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Development of secretory activity in the seminal vesicle of the male migratory grasshopper, Melanoplus sanguinipes (fabr.) (Orthoptera : Acrididae)
Int. J. lnsectMorphol. & Embryol., Vol. 17, No. 1, pp. 51-61. 1988
0020-7322/88 $3.0(I + .00 © 1988 Pergamon Press plc
Printed in Great Britain
DEVELOPMENT OF SECRETORY ACTIVITY IN THE SEMINAL VESICLE OF THE MALE MIGRATORY GRASSHOPPER, M E L A N O P L U S SANGUINIPES (FABR.) (ORTHOPTERA • ACRIDIDAE)
GRAHAM A . COUCHE a n d CEDRIC GILLOTT* Department of Biology, University of Saskatchewan, Saskatoon, SK, S7N OWO Canada
(Accepted 15 July 1987)
Abstract--This paper describes the ultrastructure of the seminal vesicle and the isoelectric focusing patterns of its secretion during sexual maturation and after allatectomy in Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae). In epithelia from seminal vesicles of newly fledged males, the rough endoplasmic reticulum is well developed, and Golgi complexes are elaborate, which indicates the gland is metabolically active. The cells also contain large glycogen deposits and the lumen microvilli are well differentiated. These ultrastructural features are more dominant in 24-hr-old adults where the cytoplasm is clearly differentiated into basal and apical regions. Basally, the cytoplasm is dominated by rough endoplasmic reticulum, large Golgi complexes, glycogen deposits and numerous mitochondria, while the apical cytoplasm is filled with large secretory and/or lysosomal vesicles. Between days 3 and 7, the ultrastructural features change little other than the rough endoplasmic reticulum cisternae, which become vesicular. Analysis by isoelectric focusing shows that the amount of secretory protein increases with age until day 3, at which time the gland contains its full complement of secretion. In seminal vesicles from allatectomized insects, ultrastructural features of cells and i8oelectric focusing patterns of the secretion are identical to those from normal males.
Index descriptors (in addition to those in title): Ultrastructure, isoelectric focusing, protein synthesis.
INTRODUCTION THE SEMINAL v e s i c l e in Melanoplus sanguinipes is a m o d i f i e d a c c e s s o r y g l a n d t u b u l e t h a t is r e a d i l y i d e n t i f i a b l e e v e n in n e w l y e m e r g e d adults. P r e v i o u s b i o c h e m i c a l s t u d i e s o f t h e a c c e s s o r y g l a n d s o f M. sanguinipes ( V e n k a t e s h a n d G i l l o t t , 1983; G i l l o t t a n d V e n k a t e s h , 1985), as w e l l as h i s t o l o g i c a l ( G i r a r d i e a n d V o g e l , 1966; C a n t a c u z G n e , 1967) a n d u l t r a s t r u c t u r a l ( O d h i a m b o , 1971) i n v e s t i g a t i o n s w i t h o t h e r A c r i d i d a e , h a v e i n d i c a t e d
* Author to whom reprint requests should be sent. Abbreviations used in Figures: G = Golgi complex; GI = glycogen deposit; N = nucleus; RER = rough endoplasmic reticulum; BL = basal lamina; L = lysosome: is = intercellular space; Lu = lumen; Mv = microvilli; SV = secretory vesicle. 51
52
GRAHAMA. COUCHEand CEDRICGILLOTT
that juvenile hormone (JH) has little influence on maturation (viz. secretory activity) of the seminal vesicle. This contrasts with the prominent role of JH in the control of secretory activity in the long hyaline gland and other accessory gland tubules (Venkatesh and Gillott, 1983; Gillott and Venkatesh, 1985; Couche and Gillott, 1987). This paper describes the ultrastructure of the seminal vesicle and the isoelectric focusing (IEF) patterns of its secretion, during maturation and after allatectomy in M. sanguinipes. By comparing our observations on the long hyaline gland (Couche and Gillot, 1987) with those of the seminal vesicle, now reported, we hope to obtain some insight into the site and mode of action of JH in the reproductive physiology of male insects.
MATERIALS AND METHODS A non-diapause strain M. sanguinipes(Pickford and Randell, 1969)was used. Males were separated from a stock culture as 5th instar nymphs and reared in isolation from females in standard cages (Pickford, 1958). Fresh barley leaves and bran were fed ad libitum. Allatectomyor sham-operationswere carried out between 12 and 24 hr postemergence as described by Gillon and Friedel (1976). After a 24 hr postoperative recovery period, allatectomizedmales were injected with juvenile hormone III (ling in 3.5 pJ paraffin oil) or paraffin oil alone. At least 10 males were sampled in each treatment group. Procedures for seminal vesicledissection, tissue preparation for light and electron microscopy,and secretory protein analysis by IEF are described in an accompanyingpaper (Couche and Gillott, 1987).
RESULTS Morphology Changes during sexual maturation. At adult emergence, the seminal vesicle has a 35-40-txm-thick columnar epithelium (Fig. 1). Cell nuclei are large and may be located basally or apically. Microvilli of the apical plasma membrane and the gland lumen are moderately well developed. Mitochondria are large and numerous, particularly in the basal cytoplasm (Fig. 2). The endoplasmic reticulum (ER) shows considerable development both as elongated rough E R cisternae and small E R vesicles. Numerous ribosomes may be found in association with rough E R or free in the cytoplasm. Large glycogen deposits and elaborate Golgi complexes are characteristic of the basal cytoplasm (Fig. 2). Lysosomes are scattered throughout. Swollen, perhaps lysosomal, vesicles are occasionally present. Intercellular spaces containing a coarse fibrous material are common in the basal regions of the lateral junctional complex (Fig. 3). Differentiation of microvilli of the apical cell surface is relatively advanced (Fig. 4). Small cytoplasmic vesicles close to the apical plasma membrane and chains of similar vesicles within bulbous apical cytoplasmic projections suggest that differentiation of microvilli involves splitting of apical projections by ordered linear fusion of chains of small vesicles. The lumen of the seminal vesicle contains a widely spread fibrous material (Fig. 4). The seminal vesicle of males 24 hr postemergence has a 35-60-p~m-thick simple columnar epithelium surrounding an empty-looking lumen, whose development is varied along the length of the gland. Central to apical nuclei, intercellular spaces, densely basophilic structures and empty-looking apical vesicles are readily observed in the light microscope (Fig. 5). As has been observed in seminal vesicles from mature insects (Couche, 1985; Couche and Gillott, unpublished), ultrastructural characteristics of the epithelial cells differ between basal and apical regions. The basal cytoplasm is dominated by highly developed
Seminal Vesicle of
Melanoplus sanguinipes
FI6. 1. Transverse section of a seminal vesicle from a newly emerged insect; toluidine blue. x 800. FIc. 2. Basal region of an epithelial cell from seminal vesicle of a newly emerged insect; black arrows indicate pinocytotic vesicles at basal plasma m e m b r a n e . × 29,100. FIG. 3. Intercellular space in the basal region of lateral cell junction, x 23,600. FI6. 4. Apical region of an epithelial cell from seminal vesicle of a newly emerged insect; white arrows indicate chains of vesicles associated with villus formation. × 34,900.
