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Vitellogenesis in the allatectomized stick insect Carausius morosus (br.) (Phasmatodea: Lonchodinae)
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Comp. Biochem. Physiol.Vol. 110B,No. 1, pp. 255-266, 1995 Copyright © 1995 ElsevierScienceLtd Printed in Great Britain.All fights 0305-0491/95 $9.50 + 0.00
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Vitellogenesis in the allatectomized stick insect C a r a u s i u s m o r o s u s (Br.) (Phasmatodea: Lonchodinae) James T. Bradley,* Massimo Masetti,1" Antonella Cecchettini: and Franco GiorgiJ; *Department of Zoology and Wildlife Science, Auburn University, Auburn, AL 36849, U.S.A.; 1-Department of Environmental Biology; and J;Department of Biomedicine, University of Pisa, Pisa, Italy Effects of allatectomy on vitellogenesis in adult Carausius morosus Br. were examined using rocket immunoelectrophoresis,polyacrylamide gel electrophoresis, fluorographicidentification and quantitation by liquid scintillation of in vivo 3SS-methionine-labeled proteins, and light microscope autoradiography. In normal adults and in adults allatectomized as last instar nymphs, Coomassie Blue-stained viteilogenin (Vg) was first detectable in the hemolymph between 3 and 5 days after adult emergence. Allatectomy of ll-18-day-old adults produced no detectable effects on subsequent Vg synthesis, secretion or uptake, but ovarian follicles in adults that had been allatectomized as nymphs were less efficient at taking up Vg than were those in sham-operated animals. Compared to sham-operated controls, adults ailatectomized as nymphs displayed an increased rate of accumulationof newly synthesized Vg in the hemolymph, a decreased rate of accumulation of vitellin in terminal follicles, a decrease in the size of yolk spheres containing newly synthesized viteliogeulc protein in the peripheral ooplasm, and alterations in the size distribution of terminal follicles. Thus, post-induction vitellogenesis and primary induction of Vg synthesis in C. morosus do not require active corpora allata (CA), but a normal rate of Vg uptake may be JH-facilitated. Key words: Vitellogenesis; Vitellogenin; Allatectomy; Corpora allata; Stick insect; Carausius rnorosus ; Phasmatodea. Comp. Biochem. Physiol. I IOB, 255-266, 1995.
Introduction Activation of the gene(s) for vitellogenin insects, the majority of Vg is synthesized by (Vg), the blood borne form of yolk protein, the fat body as pre-Vg that becomes marks the initiation of vitellogenesis. In processed intracellularly prior to its secretion into the hemolymph as phospholipoglycoprotein(s) (Kunkel and Nordin, Correspondence to: T. Bradley, Department of Zool1985; Raikhel and Dhadialla, 1992). ogy and Wildlife Science, 101 Cary Hall, Auburn Primary induction of pre-Vg synthesis in University, Auburn, A L 36849 U.S.A. Portions of Table 1 and Fig. 3 previously appeared in the fat body in most insect species is caused abstract form in: Stick Insects Phylogeny and by juvenile hormone (JH) released from Reproduction, Proceedings of the 1st International reactivated corpora allata (CA) in the adult Symposium on Stick Insects (1987) (Edited by Mazzini M. and Scali V.), Univ. of Siena and female (Engelmann, 1979, 1987; Koeppe et al., 1985). Juvenile hormone also Univ. of Bologna, 222 pp. Received 12 October 1993; accepted 20 May 1994. functions to sustain high rates of 255
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post-induction Vg synthesis (BakkerGrunwald and Applebaum, 1977) and/or vitellogenin uptake by vitellogenic follicles (Bell and Barth, 1971; Giorgi, 1979; Raikhel and Lea, 1983; Stoffolano et al., 1992; Ibanez et al., 1993). Exceptions to the exclusive control of vitellogenesis by JH include certain dipterans in which ecdysone has a key regulatory role (Hagedorn, 1985, 1989, 1990) and in moths with a non-feeding adult stage where vitellogenesis in the pharate adult stage may be developmentally programmed to occur independently of endocrine factors (Pan, 1977; Tsuchida et al., 1987; Satyanarayana et al., 1992). The stick insect Carausius morosus Br. is a parthenogenetic species with a panoistic form of ovarian development. Each ovariole consists of a graded series of developing follicles, only the terminal one being capable of undergoing vitellogenesis (Giorgi et al., 1993). Terminal follicles developing within ovarioles of the same ovary are out of phase with each other (Huebner, 1983), and oogenesis culminating in oviposition is a continuous, noncyclic process in this species. Vitellogenesis is initiated after ecdysis of the last nymphal instar to the adult form. After ovulation of the terminal follicle within a particular ovariole, the follicle previously in the subterminal position becomes a terminal follicle and enters the vitellogenic phase of oogenesis. During vitellogenesis, the hemolymph of C. morosus contains two multimeric Vgs (Vg A and Vg B) derived from pre-Vg polypeptides synthesized and secreted by the fat body (Giorgi and Macchi, 1980). These are taken up endocytically by vitellogenic follicles to become, respectively, vitellin (Vit) A and Vit B that comprise the major protein component of the yolk (Giorgi and Macchi, 1980; Giorgi et al., 1982, 1991). More than a half century ago, Pflugfelder (1937) reported that allatectomy of C. morosus during either the final nymphal instar or early adult stage had no effect on subsequent egg production in the adult, suggesting that vitellogenesis in C. morosus may be JH-independent. Since then, insect egg yolk formation has come to be understood as a complex process including the synthesis, secretion and receptor-mediated
endocytosis of Vg. Thus, a current examination of the role of the corpora allata in vitellogenesis should consider which of the above processes might be induced, maintained or modulated by the release of JH. To this end, we have studied the effects of allatectomy on certain cellular and molecular aspects of vitellogenesis in C. morosus. Insects were allatectomized either before or after initiation of vitellogenesis to obtain information about the role of the CA in regulating, respectively, the primary induction and the post-induction synthesis of Vg. The effect of allatectomy on terminal follicle morphology and Vg uptake were also examined.
Material and Methods Experimental cedures
animals
and surgical pro-
Adult and last instar nymph stage C. morosus from a laboratory colony were
allatectomized or sham-operated after being immobilized at 4°C for 20-90 min. Corpora allata were removed through a rectangular opening in the head capsule. Each pair of excised glands was placed in a watch-glass containing buffered stick insect saline (0.2N NaC1, 0.05 M Tris, pH 6.8) and identified using a stereomicroscope to confirm that both glands had been completely removed from the head capsule. After the operation, the excised cuticle was repositioned over the wound which became sealed within 10 min with coagulated hemolymph. Sham-operated females were treated similarly, the CA being left intact while a few tracheoles inside the head capsule were intentionally broken. Post-operative animals were fed fresh English ivy leaves and maintained at 20-23°C. The day of adult emergence was recorded for each animal. Approximately 30% of the operated nymphs survived to become adults. For insects allatectomized as adults, the described experiments were performed 11-18 days after the operation, and for those allatectomized as nymphs, experiments were performed 20-30 days after adult emergence. Since oogenesis is continuous and non-cyclic in C. morosus, variation in the ages of adults used in the experiments examining the effects of allate-
Vitellogenesis in allatectomized
ctomy on post-induction vitellogenesis was allowable. In vivo labeling and sample preparation Allatectomized and sham-operated insects received injections into the abdominal hemocoel of 35S-methionine (Radiochemical Centre, Amersham; 1470 Ci/mM) at the times reported in the text and containing the activities reported in the figure legends. One, 2, 4 or 8 hr later, hemolymph, fat body and ovarian follicles were collected from each animal and prepared for electrophoresis (Giorgi and Macchi, 1980) and/or light microscope autoradiography (Bradley et al., 1987). Pooled, unlabeled hemolymph samples were prepared as above by combining equal volumes of blood from three adults of the same age. When a single animal was used for more than one sampling time, at least 36hr intervened between samples.
