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Secretion in the male accessory glands of Aedes aegypti (L.) (Diptera : Culicidae)
Int. J. Insect Morphol. & Embrvol.. Vol. 12, No. 2J 3, pp. 87 to 96, 1983.
Printed in Great Britain.
0020 7322/83$03.00 + .00 1983 PergamonPresslid.
SECRETION IN THE MALE ACCESSORY G L A N D S OF A E D E S A E G Y P T I (L.) ( D I P T E R A • CULICIDAE)* S. R A M A L [ N G A M t Vector Biology Laboratory, Department of Biology, University of Notre Dame, Notre Dame, IN 46556, U.S.A. ( A c c e p t e d 26 O c t o b e r 1982)
A b s l r a e l - - T h e objective of this work is to determine the nature, the mode of synthesis, and release of the secretion of accessory glands of the male mosquito, A e d e s aegypti (Diptera : Culicidae). Cytological studies revealed that each of the 2 glands has 2 morphological types of regionally specialized secretory cells, each type differing in the nature, and the release of its products. The anterior region (AR) cells secrete small (0.3 1.3 p.m) electron-lucent granules, which during the peak period of synthesis seemed to transport the flocculent and fibrous granular substance to the apical cytoplasm of the cell by means of binary fusions. This cytoplasm, when filled with secretory granules, pinches off and falls freely into the lumen of the gland, thus, exhibiting a macroapocrine mode of secretion. The posterior region (PR) cells, however, elaborate large (mean diameter 2 lam) electron-dense granules, which are released into the lumen essentially by rupture of the cell membrane at the apical end. In males mated to depletion, the volume of the gland is reduced by 67%; there is no subsequent recovery of secretory capacity. Index descriplors (in addition to those in title): Electron microscopy, secretory cells, binary fusions. INTRODUCTION
MALE accessory glands of the mosquito, Aedes aegypti consist of 2 distinct types of secretory cells (Dapples et al., 1974; Ramalingam, 1974) that secrete by the apocrine process (Dapples et al., 1974). Earlier, Jones and Wheeler (1965) reported that the secretions from these glands are acidophilic in nature, consist of finely granular material, free nuclei, and also intact gland cells containing large granules, resulting due to holocrine mode of secretion. The secretions elicit several irreversible physiological changes in the female mosquito, and consist of at least 2 separate proteins, namely c~ and 13 (Fuchs et al., 1969). Transplantation studies showed that the secretion from the anterior cells of the glands inhibits multiple insemination, and stimulates oviposition in females, whereas the cells of the posterior region synthesize a mucous secretion that serves to bind the granules of the anterior region together, so that an effective transfer to the female would be possible during mating (Ramalingam and Craig, 1976). Thus, there is a functional difference also between the secretions of the 2 types of cells. While Dapples et al. (1974) provided preliminary information on the ultrastructure of these cells, a detailed study on this aspect is warranted. The present work is an attempt to (a) elucidate the nature of secretory cells and their products, (b) determine the mode of synthesis, transport, and release of the secretion at the ultrastructural level, and (c) relate these findings to the renewal capacity of the depleted glands. *This investigation was supported by NIH Research (;rant No. AI-02753. +Present address: Department of Biology, Canadian Union College, College Heights, Alberta T0C 0Z0, Canada. 87
88
S. RAMALINGAM
MATERIALS
AND
METHODS
The ROCK strain ofA. aegypti was used. This strain was established in Rockefeller Foundation Virus Laboratory several decades ago, and has been maintained at Notre Dame as a standard laboratory strain for over 23 years. Rearing methods used for mosquitoes are those of Craig and VandeHey (1962). Additionally, care was taken to raise the larvae in an uncrowded condition; each larva had at least 1 cm ~ of space in the rearing pan. For mating purposes, 3-day-old males were individually allowed to mate with surplus ( 15 - 20) virgin females (4 or 5day-old) for 24 hr. Then, the females were dissected, the spermathecae stained by 2% acetolactic orcein for the presence of spermatozoa, and the level of insemination was determined. Only the males, which had inseminated 6 - 8 females with the full packing of spermatozoa in at least one of the 3 spermathecae in each female, were selected for the study on the depleted glands. The dissections of the accessory glands from the male terminalia were carried out in Millonig's buffer, so that unnecessary swelling of the glands will be avoided. Glands were squashed and stained by 2% acetolactic orcein to show the regional specializations and contents of the secretory cells. Freshly dissected glands, and thin sections from the plastic embedded specimens were used for light microscopic observations. Procedures adopted for the electron microscopy were the same as reported in a previous study (Ramalingam and Craig, 1978). Approximate volume of the freshly dissected glands was estimated from the length and width measurements by assuming, in cross-section, each gland was round, and resembled a combination of a cone (in mature gland), or cylinder (in gland that was depleted by multiple matings) at the anterior part, and half-ellipsoid at the posterior part. RESULTS
AND
DISCUSSION
The accessory glands of the unmated males, 3 days post-emergence, were fully mature to their maximum, and filled with secretion in both secretory regions (Figs. 1, 5). The mean length, and width (n = 120) were 374.0 +_ 2.6 t.tm, and 139.0 +_ 2.1 I.tm, respectively. Even the glands of the 48-hr-old adult male were pear-shaped, and turgid (Fig. 4). Contrastingly, Dapples et al. (1974), using A. aegypti of the University of Georgia strain, reported incomplete maturation of 3-day-old glands. Significant variations in the length and width, 1 5 0 - 200 I.tm, and, 5 0 - 100 ~m, respectively were observed. The glands were, thus, not uniformly matured, and in addition, were not pear-shaped, and turgid (see Fig. 1 in Dapples et al., 1974), suggesting that the glands had not matured to their maximum capability within 3 days post-emergence, possibly owing to slower rate of development.