53
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GRAHAM A. COUCHE and CEDRIC GILLOTr
FIG. 5. Transverse section of a seminal vesicle from a 24-hr-old insect; toluidine blue. × 800. FIG, 6. Basal region of an epithelial cell from seminal vesicle of a 24-hr-old insect. × 33,400. FIG. 7. Apical region of an epithelial cell from seminal vesicle of a 24-hr-old insect; black arrows indicate possible coated vesicles. × 25,500.
Seminal Vesicle of
Melanoplussanguinipes
55
rough ER, whose cisternae may be formed into thin parallel or swollen vesicles of moderate size (Fig. 6). Golgi complexes are large and elaborate, containing vesicles, flattened saccules and occasional swollen but empty-looking vesicles. Mitochondria are numerous, especially near the basal plasma membrane, and small lysosomes are common throughout. Large glycogen deposits are common. The apical cytoplasm is dominated by large secretory and/or lysosomal vesicles (Fig. 7). These may contain widely dispersed flocculent material similar to that found in the lumen, or more densely packed coarse fibrous or granular material, membranous inclusions, and many small membrane-bounded vesicles reminiscent of coated vesicles. The cytoplasm surrounding the apical vesicles contains numerous smooth ER vesicles and free ribosomes. Rough ER is limited to a narrow zone near the nucleus. Small Golgi complexes and glycogen deposits, and numerous mitochondria near the apical plasma membrane are typical components of the apical cytoplasm. Differentiation of microvilli on the apical cell surface appears to be nearly complete. Chains of small vesicles within apical cytoplasmic projections as were seen in seminal vesicles from newly emerged insects are uncommon. The microvilli may be branched such as those described by Odhiambo (1969) from the seminal vesicle of Schistocerca gregaria. Seminal vesicles from 3-day-old insects have simple columnar to cuboidal epithelia of varied thickness (35-60 ~m). Toluidine blue-stained sections reveal intensely stained, irregularly shaped structures in the basal cytoplasm, central nuclei, and light- and darkstained apical vesicles. Although generally extensively developed, the lumen may occasionally be constricted (Fig. 8). Despite the limited lumen development in this region, however, the extent of gland maturation is indicated by the presence of spermatozoa. Ultrastructural features of seminal vesicle epithelial cells from 3-day-old insects differ in apical and basal regions. Numerous mitochondria near the basal plasma membrane, large Golgi complexes dominated by small vesicles, and occasional lysosomes are typical of the basal cytoplasm (Fig. 9). Membranes of the well-developed rough ER may be ordered into parallel arrays or more commonly occur in the swollen vesicular form previously described in seminal vesicles of 7-day-old insects (Couche, 1985; Couche and Gillott, unpublished). Intercellular spaces may be present in the mid-apical regions of lateral cell junctions. Major ultrastructural features of the apical cytoplasm are numerous large membranebounded vesicles, which contain coarse flocculent material and small dense inclusions that appear to be derived from coated vesicles (Fig. 10). These are presumed to be secretory vesicles as their contents are of similar appearance to that in the lumen. Other large vesicles contain finely particulate or fibrous material and sometimes membranous inclusions that resemble fragments of other cytoplasmic structures. As material similar to the contents of these vesicles is not found in the lumen, the vesicles are surmised to be lysosomes rather than secretory structures. Other cytoplasmic structures include numerous mitochondria, occasional small Golgi complexes and glycogen deposits, and vesicular ER, whose membranes are mostly free of ribosomes (Fig. 10). Small vesicles near the apical plasma membrane and chains of these vesicles in apical projections are uncommon in tissue from seminal vesicles of 3-day-old insects, which suggests that differentiation of microvilli is complete at this time.
Effects ofallatectomy. Toluidine blue-stained thick sections of seminal vesicles from 10
56
GRAHAM A. COUCHE and CEDRIC GILLOTT
FIG. 8. Transverse section of a seminal vesicle from a 3-day-old insect; toluidine blue. x 800. FIG. 9. Basal region of an adjacent epithelial cell from seminal vesicle of a 3-day-old insect. × 25,200. FI6. 10. Apical region of an epithelial cell from seminal vesicle of a 3-day-old insect; black arrows indicate possible coated vesicles, x 26,400.
Seminal Vesicle of Melanoplus sanguinipes
57
FIG. 11. Transverse section of a seminal vesicle from a 10-day-old sham-operated insect; toluidine blue. x 820. FIG. 12. Transverse section of a seminal vesicle from a 10-day-old allatectomized insect: toluidine blue. × 820. FIG. 13. Transverse section of a seminal vesicle from a 10-day-oldallatectomized insect, 8 days after injection with JH-III; toluidine blue. × 820. FIG. 14. Basal region of an epithelial cell from seminal vesicle of a 10-day-oldsham-operated insect. x 23,400. FIG. 15. Basal region of an epithelial cell from seminal vesicle of a 10-day-oldallatectomized insect. x 21,000.
day old s h a m - o p e r a t e d (Fig. 11), 10-day-old allatectomized (Fig. 12) a n d 10-day-old a l l a t e c t o m i z e d insects 8 days after i n j e c t i o n with l m g of J H - I I I (Fig. 13) differ little in a p p e a r a n c e . E p i t h e l i a are 45-60-p,m thick a n d c o m p o s e d of c o l u m n a r or c u b o i d a l cells. Lightly s t a i n e d nuclei with scattered c h r o m a t i n are centrally to apically located. Large light or dark vesicles characteristic of the apical cytoplasm of s e m i n a l vesicles from 3-day-
58
GRAHAMA. COUCHEand CEDRICGILLOTI"
old insects may be found in basal regions (Fig. 12; 13). The lumen is well developed and packed with spermatozoa. Electron micrographs confirm the similarity between seminal vesicle epithelia from sham-operated, allatectomized, and allatectomized but JH-treated insects. Characteristic basal ultrastructure includes swollen rough ER cisternae filled with coarse fibrous material, numerous small Golgi complexes and mitochondria, pinocytotic activity at the basal plasma membrane, and large glycogen deposits. The apical cytoplasm is dominated by large secretory and/or lysosomal vesicles. Examples of regions of basal cytoplasm from sham-operated and allatectomized insects are illustrated in Figs 14 and 15, respectively.
