Endocrine control of vitellogenin synthesis and vitellogenesis in Triatoma protracta

J. Insect Physiol.,

I’W,

Vol. 23, pp. 825 to 836. Pergamon

ENDOCRINE CONTROL AND VITELLOGENESIS

Press. Printed in Great Britain.

OF VITELLOGENIN SYNTHESIS IN TRIATOMA PROTRACTA

ERIC MUNDALL and FRANZ ENGELMANN

Department of Biology. University of California. Los Angeles. California 90014, U.S.A. (Received

24 January

1977)

Abstract-The corpus allatum (CA) is required for vitellogenesis in the blood-sucking reduviid, Triuron2u protructa, a:; seen by the total lack of yolk deposition in allatectomized females. Normally the CA

becomes aciive within a day after emergence. After a period of activity during which unfed virgins may mature a few eggs. the CA is inhibited via its neural connectives from the brain. The CA is activated by mating, while a blood meal provides an additional stimulus for vitellogenesis. If the ventral nerve cord (VNC) is severed within 48 hr after copulation, the mating stimulus does not get through. However. the pathway of the feeding stimulus does not involve the VNC. The brain does not have any allatotropic or gonadotropic function in this species. A female-:;pecific protein (vitellogenin) was identified by immunoelectrophoresis in the haemolymph of egg-maturing females. This protein is taken up by the oocytes immunologically unaltered and forms the bulk of the yolk. Allatectomy at emergence prevents the appearance of the vitellogenin, and the topical application of JH,,, to allatectomized females led to its synthesis de noco. as shown by the incorporation of labelled precursors into vitellogenin. From its mobility in polyacrylamide gels of different concentrations. the molecular weight of the yolk protein is estimated to be 4.37 x 10’ daltons.

WYATT, 1971). The physico-chemical characteristics of vitellogenin have likewise been investigated in only SINCE the original studies of WIGGLESWORTH(1936) a few cases (e.g. DEJMAL and BRCMXES. 1968, 1972; the corpus allatum (CA) has been found to be inBELL. 1970; PAN and WALLACE,1974; ENGELMANN volved in the control of vitellogenesis in nearly every and FRIEI)EL, 1974; ENGELMANNet ul., 1976). insect investigated (cf. ENGELMANN, 1970). However, In this study, Trinroma protructa. a bloodsucking whereas Wigglesworth had reported that the CA reduviid closely related to Rhadtzius, is used further was essential for yolk deposition in Rhocinius prolixus, to elucidate the details of the hormonal control of it was later claimec that removal of the CA did not vitellogenesis. The use of a species that feeds discontotally eliminate the deposition of yolk (DAVEY, 1967). tinuously, as does Triatonza, offers distinct advantages In those species studied in detail the brain appears over those species that require food more or less conultimately to regulate the activity of the corpus allatinuously in order to remain reproductively active. turn in the adult female, i.e.. the neurosecretory cells First, certain surgical procedures can be employed (NSC) of the pars intercerebralis seem to play an allawhich are not possible in species of the latter types totropic function in some species, whereas in others due to interference with adequate feeding, and the CA is restrained neuronally (cf. ENGELMANN, secondly. because the blood meal triggers a hormonal 1970; DE WILDE and DE LOOF, 1973). The brain reportresponse leading to vitellogenesis. the timing of exedly exerts both stirnulatory and inhibitory influences periments is facilitated. While much is known about over the activity of the CA in several hemipterans the role of the CA in yolk deposition in Rhodnius, (JOHANSSON,1958; BAEHR, 1973; BAEHR ef a/., 1973; its requirements for vitellogenesis in other reduviids FRIEDEL. 1974; HODKOVA, 1975). Thus in different speneeds to be examined. Also. whereas the ultimate concies a variety of control mechanisms may exist. trol of the CA by the brain has come under scrutiny Although in the cor!trol of the CA of adult Rhodnius (BAMR. 1973; BAEHR et al., 1973), the effects and females no r61e by the brain was apparent from the pathways of the feeding and/or mating stimuli have investigations of WIGGLESWORTH(1936, 1963) BA~.HR not been examined in detail. Furthermore, the nature and coworkers (BAEHR. 1973 ; BAEHR rt ul., 1973) have of the yolk precursor and control of its synthesis are reported that the NSC of the brain are required for investigated here. at least one hour l’ollowing feeding for the CA to become active. Synthesis of the yolk protein precursor, vitelloMATERIALS AND METHODS genin, appears to be controlled by the corpus allatum Esperimrtitc~l rlninlals hormone (JH) in most species studied, although to The insects used in this study were maintained date this is well documented for only a relatively under a 12: 12 hr light-dark cycle at a temperature small number of nsects (cf: ENGULMANN.1970; 825 IR TRODUCTION

826

ERIC MCNIIALLAND

that ranged from 21“C during the dark phase to 28°C in the light. The relative humidity was kept at approximately 70”;. Colonies were fed twice weekly on rats. Experimental animals were removed from the stock colonies on the day of emergence and kept in individual glass beakers.