FIG. 1. Male accessory glands of A. aegypti, 72 hr post-emergence. Bar = 50 lam.
Male Accessory Gland Secretion of Aedes aegypti (L.)
%
Fl(;. 2. AR secretory cells from squashed glands to show lightly stained secretory packets, and darkly stained nuclei. Stained by 2% acetolactic orcein. Bar = 50 I.tm. Fk~. 3. PR secretory cells from squashed glands; nuclei and cytoplasm are stained dark, and light, respectively. Stained by 2% acetolactic orcein. Bar = 50 lam. Fit;. 4. L.S. of glands from male, 48 hr post-emergence, showing 2 distinct secretory areas and their secretions. Note lumen is filled with secretions. Stained by Azure B at pH 4. Bar = 50 l.tm. Fl(;. 5. L S. of glands from male, 72 hr post-emergence, showing presence of AR secretory packets in PR lumen. Note darkly stained large PR granules. Stained by Azure B at pH 4. Bar - 50 lam. Fl~;. 6. L.S. of depleted glands; note empty lumen and darkly stained nuclei. Stained by Azure B at pH 4. Bar = 50 ~tm.
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S. RAMALINGAM
At the ultrastructural level, the major structural aspects of the gland concur with those of Dapples et al. (1974). However, a detailed study on the structure and functional behavior of the secretory granules suggested that the mode of synthesis, and release of secretion were different from those previously reported (Jones and Wheeler, 1965; Dapples et al., 1974). Columnar epithelial cells constituted the secretory regions of the gland in 3-day-old mosquitoes. The nuclei of the cells were present in the basal region, and found usually flattened or elliptical (Fig. 7). Although elongated cells were seen, the cells of the anterior region (AR) were mostly smaller. This was due to the macroapocrine process of secretion in which a large portion of the apical cytoplasm, when replete with secretory granules, was detached or pinched off from the cell. Squashed and stained glands showed the presence of numerous such detached cytoplasms or packets of secretory granules (Figs. 2, 4, 5) in the lumen. These granular packets had almost filled the lumen of the AR and even extended to that of the posterior region (PR) of the gland (Figs. 4, 5). A similar discharge process occurred also in the anterior accessory glands of the male A . triseriatus (Ramalingam and Craig, 1978). Numerous electron-lucent granules (sg 1) with flocculent substance at the core were synthesized in the AR cells. These granules were membrane-bounded and had a diameter range of 0.3 - 1.3 ~tm. The Golgi vesicles (Gv) were limited in number, and they occurred commonly in the immediate vicinity of the rough endoplasmic reticulum (er) (Figs. 7, 8). They were smooth-surfaced with distended saccules, possessing finely granular substance of less than 0.5 ~tm wide. It is speculated that the protein synthesized at the er was transferred to the Gv, wherein a lipid or carbohydrate moiety was added prior to the discharge. The fascinating aspect of secretion is that the secretory granules themselves were active components in the transport mechanism. The granules coalesced by forming binary vesicles (Figs. 7, 8, 1 0 - 12) in which initially the membrane of one granule formed a lingula and inserted into an adjoining granule (Figs. 10, 12). Subsequent membrane interactions at the points of contact permitted the fusion and the transfer of the granular substance into the receiver granule. Granules arrayed in a linear sequence extending from the site of origin to the cytoplasmic apex towards the lumen demonstrated fusions of apposed membranes on both ends. An extensive chain of such fusions during the peak period of synthesis suggested that the granular content could be transferred to the site of release without much dislocation of individual granules within the cell. Evidently, the granules at the distal area were larger in comparison with those at the site of origin. The occurrence of binary fusion or vesicular binesis among the AR granules suggested the active role for the granule and its membrane in particular, in the transport mechanism. Such a phenomenon was identical to the vesicular binesis in the [3 cells of the Islets of Langerhans in rats (Gabbay et al., 1975). It also pointed out to an efficient intracellular transport system during the peak synchronized synthesis of the secretory cells. Although, the chain formation and membrane interactions among the AR granules were noted in Dapples et al. (1974; in Fig. 3) study, their significance in transport was not realized. While the coalescence around the perinuclear area led to larger granules, the same in the rest of the cytoplasm aided the transport of the granular secretion to the apical end. Since the secretory mechanism in the AR is by a pinching off process, this system of coalescence and transport might be necessary to cope with the rapid synthesis of export protein. The cells of the PR differed with those of the AR in length, and the type of the granules
Male Accessory Gland Secretion of Aedes aegypti (L.)
FIG. 7. Basal region of an AR secretory cell, showing circular muscle layer (cml) with nucleus (n), Golgi vesicles (Gv), rough endoplasmic reticulum (rer), and secretory granules (sg 1) in cytoplasm. Note intercellular space (is) a m o n g cells. Bar I tam. FJ~;. 8. Portion of an AR secretory cell showing intercellular borders with tight junction (tj) and microvilli (mv). Bar = 1 tam. Fic;. 9. L.S. of circular muscle layer (ml) of gland. Notice mitochondrion (m), and basement membrane (bm) of muscle. Bar = 0.5 tam.