Electrophoresis and fluorography Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS--PAGE) was performed in 5-15% polyacrylamide gradient slab gels according to Laemmli (1970). Gels were stained with 0.1% Coomassie Brilliant Blue 250-R in 50% methanol/10% acetic acid, treated with Amplify (Radiochemical Centre, Amersham), vacuum dried onto filter paper and overlaid with Kodak SO-282 X-ray film. Fluorographs were prepared after the film was exposed for 10-15 days at -70°C. Specific activities (dpm/#g hemolymph protein) of 35S-methionine-labeled Vg A and Vg B in the hemolymph of operated adults were estimated by rocket immunoelectrophoresis (Laurell, 1966) in agarose gels containing polyclonal rabbit antisera against Vit A or Vit B from newly laid eggs (Giorgi et al., 1982). After electrophoresis, the gels were rinsed extensively in electrode buffer to remove non-immunoprecipitated label, stained with Coomassie Blue R, treated with Amplify, dried onto Gel Bond (FMC, Corp.), and fluorographed. Stained rockets containing immunoprecipitated Vg Stage/Vol. EV1 0.25-0.55
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A and Vg B were cut from the bonded gel, treated with NCS Tissue Solubilizer (Radiochemical Centre, Amersham), and counted by liquid scintillation. Background counts obtained from unstained regions of the bonded gel were subtracted from counts obtained from the excised rockets. The amount of protein in each hemolymph sample was estimated according to Lowry et al. (1951).
Light microscope autoradiography One-micrometre-thick Epon-Araldite sections of terminal follicles were mounted on gelatin-coated slides and overlaid with NTB2 Kodak Emulsion by the dipping technique. After 4 weeks of exposure, the slides were developed with a D19 Kodak Developer, fixed with Kodafix, stained in 1% Methylene Blue-1% Toluidine Blue in Borax, mounted using Permount and photographed with a Zeiss Photomicroscope III.
Morphometric analysis of developing follicles About 100 terminal vitellogenic follicles isolated from four ovaries from adults sham-operated or allatectomized as nymphs were staged according to Giorgi et al. (1993), according to size and yolk morphology. Follicle volumes were estimated by treating follicles as rotational elipsoids [ V - 4/3 (a/2)2(b/2)] where a and b are the greatest length and width of the follicle, respectively. Each follicle was assigned to one of five arbitrarily defined stages of development (early vitellogenic, EV1 and EV2; late vitellogenic, LV1 and LV2; chorionated, CH). By this classification system, EV follicles are those with oocytes containing separate, unfused yolk spheres, LV follicles are those with oocytes containing a yolk mass in the central ooplasm and also separate yolk spheres in the corical ooplasm, and CH follicles are those whose oocytes lack yolk spheres and contain a yolk mass that extends virtually up to the oolemma. The ranges of follicle volumes for the five developmental stages are as follows:
(mm 3) EV2 0.55-1.10
LV1 1.10-2.00
LV2 2.00-3.20
CH 3.20-4.25
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Results Developmental stage-specificity of hemolymph Vg in normal and allatectomized individuals Hemolymph polypeptides of individuals in the adult and last three nymphal instar stages were analyzed by SDS-PAGE in order to estimate the time of appearance of Vg in the blood. This information was needed in order to design experiments to discriminate between the CA-dependency of primary induction of vitellogenesis and post-induction maintenance of vitellogenesis. The hemolymph polypeptide patterns for two penultimate and two last instar nymphs and two adults are shown in Fig. 1. All five Vg polypeptides were present in the hemolymph of both adults, whereas, with the exception of an A3-1ike polypeptide discussed below, Vg was undetectable in the nymphs. Vitellogenin was also absent from pre-penultimate instar nymphs. We tentatively concluded that Vg first appears in the blood sometime after emergence of the adult. In order to confirm this and to better
Fig. 1. Stage-specificity of Vg in the hemolymph shown by Coomassie Blue-stained blood polypeptides separated by SDS-PAGE. Lanes 2, penultimate nymphal instar; 3, last nymphal instar; 4, adult. A1-3 and B 1-2 are Vg polypeptides present only in adult hemolymph, and lane 1 contains molecular weight markers. Each blood sample was from a different individual, and about 10/~1 of hemolymph were applied to each lane.
al.
define the developmental schedule for initiation of vitellogenesis, we collected pooled hemolymph samples from adults 1, 3, 5, 8 and 12 days after emergence. All five Vg polypeptides were present in the hemolymph of 5-day-old or older adults and undetectable in the earlier samples (Fig. 2). Also shown in Fig. 2 is a representative hemolymph polypeptide profile for an adult allatectomized during the last nymphal instar. Here too, Vg first began to accumulate in the hemolymph between days 3 and 5 of adult life. In some, but not all, hemolymph samples from nymphs or adults 3 days old or younger, there was a minor band that appeared to co-migrate with a Vg polypeptide. This is seen in the blood of three nymphs (Fig. 1) that possessed an A3-1ike polypeptide and in an allatectomized individual (Fig. 2) where a small amount of a B l-like polypeptide was present during the last nymphal instar and early adulthood. The identities of these bands are not known. They have not been detected in the blood of nymphs by Western blotting using a Vg-specific polyclonal antisera (unpublished data), so the premature "leaky" expression of Vg does not seem to be an explanation. We conclude that Vg first appears in the hemolymph of C. morosus between 72 and 120 hr after ecdysis to the adult and that its appearance on this schedule can occur in the absence of active corpora allata. Therefore, to study the CA-dependence of post-induction vitellogenesis, allatectomy was performed on adults older than 5 days, and to examine primary induction of vitellogenesis, last stage nymphs were allatectomized. In both cases, h e m o l y m p h collected just before the operation was examined for the presence of Vg by SDS-PAGE and Coomassie Blue staining. At the time of surgery, none of the nymphs contained hemolymph Vg, whereas adults contained all five Vg polypeptides.
Post-induction vitellogenesis after allatectomy To determine whether the CA are needed for maintenance of the vitellogenic state (i.e. continued synthesis, secretion and uptake of Vg), adults whose hemolymph contained Vg as determined by Coomassie
Vitellogenesis in allatectomized C.
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Allatectomized
Control
B1 A1 A2 B2 A3
Days after emergence Fig. 2. Developmental timing of appearance of Vg polypeptides in hemolymph of representative unoperated (left) and allatectomized (right) adults. The latter were allatectomized during the last nymphal instar. Gels show Coomassie Blue-stained polypeptides in hemolymph samples collected on certain days after adult emergence and separated by SDS-PAGE. P, Pre-emergence hemolymph collected at time of operation; He, control hemolymph from egg-laying adult; A1-3 and B1-2, Vg polypeptides that appear between days 3 and 5 in both groups of animals. Hemolymph volume applied per lane for control and allatectomized animals were about 10 and 3/~1, respectively. Far left lane contains molecular weight markers.
Blue staining after SDS-PAGE were allatectomized or sham-operated. Eleven to 18 days later, the animals were injected with 35S-methionine, and tissue samples were collected after various periods of incorporation. The accumulation of radiolabeled Vg A and Vg B in the hemolymph of both groups of insects was visualized using rocket immunoelectrophoresis and fluorography of the resulting precipitin lines. A representative gel and its corresponding fluorograph (Fig. 3) show that the accumulation of labeled Vg B in the hemolymph occurs at about the same rate in allatectomized and sham-operated animals. The same was observed for Vg A (fluorograph not shown). Using the same bonded agarose gels from
which the rocket fluorographs for this experiment were produced, we tested the visual impression gained from the fluorographs by quantitating via liquid scintillation the specific activities of hemolymph Vg A and Vg B for each period of incorporation (Table 1). These data confirmed that Vg A and Vg B accumulated at similar rates in the hemolymph of both groups of insects. As part of this study on post-induction vitellogenesis, we also demonstrated, by native PAGE and fluorography, that each native Vg was sequestered by terminal follicles in the ovarioles of both allatectomized and control females (data not shown). Thus, detecting no effects of allatectomy on post-induction vitellogene-sis, we
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Fig. 3. Quantitation of post-induction synthesis of Vg B by rocket immunoelectrophoresis and fluorography of hemolymph from allatectomized (CA-) and sham-operated (sham) adults. Blood was collected 1, 2, 4 and 8 hr after injection of 30/tCi of 35S-methionine/individual. Fluorograph (b) of stained gel (a) shows accumulation of newly synthesized Vg B in both CA- and sham insects. Well set on right side of gel contains isolated Vit B (8-90ug/well) as a standard.
directed our attention to the role of the CA in the primary induction of vitellogenesis.