For allatectomy. a triangular window was cut in the dorsum of the neck, exposing the retrocerebral complex (RCC), consisting of the corpus cardiacum (CC) and the corpus allaturn. For most of these operations. due to the close proximity of the CA to the CC, a portion of the latter tissue was removed along with the CA. In a number the CA alone was successfully extirpated. Before replacing the piece of cuticle over the opening, a few crystals of streptomycin were placed inside the animal. Sham operations were performed in the same manner, but a piece of fat body was taken out instead of the CA. Brain removal was done either by ligation. during the first few hours after emergence, or by decapitation in older females. using a scalpel made from a piece of razor blade. In both the RCC was left within the animal. The wound was sealed with melted paraffin. For the detachment of the CA from the brain. the RCC was exposed as for allatectomy and the corpus cardiacurn nerves severed with fine forceps. Sham operations were performed in the same manner as those for allatectomy. Severance of the ventral nerve cord (VNC) was done between the prothoracic and suboesophageal ganglia with a fine wire hook after first cutting through the soft cuticle between the neck and thorax. No paraffin was required to seal the wound. For sham operations. a piece of trachea was removed and the VNC merely touched. Ovariectomy was carried out on females approximately 6 hr after emergence when the cuticle was still transparent enough for the ovaries to be seen through the ventral abdominal surface. After making a diagonal incision through the cuticle. the ovaries were removed with forceps. The wound was then sealed with paraffin. The complete extirpation of the ovaries was easily determined by examining them under the microscope after surgery.

Antisera to the yolk of newly laid eggs of Triatorm were prepared using rabbits, after the procedure employed by ENGELMANNand PENNEY (1966). For the preparation of anti-vitellogenin, antiserum to yolk was absorbed with haemolymph from adult males until all non-female-specific antibodies were precipitated. The immunological relationship of the yolk protein and the vitellogenin found in the haemolymph was then analyzed using the specific anti-vitellogenin by means of a double diffusion test (OUCHTERLONY, 1965). Immunoelectrophoresis was carried out in lo,, agar on microscope slides as described by HIRSCHl-1:1.1)(1960).

FRAU ENGFLMANU

Electrophoresis of haemolymph and yolk proteins was carried out in Tour percent polyacrqlamide gels after DAVIS (1964). In more concentrated gels. the female-specific protein co-migrates with a protein band also found in larvae and adult males and thus cannot be detected. The gels were stained in 0.25”” Coomassie Brilliant Blue in methanol: acetic acid: water (5 : I : 5) for at least 2 hr. and then destained electrophoretically in a solution containing 7.50, acetic acid and 5”, methanol. Using the techniques of HEDRICK and SMKH (196X). polyacrylamide gel electrophoresis was used to determine the molecular weights of the principal yolk proteins of Trinkmrr. Apoferretin (MW = 4.5 x IO’ daltons). catalase (2.4 x IO’ daltons). and bovine serum albumin (6.7 x IO4 daltons) were used as protein standards.

The control of the synthesis of vitellogenin by JH was investigated in females that had been allatectomized shortly after emergence, following essentially the same procedures described by ENGELMANN(I 97 1). Allatectomized females were fed one week after emcrgence and 4 days later, JH,,, (methyl-l 0, 1I-epoxy-2,6tr.clrfs.t~~lrrrs-famesenate) dissolved in 50 1.~1 olive oil was topically applied to the abdominal tergum. After an additional 4 days 0.1 /ICi ‘“C-leucine was delivered with a glass micropipette attached to a Hamilton micro syringe. The haemolymph was collected 4 hr later. One aliquot was then treated with l0g.a TCA to precipitate total proteins, whereas a second one was incubated for 1 hr at 37°C and overnight at 4’C with anti-vitellogenin to precipitate the female-specific protein. Radioactivity in the precipitates was measured in a Nuclear Chicago scintillation counter after redissolution in 1 N NaOH. Total proteins were determined in the TCA-precipitated aliquot by the Folin-Ciocalteu assay (LOWRY et ~1.. 1951). and results of the incorporation of label are reported as counts/min/mg total proteins.