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Male Accessory Gland Secretion of Aedes aegypti (L.)
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formed. The cells were longer with relatively narrow cytoplasmic area (Figs. 3, 5). The morphological conditions of the nuclei, and other organelles involved in protein synthesis were similar to those in the AR. The secretory granules (sg 2) were larger with a mean diameter of 2 I.tm. Although most were spherical, various ovoid shapes were also encountered. Each granule was delimited by a membrane, and contained one to 6 electron-dense cores, which were more compact and homogeneous than their surroundings (Figs. 10, 12). These cores differed in size; granules with large cores had only a few of them, suggesting the possibility of coalescence. These dense cores were spherical in most, and were adhering close to the granular membrane. Cytochemicai analyses (Ramalingam, 1974) of the PR, however, indicated that the granules were homogeneous. When the granules occurred in clusters they had wide interdigitated surface contacts with adjoining ones, obviously differing from the binary fusions of the anterior granules. The few granules, which showed binary fusions, indicated coalescence to form larger granules, but there was no extensive transfer of secretion similar to those of the AR. Squash preparation of the glands showed the entire columnar cells, and no evidence of secretory packets, suggesting the absence of macroapocrine secretion in the PR (Fig. 3). The secretory process, then, appeared to be discharging single or small clusters of granules by rupture of the plasma membrane at one or more places in the apical region of the cell. Mitochondria, er, free ribosomes, and fragmented plasma membrane were also frequently seen along with the granules in the lumen. The rupture of the plasma membrane and the subsequent release process were possibly caused by the heavy accumulation of granular packets in the lumen, a n d / o r by the rhythmic contractions of the circular muscle (Fig. 9), which surrounded the secretory cells. Furthermore, the expulsion of the packets through the lumen of the PR during ejaculation would also severely detach the granules from the PR cells. This mode of release might be found appropriate when the functional role of this secretion is recognized. The PR secretion, in fact, acted as a binder of the AR secretory packets (Ramalingam and Craig, 1976), and therefore, to have the PR granules to be either loose, or in small collection is imperative. The binding of the secretory packets was evident during extrusion of the secretion, when the gland was squashed gently at the anterior end. Mature accessory glands were fully depleted with significant alterations in size and shape (Fig. 6) when the male mosquito had inseminated 6 or more virgin females within a 24-hr period. When the spermathecae of these females were examined at least one of them was fully packed with spermatozoa. The depleted glands (n = 48) on the average measured 308.0 I.tm in length, and 69.0 ram in width. When compared with the mature glands of an unmated male, these measurements represented 18070, and 50°7o reductions in length, and width, respectively. On the average, the volume of a depleted gland was
FIo. 10. Portion of cytoplasm of an A R cell displaying mitochondrion (m), ribosomes (rb), and rough endoplasmic reticulum (rer), and binary fusions among granules (sg 1). Arrow points to a lingula that indents membrane of an adjoining granule. Microvilli (my), and plasma membrane (pm) of cell, and loose granules (sg 2) of PR are also seen. Bar = 0.5 I.tm. FI(;. I I. Apical portion of an AR cell with secretory granules (sg l). Note lumen (l) of gland. Bar = 0.5 ~tm. FIG. 12. Loose secretory granules (sg 2) from a PR cell are seen adjoining a secretory packet. Bar = 0.5 I.tm.
94
S. RAMALINGAM
estimated to be 83 x 104 ~tm 3, whereas the mature gland was about 250 x 104 pm 3. Clearly, the multiple matings had caused a reduction of 167 x l04 pm 3 in volume, representing 67°7o. The volume reduced was equivalent to the amount of secretion ejected. Dapples et al. (1974), however, noted only half reduction in the glandular size following 6 or more matings. Cytological study of the depleted glands revealed the absence of entire cytoplasmic components including nuclei in most cells. Such a removal of nuclei, and intact cells in secretion confirms the findings of Jones and Wheeler (1965). In the post-mating period, the few AR cells, which retained granular cytoplasm, resorted to pinch off, and consequently, smaller secretory packets accumulated in the empty lumen of the PR (Figs. 6, 13, 14). A few cells with nuclei, nonetheless, seemed to renew secretion by forming a number of Gv (Fig. 15), with a few noticeable rer. But, there was no sign of vesicular binesis among the granules formed, suggesting only a low degree of synthetic activity. Undoubtedly, the multiple matings had caused the elimination of not only the loose secretory packets, but also much of the remaining cellular components essential for synthesis. Even when a fully depleted male was isolated for 2 weeks from females, there was not enough secretion formed to ensue successful insemination, suggesting no appreciable renewed secretory activity in the depleted gland. The renewal capability of the accessory glands in male A . aegypti has been a subject of controversy. While Jones (1967), Dapples et al. (1974), and Foster and Lea (1975) maintained that the glands had the potency to renew, and fill with secretion in 3 days after a first series of 6 - 8 matings, Hausermann and Nijhout (1975) and Ramalingam and Craig (1977) from this laboratory concluded otherwise. The present ultrastructural study reaffirms the findings of the latter authors. Topical application of a synthetic analog of a juvenile hormone (JH), however, was shown to elicit renewed secretion in depleted glands and successful insemination during matings (Ramalingam and Craig (1977). In view of this, additional work will be necessary to define the role of corpora allata in secreting JH in the post-mating period. In my opinion, partially depleted males were used in cytological and experimental studies by Dapples et al. (1974) and Foster and Lea (1975). First, in such males the glands retain a considerable proportion of secretion in the post-mating period, implying the amount transferred to females was minimal. Therefore, the reduction in volume or dimensions following depletion was not as great in comparison with that in my study. Second, the glands were not turgid with secretion prior to the initial multiple matings, as shown by Dapples et al. (1974). The males were then used for matings prior to the complete maturation of glands. Presumably, these situations allowed retention of not only a certain amount of secretion, but also the capacity to secrete by a continuous activity of JH after the initial series of matings. Had the fully mature glands, then, been depleted by 6 - 8 matings adopting the mating procedure used in this study, the "renewable fecundity" would not have been possible. The results of this study suggest that the glandular cells were nonrenewably altered, thereby losing further secretory potency following multiple matings. It is known that female mosquitoes of several species become refractory to further insemination upon receiving adequate amount of male accessory secretion during mating (Craig, 1967). The efficiency of an initial insemination to prevent a second one in females differed between the ROCK and Bangkok strains of A. aegypti (Jones and Madhukar, 1976; Williams and Hagen, 1977). In other words, the amount of secretion transferred
Male Accessory Gland Secretion of Aedes aegypti (L.)