Primary induction of vitellogenesis after allatectomy In order to determine whether removal of the CA affects primary induction of Vg synthesis and secretion in the fat body, 35S-methionine was injected into 12-40day-old adults allatectomized or sham Table 1. Specific activity of hemolymph viteilogenin after in vivo exposure of allatectomized and sham-operated females to 3SS-methionine Incorporation time (hr)
1
2 4 8
Specific activity (dpm/#g hemolymph protein) Sham-operated
Allatectomized
VgA
VgB
VgA
VgB
0.7 4.8 18.0 79.0
3.0 6.0 16.2 96.1
5.3 8.3 13.8 77.6
5.6 6.1 13.4 67.8
operated during their previous nymphal instar. Recall that oogenesis is continuous and non-cyclic in normal adults, ovulation occurring throughout adult life which lasts for several months. After 2-, 4- and 8-hr periods of incorporation, the patterns of labeled fat body and hemolymph polypeptides were examined. The results showed that the high molecular weight pre-Vg polypeptides in the fat body were synthesized in both sham-operated and allatectomized animals (Fig. 4). In both groups, the specific activity of pre-Vg appeared to decrease during the 8 hr period of incorporation. This was accompanied by the appearance of all five processed Vg polypeptides in the hemolymph of both groups of animals by 2 hr after injection of the label (Fig. 3). Far fewer hemolymph polypeptides became labeled in allatectomized females than in sham-operated females, and those which were labeled in allatectomized animals, including all five Vg polypeptides, followed a clear kinetics of accumulation (Fig. 3). By contrast, the degree of labeling of hemolymph Vg polypeptides in sham operated individuals was less after 8 than after 4 hr of incorporation. A possible explanation for the accumulation of labeled Vg in the blood of allatectomized individuals and its waning in control individuals over the 8-hr period is that Vg uptake by developing follicles was more efficient in the control animals. The appearance of highly labeled Vg in the blood by 2 hr after injection of label in Fig. 4 but not until 4 hr in Fig. 3 is probably because fewer pCi of 35S-methionine were used in the latter experiment. The earliest we have detected labeled Vg in the hemolymph of C. morosus using SDS-PAGE and fluorography is 60 min after injection of the label (unpublished data).
Uptake of labeled polypeptides by vitellogenic follicles To test whether transfer of Vg from the hemolymph to the oocyte occurred in allatectomized individuals, polypeptides in developing follicles 2-8 hr after injection of 35S-methionine were analysed electrophoretically and by fiuorography (Fig. 5). The results showed that label was present in all five Vg polypeptides in both sham-operated and allatectomized animals, and that these
Vitellogenesis in allatectomized C.
SH
CA-
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morosus
SH
CA|
~O
tt
24 8 Fat
248hr body
24
8
2 4 8hr
Hemolymph
Fig. 4. Fluorographs showing synthesis and secretion of 35S-methionine-labeled Vg in adults allatectomized (CA-) or sham-operated (SH) as last instar nymphs. PreVG, pre-vitellogenin; AI-3 and BI-2, Vg polypeptides. Hours (hr) indicate periods of in vivo incorporation after injection of 60 # Ci of label; separation was by SDS-PAGE.
polypeptides were labeled more extensively in control than in allatectomized animals. To further examine the effect of allatectomy on yolk development, terminal follicles from allatectomized and sham-operated animals exposed to 35S-methionine for 8 hr in vivo were processed for light microscope autoradiography (Fig. 6). The results clearly showed label present within the follicle cell layer and the cortical ooplasm in allatectomized animals. However, the developing yolk spheres appeared smaller and less intensely labeled in these animals than in follicles from shamoperated controls.
wished to know whether comparable numbers of follicles reached various stages of vitellogenic growth in allatectomized and sham-operated animals. To gain this information, five size classes of terminal follicles were arbitrarily defined between the minimum and maximum volumes attained by the follicles (0.25 and 4.25 mm 3, respectively). Although the low number of females available for this analysis did not allow the data to be evaluated statistically, the results (Fig. 8) showed fewer terminal follicles reaching advanced stages of vitellogenesis in allatectomized individuals compared to sham-operated females. Thus, ovaries from allatectomized animals appeared to have more follicles in the EV1, EV2 and LV1 stages of development than did ovaries Morphometric analysis of developing fol- from control animals, and this trend was licles in allatectomized individuals reversed in the late stages of development, Since data in the fluorograph of Fig. 5 LV2 and CH. Moreover, many chorionated were derived from follicles of about the follicles in allatectomized individuals were same size in early phases of vitellogenesis, smaller than those in control animals they provided no information about the size (Fig. 7), suggesting that they achieved spectrum of developing follicles engaged in full developmental maturation without vitellogenic growth. In particular, we attaining full growth.
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-
B1-
A1 , -
2
48
A2-
2
48
1985; Engelmann, 1979, 1987; Wyatt, 1991). In some cases, ovarian follicle cells are a secondary site of Vg synthesis, and regulation by JH may occur here as well (Brennan et al., 1992; Isaac and Bownes, 1982). The initial appearance of Vg in the hemolymph is an indication that the inductive events for vitellogenesis have recently occurred since the fat body of normal female insects does not store Vg but secretes it immediately after it is synthesized (Engelmann, 1979). Our observation that Vg is absent from the blood of C. m o r o s u s during nymphal instars and first becomes detectable in the blood between days 3 and 5 after ecdysis to the adult (Figs 1 and 2) suggests that the regulatory signals inducing vitellogenesis in C. m o r o s u s are normally expressed during early adulthood. Since the primary induction of pre-Vg synthesis in the fat body of C. m o r o s u s occurred in adults that had been allatectomized as nymphs before the first appearance of hemolymph Vg (Fig. 4), we conclude that Vg gene activation in this species is not dependent on a post-ecdysis burst of JH synthesis/release by the CA. The hemolymph JH titer in adult C. m o r o sus has not been measured, so the possibility that Vg is induced by JH produced by a source other than the CA cannot be ruled out, although to our knowledge there is no precedent for this. The possibility that Vg induction in C. m o r o s u s is controlled by hormones other than JH remains uninvestigated. Since all five Vg polypeptides appear in the hemolymph of adult females allatectomized as nymphs before the induction of Vg synthesis has occurred, normal Vg processing and secretion are also likely to be JHindependent. Post-induction maintenance of the vitellogenic state also appears to be CAindependent in C. m o r o s u s . Immunogenically reactive Vg was synthesized and secreted into the hemolymph of both control and allatectomized females (Fig. 3 and Table 1). Since the rate of accumulation of newly synthesized Vg in the blood was comparable in both groups (Table 1) and since the fat body is the only known biosynthetic source of Vg in C. m o r o s u s (Giorgi and Macchi, 1980), we conclude that allatet al.,
CA-
SH
hr
Fig. 5. Uptake of in vivo-labeled Vg polypeptides (al-3, B12) by follicles in adults allatectomized (CA-) or sham-operated (SH) as last instar nymphs. Hours (hr) indicate periods of incorporation; follicles were collected from the same individuals used for Fig. 4.
Discussion
This study examined the effects of allatectomy on Vg synthesis and secretion by the fat body and its uptake by developing follicles in the stick insect, C. m o r o s u s . Our findings indicate that this species may provide an exception to the usual patterns of endocrine regulation of vitellogenesis in insects. Despite the genetic and developmental diversity in reproductive strategies among insects (Engelmann, 1970), the underlying endocrine/cellular mechanisms regulating major events in egg formation remain remarkably similar across the phylogenetic spectrum. Egg yolk formation is one example. Although ecdysone induces Vg synthesis in certain dipterans (DeLoof e t ai., 1984; Hagedorn, 1990), initiation of vitellogenesis in most other insects results from reactivation of the CA that release JH which induces the fat body to begin producing prodigious quantities of Vg (Koeppe
Vitellogenesis in allatectomized
SH
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CA-
Fig. 6. Light microscope autoradiograph of terminal follicles from adults allatectomized (CA-, c-d) or sham operated (SH, a-b) at last instar nymphs. Tissue was processed after 8 hr of in vivo exposure to 35S-methionine. FC, Follicle cells; OC, oocyte; p, proteinod yolk spheres. Note that label has been incorporated into yolk spheres in the peripheral ooplasm. Scale bars represent 10/~m. Follicles were collected from the same individuals used for 8-hr samples in Figs 4 and 5.
SH
CA-
Fig. 7. Ovaries from 20-day-old adults allatectomized (CA-) or sham operated (SH) as last instar nymphs. C, Chorionated follicle; V, vitellogenic follicle. Note undersize chorionated follicles in ovary of CA- insect. Magnification: 4.2 × .