The telotrophic ovary of Triatonu contains seven ovarioles. At emergence all ovarioles are fully differentiated, and the terminal (basal) oijcytes average about 0.3 mm in length. When they have grown to a length of 0.50 to 0.55 mm. refractile yolk granules can be detected in the oBplasm under the microscope. Vitellogenic terminal oijcytes can be found in unfed virgins on the second or third day after emergence. Approximately 4 days later a few fully matured eggs 1.8to 2.2 mm long are formed. After completion, eggs are ovulated into the lateral oviducts and the next oijcyte in line. the penultimate or t-l oiicyte, commences yolk deposition. Only one oijcyte per ovariole is vitellogenic at a time. If the nutritional or hormonal

Endocrine Table

I. Effect of feeding,

control

of vitellogenin

mating, application of JH,,,. and removal vitellogenesis in 16 day-old females

Untreated virgins lunfed) Mated at 16 days ‘unfed) Fed at 16 days 3unmated) Fed-mated at 16

of the brain

Average length of terminal oiicyte

Age at dissection Category

827

(days)

(mm)t

Number of females

16

0.46 + 0.02*

9

24

0.61 k 0.05

I6

24

0.44 _+ 0.01

I5

24

1.01 * 0.08

10

30

0.65 & 0.04

11

20

0.59 * 0.07

6

20

0.41 * 0.02

7

on


5Obcg JH,,, applied ;it 16 days (unmated-unfed) Br,iin removed at 16 (unmated-unfed) Ur,operated controls (unmated-unfed)

.-No mature eggs had of 4 to 8 days.

been formed

in any

milieu is insufficient to support continued yolk deposition. the terminal oocyte undergoes resorption and the t-l oiicyte assumes the terminal position. The i@uer~ce of rr~cltirtg LIPID feedirtg o/f ddogenesis Unfed virgins of Triczrotrm matured and ovulated an average of 3.4 F 0.8 eggs (49 females checked) within the first two weeks after emergence. By day 16, yolk depositicn generally had ceased, and the average terminal oocyte length is only 0.46 f 0.02 mm. When unfed females were mated for the first time at an age of 16 days, a modest resumption of yolk deposition resulted. indicated by the increase in length of the terminal oocytes 8 days later (Table 1). It appears, therefore. that the cessation in vitellogenesis normally observed in unfed virgins 2 weeks after emergence is not due merely to a lack of nutritional reserves. If females that were mated for the first time at 16 days of age were also fed at this time. yolk deposition was enhanced. On the other hand. feeding alone failed to elicit yolk deposition in 16 day-old virgins (Table 1). The corpus allattrm ad

tlitellogrtwsis

The removal of either the CA or the CA and a portion of the corpus cardiacum (CC) within one day after emergence prevented any yolk deposition in unfed virgins (Table 2). The terminal oocytes grew to the point at which yolk is normally deposited, i.e. approximately 0.50 mm in length. and then underwent resorption. When this operation was performed on 16-day-old females 24 hr after they were fed and mated. again, no vitellogenic oocytes were seen at dissection eight days ater (Table 2). The cessation of yolk deposition in the oocytes of

case within

the experimental

periods

normal 16 day-old unfed virgins is obviously due to the inactivity of the CA. Indeed, the application of 50 pg JH,,, to the abdominal tergum of 11 unfed virgins 16 days after emergence resulted in a resumption of yolk deposition in 9 of the I I females. The average terminal oocyte length for all I I females was 0.65 + 0.04mm which is similar in magnitude to the level of vitellogenesis induced by mating (Table I ). Further details of the role of JH in vitellogenesis are reported in a later section. Control oj’ corpus allaturn actiaity bra the brain Vitellogenesis was not impaired in virgin Triarottla females that were decapitated (leaving the retrocerebra1 complex within the animal) from 15 min to 2 hr after emergence. as seen at dissection 20 days later Table alone

2. Effect of removal of the corpus allaturn. either (CA-), or together with a portion of the corpus cardiacum (CA-CC-), on vitellogenesis

Category Unfed virgins: CA at emergence CA-CC at emerg. Sham-operated Unoperated Fed-mated at 16 days: CA- one day later CA-CCone day later Sham-operated Unoperated

Females with vitellogenic oocytes 8 days later

Number of females

0 0 93.8 90.9

8 26 16 II

0 0 81.R 90.0

9 24 II IO

82X

ERIC MUNDALL Table

3. Effect of removal

of brain

Br- at emergence (unmated-unfed) Unoperated (unmated -unfed) Unoperated (mated- unfed)

(Brr)

on vitcllogenesis

6.9 * 0.9*

0.81 & 0.07

II

2.6 k 1.5

0.41 i_ 0.03

7

7.6 + 0.8

0.46 k 0.02

5

20 days after emergence.