~,
95
o.s
l~JcJ. 13. Portion of a depleted gland a day after multiple matings. Cytoplasm has vacuoles (v), nucleus (n), and secretory granules (sg 1). Lumen (1), and disruption in muscle layer (ml) are evident. Bar = 0.5 ~m. Fit;. 14. Arrow points to a secretory packet surrounded by an empty lumen (1) in process of being detached in gland a day after multiple matings. Bar = 0.5 ~tm. FIG. 15. Depleted gland 3 days after multiple matings shows reorganization of rough endoplasmic reticulum (rer), and Golgi vesicles (Gv). A few secretory granules of PR (sg 2) are also seen. Bar = 0.5 pm.
96
S. RAMALINGAM
during mating varied between strains. This could be attributed to the variations in the level of maturation or contents of the glands. It is possible then that genetic differences and their interactions with the differences in the procedures adopted for raising larvae, and adults, as well as mating of adults would have accounted for the variations in the maturation level, and renewal of secretion. The level of glandular maturation attained just prior to multiple matings, the amount of secretion transferred in each mating, the extent of damage done to the cells during the mating events, and the influence of JH activity in the post-mating period determined in essence the renewal of secretion by the depleted glands. Acknowledgement--I am greatly indebted to Prof. George B. Craig, Jr. for his enthusiasm, criticism, and support in the course of this study. REFERENCES CRAIG, G. B., JR. 1967. Mosquito: female m o n o g a m y induced by male accessory gland substance. Science (Wash., D.C.) 156: 1499- 501. CRAIG, G. B., JR. and R. C. VANDEHEY. 1962. Genetic variability in Aedes aegypti--l. Mutations affecting color pattern. Ann. Entornol. Soc. Amer. 55: 4 7 - 58. DAPPLES, C. C., W. A. FOSTER and A. O. LEA. 1974. Ultrastrueture of the accessory gland of the male mosquito, Aedes aegypti (L.) (Diptera : Culicidae). Int. J. Insect Morphol. Embryol. 3 (2): 2 7 9 - 9 1 . FOSTER, W. A. and A. O. LEA. 1975. Renewable fecundity of male Aedes aegypti following replenishment of seminal vesicles and accessory glands. J. Insect Physiol. 21: 1 0 8 5 - 90. FUCHS, M. S., G. B. CRAIG, JR. and D. D. DESPOMMIER. 1969. The protein nature of the substance inducing female m o n o g a m y in Aedes aegypti. J. Insect Physiol. 15:701 - 9 . GABBAY, K. H., J. KORFF and E. E. SCHNEEBERGER. 1975. Vesicular binesis: glucose effect on insulin secretory vesicles. Science (Wash., D.C.) 187: 1 7 7 - 9 . HAUSERMANN, W. and F. NIJHOUT. 1975. Permanent loss of male fecundity following sperm depletion in Aedes aegypti (L.). J. Med. Entomol. 6: 7 0 7 - 15. JONES, J. C. 1967. Spermatocysts in Aedes aegypti L. Biol. Bull. (Woods Hole) 132:23 - 33. JONES, J. C. and B. V. MADHUKAR. 1976. The potency of male accessory gland material in the mosquito (Aedes aegypti). Experientia 32:452 - 3. JONES, J. C. and R. E. WHEELER. 1965. Studies on spermathecal filling in Aedes aegypti L. 1. Description. Biol. Bull. (Woods Hole) 129: 1 3 4 - 5 0 . RAMALINGAM, S. 1974. Structure and function of the male accessory glands of Aedes mosquitoes. Ph.D. Dissertation, University of Notre Dame. RAMALINGAM, S. and G. B. CRAIG, JR. 1976. Functions of the male accessory gland secretions of Aedes mosquitoes (Diptera : Culicidae): Transplantation studies. Can. Entomol. 108:955 - 60. RAMALINGAM, S. and G. B. CRAIG, JR. 1977. The effects of a JH mimic and cauterization of the corpus allatum complex on the male accessory glands of Aedes aegypti (Diptera : Culicidae). Can. Entomol. 109: 897 - 906. RAMALINGAM, S. and G. B. CRAIG, JR. 1978. Fine structure of the male accessory gland in Aedes triseriatus. J. Insect Physiol. 2 4 : 2 5 1 - 9 . WILLIAMS, R. W. and N. K. B. HAGAN. 1977. Efficiency of a single insemination in preventing a second in the ROCK strain of the mosquito, Aedes aegypti. J. Insect Physiol. 23: 1205- 7.
