Archives of Insect Biochemistry and Physiology 16:221-234 (1 991) Development of Male-Incubated Ovaries in the Gypsy Moth, Lymnntria dispar JoanneBallarino, M i c h a e l Ma, Tsuey Ding, and Craig Lamison Department of Entomology (1.B., M.M.) and Center for Agricultural Biotechnology, Ma yland Biotechnology lnstitute (M.M.), University of Ma yland, College Park, Maryland; Institute of Zoology, Academia Sinica, Beijing, People's Republic of China (T.D.); and E.I. Du Pont De Nemours & Co., Ag Diagnostics, Newark, Delaware (C.L.) Ovaries from Lymantria disparfemales were transplanted into an environment lacking vitellogenin, the male milieu, in order to determine how the presence of vitellogenin i n the hemolymph affects the process of protein uptake by gypsy moth oocytes. When undeveloped ovaries from newly ecdysed last instar females were transplanted into males of the same stage, follicles detached from the germarium and increased i n size, but the growth of oocytes proceeded more slowly than those from female controls. Although chorion formation was delayed i n male-grown ovaries, scanning electron microscopy of chorionated eggs recovered from adult males showed that a chorion with normal surface architecture was formed by the adult stage. SDS-PACE analysis of the male-grown ovaries and hemolymph from males receiving ovaries showed that vitellogenin production was not stimulated by the organ transplant and only male hemolymph proteins were internalized by the maleincubated ovaries. Thus, in the absence of vitellogenin, endocytosis of male hemolymph proteins occurred, but the rate of oocyte growth was slowed. Key words: vitellogenesis, hernolymph proteins, transplanted ovaries, follicle growth INTRODUCTION In egg-laying species, the rapid growth of the oocyte is due predominantly to the storage of large amounts of yolk over a relatively short period of time. Acknowledgments: We thank Dr. Robert Bell of the Insect Reproduction Laboratory at USDA, Beltsville, M D for providing us with gypsy moths. Timothy Mau el of the Laboratory for Biological Ultrastructure at the University of Maryland provided help uI suggestions on the scanning electron microscopy. Professor William Telfer (Univ. Pennsylvania), Professor M.L. Pan (Univ. Tennessee), Michael Blackburn (Univ. Maryland), and Dr. David Dussourd (Univ. Maryland) provided us with helpful comments on the manuscript. This research was supported by the College of Agriculture and Life Sciences, University of Maryland at College Park, and by the Maryland Experiment Station. This paper is Scientific Article No. A6061, Contribution No. 8223 and it i s contribution No. 57 from the Laboratory for Biological Ultrastructure, Universify of Maryland. B Received July27,1990; accepted December 13,1990. Address reprint requests to Dr. Michael Ma, Department of Entomology, University of Maryland, College Park, M D 20742. 0 1991 Wiley-Liss, Inc, 222 Ballarino et al. In most insects the yolk proteins, or vitellins, are synthesized not by the oocyte, but by the fat body. The form synthesized by the fat body is termed vitellogenin, and is secreted into the hemolymph and incorporated into the oocytes by endocytosis. The timing and hormonal regulation of vitellogenic events is correlated with the feeding habits of the adult stage insect. In insect species which feed as adults, vitellogenesis generally takes place in the adult stage [1,2], while in insects which do not feed as adults, vitellogenesis takes place in the pharate adult stage [3-61. The endocytotic uptake of vitellogenin appears to be receptor-mediated as evidenced by the presence in the oocyte of clathrin-coatedpits and vesicles to which vitellogenin adheres 17-91 and by kinetic binding studies demonstrating saturable uptake of radiolabeled vitellogenin by follicles cultured in vitro [lo-151. Is the binding of the vitellogenin ligand to its receptor necessary to the process of endocytosis in oocytes? In many membrane transport systems which utilize receptor-mediated endocytosis to transport macromolecules, the presence of the macromolecular ligand is not a necessary condition fox endocytosis to occur . From the evidence provided by studies of ovarian development in the male milieu, this appears to be the case in several Lepidoptera with nonfeeding adults 1171. Transplantation of ovaries into male Hyalophora cecropia [18,19]and Bombyx mori [20,21]results in the uptake of male hemolymph proteins in the absence of vitellogenins. Although the male-grown eggs are less numerous in B. mori, they reach nearly normal size and can be stimulated parthenogenetically to develop and hatch into larvae . The gypsy moth, Lymantria dispar, does not feed as an adult, and like other lepidopteran species with nonfeeding adults ( H . cecropia, B. mori),vitellogenin synthesis and uptake occurs before adult emergence 1231. We have found, however, that the gypsy moth differs from other lepidopterans with nonfeeding adults in the timing of follicle detachment, vitellogenin synthesis, and