Table 4. Effect of removal

Category

(b)

at emergence

Number of females

of

(Table 3). Apparently the brain is not required for either CA activation or yolk deposition in females of this species. Also. the enhanced level of vitellogenesis compared to that of unoperated virgins, and the continuing yolk deposition when it normally had ceased in virgins or mated females (Table 3) suggests that in normal females the CA is restrained by the brain. When the brain was removed in 16-day-old unfed virgins, after the inactivation of the CA had presumably already commenced. a modest renewal of CA activity and yolk desposition resulted (Table 1). In Rho&us the brain appears to be required for the activation of the CA when females were fed after lengthy starvation (BAEHR, 1973; BAEHR et uI., 1973). In order to determine whether in Triatoma the brain

(a)

FRANZ EKELMANN

Average length of terminal oiicytes (mm)

Average number eggs matured?

Category

t At dissection * _+ S.E.M.

A&D

Fed-unmated: Br- immediately after feeding Br- 24 hr after feeding NCCI- 24 hr after feeding Sham-operated controls Unoperated controls Fed-mated: Br- immediately after feeding NCCI- 24 hrf after feeding Sham-operated controls Unoperated controls

is likewise

required then, it was removed by decapitation in four week old females immediately after feeding. At the time of the meal (which was the first since emergence). the females were reproductively inactive. as verified by dissection of additional females at this time. Two weeks after the operation it was found that vitellogenesis had resumed, both in virgin and mated females (Table 4). In both cases the level of yolk deposition was enhanced compared to that of the unoperated and sham-operated controls. A delay of 24 hr before brain removal did not yield a greater egg output in unmated females than that of those decerebrated (Br-) immediately after feeding. Thus, for Triatonzu, no evidence has been found of a transitory allatotropic function of the brain, as

of the brain (Br ) and severance nerves (NCCl-) on vitellogenesist

Average number eggs matured

of

of the corpus

Average length of terminal odcytes

(mm)

7.3 & 1.0*

0.85 * 0.12

7.0 * 1.2

0.66 * 0.09

6.3 + 0.7 .-

0.73 * 0.09

2.6 k 0.7

0.43 + 0.02

2.9 + 1.3

0.45 If: 0.01

1.4

1.20 f 0.06

8.6 k 1.5

0.94 ri: 0.07

7.5 & 0.8

0.70 * 0.09

6.9 f 1.1

0.71 * 0.07

11.5 f

cardiacum

Number of females

* 2 S.E.M. t Females were fed once four weeks after emergence and dissected 2 weeks later. $ NCCI- females were mated one day after the operation; all others were mated 2 weeks beforehand.

Adult

New I y emerged

CA-at

Fifth

male

instar

female

female

emergence

Egg Extract (Male-absorbed antiserum)

I.

Egg extract

2.

d-Absorbed

EM?

Adult8

5th?

Antiserum

Fig. I. lmmunoelectrophoresis of Triurorm haemolymph and saline extracts of newly laid eggs. The upper half of each slide displays the antigens of egg-maturing females, while the lower half shows the antigens as indicated. After electrophoresis (anode to the right). antiserum to the saline extracts of eggs was filled into the center trough and allowed to diffuse overnight. In the bottom slide the antiserum was first absorbed with haemolymph from adult males. Fig. 2. Diffusion of haemolymph proteins and yolk extracts of Triufom against anti-vitellogenin paredbyal)sorptiontoyolkantiserumwithadultmalehaemolymph).Thecompletefusionoftheprecipitation arcs indicates immttnological identity of the yolk protein and the vitellogenin.