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°t
•
CA-OPERA'I~D
•
SHAM-OPERATED
[kd
10'
EVt
EV2
LVI
LV2
CH
Size Class
Fig. 8. Histogram showing numbers of vitellogenic follicles attaining certain stages of development in allatectomized and sham-operated individuals. EV, Early vitellogenin;LV, late vitellogenic;CH, chorionated. Please see Methods for morphological criteria for staging and key to size classes.
ectomy has no detectable effect on the rate at which pre-Vg traverses the processing/secretory pathway within the fat body cells. Accumulation of 35S-methioninelabeled Vit in terminal follicles of allatectomized adults whose hemolymph contains in vivo labeled Vg and the autoradiographic demonstration of labeled yolk spheres in the peripheral ooplasm of these animals (Bradley et al., 1989) indicate that continued uptake of Vg by developing follicles can occur in the absence of the CA. Thus, our results show that both primary and postinduction Vg synthesis and secretion in C. morosus are independent of active CA. Other parts of our data indicate that a normal rate of Vg uptake by developing follicles requires that CA be present during the adult stage. The first indication of this was the fluorographs of in vivo labeled hemolymph protein showing an accumulation of label ]n Vg polypeptides in allatectomized females and a decrease over time in the degree of labeling of the same polypeptides in control animals (Fig. 4). From this, we hypothesized that allatectomy may adversely affect the hemolymphoocyte transfer of Vg and thereby cause an elevation in the hemolymph titer of Vg. Further evidence for the hypothesis was gained from fluorographs of in vivo labeled Vit from terminal follicles (Fig. 5) showing a marked depression in the rate of accumulation of labeled Vit in the follicles of adult females allatectomized as nymphs. This and
the smaller, labeled yolk spheres in the cortical ooplasm of follicles from allatectomized females (Fig. 6) strongly suggest that the Vg uptake mechanism operates less efficiently in these animals. Finally, the presence of undersized, chorionated follicles in allatectomized females (Fig. 7) and our morphometric analysis showing a higher proportion of early and intermediate-stage vitellogenic follicles in the ovaries of allatectomized females suggest that follicles in allatectomized animals may incorporate less than the normal amount of Vg. Based on these results, we propose that Vg uptake in C. morosus is JH-facilitated but not JHdependent as observed in other species including Phormia regina (Yin et al., 1989; Stoffolano et al., 1992), Drosophila melanogaster (Wilson, 1982), Manduca sexta (Kawooya and Law, 1983), and Spilostethus pandurus (Ibanez et al., 1993). Hemolymph Vg gains access to the oolemma through spaces between follicle cells and enters the ooplasm by receptormediated endocytosis (Telfer et al., 1982; Ferenz, 1990; Raikhel and Dhadialla, 1992). Other than in Rhodnius prolixus where JH regulates vitellogenic patency of follicles via action on a subunit of Na+/K + ATPase (Davey, 1981; Raikhel and Dhadialla, 1992) and may enhance endocytic uptake by increasing the number of Vg receptors (Wang and Davey, 1991), the role of hormones in regulating Vg uptake in insects is not well understood. In summary, we have demonstrated that the primary induction of vitellogenesis and the post-induction maintenance of vitellogenesis in the stick insect, C. morosus, can occur after removal of the CA. Correlated biochemical and morphological data show that CA-independent vitellogenesis can occur at the level of Vg synthesis, processing, and secretion, but that a normal rate of Vg uptake may require the presence of CA and therefore be JH-facilitated. Our results provide an explanation at the molecular and cellular levels for the earlier observation by Pflugfelder (1937) that allatectomy during the last nymphal instar does not affect egg production in this species. Acknowledgements--This work was done while JTB was on professionalimprovementleavein the laboratory of F.G. Support was from the Italian Ministry
Vitellogenesis in allateetomized C. morosus of Education to FG and NCRS and NSF/EPSCoR (3R11-8610669) funds to JTB.
References Bakker-Grunwald T. and Applebaum S. W. (1977) A quantitative description of vitellogenesis in Locusta migratoria migratorioides. J. Insect Physiol. 23, 259-263. Bell W. J. and Barth R. H. Jr (1971) Initiation of yolk deposition by juvenile hormone. Nature New Biol. 230, 220-221. Bradley J. T., Masetti M. and Giorgi F. (1989) Vitellogenesis in allatectomized Carausius morosus. J. Cell Biol. 230, 32a. Bradley J. T., Mazzini M. and Giorgi F. (1987) Vitellogenesis in the stick insect Bacillus rossius (Phasmatodea, Bacillidae)--VI. Kinetics of viteliogenic development. Mon. Zool. Ital. 21, 99-115. Brennan M. D., Weiner A. J., Gosalski T. J. and Mahowald A. P. (1982) The follicle cells are a major site of vitellogenin synthesis in Drosophila melanogaster. Devl Biol. 89, 225-236. Davey K. G. (1981) Hormonal control of viteilogenin uptake in Rhodnius prolixus. Stal. Am. Zool. 21, 701-705. De Loof A., Briers T. and Huybrechts R. (1984) Presence and function of ecdysteroids in adult insects. Comp. Biochem. Physiol. 79B, 505-509. Engelmann F. (1970) The physiology of insect reproduction. Internation Series of Monographs in Pure and Applied Biology (Edited by Kerkut G. A.). Pergamon Press, Oxford. Engelmann F. (1979) Insect vitellogenin: identification, biosynthesis, and role in vitellogenesis. Adv. Insect Physiol. 14, 49-108. Engelmann F. (1987) The pleiotropic action of juvenile hormone in vitellogenin synthesis. Mitt. Dtsch. Ges. Angew. Entomol. 5, 186-194. Ferenz H.-J. (1990) Receptor-mediated endocytosis of insect yolk proteins. In Molecular Insect Science (Edited by Hagedorn, H. H., Hildebrand J. C., Kidwell M. G. and Law J. H.), pp. 131-138. Plenum Press, New York. Giorgi F. (1979) In vitro induced pinocytotic activity by a juvenile hormone analogue in oocytes of Drosophila melanogaster. Cell Tissue Res. 203, 241-247. Giorgi F. and Macchi F. (1980) Vitellogenesis in the stick insect Carausius morosus--I. Specific protein synthesis during ovarian development. J. Cell Sci. 46, 1-16. Giorgi F., Baldini G., Simonini A. L. and Mengheri M. (1982) Vitellogenesis in the stick insect Carausius morosus--II. Purification and biochemical characterization of two vitellins from eggs. Insect Biochem. 12, 553-562. Giorgi F., Masetti M., Ignacchiti V., Cecchettini A., Estridge B. and Bradley J. T. (1991) Vitellin polypeptides undergo post-endocytic processing in ovarian follicles of the stick insect Carausius morosus. Br. J. Cell Biol. 115, 48a. Giorgi F., Cecchettini A., Lucchesi P. and Mazzini M. (1993) Oocyte growth, follicle cell differentiation and vitellin processing in the stick insect,
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Carausius morosus Br. (Phasmatodea). Int. J. Insect Morphol. Embryol. 22, 271-292. Giorgi F., Masetti M., Ignacchiti V., Cecchettini A. and Bradley J. T. (1993) Postendocytic vitellin processing in ovarian follicles of the stick insect Carausius morosus (Br.). Archs Insect Biochem. Physiol. 24, 93-111. Hagedorn H. H. (1985) The role of ecdysteroids in reproduction. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 8, pp. 205-262. Pergamon Press, Oxford. Hagedorn H. H. (1989) Physiological roles of hemolymph ecdysteroids in the adult insect. In Ecdysone, from Chemistry to Mode of Action (Edited by Koolman J.), pp. 279-289. Thieme, Stuttgart. Hagedorn H. H. (1990) In search of functions for ecdysone in the female insect. Progr. clin. Biol. Res. 342, 365-371. Huebner E. (1983) Oostatic hormone antigonadotropin and reproduction. In Endocrinology of Insects (Edited by Dower R. G. H. and Laufer H.), pp. 319-329. Alan R. Liss, New York. Ibanez P., Garcera M. D., Alcacer E., Conill F. and Martinez R. (1993) Effect of starvation on haemolymph vitellogenins and ovary uptake in Spilosterthus pandurus. Comp. Biochem. Physiol. 104B, 531-536. Isaac P. and Bownes M. (1982) Ovarian and fat body vitellogenin synthesis in Drosophila melanogaster. Eur. J. Biochem. 123, 527-536. Kawooya J. K. and Law J. H. (1983) Purification and properties of microviteUogenin of Manduca sexta--role of juvenile hormone in appearance and uptake. Biochem. biophys. Res. Commun. 117, 643-650. Koeppe J. K., Fuchs M., Chen T. T., Hunt L. M., Kovlalick G. E. and Briers T. (1985) In Comprehensive Insect Physiology, Biochemistry, and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 8, pp. 165-203. Pergamon, New York. Kunkel J. G. and Nordin J. H. (1985) Yolk proteins. In Comprehensive Insect Physiology, Biochemistry, and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 1, pp. 83-111. Pergamon, New York. Laurell C. B. (1966) Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analyt. Biochem. 15, 45-52. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-685. Lowry O. H., Rosebrough H. J., Far A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Pan M. L. (1977) Juvenile hormone and vitellogenin synthesis in the cecropia silkworm. Biol. Bull. 153, 336-345. Pflugfelder O. (1937) Bau, Entwicklung und Funktion der corpora allata und cardiaca von Dixippus morosus Br. Z. Wiss. Zool. 249, 477-512. Raikhel A. S. and Dhadialla T. S. (1992) Accumulation of yolk proteins in insect oocytes. Ann. Rev. Entomol. 37, 217-251. Raikhel A. S. and Lea A. O. (1983) Juvenile hormone