Proteins of the secretion Changes during sexual maturation. Samples of secretion from seminal vesicles of newly emerged insects contain little soluble protein. In Fig. 16, lane 2, a trace of protein is present at pH 6.33. IEF gels resolve 5-10 acidic protein bands from secretion samples of seminal vesicles from 24-hr-old insects (Fig. 16, lane 3). Minor bands are present at pH 6.33, 5.36 and 4.80. The remaining proteins are present in trace amounts. By 3 days of age, the secretory protein content of seminal vesicles has increased significantly. Seminal vesicle secretions electrofocus to yield major proteins at pH 6.33 and 4.80. Other bands include minor proteins with isoelectric points (pls) of 5.25, 5.23, 5.12, 4.98 and trace proteins with pls ranging from 5.65-4.75 (Fig. 16, lane 4). 1
2
3
4
5
6
3"5 4"5 ~
.~a
5.2
5"8
Q
,~b
,5
~
6"8
7"3
8"1 8" 4 8"6 9"3
. ---
FIG. 16. lsoelectric focusing profiles of proteins from secretions of a seminal vesicle at various times during maturation, and effect of allatectomy. Lane 1, pI marker proteins; lane 2, newly emerged insect; lane 3, 24-hr-old insect; lane 4, 3-day-old insect; lane 5, 10-day-old sham-operated insect; lane 6, 10-day-old allatectomized insect. Numbers to the left indicate approximate pl of marker proteins; arrow on lane 2 indicates trace band at pI 6.33; arrows on lane 6 (a and b) indicate major bands at pl 4.80 and 6.33, respectively; insoluble material at site of sample application (*).
Seminal Vesicle of
Melanoplus sanguinipes
59
Effects of allatectomy. Protein maps of seminal vesicle secretions from 10-day-old sham-operated and 10-day-old allatectomized insects are indistinguishable from those of 3-day-old (Fig. 16, lanes 4, 5 and 6), and 7-day-old insects (Couche, 1985). DISCUSSION In contrast to the long hyaline gland (Couche and Gillott, 1987) differentiation of the lumen and cytoplasmic structures is well advanced in seminal vesicles of newly emerged insects. The relatively high degree of gland development and the presence of secretion in the lumen suggest that limited secretory activity is occurring at this time. IEF analysis of proteins from the lumen confirms the presence of secretory activity in seminal vesicles of newly emerged insects. Further differentiation of seminal vesicles is rapid for at least 24 hr, at which time the epithelial cells appear structurally capable of extensive secretory activity. The density of ribosomes, extent of rough ER proliferation, and elaborate Golgi complexes contrast with their counterparts in epithelia of seminal vesicles for 7-day-old insects (Couche, 1985; Couche and Gillott, unpublished) or 10-day sham-operated insects where ultrastructural features indicate minimal metabolic activity. Analyses of secretory proteins demonstrate a quantitative deficiency, but no qualitative differences in seminal vesicle secretions of 24-hr-old insects as compared to those from 3-day or older insects. By day 3, however, vesicularisation of the rough ER and reductions in Golgi size and complexity suggest that seminal vesicles at this time are relatively less active than those of 24-hr insects. Qualitative and quantitative similarities between secretions of seminal vesicles from day-3 and day-10 sham-operated insects indicate that these glands are functionally mature as early as 3 days after final ecdysis. Such rapid maturation is to be expected, as sperm descent from the testes can occur at this time (Fig. 8). Similarly, rapid seminal vesicle maturation has been reported to occur in S. gregaria (Odhiambo, 1971). Venkatesh and Gillott (1983) observed a continuous increase in protein content of the seminal vesicle of M. sanguinipes up to 10 days postemergence. The present isoelectric focusing study suggests little difference between protein quantities at 3 days and those at 10 days postemergence. This apparent discrepancy may be explained by the different methods used for analysis. Venkatesh and Gillott (1983) measured the entire protein content of the seminal vesicle (including tissue and secretion) whereas for the present isoelectric focusing study, only distilled water-soluble secretory proteins oozing from the cut gland were extracted. Thus, the discrepancy may reflect an increasing proportion of insoluble protein in the seminal vesicle secretion as insects age. Of the 16 accessory gland tubules, only the seminal vesicle of mature insects displays morphological features compatible with mechanisms for uptake of extraglandular proteins (Couche, 1985; Couche and Gillott, unpublished). The present study provides evidence for both extracellular accumulation, via intercellular channels in the seminal vesicle epithelium, and intracellular uptake (pinocytosis at the basal plasma membrane). Intracellular processing of materials derived from the hemolymph has been suggested previously (Cantacuz~ne, 1972) to explain apparent accumulation of coated vesicles in large apical vacuoles common in epithelial cells from the seminal vesicle of Locusta
migratoria. Ultrastructurai studies of insect accessory glands during imaginal maturation are limited to reports on gland 1 and the seminal vesicle ofS. gregaria (Odhiambo, 1971), the
6(1
GRAHAMA. COUCHEand CEDRICGILLO11"
tubular gland of Tenebrio molitor (Gadzama et al., 1977), the D-tubules of Acheta domesticus (Kaulenas et al., 1979), and the accessory gland of Drosophila funebris (Federer and Chen, 1982). In each of these studies, as is the case in long hyaline gland and seminal vesicle of M. sanguinipes, proliferation of ultrastructural features associated with a presumptive secretory function is a rapid process initiated at or shortly after the imaginal moult and completed by 3 days postemergence. However, quantitative assessments of gland maturation based on general features such as protein content, wet or dry weight, and rate of incorporation of radiolabelled amino acids, indicate that growth continues after maximal ultrastructural differentiation has been attained. Such growth is thought to reflect synthesis and storage of secretions within the gland (Odhiambo, 1966; 1971; Kaulenas et al., 1975; 1979; Happ et al., 1977; Gillott and Friedel, 1976; Gillott and Venkatesh, 1985). Thus, maturation of the accessory glands in M. sanguinipes and other insects exhibits characteristics of terminally differentiating systems, which typically involve programmed differentiation, culminating in massive synthesis of cell-specific proteins (Kafatos, 1972). In the accessory glands, however, this system has been modified such that the secretion may occur concurrently with or following differentiation, depending on the gland and the component of the secretion in question (Happ et al., 1977; Kaulenas et al., 1979; Gillott and Venkatesh, 1985). The seminal vesicle of M. sanguinipes and S. gregaria (Odhiambo, 1971) appears to represent a highly modified system in which the terminal differentiation state is associated with cessation of secretory activity. Although JH has been implicated in the control of adult male accessory gland maturation in M. sanguinipes (Gillott and Friedel, 1976) and many other species (Chen, 1984), this conclusion is based entirely on in vivo biochemical comparisons of whole glands from normal and allatectomized insects. Until recently, a precise role for JH has remained unclear. Ultrastructural studies of changes occurring during sexual maturation (Odhiambo, 1971; De Loof and Lagasse, 1972; Couche and Gillott, 1987; present observations) show that differentiation of accessory gland cells occurs during the first few days of adult life, a period when JH biosynthesis is known to be minimal (Avruch and Tobe, 1978; Couche et al., 1985). This is confirmed by observations of allatectomized S. gregaria (Odhiambo, 1966) and M. sanguinipes where accessory gland differentiation occurs in a manner similar to that of normal insects. Our studies (Couche and Gillott, 1987; present observations) show that the action of JH is tissue-specific, affecting the long hyaline gland but not the seminal vesicle. JH seems to be almost without effect, either qualitative or quantitative, on the production of secretion in the seminal vesicle (Venkatesh and Gillott, 1983; Lange et al., 1983; Giliott and Venkatesh, 1985; present observations). In contrast, as first indicated by Friedel and Gillott (1976), in the long hyaline gland, JH promotes the synthesis of specific secretory proteins thus having a role comparable to that of promoting vitellogenin synthesis in many female insects. Acknowledgements--This work was supported by Natural Sciences and Engineering Research Council of Canada grants to C. Gillott and University of Saskatchewan graduate scholarships to G. A. Couche.