(pre-

.Origin

Vitel

8.-r

Egg extract

’

logenin

-Front

EM

CA-

0

Fig. 3. Polyacrylamide gel electrophoresis (40, gels) of Triarormr haemolymph Allatectomy was performed at emergence. As seen, the application of 5.0/g dr NILVJsynthesis of vitellogenin.

and saline egg extracts. JH,,, is followed by the

Endocrine control of vitellogenin Baehr had reportt,d for Rhodrzius. Furthermore, judging from the enhanced level of vitellogenesis in the mated-BY females compared to normal mated females. it appears that copulation does not completely lift the inhibition of the CA by the brain (Table 4b). Severance of the corpus cardiacum nerves of virgins 24 hr after feeding (again, after four weeks starvation) resulted in a resumption of vitellogenesis that was similar in magnitude to that of the Br- females (Table 4a) which indicatt:s that the brain inhibits the CA via the neuronal connections. In order to see if mating could stimulate C4 activity after it has been disconnected from the brain. 5 additional females were mated one day after this operation. Egg maturation was not significantly enhanced (Table 4b).

In RhodrCus,ju:jt as feeding initiates CA activity and yolk deposition in the adult female. so does it initiate molting cycles in larval instars (WIGGLESWORTH, 1934. 193t). Was the VNC involved in transmitting the feeding stimulus in the adult as it had been shown to be in larvae? This possibility was investigated in Trictromuby the severance of the VNC in front of the pro) horacic ganglion in reproductively inactive females immediately after the completion of feeding. Females that had been mated two weeks before the severance of the VNC deposited yolk norinally compared to the sham-operated controls, as seen at dissection t NO weeks later (Table 5a, b). Those that were unmatec, again compared well to the respective sham-operated females (Table SC. d). Thus the operation itself did not stimulate vitellogenesis. Puthwuy of’ the rnu:irlgstirdus

It was of interes: to determine

if the ventral

nerve

cord was involved in transmitting the mating information that results in a reduced restraint of the CA by the brain. Four week-old starved virgin females were fed, then allowed to copulate. From 12 to 48 hr later the VNC was severed. At dissection two weeks after mating, no renewed vitellogenesis was evident in those females whose VNC was severed 12 hr after mating, only 1 of 3 females operated on after 34 hr had a few vitellogenic oocytes. and of the 11 females that had an intact VNC for 48 hr after copulation. only three deposited small amounts of yolk. The average terminal oijcyte length and number of eggs matured by the females from all three groups were not significantly different from those of the operated virgins (Table 5c, e). Thus it appears that the VNC must remain intact for at least 48 hr after mating in order for the copulatory stimulus to get through to the brain. In those females that had mated two weeks before the blood meal, severance of the VNC after feeding did not impair vitellogenesis. as reported in the preceding section (Table 5a). This result suggests that once the mating stimulus has been received by the brain an intact VNC is no longer required for the activation of the CA and ensuing vitellogenesis that normally follows feeding in mated females.

Identification of uite/iogeriirz ~JJirnnw~oekctrophoresis. With antiserum prepared against the yolk of newly laid eggs of Trintonzu. immunoelectrophoresis was used to analyze the relationship between the yolk proteins and those of the haemolymph. As many as seven antigens were precipitated from the haemolymph of egg maturing (EM) females by the yolk antiserum, one of which was absent from adult males and fifth instar females (Fig. 1). This antigen was also absent from the haemolymph of newly emerged

Table 5. Effect of severance of the ventral nerve cord (VNC)

Category (a)

(b) (c) (d) (e)

VNC- immed. after feeding (mated) Sham-operated (mated-fed) VNC 24 hr after feeding (unmated) Sham-operated (unmated-fed) Mated 24 hr after feeding. VNC- 12 hr later VNC- 24 hr later VNC 48 hr later

831

on vitellogenesis

Average number of eggs matured?

Average length of terminal oocytes (mm)

Number of females

7.8 + ox*

0.70 * 0.08

8

9.8 + I.2

0.64 + 0.04

12

1.9 * 0.9

0.45 + 0.02

17

3.4 * 1.2

0.44 + 0.02

8

1.6 + 1.0 0.7 & 0.5 3.9 + 0.9

0.44 f 0.02 0.50 k 0.07 0.48 f 0.02

9 3 II

* + S.E.M. t Cemales were fed once 4 weeks after emergence and dissected 2 weeks later.