Printed in Great Britain.
0020 7322/83$03.00 + .00 1983 PergamonPresslid.
SECRETION IN THE MALE ACCESSORY G L A N D S OF A E D E S A E G Y P T I (L.) ( D I P T E R A • CULICIDAE)* S. R A M A L [ N G A M t Vector Biology Laboratory, Department of Biology, University of Notre Dame, Notre Dame, IN 46556, U.S.A. ( A c c e p t e d 26 O c t o b e r 1982)
A b s l r a e l - - T h e objective of this work is to determine the nature, the mode of synthesis, and release of the secretion of accessory glands of the male mosquito, A e d e s aegypti (Diptera : Culicidae). Cytological studies revealed that each of the 2 glands has 2 morphological types of regionally specialized secretory cells, each type differing in the nature, and the release of its products. The anterior region (AR) cells secrete small (0.3 1.3 p.m) electron-lucent granules, which during the peak period of synthesis seemed to transport the flocculent and fibrous granular substance to the apical cytoplasm of the cell by means of binary fusions. This cytoplasm, when filled with secretory granules, pinches off and falls freely into the lumen of the gland, thus, exhibiting a macroapocrine mode of secretion. The posterior region (PR) cells, however, elaborate large (mean diameter 2 lam) electron-dense granules, which are released into the lumen essentially by rupture of the cell membrane at the apical end. In males mated to depletion, the volume of the gland is reduced by 67%; there is no subsequent recovery of secretory capacity. Index descriplors (in addition to those in title): Electron microscopy, secretory cells, binary fusions. INTRODUCTION
MALE accessory glands of the mosquito, Aedes aegypti consist of 2 distinct types of secretory cells (Dapples et al., 1974; Ramalingam, 1974) that secrete by the apocrine process (Dapples et al., 1974). Earlier, Jones and Wheeler (1965) reported that the secretions from these glands are acidophilic in nature, consist of finely granular material, free nuclei, and also intact gland cells containing large granules, resulting due to holocrine mode of secretion. The secretions elicit several irreversible physiological changes in the female mosquito, and consist of at least 2 separate proteins, namely c~ and 13 (Fuchs et al., 1969). Transplantation studies showed that the secretion from the anterior cells of the glands inhibits multiple insemination, and stimulates oviposition in females, whereas the cells of the posterior region synthesize a mucous secretion that serves to bind the granules of the anterior region together, so that an effective transfer to the female would be possible during mating (Ramalingam and Craig, 1976). Thus, there is a functional difference also between the secretions of the 2 types of cells. While Dapples et al. (1974) provided preliminary information on the ultrastructure of these cells, a detailed study on this aspect is warranted. The present work is an attempt to (a) elucidate the nature of secretory cells and their products, (b) determine the mode of synthesis, transport, and release of the secretion at the ultrastructural level, and (c) relate these findings to the renewal capacity of the depleted glands. *This investigation was supported by NIH Research (;rant No. AI-02753. +Present address: Department of Biology, Canadian Union College, College Heights, Alberta T0C 0Z0, Canada. 87
88
S. RAMALINGAM
MATERIALS
AND
METHODS
The ROCK strain ofA. aegypti was used. This strain was established in Rockefeller Foundation Virus Laboratory several decades ago, and has been maintained at Notre Dame as a standard laboratory strain for over 23 years. Rearing methods used for mosquitoes are those of Craig and VandeHey (1962). Additionally, care was taken to raise the larvae in an uncrowded condition; each larva had at least 1 cm ~ of space in the rearing pan. For mating purposes, 3-day-old males were individually allowed to mate with surplus ( 15 - 20) virgin females (4 or 5day-old) for 24 hr. Then, the females were dissected, the spermathecae stained by 2% acetolactic orcein for the presence of spermatozoa, and the level of insemination was determined. Only the males, which had inseminated 6 - 8 females with the full packing of spermatozoa in at least one of the 3 spermathecae in each female, were selected for the study on the depleted glands. The dissections of the accessory glands from the male terminalia were carried out in Millonig's buffer, so that unnecessary swelling of the glands will be avoided. Glands were squashed and stained by 2% acetolactic orcein to show the regional specializations and contents of the secretory cells. Freshly dissected glands, and thin sections from the plastic embedded specimens were used for light microscopic observations. Procedures adopted for the electron microscopy were the same as reported in a previous study (Ramalingam and Craig, 1978). Approximate volume of the freshly dissected glands was estimated from the length and width measurements by assuming, in cross-section, each gland was round, and resembled a combination of a cone (in mature gland), or cylinder (in gland that was depleted by multiple matings) at the anterior part, and half-ellipsoid at the posterior part. RESULTS
AND
DISCUSSION
The accessory glands of the unmated males, 3 days post-emergence, were fully mature to their maximum, and filled with secretion in both secretory regions (Figs. 1, 5). The mean length, and width (n = 120) were 374.0 +_ 2.6 t.tm, and 139.0 +_ 2.1 I.tm, respectively. Even the glands of the 48-hr-old adult male were pear-shaped, and turgid (Fig. 4). Contrastingly, Dapples et al. (1974), using A. aegypti of the University of Georgia strain, reported incomplete maturation of 3-day-old glands. Significant variations in the length and width, 1 5 0 - 200 I.tm, and, 5 0 - 100 ~m, respectively were observed. The glands were, thus, not uniformly matured, and in addition, were not pear-shaped, and turgid (see Fig. 1 in Dapples et al., 1974), suggesting that the glands had not matured to their maximum capability within 3 days post-emergence, possibly owing to slower rate of development.