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lation of yolk proteins in insect oocytes. Ann. Rev. Entomol. 37, 217-251. Raikhel A. S. and Lea A. O. (1983) Juvenile hormone dependent formation of endocytotic organelles and ligand dependent stimulation of the vitellogenin accumulative pathway in mosquito Aedes aegypti oocytes. Am. Zool. 23, 984. Satyanarayana K., Bhaskaran G., Dahm K. H. and Meola R. (1992) Regulation of viteliogenin synthesis by juvenile hormone in the corn earworm, Helicoverpa zea. Invert. Reprod. Devl. 21, 169-178. Stoffolano J. G. Jr, Li M.-F., Zou B.-X. and Yin C.-M. (1992) Vitellogenin uptake, not synthesis, is dependent on juvenile hormone in adults of Phormia regina (Meigen). J. Insect Physiol. 38, 839-845. Telfer W. H., Huebner E. and Smith S. D. (1982) The cell biology of vitellogenic follicles in Hyalophora and Rhodnius. In Insect Ultrastructure (Edited by
King R. C. and Akai H.), pp. 118-149. Plenum Press, New York. Tsuchida K., Nagata M. and Suzuki A. (1987) Hormonal control of ovarian development in the silkworm, Bombyx mori. Archs Insect Biochem. Physiol. 5, 167-177. Wang Z. and Davey K. G. (1992) Characterization of yolk protein and its receptor on the oocyte membrane in Rhodnius prolixus. Insect Biochem. molec. Biol. 22, 757-767. Wilson T. G. (1982) A correlation between juvenile hormone deficiency and vitellogenic oocyte degeneration in Drosophila melanogaster. Wilhelm Roux's Archs Devl. Biol. 191, 257-263. Wyatt G. R. (1991) Gene regulation in insect reproduction. Invert. Reprod. Devl. 20, 1-35. Yin C.-M., Zou B.-X. and Stoffolano Jr J. G. (1989) Precocene II treatment inhibits terminal oocyte development but not vitellogenin synthesis and release in the black blowfly Phormia regina Meigen. J. Insect Physiol. 35, 465-474.
Comp. Biochem. Physiol.Vol. 110B,No. 1, pp. 255-266, 1995 Copyright © 1995 ElsevierScienceLtd Printed in Great Britain.All fights 0305-0491/95 $9.50 + 0.00
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Vitellogenesis in the allatectomized stick insect C a r a u s i u s m o r o s u s (Br.) (Phasmatodea: Lonchodinae) James T. Bradley,* Massimo Masetti,1" Antonella Cecchettini: and Franco GiorgiJ; *Department of Zoology and Wildlife Science, Auburn University, Auburn, AL 36849, U.S.A.; 1-Department of Environmental Biology; and J;Department of Biomedicine, University of Pisa, Pisa, Italy Effects of allatectomy on vitellogenesis in adult Carausius morosus Br. were examined using rocket immunoelectrophoresis,polyacrylamide gel electrophoresis, fluorographicidentification and quantitation by liquid scintillation of in vivo 3SS-methionine-labeled proteins, and light microscope autoradiography. In normal adults and in adults allatectomized as last instar nymphs, Coomassie Blue-stained viteilogenin (Vg) was first detectable in the hemolymph between 3 and 5 days after adult emergence. Allatectomy of ll-18-day-old adults produced no detectable effects on subsequent Vg synthesis, secretion or uptake, but ovarian follicles in adults that had been allatectomized as nymphs were less efficient at taking up Vg than were those in sham-operated animals. Compared to sham-operated controls, adults ailatectomized as nymphs displayed an increased rate of accumulationof newly synthesized Vg in the hemolymph, a decreased rate of accumulation of vitellin in terminal follicles, a decrease in the size of yolk spheres containing newly synthesized viteliogeulc protein in the peripheral ooplasm, and alterations in the size distribution of terminal follicles. Thus, post-induction vitellogenesis and primary induction of Vg synthesis in C. morosus do not require active corpora allata (CA), but a normal rate of Vg uptake may be JH-facilitated. Key words: Vitellogenesis; Vitellogenin; Allatectomy; Corpora allata; Stick insect; Carausius rnorosus ; Phasmatodea. Comp. Biochem. Physiol. I IOB, 255-266, 1995.
Introduction Activation of the gene(s) for vitellogenin insects, the majority of Vg is synthesized by (Vg), the blood borne form of yolk protein, the fat body as pre-Vg that becomes marks the initiation of vitellogenesis. In processed intracellularly prior to its secretion into the hemolymph as phospholipoglycoprotein(s) (Kunkel and Nordin, Correspondence to: T. Bradley, Department of Zool1985; Raikhel and Dhadialla, 1992). ogy and Wildlife Science, 101 Cary Hall, Auburn Primary induction of pre-Vg synthesis in University, Auburn, A L 36849 U.S.A. Portions of Table 1 and Fig. 3 previously appeared in the fat body in most insect species is caused abstract form in: Stick Insects Phylogeny and by juvenile hormone (JH) released from Reproduction, Proceedings of the 1st International reactivated corpora allata (CA) in the adult Symposium on Stick Insects (1987) (Edited by Mazzini M. and Scali V.), Univ. of Siena and female (Engelmann, 1979, 1987; Koeppe et al., 1985). Juvenile hormone also Univ. of Bologna, 222 pp. Received 12 October 1993; accepted 20 May 1994. functions to sustain high rates of 255
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post-induction Vg synthesis (BakkerGrunwald and Applebaum, 1977) and/or vitellogenin uptake by vitellogenic follicles (Bell and Barth, 1971; Giorgi, 1979; Raikhel and Lea, 1983; Stoffolano et al., 1992; Ibanez et al., 1993). Exceptions to the exclusive control of vitellogenesis by JH include certain dipterans in which ecdysone has a key regulatory role (Hagedorn, 1985, 1989, 1990) and in moths with a non-feeding adult stage where vitellogenesis in the pharate adult stage may be developmentally programmed to occur independently of endocrine factors (Pan, 1977; Tsuchida et al., 1987; Satyanarayana et al., 1992). The stick insect Carausius morosus Br. is a parthenogenetic species with a panoistic form of ovarian development. Each ovariole consists of a graded series of developing follicles, only the terminal one being capable of undergoing vitellogenesis (Giorgi et al., 1993). Terminal follicles developing within ovarioles of the same ovary are out of phase with each other (Huebner, 1983), and oogenesis culminating in oviposition is a continuous, noncyclic process in this species. Vitellogenesis is initiated after ecdysis of the last nymphal instar to the adult form. After ovulation of the terminal follicle within a particular ovariole, the follicle previously in the subterminal position becomes a terminal follicle and enters the vitellogenic phase of oogenesis. During vitellogenesis, the hemolymph of C. morosus contains two multimeric Vgs (Vg A and Vg B) derived from pre-Vg polypeptides synthesized and secreted by the fat body (Giorgi and Macchi, 1980). These are taken up endocytically by vitellogenic follicles to become, respectively, vitellin (Vit) A and Vit B that comprise the major protein component of the yolk (Giorgi and Macchi, 1980; Giorgi et al., 1982, 1991). More than a half century ago, Pflugfelder (1937) reported that allatectomy of C. morosus during either the final nymphal instar or early adult stage had no effect on subsequent egg production in the adult, suggesting that vitellogenesis in C. morosus may be JH-independent. Since then, insect egg yolk formation has come to be understood as a complex process including the synthesis, secretion and receptor-mediated
endocytosis of Vg. Thus, a current examination of the role of the corpora allata in vitellogenesis should consider which of the above processes might be induced, maintained or modulated by the release of JH. To this end, we have studied the effects of allatectomy on certain cellular and molecular aspects of vitellogenesis in C. morosus. Insects were allatectomized either before or after initiation of vitellogenesis to obtain information about the role of the CA in regulating, respectively, the primary induction and the post-induction synthesis of Vg. The effect of allatectomy on terminal follicle morphology and Vg uptake were also examined.