x32

ERIC MUNDALL ANU FRANZ ENGELMANN

fcmalcs. and first appeared on the 2nd or 3rd day. before the onset of vitellogenesis. The female-specific antibody. anti-vitellogenin. was obtained by absorption of the yolk antiserum with adult male haemolymph. Using this male-absorbed antiserum. only one precipitation arc is formed when tested against haemolymph from EM females and the yolk extract (Fig. I). The precipitation arcs of the EM female haemolymph protein and the yolk antigen fused completely without the formation of spurs in a double diffusion Ouchterlony test (Fig. 7). Thus. it appears that the female-specific protein found in the haemolymph of EM females (vitellogenin) is immunologically identical to the principal yolk protein. Cor~rrol q/ ~‘it~lloye~ri~~ .s~r~thesis. The removal of the corpus allatum (and a portion of the CC) within 6 hr after emergence prevented the appearance of vitellogenin in nearly W’,, of the females, as tested by immunoelectrophoresis of the haemolymph obtained X days later (Fig. 1). In order to determine if the rlc WPU synthesis of vitellogenin was under the control of the corpus allatum hormone (JH). allatectomized females were fed one week after emergence and 4 days later. JH,,, dissolved in olive oil was topically applied to the abdominal tergum. After a 4 hr pulse of 0.1 PCi “C-1eucine four days later the haemolymph was collected. Incorporation of the label into vttellogenin was found to be related to the dose of JH applied (Table 6). This suggested that the de hero synthesis of vitellogenin was indeed under the control of the CA hormone. To see if the ovaries play a part in the appearance of vitellogenin in TV~LI~OWI. as they apparently do in At&s mqypri (HAGEDORN and FALLON, l973), ovariectomy was performed within 6 hr after emergence in 7 females. Immunoelectrophoresis of the haemolymph obtained 8 days later revealed that ample amounts of vitellogenin had appeared in 6 of the 7 females (Fig. I). Thus, vitellogenin synthesis is not influenced by the ovaries. Dr,llorlstr.Lltiori Lltld I.llar.Llcter.i_cltiorl of’ rirellogrrrirt hy poI~~~.~I~~~nirivqcll rlrctropho~sis. The vitellogenin from the haemolymph of EM females can also be identified from among the other haemolymph proteins by electrophoresis on polyacrylamide gels, Four

per cent gels were used to optimally visualize the vitellogenin. After allatectomy at emergence, no vitellogenin band was seen in haemolymph of adult females collected 8 days later (Fig. 3). No effect on other haemolymph proteins was apparent. Application of 5.Opg JH,,, caused the appearance of the vitcllogenin in haemolymph of females that had been allatectomized at emergence (Fig. 3). The yolk of T~iurontc~ eggs is composed almost entirely of two soluble proteins. The more slowly migrating major component has an identical electrophoretic mobility as the haemolymph vitellogenin. The faster migrating yolk component has no haemolymph counterpart. The molecular weights of the two principal yolk constituents were determined by using polyacrylamide gel electrophoresis. after the methods of HEDKICK and SMITH (1968). The relative mobilities were

TP,

1.2 -

1.0 Tp2

i

0.8 -

lCOt.

I

L

i

IO 20 Molecular

I

I

I

30

40

50

weight.

x 10m4

Fig. 4. Determination of the molecular weights of the prmcipal yolk components of Triutotuu eggs. by the use of the method given by HEDRICK and SMITH (1968). Slopes for standards and unknowns were obtained from the distance of migration in polyacrylamide gels of concentrations from 4 to 9”,. Abbreviations used: T,r = the major yolk protein; T,,2 = the minor yolk protein; Apo = apoferretin; Cat = catalase: BSA = bovine serum albumin.

Table 6. Incorporation of “C-1eucine into TCA- and anti-vitellogenin-precipitated proteins from the hemolymph of CA- females 4 days after application of JH,,,t

Treatment CA--+: 50 ,uI olive oil 0.5 /Lg JH,,, 5.0 /‘g JH,,, 5Ofig JH,,, * + S.E.M. + For details

Specific activity of the TCA-precipitated protein (CPM/mg protein)

1497 1582 3874 3804

see Materials

k * 5 *

216* 314 977 792

and Methods.

APO.

Fraction of label precipitated with anti-titellogenin (CPM)

100 197 1309 2226

+ + + + _

29 25 369 371

= = = =

8.00, 15.611 33.81; 60.9’;

Number of females

4 3 5 Y

Endocrine control of vitellogenin determined at ge; concentrations ranging from 4 to So<, and the resulting slopes were replotted against molecular weight (Fig. 4). From three such determinations (each with duplicates). the molecular weight of the principal yolk component (T,,) was estimated to be 4.37 & 0.06 x lo5 daltons, and that of the second component (TpL), 2.92 + 0.09 x lo5 daltons (Fig. 4). Sohrhility of tile yolk protrim In a number of insects. the major yolk component has been found to be virtually insoluble in media of low ionic strength (e.g. DE~MAL and BRCHKES, 1968 ; BELL, 1969). The yolk proteins of Tricltana. in contrast. are equally soluble in distilled water as they are in 0.4 M NaCl.