FIG. 1. Male accessory glands of A. aegypti, 72 hr post-emergence. Bar = 50 lam.
Male Accessory Gland Secretion of Aedes aegypti (L.)
%
Fl(;. 2. AR secretory cells from squashed glands to show lightly stained secretory packets, and darkly stained nuclei. Stained by 2% acetolactic orcein. Bar = 50 I.tm. Fk~. 3. PR secretory cells from squashed glands; nuclei and cytoplasm are stained dark, and light, respectively. Stained by 2% acetolactic orcein. Bar = 50 lam. Fit;. 4. L.S. of glands from male, 48 hr post-emergence, showing 2 distinct secretory areas and their secretions. Note lumen is filled with secretions. Stained by Azure B at pH 4. Bar = 50 l.tm. Fl(;. 5. L S. of glands from male, 72 hr post-emergence, showing presence of AR secretory packets in PR lumen. Note darkly stained large PR granules. Stained by Azure B at pH 4. Bar - 50 lam. Fl~;. 6. L.S. of depleted glands; note empty lumen and darkly stained nuclei. Stained by Azure B at pH 4. Bar = 50 ~tm.
89
90
S. RAMALINGAM
At the ultrastructural level, the major structural aspects of the gland concur with those of Dapples et al. (1974). However, a detailed study on the structure and functional behavior of the secretory granules suggested that the mode of synthesis, and release of secretion were different from those previously reported (Jones and Wheeler, 1965; Dapples et al., 1974). Columnar epithelial cells constituted the secretory regions of the gland in 3-day-old mosquitoes. The nuclei of the cells were present in the basal region, and found usually flattened or elliptical (Fig. 7). Although elongated cells were seen, the cells of the anterior region (AR) were mostly smaller. This was due to the macroapocrine process of secretion in which a large portion of the apical cytoplasm, when replete with secretory granules, was detached or pinched off from the cell. Squashed and stained glands showed the presence of numerous such detached cytoplasms or packets of secretory granules (Figs. 2, 4, 5) in the lumen. These granular packets had almost filled the lumen of the AR and even extended to that of the posterior region (PR) of the gland (Figs. 4, 5). A similar discharge process occurred also in the anterior accessory glands of the male A . triseriatus (Ramalingam and Craig, 1978). Numerous electron-lucent granules (sg 1) with flocculent substance at the core were synthesized in the AR cells. These granules were membrane-bounded and had a diameter range of 0.3 - 1.3 ~tm. The Golgi vesicles (Gv) were limited in number, and they occurred commonly in the immediate vicinity of the rough endoplasmic reticulum (er) (Figs. 7, 8). They were smooth-surfaced with distended saccules, possessing finely granular substance of less than 0.5 ~tm wide. It is speculated that the protein synthesized at the er was transferred to the Gv, wherein a lipid or carbohydrate moiety was added prior to the discharge. The fascinating aspect of secretion is that the secretory granules themselves were active components in the transport mechanism. The granules coalesced by forming binary vesicles (Figs. 7, 8, 1 0 - 12) in which initially the membrane of one granule formed a lingula and inserted into an adjoining granule (Figs. 10, 12). Subsequent membrane interactions at the points of contact permitted the fusion and the transfer of the granular substance into the receiver granule. Granules arrayed in a linear sequence extending from the site of origin to the cytoplasmic apex towards the lumen demonstrated fusions of apposed membranes on both ends. An extensive chain of such fusions during the peak period of synthesis suggested that the granular content could be transferred to the site of release without much dislocation of individual granules within the cell. Evidently, the granules at the distal area were larger in comparison with those at the site of origin. The occurrence of binary fusion or vesicular binesis among the AR granules suggested the active role for the granule and its membrane in particular, in the transport mechanism. Such a phenomenon was identical to the vesicular binesis in the [3 cells of the Islets of Langerhans in rats (Gabbay et al., 1975). It also pointed out to an efficient intracellular transport system during the peak synchronized synthesis of the secretory cells. Although, the chain formation and membrane interactions among the AR granules were noted in Dapples et al. (1974; in Fig. 3) study, their significance in transport was not realized. While the coalescence around the perinuclear area led to larger granules, the same in the rest of the cytoplasm aided the transport of the granular secretion to the apical end. Since the secretory mechanism in the AR is by a pinching off process, this system of coalescence and transport might be necessary to cope with the rapid synthesis of export protein. The cells of the PR differed with those of the AR in length, and the type of the granules
Male Accessory Gland Secretion of Aedes aegypti (L.)
FIG. 7. Basal region of an AR secretory cell, showing circular muscle layer (cml) with nucleus (n), Golgi vesicles (Gv), rough endoplasmic reticulum (rer), and secretory granules (sg 1) in cytoplasm. Note intercellular space (is) a m o n g cells. Bar I tam. FJ~;. 8. Portion of an AR secretory cell showing intercellular borders with tight junction (tj) and microvilli (mv). Bar = 1 tam. Fic;. 9. L.S. of circular muscle layer (ml) of gland. Notice mitochondrion (m), and basement membrane (bm) of muscle. Bar = 0.5 tam.
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Male Accessory Gland Secretion of Aedes aegypti (L.)