Material and Methods Experimental cedures
animals
and surgical pro-
Adult and last instar nymph stage C. morosus from a laboratory colony were
allatectomized or sham-operated after being immobilized at 4°C for 20-90 min. Corpora allata were removed through a rectangular opening in the head capsule. Each pair of excised glands was placed in a watch-glass containing buffered stick insect saline (0.2N NaC1, 0.05 M Tris, pH 6.8) and identified using a stereomicroscope to confirm that both glands had been completely removed from the head capsule. After the operation, the excised cuticle was repositioned over the wound which became sealed within 10 min with coagulated hemolymph. Sham-operated females were treated similarly, the CA being left intact while a few tracheoles inside the head capsule were intentionally broken. Post-operative animals were fed fresh English ivy leaves and maintained at 20-23°C. The day of adult emergence was recorded for each animal. Approximately 30% of the operated nymphs survived to become adults. For insects allatectomized as adults, the described experiments were performed 11-18 days after the operation, and for those allatectomized as nymphs, experiments were performed 20-30 days after adult emergence. Since oogenesis is continuous and non-cyclic in C. morosus, variation in the ages of adults used in the experiments examining the effects of allate-
Vitellogenesis in allatectomized
ctomy on post-induction vitellogenesis was allowable. In vivo labeling and sample preparation Allatectomized and sham-operated insects received injections into the abdominal hemocoel of 35S-methionine (Radiochemical Centre, Amersham; 1470 Ci/mM) at the times reported in the text and containing the activities reported in the figure legends. One, 2, 4 or 8 hr later, hemolymph, fat body and ovarian follicles were collected from each animal and prepared for electrophoresis (Giorgi and Macchi, 1980) and/or light microscope autoradiography (Bradley et al., 1987). Pooled, unlabeled hemolymph samples were prepared as above by combining equal volumes of blood from three adults of the same age. When a single animal was used for more than one sampling time, at least 36hr intervened between samples.
Electrophoresis and fluorography Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS--PAGE) was performed in 5-15% polyacrylamide gradient slab gels according to Laemmli (1970). Gels were stained with 0.1% Coomassie Brilliant Blue 250-R in 50% methanol/10% acetic acid, treated with Amplify (Radiochemical Centre, Amersham), vacuum dried onto filter paper and overlaid with Kodak SO-282 X-ray film. Fluorographs were prepared after the film was exposed for 10-15 days at -70°C. Specific activities (dpm/#g hemolymph protein) of 35S-methionine-labeled Vg A and Vg B in the hemolymph of operated adults were estimated by rocket immunoelectrophoresis (Laurell, 1966) in agarose gels containing polyclonal rabbit antisera against Vit A or Vit B from newly laid eggs (Giorgi et al., 1982). After electrophoresis, the gels were rinsed extensively in electrode buffer to remove non-immunoprecipitated label, stained with Coomassie Blue R, treated with Amplify, dried onto Gel Bond (FMC, Corp.), and fluorographed. Stained rockets containing immunoprecipitated Vg Stage/Vol. EV1 0.25-0.55
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A and Vg B were cut from the bonded gel, treated with NCS Tissue Solubilizer (Radiochemical Centre, Amersham), and counted by liquid scintillation. Background counts obtained from unstained regions of the bonded gel were subtracted from counts obtained from the excised rockets. The amount of protein in each hemolymph sample was estimated according to Lowry et al. (1951).
Light microscope autoradiography One-micrometre-thick Epon-Araldite sections of terminal follicles were mounted on gelatin-coated slides and overlaid with NTB2 Kodak Emulsion by the dipping technique. After 4 weeks of exposure, the slides were developed with a D19 Kodak Developer, fixed with Kodafix, stained in 1% Methylene Blue-1% Toluidine Blue in Borax, mounted using Permount and photographed with a Zeiss Photomicroscope III.
Morphometric analysis of developing follicles About 100 terminal vitellogenic follicles isolated from four ovaries from adults sham-operated or allatectomized as nymphs were staged according to Giorgi et al. (1993), according to size and yolk morphology. Follicle volumes were estimated by treating follicles as rotational elipsoids [ V - 4/3 (a/2)2(b/2)] where a and b are the greatest length and width of the follicle, respectively. Each follicle was assigned to one of five arbitrarily defined stages of development (early vitellogenic, EV1 and EV2; late vitellogenic, LV1 and LV2; chorionated, CH). By this classification system, EV follicles are those with oocytes containing separate, unfused yolk spheres, LV follicles are those with oocytes containing a yolk mass in the central ooplasm and also separate yolk spheres in the corical ooplasm, and CH follicles are those whose oocytes lack yolk spheres and contain a yolk mass that extends virtually up to the oolemma. The ranges of follicle volumes for the five developmental stages are as follows:
(mm 3) EV2 0.55-1.10
LV1 1.10-2.00
LV2 2.00-3.20
CH 3.20-4.25
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Results Developmental stage-specificity of hemolymph Vg in normal and allatectomized individuals Hemolymph polypeptides of individuals in the adult and last three nymphal instar stages were analyzed by SDS-PAGE in order to estimate the time of appearance of Vg in the blood. This information was needed in order to design experiments to discriminate between the CA-dependency of primary induction of vitellogenesis and post-induction maintenance of vitellogenesis. The hemolymph polypeptide patterns for two penultimate and two last instar nymphs and two adults are shown in Fig. 1. All five Vg polypeptides were present in the hemolymph of both adults, whereas, with the exception of an A3-1ike polypeptide discussed below, Vg was undetectable in the nymphs. Vitellogenin was also absent from pre-penultimate instar nymphs. We tentatively concluded that Vg first appears in the blood sometime after emergence of the adult. In order to confirm this and to better
Fig. 1. Stage-specificity of Vg in the hemolymph shown by Coomassie Blue-stained blood polypeptides separated by SDS-PAGE. Lanes 2, penultimate nymphal instar; 3, last nymphal instar; 4, adult. A1-3 and B 1-2 are Vg polypeptides present only in adult hemolymph, and lane 1 contains molecular weight markers. Each blood sample was from a different individual, and about 10/~1 of hemolymph were applied to each lane.
al.
define the developmental schedule for initiation of vitellogenesis, we collected pooled hemolymph samples from adults 1, 3, 5, 8 and 12 days after emergence. All five Vg polypeptides were present in the hemolymph of 5-day-old or older adults and undetectable in the earlier samples (Fig. 2). Also shown in Fig. 2 is a representative hemolymph polypeptide profile for an adult allatectomized during the last nymphal instar. Here too, Vg first began to accumulate in the hemolymph between days 3 and 5 of adult life. In some, but not all, hemolymph samples from nymphs or adults 3 days old or younger, there was a minor band that appeared to co-migrate with a Vg polypeptide. This is seen in the blood of three nymphs (Fig. 1) that possessed an A3-1ike polypeptide and in an allatectomized individual (Fig. 2) where a small amount of a B l-like polypeptide was present during the last nymphal instar and early adulthood. The identities of these bands are not known. They have not been detected in the blood of nymphs by Western blotting using a Vg-specific polyclonal antisera (unpublished data), so the premature "leaky" expression of Vg does not seem to be an explanation. We conclude that Vg first appears in the hemolymph of C. morosus between 72 and 120 hr after ecdysis to the adult and that its appearance on this schedule can occur in the absence of active corpora allata. Therefore, to study the CA-dependence of post-induction vitellogenesis, allatectomy was performed on adults older than 5 days, and to examine primary induction of vitellogenesis, last stage nymphs were allatectomized. In both cases, h e m o l y m p h collected just before the operation was examined for the presence of Vg by SDS-PAGE and Coomassie Blue staining. At the time of surgery, none of the nymphs contained hemolymph Vg, whereas adults contained all five Vg polypeptides.