833

another (N. T. DAVIS, 1964), while in Dirldyfnus, the neuronal connections from the brain were severed (FRIEI)EL. 1974). Baehr and co-workers (BAEHR, 1973. 1974; BAEHR et crl., 1973) found that in Rho&Gus females that were reproductively inactive due to lengthy starvation, cautery of the neurosecretory cells of the brain within I hr after feeding prevented subsequent yolk deposition. If the operation was delayed for 24 hr, yolk deposition was unimpaired; indeed. it appeared to be elevated somewhat, compared to the unoperated controls. Thrse findings led Baehr and co-workers to postulate a transitory neurosecretory stimulation that was required for CA activation when females were fed after a period of starvation, and a neuronal inhibiDISCLISSION tory influence that mildly restrains the CA conIn Rhodnius. \VIGGL~SWORTH (1936) found that tinuously. Recently BAEHR (1976) has obtained evidecapitation, which included the removal of the CA, dence for a similar control of the CA following feed24 hr after a blood meal. prevented yolk deposition ing in larval instars of RhodrAs. in the oiicytes. Parabiosis of decapitated females Dual systems of control of the CA. analogous to with larvae or adults with active CA reestablished that found in Rhodrtius. have now been reported in vitellogenesis, as did implantation of active glands studies on several Hemiptera: O~~copvltus j2uscinru.s (WIGGIXSWORTH,1948). and topical application or in(JOHANSSON. 1958). D~&IUIIS cersicolor (FRIEDEL, jection of compounds with juvenile hormone activity 1974). and P~whocoris aptma (HODKOV~~,1975). In (WIGG~.~SW~RTK 196 I. 1963). That the CA is required Triatonm. on the other hand, no evidence has been obtained for a transitory allatotropic function by the for yolk deposition in R/d/Gus was confirmed by brain following feeding. as in Rhodtlim. Instead, the VANDERBERG(1961;) and BAEHR (1.973). Contradictory brain appears to exert a purely inhibitory control findings were reported by DAVEY and co-workers over the activity of the CA via the neuronal connec(DAVE).. 1967; PA-I~HIN and DAVEY, 1968; PRATT and tions. DAVL:Y. 1972). who claimed that the removal of the In larval instars of Rhoduius. WIGGLESWORTH(1934) CA did not totally prevent the formation of mature established that apolysis was initiated by the blood eggs. In the present paper we show that allatectomy prevented any yolk deposition in Triatom (Table 2). meal. If the larvae were given several small meals in succession, that equalled one normal meal in terms thus making it likely that in reduviid Hemiptera the of their nutrient content. but did not cause the same CA is generally es,jential for vitellogenesis. degree of abdominal distension. they failed to apolyse. Following a period of reproductive activity in the Also. the severance of the VNC 24 hr after the meal unfed virgin female. during which a few eggs may prevented apolysis. Apparently the abdominal distenbe matured, vitellogenesis ceases by about the 16th sion associated with the large volume of blood inday after emergence (Table 1). The cessation of yolk gested during feeding initiates nervous impulses from deposition was apparently due to the inhibition of stretch receptors in the abdomen; these impulses CA activity by the brain. rather than being caused by the unavailabihty of yolk precursors. The CA of travel up the VNC to the brain, causing the release of the prothoracotropic hormone and activation of starving females of O~coprltus falsciutus is similarly the prothoracic glands. restrained by the brain. which could be overcome by Since the blood meal initiates CA activity in the severance of the neuronal connections (JOHANSSON. adult female in an analogous manner as it triggers 19%). Whereas in Ot~cop~lt~s. the inactivation of the the activation of the prothoracic glands in larvae it CA was clearly attt,ibutable to the lack of nutritional was of interest to determine if the VNC was similarly reserves, and could be overcome by feeding, in Triuinvolved in the pathway of the feeding stimulus in fom~~, the CA of the unfed virgins could be reactivated adults. The finding that vitellogenesis was not imby the copulatory stimulus alone. Feeding. on the paired in mated Tritrronru females when the VNC was other hand. was not sufficient to overcome the inhibisevered within 15 min after the blood meal (Table tion placed on the CA by the brain unless mating 5(a)). suggests that in the adult of this species the had taken place a:; well (Table 1). In Cirnev lectuVNC does not constitute the pathway necessary to luritts (MELLAKBY. ,939: N. T. DAVIS. 1964) and Dilleffect the feeding response. As a likely alternative it ~~I~ILIS wrsicolov (FfuEDEL. 1974). the CA of fed virgins is suggested that the CA or a controlling center in also remained inactive. It was experimentally actithe brain responds directly to the nutritional milieu, vated in these species, as done here in Triatornu, by as was proposed for C’alliphoxr erythrocephnlrr removing the inhibition exerted by the brain. In (STRANGWAYS-DIXON,1962) and L~UCO~~LI~~J mudercw Ciws, this was accomplished by removal of the CA (ENGELMANN.1962). from one virgin female and its implantafion into