REFERENCES AVRUCH, L. I. and S. S. TOBE. 1978. Juvenile hormone biosynthesis by the corpora allata of the male desert locust, Schistocerca gregaria during sexual maturation. Can. J. Zool. 56: 2097-2102.
Seminal Vesicle of Melanoplus sanguinipes
61
CANTACUZI~NE,A. M. 1967. Histologie des glandes annexes mfiles de Schistocerca gregaria F. (Orthopt~re). Effet de l'allatectomie sur leur activit6. C. R. Acad. Sci. Paris Sdr. D. 264: 93-96. CANTACUZ~NE,A. M. 1972. Recherches morphologiques et physiologiques sur les glandes annexes mfiles des Orthopt~res. IV. Ultrastructure de la v6sicule s6minale de Locusta migratoria migratorioides L. Ann. Sci. Nat. Zool. Paris. 14: 389-410. CrtEN, P. S. 1984. The functional morphology and biochemistry of insect male accessory glands and their secretions. Annu. Rev. Entomol. 29: 233-55. COUCHE, G. A. 1985. Accessory reproductive glands in male Melanoplus sanguinipes (Fabr.): structure and the influence of juvenile hormone during maturation of selected glands. Ph.D. thesis, University of Saskatchewan. COUCHE,G. A. and C. GILLOTT. 1987. Development of secretory activity in the long hyaline gland of the male migratory grasshopper, Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae). Int. J. Insect Morphol. EmbryoL 16: 355-69. COUCHE, G. A., C. GILLOTr, S. S. TOaE and R. FEYEREISEN. 1985. Juvenile hormone biosynthesis during sexual maturation and after mating in the adult male migratory grasshopper, Melanoplus sanguinipes. Can. J. Zool. 63: 2789-92. DE LooF, A. and A. LAGASSE. 1972. The ultrastructure of the male accessory reproductive glands of the Colorado potato beetle. Z. Zellforsch. 130: 545-52. FEDERER, H. and P. S. CHEN. 1982. Ultrastructure and nature of secretory proteins in the male accessory gland of Drosophila funebris. J. Insect Physiol. 28: 743-51. FRIEDEL, T. and C. GILLOTr. 1976. Male accessory gland substance of Melanoplus sanguinipes: an oviposition stimulant under the control of the corpus allatum. J. Insect Physiol. 22: 489-95. GADZAMA, N., C. M. HAPP and G. M. HAPP. 1977. Cytodifferentiation in the accessory glands of Tenebrio molitor, I. Ultrastructure of the tubular gland in the post-ecdysial adult male. J. Exp. Zool. 200: 211-22. GILLOTr, C. and T. FRIEDEL. 1976. Development of accessory reproductive glands and its control by the corpus allatum in adult male Melanoplus sanguinipes. J. Insect Physiol. 22: 365-72. GILLOTT, C. and K. VENKATESH. 1985. Accumulation of secretory proteins in the accessory reproductive glands of the male migratory grasshopper, Melanoplus sanguinipes: a developmental study. J. Insect Physiol, 31: 195-204. GIRARDIE, A. and A. VOGEL. 1966. t~tude du contr61e neuro-humoral de l'activit6 sexuelle mhle de Locusta migratoria (L.) C. R. Acad. Sci. Paris Sdr. D. 263: 543-46. HAPP, G. M., C. YUNKER, and S. A. HUFFMIRE. 1977. Cytodifferentiation in the accessory glands of Tenebrio molitor. II. Patterns of leucine incorporation in the tubular glands of post-ecdysial adult males. J. Exp. Zool. 200: 223-36. KAFAIOS,F. C. 1972. The cocoonase zymogen cells of silk moths: a model of terminal cell differentiation for specific protein synthesis. Curt. Top. Dev. Biol. 1: 125-91. KAULENAS, M. S., R. L. YENOFSKY, H. E. POTSWALD and A. L. BURNS. 1975. Protein synthesis by the accessory gland of the male house cricket, Acheta domesticus. J. Exp. Zool. 193: 21-36. KAULENAS,M. S., H. E. POTSWALD,A. L. BURNSand R. L. YENOFSKY. 1979. Development of structural and functional specialisations for export protein synthesis by the accessory gland of the male cricket, Acheta domesticus. Int. J. Insect Morphol. Embrvol. 8: 3349. LANGE, A. B., D. R. PHILLIPS and B. G. LOUGrfTON. 1983. The effects of precocene II on early adult development in male Locusta. J. Insect Physiol. 29: 73-81. ODHIAMBO, T. R. 1966. Growth and the hormonal control of sexual maturation in the male desert locust, Schistocerca gregaria (Forskhl). Trans. R. Entomol. Soc. Lond. ll8: 393412. ODHIAMBO, T. R. 1969. The architecture of the accessory reproductive glands of the desert locust. IV. Fine structure of the glandular epithelium. Philos. Trans. R. Soc. Set. B. 256: 85-114. OOHiAMBO, T. R. 1971. The architecture of the accessory reproductive glands of the male desert locust. V. UItrastructure during maturation. Tissue Cell 3: 309-24. PICKFORD, R. 1958. Observations on the reproductive potential of Melanoplus bilituratus (Orthoptera : Acrididae) reared on different food plants in the laboratory. Can. Entomol. 90: 483-85. PICKFORO, R. and R. L. RANDELL. 1969. A non-diapause strain of the migratory grasshopper, Melanoplus sanguinipes (Orthoptera : Acrididae). Can. Entomol. 101: 894-96. VENKATESH, K. and C. GILLOTT. 1983. Protein production in components of the accessory gland complex of male Melanoplus sanguinipes. Int. J. lnvertebr. Reprod. 6: 317--25.