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formed. The cells were longer with relatively narrow cytoplasmic area (Figs. 3, 5). The morphological conditions of the nuclei, and other organelles involved in protein synthesis were similar to those in the AR. The secretory granules (sg 2) were larger with a mean diameter of 2 I.tm. Although most were spherical, various ovoid shapes were also encountered. Each granule was delimited by a membrane, and contained one to 6 electron-dense cores, which were more compact and homogeneous than their surroundings (Figs. 10, 12). These cores differed in size; granules with large cores had only a few of them, suggesting the possibility of coalescence. These dense cores were spherical in most, and were adhering close to the granular membrane. Cytochemicai analyses (Ramalingam, 1974) of the PR, however, indicated that the granules were homogeneous. When the granules occurred in clusters they had wide interdigitated surface contacts with adjoining ones, obviously differing from the binary fusions of the anterior granules. The few granules, which showed binary fusions, indicated coalescence to form larger granules, but there was no extensive transfer of secretion similar to those of the AR. Squash preparation of the glands showed the entire columnar cells, and no evidence of secretory packets, suggesting the absence of macroapocrine secretion in the PR (Fig. 3). The secretory process, then, appeared to be discharging single or small clusters of granules by rupture of the plasma membrane at one or more places in the apical region of the cell. Mitochondria, er, free ribosomes, and fragmented plasma membrane were also frequently seen along with the granules in the lumen. The rupture of the plasma membrane and the subsequent release process were possibly caused by the heavy accumulation of granular packets in the lumen, a n d / o r by the rhythmic contractions of the circular muscle (Fig. 9), which surrounded the secretory cells. Furthermore, the expulsion of the packets through the lumen of the PR during ejaculation would also severely detach the granules from the PR cells. This mode of release might be found appropriate when the functional role of this secretion is recognized. The PR secretion, in fact, acted as a binder of the AR secretory packets (Ramalingam and Craig, 1976), and therefore, to have the PR granules to be either loose, or in small collection is imperative. The binding of the secretory packets was evident during extrusion of the secretion, when the gland was squashed gently at the anterior end. Mature accessory glands were fully depleted with significant alterations in size and shape (Fig. 6) when the male mosquito had inseminated 6 or more virgin females within a 24-hr period. When the spermathecae of these females were examined at least one of them was fully packed with spermatozoa. The depleted glands (n = 48) on the average measured 308.0 I.tm in length, and 69.0 ram in width. When compared with the mature glands of an unmated male, these measurements represented 18070, and 50°7o reductions in length, and width, respectively. On the average, the volume of a depleted gland was
FIo. 10. Portion of cytoplasm of an A R cell displaying mitochondrion (m), ribosomes (rb), and rough endoplasmic reticulum (rer), and binary fusions among granules (sg 1). Arrow points to a lingula that indents membrane of an adjoining granule. Microvilli (my), and plasma membrane (pm) of cell, and loose granules (sg 2) of PR are also seen. Bar = 0.5 I.tm. FI(;. I I. Apical portion of an AR cell with secretory granules (sg l). Note lumen (l) of gland. Bar = 0.5 ~tm. FIG. 12. Loose secretory granules (sg 2) from a PR cell are seen adjoining a secretory packet. Bar = 0.5 I.tm.
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estimated to be 83 x 104 ~tm 3, whereas the mature gland was about 250 x 104 pm 3. Clearly, the multiple matings had caused a reduction of 167 x l04 pm 3 in volume, representing 67°7o. The volume reduced was equivalent to the amount of secretion ejected. Dapples et al. (1974), however, noted only half reduction in the glandular size following 6 or more matings. Cytological study of the depleted glands revealed the absence of entire cytoplasmic components including nuclei in most cells. Such a removal of nuclei, and intact cells in secretion confirms the findings of Jones and Wheeler (1965). In the post-mating period, the few AR cells, which retained granular cytoplasm, resorted to pinch off, and consequently, smaller secretory packets accumulated in the empty lumen of the PR (Figs. 6, 13, 14). A few cells with nuclei, nonetheless, seemed to renew secretion by forming a number of Gv (Fig. 15), with a few noticeable rer. But, there was no sign of vesicular binesis among the granules formed, suggesting only a low degree of synthetic activity. Undoubtedly, the multiple matings had caused the elimination of not only the loose secretory packets, but also much of the remaining cellular components essential for synthesis. Even when a fully depleted male was isolated for 2 weeks from females, there was not enough secretion formed to ensue successful insemination, suggesting no appreciable renewed secretory activity in the depleted gland. The renewal capability of the accessory glands in male A . aegypti has been a subject of controversy. While Jones (1967), Dapples et al. (1974), and Foster and Lea (1975) maintained that the glands had the potency to renew, and fill with secretion in 3 days after a first series of 6 - 8 matings, Hausermann and Nijhout (1975) and Ramalingam and Craig (1977) from this laboratory concluded otherwise. The present ultrastructural study reaffirms the findings of the latter authors. Topical application of a synthetic analog of a juvenile hormone (JH), however, was shown to elicit renewed secretion in depleted glands and successful insemination during matings (Ramalingam and Craig (1977). In view of this, additional work will be necessary to define the role of corpora allata in secreting JH in the post-mating period. In my opinion, partially depleted males were used in cytological and experimental studies by Dapples et al. (1974) and Foster and Lea (1975). First, in such males the glands retain a considerable proportion of secretion in the post-mating period, implying the amount transferred to females was minimal. Therefore, the reduction in volume or dimensions following depletion was not as great in comparison with that in my study. Second, the glands were not turgid with secretion prior to the initial multiple matings, as shown by Dapples et al. (1974). The males were then used for matings prior to the complete maturation of glands. Presumably, these situations allowed retention of not only a certain amount of secretion, but also the capacity to secrete by a continuous activity of JH after the initial series of matings. Had the fully mature glands, then, been depleted by 6 - 8 matings adopting the mating procedure used in this study, the "renewable fecundity" would not have been possible. The results of this study suggest that the glandular cells were nonrenewably altered, thereby losing further secretory potency following multiple matings. It is known that female mosquitoes of several species become refractory to further insemination upon receiving adequate amount of male accessory secretion during mating (Craig, 1967). The efficiency of an initial insemination to prevent a second one in females differed between the ROCK and Bangkok strains of A. aegypti (Jones and Madhukar, 1976; Williams and Hagen, 1977). In other words, the amount of secretion transferred
Male Accessory Gland Secretion of Aedes aegypti (L.)
