live mnc. In either sugar-fed autogenous taeniorhynchus or blood-fed Aedes spp., egg development is arrested by allatectomy but can be restored by transplantation of corpora allata (LEA, 1963). Thus, both a hormone from the corpora allata and a hormone from the mnc are required for egg maturation in mosquitoes. In other insects, it has been postulated that the mnc regulate the production of hormone by the corpora allata (for review see HIGHNAM, 1964), and that the corpora allata influence synthesis of hormone by the mnc (LEA and THOMSEN, 1962). Thus, the arrest of egg maturation, by allatectomy for example, might be due to the failure of the corpora allata to stimulate the mnc. Because the effect of both allatectomy and mnc-ablation was reversible in taeniorhynchus, this mosquito could be used to determine whether the corpora allata and the mnc also have independent functions in egg development. Table 6 showed that the corpora allata and the mnc were not interchangeable, and therefore, irrespective of their possible influence on each other, they regulate different processes essential for egg development. Acknowledgements-The assistance of Mr. GORDONEVANSwith adult feeding and ovarian examination is acknowledged with thanks. This work was supported by Public Health Service Research Grant No. AI-06587. REFERENCES CHRISTOPHER~ S. R. (1911) Development of the egg follicle in anophelines. P&d&m 2, 73-88. EPHRUSSIB. and BEADLEG. W. (1936) A technique of transplantation for Drosophila. Am. Nat. 70, 218-225. HAYES R. 0. and MORLAN H. B. (1957) Notes on Aedes triseriutus egg incubation and colonization. Mosquito News 17, 33-36. HIGHNAM K. C. (1962) Neurosecretory control of ovarian development in Schistocercu gregaria. Quart. J. micr. Sci. 103, 57-72. HIGHNAMK. C. (1964) Endocrine relationships in insect reproduction. Insect Reproduction. Symp. R. ent. Sot. Lond. 2, 26-42. HIGHNAMK. C., LUSISO., and HILL L. (1963) The role of the corpora allata during oijcyte growth in the desert locust, Schistocerca gregaria Forsk. .J. Insect Physiol. 9, 587-596. LEA A. 0. (1963) Some relationships between environment, corpora allata, and egg maturation in aedine mosquitoes. J. Insect Physiol. 9, 793-809. LEA A. 0. (1964) Studies on the dietary and endocrine regulation of autogenous reproduction in Aedes taeniorhynchus @Vied.). J. med. Ent. 1, 4044. LEA A. 0. and THOMSENE. (1962) Cycles in the synthetic activity of the medial neurosecretory cells of Calliphora erythrocephala and their regulation. Mem. sot. Endow. 12, 345-347. MORDUE W. (1965) The neuro-endocrine control of oijcyte development in Tenebrio molitor L. J. Insect Physiol. 11, 505-511. STRANGWAYS-DIXON J. (1961) The relationship between nutrition, hormones, and reproduction in the blowfly Calliphora erythrocephala (Meig.)-II. The effect of removing the ovaries, the corpus allatum and the median neurosecretory cells upon selective feeding, and the demonstration of the corpus allatum cycle. 3. exp. Biol. 38, 637-646.
MEDIALNEUROSECRRTORY CELLSAND EGGMATURATION IN MOSQUITOES
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STRONG L. (1965) The relationships between the brain, corpora allata, and oocyte growth in the Central American locust, Schistocercu sp.-I. The cerebral neurosecretory system, the corpora allata, and oijcyte growth. J. Insect Physiol. 11, 135-146. THOMSRNE. (1942) An experimental and anatomical study of the corpus allatum in the blow-fly, Calliphora erythrocephala Meig. V&n&. Me&r dansk naturh. Foren. 106, 320-405. THOMSENE. (1952) Ftmctional significance of the neurosecretory brain cells and the corpus cardiacurn in the female blow-fly, Calliphora erythrocephala Meig. J. exp. Biol. 29, 137-172. THOMSENE. and MP)LLRRI. (1963) Influence of neurosecretory cells and of corpus allatum on intestinal protease activity in the adult Calliphora erythrocephala Meig. J. exp. Biol. 40, 301-321. VAN HANDELE. (1961) Suggested modifications of the micro determination of triglycerides. Clin. Chem. 7, 249-25 1. VAN HANDELE. (1965) The obese mosquito. J. Physiol. 181,478-486. VAN HANDELE. and LRA A. 0. (1965) Medial neurosecretory cells as regulators of glycogen and triglyceride synthesis. Science, N. Y. 149,298-300.