0020-7322/88 $3.0(I + .00 © 1988 Pergamon Press plc
Printed in Great Britain
DEVELOPMENT OF SECRETORY ACTIVITY IN THE SEMINAL VESICLE OF THE MALE MIGRATORY GRASSHOPPER, M E L A N O P L U S SANGUINIPES (FABR.) (ORTHOPTERA • ACRIDIDAE)
GRAHAM A . COUCHE a n d CEDRIC GILLOTT* Department of Biology, University of Saskatchewan, Saskatoon, SK, S7N OWO Canada
(Accepted 15 July 1987)
Abstract--This paper describes the ultrastructure of the seminal vesicle and the isoelectric focusing patterns of its secretion during sexual maturation and after allatectomy in Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae). In epithelia from seminal vesicles of newly fledged males, the rough endoplasmic reticulum is well developed, and Golgi complexes are elaborate, which indicates the gland is metabolically active. The cells also contain large glycogen deposits and the lumen microvilli are well differentiated. These ultrastructural features are more dominant in 24-hr-old adults where the cytoplasm is clearly differentiated into basal and apical regions. Basally, the cytoplasm is dominated by rough endoplasmic reticulum, large Golgi complexes, glycogen deposits and numerous mitochondria, while the apical cytoplasm is filled with large secretory and/or lysosomal vesicles. Between days 3 and 7, the ultrastructural features change little other than the rough endoplasmic reticulum cisternae, which become vesicular. Analysis by isoelectric focusing shows that the amount of secretory protein increases with age until day 3, at which time the gland contains its full complement of secretion. In seminal vesicles from allatectomized insects, ultrastructural features of cells and i8oelectric focusing patterns of the secretion are identical to those from normal males.
Index descriptors (in addition to those in title): Ultrastructure, isoelectric focusing, protein synthesis.
INTRODUCTION THE SEMINAL v e s i c l e in Melanoplus sanguinipes is a m o d i f i e d a c c e s s o r y g l a n d t u b u l e t h a t is r e a d i l y i d e n t i f i a b l e e v e n in n e w l y e m e r g e d adults. P r e v i o u s b i o c h e m i c a l s t u d i e s o f t h e a c c e s s o r y g l a n d s o f M. sanguinipes ( V e n k a t e s h a n d G i l l o t t , 1983; G i l l o t t a n d V e n k a t e s h , 1985), as w e l l as h i s t o l o g i c a l ( G i r a r d i e a n d V o g e l , 1966; C a n t a c u z G n e , 1967) a n d u l t r a s t r u c t u r a l ( O d h i a m b o , 1971) i n v e s t i g a t i o n s w i t h o t h e r A c r i d i d a e , h a v e i n d i c a t e d
* Author to whom reprint requests should be sent. Abbreviations used in Figures: G = Golgi complex; GI = glycogen deposit; N = nucleus; RER = rough endoplasmic reticulum; BL = basal lamina; L = lysosome: is = intercellular space; Lu = lumen; Mv = microvilli; SV = secretory vesicle. 51
52
GRAHAMA. COUCHEand CEDRICGILLOTT
that juvenile hormone (JH) has little influence on maturation (viz. secretory activity) of the seminal vesicle. This contrasts with the prominent role of JH in the control of secretory activity in the long hyaline gland and other accessory gland tubules (Venkatesh and Gillott, 1983; Gillott and Venkatesh, 1985; Couche and Gillott, 1987). This paper describes the ultrastructure of the seminal vesicle and the isoelectric focusing (IEF) patterns of its secretion, during maturation and after allatectomy in M. sanguinipes. By comparing our observations on the long hyaline gland (Couche and Gillot, 1987) with those of the seminal vesicle, now reported, we hope to obtain some insight into the site and mode of action of JH in the reproductive physiology of male insects.
MATERIALS AND METHODS A non-diapause strain M. sanguinipes(Pickford and Randell, 1969)was used. Males were separated from a stock culture as 5th instar nymphs and reared in isolation from females in standard cages (Pickford, 1958). Fresh barley leaves and bran were fed ad libitum. Allatectomyor sham-operationswere carried out between 12 and 24 hr postemergence as described by Gillon and Friedel (1976). After a 24 hr postoperative recovery period, allatectomizedmales were injected with juvenile hormone III (ling in 3.5 pJ paraffin oil) or paraffin oil alone. At least 10 males were sampled in each treatment group. Procedures for seminal vesicledissection, tissue preparation for light and electron microscopy,and secretory protein analysis by IEF are described in an accompanyingpaper (Couche and Gillott, 1987).
RESULTS Morphology Changes during sexual maturation. At adult emergence, the seminal vesicle has a 35-40-txm-thick columnar epithelium (Fig. 1). Cell nuclei are large and may be located basally or apically. Microvilli of the apical plasma membrane and the gland lumen are moderately well developed. Mitochondria are large and numerous, particularly in the basal cytoplasm (Fig. 2). The endoplasmic reticulum (ER) shows considerable development both as elongated rough E R cisternae and small E R vesicles. Numerous ribosomes may be found in association with rough E R or free in the cytoplasm. Large glycogen deposits and elaborate Golgi complexes are characteristic of the basal cytoplasm (Fig. 2). Lysosomes are scattered throughout. Swollen, perhaps lysosomal, vesicles are occasionally present. Intercellular spaces containing a coarse fibrous material are common in the basal regions of the lateral junctional complex (Fig. 3). Differentiation of microvilli of the apical cell surface is relatively advanced (Fig. 4). Small cytoplasmic vesicles close to the apical plasma membrane and chains of similar vesicles within bulbous apical cytoplasmic projections suggest that differentiation of microvilli involves splitting of apical projections by ordered linear fusion of chains of small vesicles. The lumen of the seminal vesicle contains a widely spread fibrous material (Fig. 4). The seminal vesicle of males 24 hr postemergence has a 35-60-p~m-thick simple columnar epithelium surrounding an empty-looking lumen, whose development is varied along the length of the gland. Central to apical nuclei, intercellular spaces, densely basophilic structures and empty-looking apical vesicles are readily observed in the light microscope (Fig. 5). As has been observed in seminal vesicles from mature insects (Couche, 1985; Couche and Gillott, unpublished), ultrastructural characteristics of the epithelial cells differ between basal and apical regions. The basal cytoplasm is dominated by highly developed
Seminal Vesicle of
Melanoplus sanguinipes
FI6. 1. Transverse section of a seminal vesicle from a newly emerged insect; toluidine blue. x 800. FIc. 2. Basal region of an epithelial cell from seminal vesicle of a newly emerged insect; black arrows indicate pinocytotic vesicles at basal plasma m e m b r a n e . × 29,100. FIG. 3. Intercellular space in the basal region of lateral cell junction, x 23,600. FI6. 4. Apical region of an epithelial cell from seminal vesicle of a newly emerged insect; white arrows indicate chains of vesicles associated with villus formation. × 34,900.