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l~JcJ. 13. Portion of a depleted gland a day after multiple matings. Cytoplasm has vacuoles (v), nucleus (n), and secretory granules (sg 1). Lumen (1), and disruption in muscle layer (ml) are evident. Bar = 0.5 ~m. Fit;. 14. Arrow points to a secretory packet surrounded by an empty lumen (1) in process of being detached in gland a day after multiple matings. Bar = 0.5 ~tm. FIG. 15. Depleted gland 3 days after multiple matings shows reorganization of rough endoplasmic reticulum (rer), and Golgi vesicles (Gv). A few secretory granules of PR (sg 2) are also seen. Bar = 0.5 pm.
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during mating varied between strains. This could be attributed to the variations in the level of maturation or contents of the glands. It is possible then that genetic differences and their interactions with the differences in the procedures adopted for raising larvae, and adults, as well as mating of adults would have accounted for the variations in the maturation level, and renewal of secretion. The level of glandular maturation attained just prior to multiple matings, the amount of secretion transferred in each mating, the extent of damage done to the cells during the mating events, and the influence of JH activity in the post-mating period determined in essence the renewal of secretion by the depleted glands. Acknowledgement--I am greatly indebted to Prof. George B. Craig, Jr. for his enthusiasm, criticism, and support in the course of this study. REFERENCES CRAIG, G. B., JR. 1967. Mosquito: female m o n o g a m y induced by male accessory gland substance. Science (Wash., D.C.) 156: 1499- 501. CRAIG, G. B., JR. and R. C. VANDEHEY. 1962. Genetic variability in Aedes aegypti--l. Mutations affecting color pattern. Ann. Entornol. Soc. Amer. 55: 4 7 - 58. DAPPLES, C. C., W. A. FOSTER and A. O. LEA. 1974. Ultrastrueture of the accessory gland of the male mosquito, Aedes aegypti (L.) (Diptera : Culicidae). Int. J. Insect Morphol. Embryol. 3 (2): 2 7 9 - 9 1 . FOSTER, W. A. and A. O. LEA. 1975. Renewable fecundity of male Aedes aegypti following replenishment of seminal vesicles and accessory glands. J. Insect Physiol. 21: 1 0 8 5 - 90. FUCHS, M. S., G. B. CRAIG, JR. and D. D. DESPOMMIER. 1969. The protein nature of the substance inducing female m o n o g a m y in Aedes aegypti. J. Insect Physiol. 15:701 - 9 . GABBAY, K. H., J. KORFF and E. E. SCHNEEBERGER. 1975. Vesicular binesis: glucose effect on insulin secretory vesicles. Science (Wash., D.C.) 187: 1 7 7 - 9 . HAUSERMANN, W. and F. NIJHOUT. 1975. Permanent loss of male fecundity following sperm depletion in Aedes aegypti (L.). J. Med. Entomol. 6: 7 0 7 - 15. JONES, J. C. 1967. Spermatocysts in Aedes aegypti L. Biol. Bull. (Woods Hole) 132:23 - 33. JONES, J. C. and B. V. MADHUKAR. 1976. The potency of male accessory gland material in the mosquito (Aedes aegypti). Experientia 32:452 - 3. JONES, J. C. and R. E. WHEELER. 1965. Studies on spermathecal filling in Aedes aegypti L. 1. Description. Biol. Bull. (Woods Hole) 129: 1 3 4 - 5 0 . RAMALINGAM, S. 1974. Structure and function of the male accessory glands of Aedes mosquitoes. Ph.D. Dissertation, University of Notre Dame. RAMALINGAM, S. and G. B. CRAIG, JR. 1976. Functions of the male accessory gland secretions of Aedes mosquitoes (Diptera : Culicidae): Transplantation studies. Can. Entomol. 108:955 - 60. RAMALINGAM, S. and G. B. CRAIG, JR. 1977. The effects of a JH mimic and cauterization of the corpus allatum complex on the male accessory glands of Aedes aegypti (Diptera : Culicidae). Can. Entomol. 109: 897 - 906. RAMALINGAM, S. and G. B. CRAIG, JR. 1978. Fine structure of the male accessory gland in Aedes triseriatus. J. Insect Physiol. 2 4 : 2 5 1 - 9 . WILLIAMS, R. W. and N. K. B. HAGAN. 1977. Efficiency of a single insemination in preventing a second in the ROCK strain of the mosquito, Aedes aegypti. J. Insect Physiol. 23: 1205- 7.