vitellogenin uptake . Follicle detachment from the germarium begins on day 4 of the last larval instar (apolysis begins on day 8 of the last larval instar). Vitellogenin production and secretion into the hemolymph is initiated even earlier, on day 2 of the last larval instar in gypsy moths, notably prior to pharate pupa formation. This is earlier than in B. mori in which the presence of vitellogenin has been reported in the pharate adult stage  or H. cecropia in which vitellogenin has been detected during the larvel-pupal molt [5,18]. Although vitellogenin synthesis begins early in the last larval instar in gypsy moths, vitellogenin uptake does not begin until day 3 after pupal ecdysis , when pharate adult development has begun. Since the final larval stadium lasts approximately 10 days in female gypsy moths, and uptake does not begin until day 3 after pupal ecdysis, this results in a long period during which vitellogenin titers are high in the hemolymph. Why does vitellogenin synthesis begin so early in the gypsy moth? Certainly there must be a cost to maintaining high vitellogenin titers over a long period of time. Is this prolonged period of high vitellogenin titers in the hemolymph necessary for the onset of patency and endocytosis? Does vitellogenin cue the onset of these processes? Or would these processes proceed normally in the absence of vitellogenin? We felt that answers to these questions might be provided by studying ovarian development in an environment lacking vitellogenin: the male milieu. Our objectives for this study were to monitor possible differences in the develop- Development of Male-IncubatedOvaries 223 ment of male-incubated ovaries with respect to 1)the number of folicles formed; 2) the size of the follicles (i.e., the degree of growth and uptake); 3) the formation of the chorion; and 4) the protein composition of mature eggs. MATERIALS AND METHODS Insect Rearing Gypsy moths (L. dispur, NJSS-22 strain) were received as 4th stadium larvae from Dr. R.A. Bell of the Insect Reproduction Laboratory at USDA, Beltsville, Maryland. The larvae were maintained on a modified wheat germ diet  at 26 ? 1°Cunder a 16:8 L:D photoperiod. Microsurgery Ovarian cysts were removed from newly ecdysed last larval instar females and transplanted into newly ecdysed last larval instar males, or into female controls from which the ovarian cysts had just been removed. Larvae were placed on ice prior to surgery and pinned down with 2 pieces of flexible tubing which were cut in half lengthwise. A window was cut from one of the pieces of tubing and placed over the midpoint of the fifth tergum. A small longitudinal incision was made at the midpoint of the fifth tergum and the ovaries were removed and placed in Insect Ringer solution. A similar incision was made at the fifth tergum in newly ecdysed last instar males and the ovarian cysts were implanted into the male abdominal cavity, or into the abdominal cavity of other females from which the ovarian cysts had been removed. Wounds were blotted dry with sterile Kimwipesm, dusted with penicillin and streptomycin, and sealed with dental wax. Monitoring the Production and Growth of Ovarian Follicles Male-grown, female-operated control and normal control ovaries were dissected from day 5 last instar larvae, from day 1, day 4, day 6, and day 8 pupae, and from newly emerged adults. A minimum of 5 insects was used for each time period and experimental group. Ovarian cysts or ovaries were removed, and the ovarioles were dissected free of fat body, connective tissue, and ovariole membranes, and observed under a dissecting microscope with oblique lighting. The number of follicles per ovariole that were detached from the germarium was counted for each time period. A follicle was considered "detached" when its opalescent oocyte lined up with the other oocytes like a string of pearls. Growth of the ovarian follicles was monitored by measuring the diameters of the terminal (closest to the oviduct) follicles (including oocytes, nurse cells, and follicular epithelium) with an ocular micrometer, In addition, the number of oocytes (not follicles) per ovariole which had reached an arbitrary width of 100 pm was counted for each time period. This number was converted to percent to eliminate the effect of the reduced number of follicles in transplanted ovaries. This latter measurement was made to determine whether follicles other than the terminal follicle exhibited a reduced rate of growth in male-grown ovaries. For day 8 pupae and adults, the number of chorionated eggs was counted, and the diameters of the terminal chorionated eggs were measured with an ocular micrometer. 