53
54
GRAHAM A. COUCHE and CEDRIC GILLOTr
FIG. 5. Transverse section of a seminal vesicle from a 24-hr-old insect; toluidine blue. × 800. FIG, 6. Basal region of an epithelial cell from seminal vesicle of a 24-hr-old insect. × 33,400. FIG. 7. Apical region of an epithelial cell from seminal vesicle of a 24-hr-old insect; black arrows indicate possible coated vesicles. × 25,500.
Seminal Vesicle of
Melanoplussanguinipes
55
rough ER, whose cisternae may be formed into thin parallel or swollen vesicles of moderate size (Fig. 6). Golgi complexes are large and elaborate, containing vesicles, flattened saccules and occasional swollen but empty-looking vesicles. Mitochondria are numerous, especially near the basal plasma membrane, and small lysosomes are common throughout. Large glycogen deposits are common. The apical cytoplasm is dominated by large secretory and/or lysosomal vesicles (Fig. 7). These may contain widely dispersed flocculent material similar to that found in the lumen, or more densely packed coarse fibrous or granular material, membranous inclusions, and many small membrane-bounded vesicles reminiscent of coated vesicles. The cytoplasm surrounding the apical vesicles contains numerous smooth ER vesicles and free ribosomes. Rough ER is limited to a narrow zone near the nucleus. Small Golgi complexes and glycogen deposits, and numerous mitochondria near the apical plasma membrane are typical components of the apical cytoplasm. Differentiation of microvilli on the apical cell surface appears to be nearly complete. Chains of small vesicles within apical cytoplasmic projections as were seen in seminal vesicles from newly emerged insects are uncommon. The microvilli may be branched such as those described by Odhiambo (1969) from the seminal vesicle of Schistocerca gregaria. Seminal vesicles from 3-day-old insects have simple columnar to cuboidal epithelia of varied thickness (35-60 ~m). Toluidine blue-stained sections reveal intensely stained, irregularly shaped structures in the basal cytoplasm, central nuclei, and light- and darkstained apical vesicles. Although generally extensively developed, the lumen may occasionally be constricted (Fig. 8). Despite the limited lumen development in this region, however, the extent of gland maturation is indicated by the presence of spermatozoa. Ultrastructural features of seminal vesicle epithelial cells from 3-day-old insects differ in apical and basal regions. Numerous mitochondria near the basal plasma membrane, large Golgi complexes dominated by small vesicles, and occasional lysosomes are typical of the basal cytoplasm (Fig. 9). Membranes of the well-developed rough ER may be ordered into parallel arrays or more commonly occur in the swollen vesicular form previously described in seminal vesicles of 7-day-old insects (Couche, 1985; Couche and Gillott, unpublished). Intercellular spaces may be present in the mid-apical regions of lateral cell junctions. Major ultrastructural features of the apical cytoplasm are numerous large membranebounded vesicles, which contain coarse flocculent material and small dense inclusions that appear to be derived from coated vesicles (Fig. 10). These are presumed to be secretory vesicles as their contents are of similar appearance to that in the lumen. Other large vesicles contain finely particulate or fibrous material and sometimes membranous inclusions that resemble fragments of other cytoplasmic structures. As material similar to the contents of these vesicles is not found in the lumen, the vesicles are surmised to be lysosomes rather than secretory structures. Other cytoplasmic structures include numerous mitochondria, occasional small Golgi complexes and glycogen deposits, and vesicular ER, whose membranes are mostly free of ribosomes (Fig. 10). Small vesicles near the apical plasma membrane and chains of these vesicles in apical projections are uncommon in tissue from seminal vesicles of 3-day-old insects, which suggests that differentiation of microvilli is complete at this time.
Effects ofallatectomy. Toluidine blue-stained thick sections of seminal vesicles from 10
56
GRAHAM A. COUCHE and CEDRIC GILLOTT
FIG. 8. Transverse section of a seminal vesicle from a 3-day-old insect; toluidine blue. x 800. FIG. 9. Basal region of an adjacent epithelial cell from seminal vesicle of a 3-day-old insect. × 25,200. FI6. 10. Apical region of an epithelial cell from seminal vesicle of a 3-day-old insect; black arrows indicate possible coated vesicles, x 26,400.
Seminal Vesicle of Melanoplus sanguinipes
57
FIG. 11. Transverse section of a seminal vesicle from a 10-day-old sham-operated insect; toluidine blue. x 820. FIG. 12. Transverse section of a seminal vesicle from a 10-day-old allatectomized insect: toluidine blue. × 820. FIG. 13. Transverse section of a seminal vesicle from a 10-day-oldallatectomized insect, 8 days after injection with JH-III; toluidine blue. × 820. FIG. 14. Basal region of an epithelial cell from seminal vesicle of a 10-day-oldsham-operated insect. x 23,400. FIG. 15. Basal region of an epithelial cell from seminal vesicle of a 10-day-oldallatectomized insect. x 21,000.
day old s h a m - o p e r a t e d (Fig. 11), 10-day-old allatectomized (Fig. 12) a n d 10-day-old a l l a t e c t o m i z e d insects 8 days after i n j e c t i o n with l m g of J H - I I I (Fig. 13) differ little in a p p e a r a n c e . E p i t h e l i a are 45-60-p,m thick a n d c o m p o s e d of c o l u m n a r or c u b o i d a l cells. Lightly s t a i n e d nuclei with scattered c h r o m a t i n are centrally to apically located. Large light or dark vesicles characteristic of the apical cytoplasm of s e m i n a l vesicles from 3-day-
58
GRAHAMA. COUCHEand CEDRICGILLOTI"
old insects may be found in basal regions (Fig. 12; 13). The lumen is well developed and packed with spermatozoa. Electron micrographs confirm the similarity between seminal vesicle epithelia from sham-operated, allatectomized, and allatectomized but JH-treated insects. Characteristic basal ultrastructure includes swollen rough ER cisternae filled with coarse fibrous material, numerous small Golgi complexes and mitochondria, pinocytotic activity at the basal plasma membrane, and large glycogen deposits. The apical cytoplasm is dominated by large secretory and/or lysosomal vesicles. Examples of regions of basal cytoplasm from sham-operated and allatectomized insects are illustrated in Figs 14 and 15, respectively.