Post-induction vitellogenesis after allatectomy To determine whether the CA are needed for maintenance of the vitellogenic state (i.e. continued synthesis, secretion and uptake of Vg), adults whose hemolymph contained Vg as determined by Coomassie
Vitellogenesis in allatectomized C.
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morosus
Allatectomized
Control
B1 A1 A2 B2 A3
Days after emergence Fig. 2. Developmental timing of appearance of Vg polypeptides in hemolymph of representative unoperated (left) and allatectomized (right) adults. The latter were allatectomized during the last nymphal instar. Gels show Coomassie Blue-stained polypeptides in hemolymph samples collected on certain days after adult emergence and separated by SDS-PAGE. P, Pre-emergence hemolymph collected at time of operation; He, control hemolymph from egg-laying adult; A1-3 and B1-2, Vg polypeptides that appear between days 3 and 5 in both groups of animals. Hemolymph volume applied per lane for control and allatectomized animals were about 10 and 3/~1, respectively. Far left lane contains molecular weight markers.
Blue staining after SDS-PAGE were allatectomized or sham-operated. Eleven to 18 days later, the animals were injected with 35S-methionine, and tissue samples were collected after various periods of incorporation. The accumulation of radiolabeled Vg A and Vg B in the hemolymph of both groups of insects was visualized using rocket immunoelectrophoresis and fluorography of the resulting precipitin lines. A representative gel and its corresponding fluorograph (Fig. 3) show that the accumulation of labeled Vg B in the hemolymph occurs at about the same rate in allatectomized and sham-operated animals. The same was observed for Vg A (fluorograph not shown). Using the same bonded agarose gels from
which the rocket fluorographs for this experiment were produced, we tested the visual impression gained from the fluorographs by quantitating via liquid scintillation the specific activities of hemolymph Vg A and Vg B for each period of incorporation (Table 1). These data confirmed that Vg A and Vg B accumulated at similar rates in the hemolymph of both groups of insects. As part of this study on post-induction vitellogenesis, we also demonstrated, by native PAGE and fluorography, that each native Vg was sequestered by terminal follicles in the ovarioles of both allatectomized and control females (data not shown). Thus, detecting no effects of allatectomy on post-induction vitellogene-sis, we
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Fig. 3. Quantitation of post-induction synthesis of Vg B by rocket immunoelectrophoresis and fluorography of hemolymph from allatectomized (CA-) and sham-operated (sham) adults. Blood was collected 1, 2, 4 and 8 hr after injection of 30/tCi of 35S-methionine/individual. Fluorograph (b) of stained gel (a) shows accumulation of newly synthesized Vg B in both CA- and sham insects. Well set on right side of gel contains isolated Vit B (8-90ug/well) as a standard.
directed our attention to the role of the CA in the primary induction of vitellogenesis.
Primary induction of vitellogenesis after allatectomy In order to determine whether removal of the CA affects primary induction of Vg synthesis and secretion in the fat body, 35S-methionine was injected into 12-40day-old adults allatectomized or sham Table 1. Specific activity of hemolymph viteilogenin after in vivo exposure of allatectomized and sham-operated females to 3SS-methionine Incorporation time (hr)
1
2 4 8
Specific activity (dpm/#g hemolymph protein) Sham-operated
Allatectomized
VgA
VgB
VgA
VgB
0.7 4.8 18.0 79.0
3.0 6.0 16.2 96.1
5.3 8.3 13.8 77.6
5.6 6.1 13.4 67.8
operated during their previous nymphal instar. Recall that oogenesis is continuous and non-cyclic in normal adults, ovulation occurring throughout adult life which lasts for several months. After 2-, 4- and 8-hr periods of incorporation, the patterns of labeled fat body and hemolymph polypeptides were examined. The results showed that the high molecular weight pre-Vg polypeptides in the fat body were synthesized in both sham-operated and allatectomized animals (Fig. 4). In both groups, the specific activity of pre-Vg appeared to decrease during the 8 hr period of incorporation. This was accompanied by the appearance of all five processed Vg polypeptides in the hemolymph of both groups of animals by 2 hr after injection of the label (Fig. 3). Far fewer hemolymph polypeptides became labeled in allatectomized females than in sham-operated females, and those which were labeled in allatectomized animals, including all five Vg polypeptides, followed a clear kinetics of accumulation (Fig. 3). By contrast, the degree of labeling of hemolymph Vg polypeptides in sham operated individuals was less after 8 than after 4 hr of incorporation. A possible explanation for the accumulation of labeled Vg in the blood of allatectomized individuals and its waning in control individuals over the 8-hr period is that Vg uptake by developing follicles was more efficient in the control animals. The appearance of highly labeled Vg in the blood by 2 hr after injection of label in Fig. 4 but not until 4 hr in Fig. 3 is probably because fewer pCi of 35S-methionine were used in the latter experiment. The earliest we have detected labeled Vg in the hemolymph of C. morosus using SDS-PAGE and fluorography is 60 min after injection of the label (unpublished data).
Uptake of labeled polypeptides by vitellogenic follicles To test whether transfer of Vg from the hemolymph to the oocyte occurred in allatectomized individuals, polypeptides in developing follicles 2-8 hr after injection of 35S-methionine were analysed electrophoretically and by fiuorography (Fig. 5). The results showed that label was present in all five Vg polypeptides in both sham-operated and allatectomized animals, and that these
Vitellogenesis in allatectomized C.
SH
CA-
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morosus
SH
CA|
~O
tt
24 8 Fat
248hr body
24
8
2 4 8hr
Hemolymph
Fig. 4. Fluorographs showing synthesis and secretion of 35S-methionine-labeled Vg in adults allatectomized (CA-) or sham-operated (SH) as last instar nymphs. PreVG, pre-vitellogenin; AI-3 and BI-2, Vg polypeptides. Hours (hr) indicate periods of in vivo incorporation after injection of 60 # Ci of label; separation was by SDS-PAGE.
polypeptides were labeled more extensively in control than in allatectomized animals. To further examine the effect of allatectomy on yolk development, terminal follicles from allatectomized and sham-operated animals exposed to 35S-methionine for 8 hr in vivo were processed for light microscope autoradiography (Fig. 6). The results clearly showed label present within the follicle cell layer and the cortical ooplasm in allatectomized animals. However, the developing yolk spheres appeared smaller and less intensely labeled in these animals than in follicles from shamoperated controls.
wished to know whether comparable numbers of follicles reached various stages of vitellogenic growth in allatectomized and sham-operated animals. To gain this information, five size classes of terminal follicles were arbitrarily defined between the minimum and maximum volumes attained by the follicles (0.25 and 4.25 mm 3, respectively). Although the low number of females available for this analysis did not allow the data to be evaluated statistically, the results (Fig. 8) showed fewer terminal follicles reaching advanced stages of vitellogenesis in allatectomized individuals compared to sham-operated females. Thus, ovaries from allatectomized animals appeared to have more follicles in the EV1, EV2 and LV1 stages of development than did ovaries Morphometric analysis of developing fol- from control animals, and this trend was licles in allatectomized individuals reversed in the late stages of development, Since data in the fluorograph of Fig. 5 LV2 and CH. Moreover, many chorionated were derived from follicles of about the follicles in allatectomized individuals were same size in early phases of vitellogenesis, smaller than those in control animals they provided no information about the size (Fig. 7), suggesting that they achieved spectrum of developing follicles engaged in full developmental maturation without vitellogenic growth. In particular, we attaining full growth.