224 Ballarino et al. Gel Electrophoresis Adult ovaries from males, female-operated controls, and normal controls were removed and the follicles were dissected free of fat body, connective tissue, and ovariole membranes. Follicles were washed 5 times in phosphatebuffered saline (137 mM NaCl, 8 mM Na2HP04, 1.5 mM KHzPO3, 2.7 mM KCl, pH 7.4). They were then homogenized in phosphate-buffered saline with 1 mM PMSF" and saturated PTU, and centrifuged for 5 rnin at 12,OOOg. The soluble supernatants were collected and frozen with PMSF and PTU at - 70°C for no more than 1week. Previtellogenic ovaries from day 8 last larval stadium of normal insects were prepared in the same manner. Hemolymph was collected from day 1 pupae of normal males, normal females, males receiving ovary transplants, and female operated-controls receiving ovary implants. An incision was made in the pupal case at the first abdominal segment and a Gilson micropipettor was used to collect the hemolymph. The hemolymph was added to microfuge tubes containing PMSF and PTU, on ice, and was centrifuged at 4°C for 5 min at 12,OOOg to remove hemocytes. They were stored with PMSF and PTU at - 70°C for no more than 2 weeks. SDS-PAGE was performed according to the methods of Laemmli . For each hemolymph sample, 1 pl of hemolymph was loaded. For each ovary extract sample, except the previtellogenic ovary, extract from 4 terminal (closest to the oviduct) follicles of adult insects was loaded. For the previtellogenic ovary, extract from 4 whole ovarioles from the 8 day last larval stadium was loaded. Scanning Electron Microscopy Chorionated eggs were dissected from males, female-operatedcontrols, and normal controls and fixed in 4% glutaraldehyde in Millonig's buffer pH 7.3  for 24 h at 4°C. They were washed 3 times in buffer and then post-fixed in 1% osmium tetroxide in Millonig's buffer for 1.5 h. The eggs were then dehydrated in an ethanol series, critical point dried, mounted on stubs, and coated with gold-palladium. They were observed under an AMR 1820 scanning electron microscope at 20 kV. RESULTS After transplantation of ovaries, female operated-control and male larvae fed, pupated, and eclosed, with a 20% mortality rate. At all stages, the number of follicles detached from the germarium per ovariole was lower in transplanted ovaries from both males and female operated-controls when compared to ovaries from normal females (Figs. 1, 2). There was no significant difference between males and female operated-controls in the number of follicles detachediovariole. The diameter of the terminal follicle (the follicle closest to the oviduct) increased at a greater rate in normal control ovaries and female operated-control ovaries than in male-grown ovaries (Fig. 3). The terminal follicles from normal ovaries were significantly larger than those from male ovaries at all stages except day 1 pupa. Terminal follicles from female operated-control ovaries were significantly larger than those from male-grown ovaries at and following the 'Abbreviations used: k D = kilodalton; PMSF = phenylmethylsulfonyl fluoride; PTU = phenylthiourea; SDS = sodium dodecyl sulfate. Development of Male-IncubatedOvaries 225 Fig. 1. Ovarioles dissected from normal control and male-grown ovaries at various times in the pupal stadium. Male-incubated ovaries appear in A-D and normal control ovaries in E-H. Ovariole age increases from top to bottom, with ovarioles from 4 day pupae shown in A and E, from 6 day pupae in B and F, from 8 day pupae in C and G, and from newly emerged adults in D and H. Bar length = 1crn. The bar i s shown in H, but is the same for all micrographs. day 6 pupa. Prior to day 6, there was no significant difference between female operated-controls and males in the size of the terminal follicle. A greater percent of the oocytes in normal ovaries and female operatedcontrol ovaries reached an arbitrary width of 100 km than in male-grown ovaries (Fig. 4). This difference between normal and male-grown ovaries in the percent of oocytes that reached a width of 100 pm was significant at and fol- 226 Ballarino et al. -0- normal 200 - 150 - 100 - 0 5d L -0- - . A - -male ld P 4d P op ctrl 64 P 8d P Adult Developmental Stage Fig. 2. The number of follicles detached from the germarium per ovariole as a function of insect age. Each point represents the mean number of follicles detached per ovariole in ovarioles from at least 5 different insects. At least 3 ovarioles were examined per insect. The bars represent the standard error around the mean. Where bars do not appear, the standard error was smaller than the size of the marker. Significant difference from male-incubatedovaries, as determined with Student's t-test, i s indicated with an asterisk. -0- normal - 0- -.4-. male OD ctrl 1.25 1.00 0.75 0.50 0.25 0.00 0 5d L id P 46 P 6d P 8d P Adult Developmental Stage Fig. 3. The diameter of the terminal follicle (the follicle closest to the oviduct) as a function of insect age. Each point represents the mean diameter of the terminal follicle of ovarioles from at lest 5 different insects. At least 3 ovarioles were examined per insect. The bars represent the standard error around the mean. Where bars do not appear, the standard error was smaller than the size of the marker. Significant difference from male-incubatedovaries, as determined with Student's t-test, i s indicated with an