Proteins of the secretion Changes during sexual maturation. Samples of secretion from seminal vesicles of newly emerged insects contain little soluble protein. In Fig. 16, lane 2, a trace of protein is present at pH 6.33. IEF gels resolve 5-10 acidic protein bands from secretion samples of seminal vesicles from 24-hr-old insects (Fig. 16, lane 3). Minor bands are present at pH 6.33, 5.36 and 4.80. The remaining proteins are present in trace amounts. By 3 days of age, the secretory protein content of seminal vesicles has increased significantly. Seminal vesicle secretions electrofocus to yield major proteins at pH 6.33 and 4.80. Other bands include minor proteins with isoelectric points (pls) of 5.25, 5.23, 5.12, 4.98 and trace proteins with pls ranging from 5.65-4.75 (Fig. 16, lane 4). 1
2
3
4
5
6
3"5 4"5 ~
.~a
5.2
5"8
Q
,~b
,5
~
6"8
7"3
8"1 8" 4 8"6 9"3
. ---
FIG. 16. lsoelectric focusing profiles of proteins from secretions of a seminal vesicle at various times during maturation, and effect of allatectomy. Lane 1, pI marker proteins; lane 2, newly emerged insect; lane 3, 24-hr-old insect; lane 4, 3-day-old insect; lane 5, 10-day-old sham-operated insect; lane 6, 10-day-old allatectomized insect. Numbers to the left indicate approximate pl of marker proteins; arrow on lane 2 indicates trace band at pI 6.33; arrows on lane 6 (a and b) indicate major bands at pl 4.80 and 6.33, respectively; insoluble material at site of sample application (*).
Seminal Vesicle of
Melanoplus sanguinipes
59
Effects of allatectomy. Protein maps of seminal vesicle secretions from 10-day-old sham-operated and 10-day-old allatectomized insects are indistinguishable from those of 3-day-old (Fig. 16, lanes 4, 5 and 6), and 7-day-old insects (Couche, 1985). DISCUSSION In contrast to the long hyaline gland (Couche and Gillott, 1987) differentiation of the lumen and cytoplasmic structures is well advanced in seminal vesicles of newly emerged insects. The relatively high degree of gland development and the presence of secretion in the lumen suggest that limited secretory activity is occurring at this time. IEF analysis of proteins from the lumen confirms the presence of secretory activity in seminal vesicles of newly emerged insects. Further differentiation of seminal vesicles is rapid for at least 24 hr, at which time the epithelial cells appear structurally capable of extensive secretory activity. The density of ribosomes, extent of rough ER proliferation, and elaborate Golgi complexes contrast with their counterparts in epithelia of seminal vesicles for 7-day-old insects (Couche, 1985; Couche and Gillott, unpublished) or 10-day sham-operated insects where ultrastructural features indicate minimal metabolic activity. Analyses of secretory proteins demonstrate a quantitative deficiency, but no qualitative differences in seminal vesicle secretions of 24-hr-old insects as compared to those from 3-day or older insects. By day 3, however, vesicularisation of the rough ER and reductions in Golgi size and complexity suggest that seminal vesicles at this time are relatively less active than those of 24-hr insects. Qualitative and quantitative similarities between secretions of seminal vesicles from day-3 and day-10 sham-operated insects indicate that these glands are functionally mature as early as 3 days after final ecdysis. Such rapid maturation is to be expected, as sperm descent from the testes can occur at this time (Fig. 8). Similarly, rapid seminal vesicle maturation has been reported to occur in S. gregaria (Odhiambo, 1971). Venkatesh and Gillott (1983) observed a continuous increase in protein content of the seminal vesicle of M. sanguinipes up to 10 days postemergence. The present isoelectric focusing study suggests little difference between protein quantities at 3 days and those at 10 days postemergence. This apparent discrepancy may be explained by the different methods used for analysis. Venkatesh and Gillott (1983) measured the entire protein content of the seminal vesicle (including tissue and secretion) whereas for the present isoelectric focusing study, only distilled water-soluble secretory proteins oozing from the cut gland were extracted. Thus, the discrepancy may reflect an increasing proportion of insoluble protein in the seminal vesicle secretion as insects age. Of the 16 accessory gland tubules, only the seminal vesicle of mature insects displays morphological features compatible with mechanisms for uptake of extraglandular proteins (Couche, 1985; Couche and Gillott, unpublished). The present study provides evidence for both extracellular accumulation, via intercellular channels in the seminal vesicle epithelium, and intracellular uptake (pinocytosis at the basal plasma membrane). Intracellular processing of materials derived from the hemolymph has been suggested previously (Cantacuz~ne, 1972) to explain apparent accumulation of coated vesicles in large apical vacuoles common in epithelial cells from the seminal vesicle of Locusta
migratoria. Ultrastructurai studies of insect accessory glands during imaginal maturation are limited to reports on gland 1 and the seminal vesicle ofS. gregaria (Odhiambo, 1971), the
6(1
GRAHAMA. COUCHEand CEDRICGILLO11"
tubular gland of Tenebrio molitor (Gadzama et al., 1977), the D-tubules of Acheta domesticus (Kaulenas et al., 1979), and the accessory gland of Drosophila funebris (Federer and Chen, 1982). In each of these studies, as is the case in long hyaline gland and seminal vesicle of M. sanguinipes, proliferation of ultrastructural features associated with a presumptive secretory function is a rapid process initiated at or shortly after the imaginal moult and completed by 3 days postemergence. However, quantitative assessments of gland maturation based on general features such as protein content, wet or dry weight, and rate of incorporation of radiolabelled amino acids, indicate that growth continues after maximal ultrastructural differentiation has been attained. Such growth is thought to reflect synthesis and storage of secretions within the gland (Odhiambo, 1966; 1971; Kaulenas et al., 1975; 1979; Happ et al., 1977; Gillott and Friedel, 1976; Gillott and Venkatesh, 1985). Thus, maturation of the accessory glands in M. sanguinipes and other insects exhibits characteristics of terminally differentiating systems, which typically involve programmed differentiation, culminating in massive synthesis of cell-specific proteins (Kafatos, 1972). In the accessory glands, however, this system has been modified such that the secretion may occur concurrently with or following differentiation, depending on the gland and the component of the secretion in question (Happ et al., 1977; Kaulenas et al., 1979; Gillott and Venkatesh, 1985). The seminal vesicle of M. sanguinipes and S. gregaria (Odhiambo, 1971) appears to represent a highly modified system in which the terminal differentiation state is associated with cessation of secretory activity. Although JH has been implicated in the control of adult male accessory gland maturation in M. sanguinipes (Gillott and Friedel, 1976) and many other species (Chen, 1984), this conclusion is based entirely on in vivo biochemical comparisons of whole glands from normal and allatectomized insects. Until recently, a precise role for JH has remained unclear. Ultrastructural studies of changes occurring during sexual maturation (Odhiambo, 1971; De Loof and Lagasse, 1972; Couche and Gillott, 1987; present observations) show that differentiation of accessory gland cells occurs during the first few days of adult life, a period when JH biosynthesis is known to be minimal (Avruch and Tobe, 1978; Couche et al., 1985). This is confirmed by observations of allatectomized S. gregaria (Odhiambo, 1966) and M. sanguinipes where accessory gland differentiation occurs in a manner similar to that of normal insects. Our studies (Couche and Gillott, 1987; present observations) show that the action of JH is tissue-specific, affecting the long hyaline gland but not the seminal vesicle. JH seems to be almost without effect, either qualitative or quantitative, on the production of secretion in the seminal vesicle (Venkatesh and Gillott, 1983; Lange et al., 1983; Giliott and Venkatesh, 1985; present observations). In contrast, as first indicated by Friedel and Gillott (1976), in the long hyaline gland, JH promotes the synthesis of specific secretory proteins thus having a role comparable to that of promoting vitellogenin synthesis in many female insects. Acknowledgements--This work was supported by Natural Sciences and Engineering Research Council of Canada grants to C. Gillott and University of Saskatchewan graduate scholarships to G. A. Couche.