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-
B1-
A1 , -
2
48
A2-
2
48
1985; Engelmann, 1979, 1987; Wyatt, 1991). In some cases, ovarian follicle cells are a secondary site of Vg synthesis, and regulation by JH may occur here as well (Brennan et al., 1992; Isaac and Bownes, 1982). The initial appearance of Vg in the hemolymph is an indication that the inductive events for vitellogenesis have recently occurred since the fat body of normal female insects does not store Vg but secretes it immediately after it is synthesized (Engelmann, 1979). Our observation that Vg is absent from the blood of C. m o r o s u s during nymphal instars and first becomes detectable in the blood between days 3 and 5 after ecdysis to the adult (Figs 1 and 2) suggests that the regulatory signals inducing vitellogenesis in C. m o r o s u s are normally expressed during early adulthood. Since the primary induction of pre-Vg synthesis in the fat body of C. m o r o s u s occurred in adults that had been allatectomized as nymphs before the first appearance of hemolymph Vg (Fig. 4), we conclude that Vg gene activation in this species is not dependent on a post-ecdysis burst of JH synthesis/release by the CA. The hemolymph JH titer in adult C. m o r o sus has not been measured, so the possibility that Vg is induced by JH produced by a source other than the CA cannot be ruled out, although to our knowledge there is no precedent for this. The possibility that Vg induction in C. m o r o s u s is controlled by hormones other than JH remains uninvestigated. Since all five Vg polypeptides appear in the hemolymph of adult females allatectomized as nymphs before the induction of Vg synthesis has occurred, normal Vg processing and secretion are also likely to be JHindependent. Post-induction maintenance of the vitellogenic state also appears to be CAindependent in C. m o r o s u s . Immunogenically reactive Vg was synthesized and secreted into the hemolymph of both control and allatectomized females (Fig. 3 and Table 1). Since the rate of accumulation of newly synthesized Vg in the blood was comparable in both groups (Table 1) and since the fat body is the only known biosynthetic source of Vg in C. m o r o s u s (Giorgi and Macchi, 1980), we conclude that allatet al.,
CA-
SH
hr
Fig. 5. Uptake of in vivo-labeled Vg polypeptides (al-3, B12) by follicles in adults allatectomized (CA-) or sham-operated (SH) as last instar nymphs. Hours (hr) indicate periods of incorporation; follicles were collected from the same individuals used for Fig. 4.
Discussion
This study examined the effects of allatectomy on Vg synthesis and secretion by the fat body and its uptake by developing follicles in the stick insect, C. m o r o s u s . Our findings indicate that this species may provide an exception to the usual patterns of endocrine regulation of vitellogenesis in insects. Despite the genetic and developmental diversity in reproductive strategies among insects (Engelmann, 1970), the underlying endocrine/cellular mechanisms regulating major events in egg formation remain remarkably similar across the phylogenetic spectrum. Egg yolk formation is one example. Although ecdysone induces Vg synthesis in certain dipterans (DeLoof e t ai., 1984; Hagedorn, 1990), initiation of vitellogenesis in most other insects results from reactivation of the CA that release JH which induces the fat body to begin producing prodigious quantities of Vg (Koeppe
Vitellogenesis in allatectomized
SH
263
C. morosus
CA-
Fig. 6. Light microscope autoradiograph of terminal follicles from adults allatectomized (CA-, c-d) or sham operated (SH, a-b) at last instar nymphs. Tissue was processed after 8 hr of in vivo exposure to 35S-methionine. FC, Follicle cells; OC, oocyte; p, proteinod yolk spheres. Note that label has been incorporated into yolk spheres in the peripheral ooplasm. Scale bars represent 10/~m. Follicles were collected from the same individuals used for 8-hr samples in Figs 4 and 5.
SH
CA-
Fig. 7. Ovaries from 20-day-old adults allatectomized (CA-) or sham operated (SH) as last instar nymphs. C, Chorionated follicle; V, vitellogenic follicle. Note undersize chorionated follicles in ovary of CA- insect. Magnification: 4.2 × .
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°t
•
CA-OPERA'I~D
•
SHAM-OPERATED
[kd
10'
EVt
EV2
LVI
LV2
CH
Size Class
Fig. 8. Histogram showing numbers of vitellogenic follicles attaining certain stages of development in allatectomized and sham-operated individuals. EV, Early vitellogenin;LV, late vitellogenic;CH, chorionated. Please see Methods for morphological criteria for staging and key to size classes.
ectomy has no detectable effect on the rate at which pre-Vg traverses the processing/secretory pathway within the fat body cells. Accumulation of 35S-methioninelabeled Vit in terminal follicles of allatectomized adults whose hemolymph contains in vivo labeled Vg and the autoradiographic demonstration of labeled yolk spheres in the peripheral ooplasm of these animals (Bradley et al., 1989) indicate that continued uptake of Vg by developing follicles can occur in the absence of the CA. Thus, our results show that both primary and postinduction Vg synthesis and secretion in C. morosus are independent of active CA. Other parts of our data indicate that a normal rate of Vg uptake by developing follicles requires that CA be present during the adult stage. The first indication of this was the fluorographs of in vivo labeled hemolymph protein showing an accumulation of label ]n Vg polypeptides in allatectomized females and a decrease over time in the degree of labeling of the same polypeptides in control animals (Fig. 4). From this, we hypothesized that allatectomy may adversely affect the hemolymphoocyte transfer of Vg and thereby cause an elevation in the hemolymph titer of Vg. Further evidence for the hypothesis was gained from fluorographs of in vivo labeled Vit from terminal follicles (Fig. 5) showing a marked depression in the rate of accumulation of labeled Vit in the follicles of adult females allatectomized as nymphs. This and
the smaller, labeled yolk spheres in the cortical ooplasm of follicles from allatectomized females (Fig. 6) strongly suggest that the Vg uptake mechanism operates less efficiently in these animals. Finally, the presence of undersized, chorionated follicles in allatectomized females (Fig. 7) and our morphometric analysis showing a higher proportion of early and intermediate-stage vitellogenic follicles in the ovaries of allatectomized females suggest that follicles in allatectomized animals may incorporate less than the normal amount of Vg. Based on these results, we propose that Vg uptake in C. morosus is JH-facilitated but not JHdependent as observed in other species including Phormia regina (Yin et al., 1989; Stoffolano et al., 1992), Drosophila melanogaster (Wilson, 1982), Manduca sexta (Kawooya and Law, 1983), and Spilostethus pandurus (Ibanez et al., 1993). Hemolymph Vg gains access to the oolemma through spaces between follicle cells and enters the ooplasm by receptormediated endocytosis (Telfer et al., 1982; Ferenz, 1990; Raikhel and Dhadialla, 1992). Other than in Rhodnius prolixus where JH regulates vitellogenic patency of follicles via action on a subunit of Na+/K + ATPase (Davey, 1981; Raikhel and Dhadialla, 1992) and may enhance endocytic uptake by increasing the number of Vg receptors (Wang and Davey, 1991), the role of hormones in regulating Vg uptake in insects is not well understood. In summary, we have demonstrated that the primary induction of vitellogenesis and the post-induction maintenance of vitellogenesis in the stick insect, C. morosus, can occur after removal of the CA. Correlated biochemical and morphological data show that CA-independent vitellogenesis can occur at the level of Vg synthesis, processing, and secretion, but that a normal rate of Vg uptake may require the presence of CA and therefore be JH-facilitated. Our results provide an explanation at the molecular and cellular levels for the earlier observation by Pflugfelder (1937) that allatectomy during the last nymphal instar does not affect egg production in this species. Acknowledgements--This work was done while JTB was on professionalimprovementleavein the laboratory of F.G. Support was from the Italian Ministry
Vitellogenesis in allateetomized C. morosus of Education to FG and NCRS and NSF/EPSCoR (3R11-8610669) funds to JTB.
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King R. C. and Akai H.), pp. 118-149. Plenum Press, New York. Tsuchida K., Nagata M. and Suzuki A. (1987) Hormonal control of ovarian development in the silkworm, Bombyx mori. Archs Insect Biochem. Physiol. 5, 167-177. Wang Z. and Davey K. G. (1992) Characterization of yolk protein and its receptor on the oocyte membrane in Rhodnius prolixus. Insect Biochem. molec. Biol. 22, 757-767. Wilson T. G. (1982) A correlation between juvenile hormone deficiency and vitellogenic oocyte degeneration in Drosophila melanogaster. Wilhelm Roux's Archs Devl. Biol. 191, 257-263. Wyatt G. R. (1991) Gene regulation in insect reproduction. Invert. Reprod. Devl. 20, 1-35. Yin C.-M., Zou B.-X. and Stoffolano Jr J. G. (1989) Precocene II treatment inhibits terminal oocyte development but not vitellogenin synthesis and release in the black blowfly Phormia regina Meigen. J. Insect Physiol. 35, 465-474.