834 In a number

ERIC MUNDALLAND

of insect species mating plays a stimurole in the activation of the CA (cf. ENGELMANN. 1970). Severance of the VNC prevented the transmission of the copulatory stimulus in several different species (ENGELMANN, 1959, 1960; ROTH and STAY. 1961: ROTH, 1964; DAVIS, 1965a, b; FRIEDEL. 1974). While in Cimes lect~durius. an intact VNC was required for only 3 hr after insemination (DAVIS. 1965b). in the cockroaches, Leucoykarcr rnaderne (ENGELMANN. 1960) and Diploptrrcl ptmctata (ROTH and STAY, 1961). the VNC was required intact for two days or longer after the completion of mating. In Triutornu, as in these species, the severance of the VNC as late as 2 days after copulation had taken place prevented CA activation (Table 5e). Once the “matedness” signal gets through to the brain, as seen in females mated two weeks prior to the severance of the VNC, an intact nerve cord is no longer required (Table 5a) and the Ca is activated by subsequent feeding. Using evidence obtained by immunological techniques. it has now been established in several species, including Tricttow in the present study, that the CA or JH controls the appearance of the principal yolk protein precursor. vitellogenin (ENGELMANNand PENNEY. 1966; ENGELMANN. 1969, 1971. 1972; ENGELMANN, et NI., 1971; BROOKES. 1969; BELL, 1969. 1970; BELL and BARTH. 1970; WILKENS, 1969; DE LOOF and DE WILDE. 1970: PAN and WYATT. 1971; LANZREIN, 1974). to allatectomized Triatorna JH,,, application females resulted in stimulated incorporation of “C-leucine into vitellogenin (Table 6). suggesting that the CA hormone controls the & nono synthesis of the yolk protein precursor. just as had been previously demonstrated in Lrucopl~necl rwderue (ENGELMANN, 1969, 1971, 1972: BROOKES. 1969) and Danaus plerippus (PAN and WYATT, 1971). Unlike Aedes ctegJ>pri (HAGEDORN and FALLON. 1973). in Triutorm, the ovaries clearly do not play a part in control of vitellogenin synthesis (Fig. 1). In Periplaneta mwicmu, the synthesis of two viteliogenic proteins is apparently under the control of the CA (BELL. 1969. 1970). This is in contrast to most other species in which only one vitellogenin protein has been found. COLES (1964, 1965a, b) similarly reported that there were two vitellogenins in Rhodnius. providing evidence obtained from starch-gel electrophoresis. Using polyacrylamide gel electrophoresis. however, BAEHR (1974) found only one yolk protein precursor in the hemolymph. Although he did report that there were two major yolk components in the extracts of eggs, only the more heavily staining of the two had a hemolymph counterpart. In Trictrotnu, the yolk is similarly composed almost entirely of two components: a major one with a molecular weight of approximately 4.37 x IO5 daltons, and with an identical mobility on polyacrylamide gels as the vitellogenin in the haemolymph of EM females; and a minor band of 2.92 x lo5 daltons that had no latory

FRANZ ENGELMANN haemolymph counterpart, in terms of electrophoretic mobility. The strength of this second band is highly variable. and most often is very light. Proteins were detected by immunoelectrophoresis in the yolk of Hyulophoru cecropia (TELFER, 1960) and Periplurreta americutm (BELL. 1970) that did not appear in the haemolymph. These authors suggested that the ovary may have been the source of these proteins. This cannot explain the appearance of the second yolk component in Triutomu, since with antiserum to yolk that had been absorbed with haemolymph of adult males, a single precipitation arc appears both in the yolk extracts and haemolymph of egg maturing females (Fig. I ). An explanation for the presence of the additional yolk component may be that it is a breakdown product of the principal yolk protein, as is reported for Leucophcm tnudrruc? (ENGELMANW tar al.. 1976). Ackrlowlrdgrme,lt-The equipment was purchased with funds obtained GB 14965. This support is gratefully

used in this study through NSF Grant acknowledged.

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