REFERENCES AVRUCH, L. I. and S. S. TOBE. 1978. Juvenile hormone biosynthesis by the corpora allata of the male desert locust, Schistocerca gregaria during sexual maturation. Can. J. Zool. 56: 2097-2102.
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61
CANTACUZI~NE,A. M. 1967. Histologie des glandes annexes mfiles de Schistocerca gregaria F. (Orthopt~re). Effet de l'allatectomie sur leur activit6. C. R. Acad. Sci. Paris Sdr. D. 264: 93-96. CANTACUZ~NE,A. M. 1972. Recherches morphologiques et physiologiques sur les glandes annexes mfiles des Orthopt~res. IV. Ultrastructure de la v6sicule s6minale de Locusta migratoria migratorioides L. Ann. Sci. Nat. Zool. Paris. 14: 389-410. CrtEN, P. S. 1984. The functional morphology and biochemistry of insect male accessory glands and their secretions. Annu. Rev. Entomol. 29: 233-55. COUCHE, G. A. 1985. Accessory reproductive glands in male Melanoplus sanguinipes (Fabr.): structure and the influence of juvenile hormone during maturation of selected glands. Ph.D. thesis, University of Saskatchewan. COUCHE,G. A. and C. GILLOTT. 1987. Development of secretory activity in the long hyaline gland of the male migratory grasshopper, Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae). Int. J. Insect Morphol. EmbryoL 16: 355-69. COUCHE, G. A., C. GILLOTr, S. S. TOaE and R. FEYEREISEN. 1985. Juvenile hormone biosynthesis during sexual maturation and after mating in the adult male migratory grasshopper, Melanoplus sanguinipes. Can. J. Zool. 63: 2789-92. DE LooF, A. and A. LAGASSE. 1972. The ultrastructure of the male accessory reproductive glands of the Colorado potato beetle. Z. Zellforsch. 130: 545-52. FEDERER, H. and P. S. CHEN. 1982. Ultrastructure and nature of secretory proteins in the male accessory gland of Drosophila funebris. J. Insect Physiol. 28: 743-51. FRIEDEL, T. and C. GILLOTr. 1976. Male accessory gland substance of Melanoplus sanguinipes: an oviposition stimulant under the control of the corpus allatum. J. Insect Physiol. 22: 489-95. GADZAMA, N., C. M. HAPP and G. M. HAPP. 1977. Cytodifferentiation in the accessory glands of Tenebrio molitor, I. Ultrastructure of the tubular gland in the post-ecdysial adult male. J. Exp. Zool. 200: 211-22. GILLOTr, C. and T. FRIEDEL. 1976. Development of accessory reproductive glands and its control by the corpus allatum in adult male Melanoplus sanguinipes. J. Insect Physiol. 22: 365-72. GILLOTT, C. and K. VENKATESH. 1985. Accumulation of secretory proteins in the accessory reproductive glands of the male migratory grasshopper, Melanoplus sanguinipes: a developmental study. J. Insect Physiol, 31: 195-204. GIRARDIE, A. and A. VOGEL. 1966. t~tude du contr61e neuro-humoral de l'activit6 sexuelle mhle de Locusta migratoria (L.) C. R. Acad. Sci. Paris Sdr. D. 263: 543-46. HAPP, G. M., C. YUNKER, and S. A. HUFFMIRE. 1977. Cytodifferentiation in the accessory glands of Tenebrio molitor. II. Patterns of leucine incorporation in the tubular glands of post-ecdysial adult males. J. Exp. Zool. 200: 223-36. KAFAIOS,F. C. 1972. The cocoonase zymogen cells of silk moths: a model of terminal cell differentiation for specific protein synthesis. Curt. Top. Dev. Biol. 1: 125-91. KAULENAS, M. S., R. L. YENOFSKY, H. E. POTSWALD and A. L. BURNS. 1975. Protein synthesis by the accessory gland of the male house cricket, Acheta domesticus. J. Exp. Zool. 193: 21-36. KAULENAS,M. S., H. E. POTSWALD,A. L. BURNSand R. L. YENOFSKY. 1979. Development of structural and functional specialisations for export protein synthesis by the accessory gland of the male cricket, Acheta domesticus. Int. J. Insect Morphol. Embrvol. 8: 3349. LANGE, A. B., D. R. PHILLIPS and B. G. LOUGrfTON. 1983. The effects of precocene II on early adult development in male Locusta. J. Insect Physiol. 29: 73-81. ODHIAMBO, T. R. 1966. Growth and the hormonal control of sexual maturation in the male desert locust, Schistocerca gregaria (Forskhl). Trans. R. Entomol. Soc. Lond. ll8: 393412. ODHIAMBO, T. R. 1969. The architecture of the accessory reproductive glands of the desert locust. IV. Fine structure of the glandular epithelium. Philos. Trans. R. Soc. Set. B. 256: 85-114. OOHiAMBO, T. R. 1971. The architecture of the accessory reproductive glands of the male desert locust. V. UItrastructure during maturation. Tissue Cell 3: 309-24. PICKFORD, R. 1958. Observations on the reproductive potential of Melanoplus bilituratus (Orthoptera : Acrididae) reared on different food plants in the laboratory. Can. Entomol. 90: 483-85. PICKFORO, R. and R. L. RANDELL. 1969. A non-diapause strain of the migratory grasshopper, Melanoplus sanguinipes (Orthoptera : Acrididae). Can. Entomol. 101: 894-96. VENKATESH, K. and C. GILLOTT. 1983. Protein production in components of the accessory gland complex of male Melanoplus sanguinipes. Int. J. lnvertebr. Reprod. 6: 317--25.