asterisk. Development of Male-Incubated Ovaries -0- normal 56 L - . A - . male 227 -0- op ctrl 4d P 66 P Developmental Stage Id P 8d P Adult Fig. 4. The percent of oocytes that reached a width greater than 100 pn as a function of insect age. Each point represents the mean percent of oocytes that reached a width greater than 100 p m in ovarioles from at least 6 different insects. At least 3 ovarioles were examined per insect. The bars represent the standard error around the mean. Where bars d o not appear, the standard error was smaller than the size of the marker. Significant difference from male-incubated ovaries, as determined with Student’s t-test, is indicated with an asterisk. -0- normal -.A-. male - 0- op ctrl r 0 Sd L ld P 4d P 66 P 8d P Adult Developmental Stage Fig. 5. The percent of chorionated eggs as a function of insect age. Each point represents the mean percent of chorionated eggs in ovarioles from at least 7 different insects. At least 3 ovarioles were examined per insect. The bars represent the standard error around the mean. Where bars d o not appear, the standard error was smaller than the size of the marker. Significant difference from male-incubated ovaries, as determined with Student’s t-test, is indicated with an asterisk. 228 Ballarino et al. lowing the day 4 pupal stage. The difference between female operated-control and male ovaries was significant at and following the day 6 pupal stage. Percentages were used to eliminate the effect of the reduced number of follicles in transplanted ovaries. A significantly greater percent of chorionated eggs was found in normal and female-operated control ovaries than in male ovaries at the day 8 pupal stage and the adult stage (Fig. 5). In adult male-grown ovaries some ovarioles contained chorionated eggs, while other ovarioles within the same ovary contained smaller follicles with no evidence of chorion formation at the light microscope level. Chorionated eggs from adult males reached 95.0% of the diameter of normal chorionated eggs and 94.2% of the diameter of female operated-control chorionated eggs. This difference between the chorionated eggs of males and those of normals and female operated-controls, though small (5% and 5.8% respectively), was significant. The variance in the diameter of chorionated eggs was small within experimental groups, with a mean and standard deviation of 1.20 + / - 0.03 mm for normals, 1.21 + / - 0.03 mm for female operated-controls, and 1.14 / - 0.07 mm for males. Scanning electron microscopy showed that the surface architecture of the chorion of adult eggs is similar in males, female operated-controls, and normal controls (Figs. 6, 7). Both the micropyle region (Figs. 68, 78) and the honeycomb-like region (Figs. 6C, 7C) developed normally in male-grown ovaries. SDS-PAGE analysis of hemolymph from normal females and normal males, and from experimental females and males receiving ovary transplants’ showed that vitellogenin production was not stimulated by the ovary transplant (Fig. 8; arrows indicate vitellogenin subunits). SDS-PAGE analysis comparing adult male-grown ovaries to normal female larval ovaries (previtellogenic),normal female adult ovaries, and female-operated control adult ovaries, showed that male-grown ovaries lacked vitellogenin, but did internalize other hernolymph proteins (Fig. 8). The arrowheads in Figure 8 indicate protein subunits which are found in the male hemolymph and in adult male-incubated ovaries, but which are not found in pre-vitellogenic ovaries. The three male-grown ovary samples contain different quantities of protein because the terminal follicles were of different sizes. + DISCUSSION When ovarian cysts from last instar female larvae were transplanted into last instar male larvae, follicles detached from the germarium during the larval stage (Fig. 2), increased in size during pharate adult development (Fig. 3), and formed a chorion with normal surface architecture (Figs. 6, 7). However, the transplanted ovaries from both males and female-operated controls exhibFig. 6. Scanning electron micrograph of a chorionated egg from a normal adult ovary showing the surface architecture of the chorion. A: Whole view. Arrow 1 indicates the micropile region and arrow 2 indicates the honeycomb region. Bar length: 100 pm. 6: Enlargement of the micropile region. Bar length: 10 pm. C: Enlargement of the honeycomb region. Bar length: 10 pm. Development of Male-IncubatedOvaries Fig. 6 229 230 Ballarino et al. Fig. 7 231 Development of Male-Incubated Ovaries c P C E"* 0 E Q c 6 b E P * -E* e I E 0 0 L L C b OL clr P 0 n -* E Q) '0 c E E Q 0 E Q 0 200, - 116- 97 66 + 43- Fig. 8. SDS-PAGE analysis of hemolymph and soluble ovary extracts. For each hemolymph sample, 1 pI of hemolymph was loaded. For each ovary extract sample, except the previtellogenic ovary, extract from 4 adult eggs was loaded. For the previtellogenic ovary, extract from 4 whole ovarioles from the 8 day last larval instar was loaded. Molecular weight standards of 200, 116, 97,66, and 43 kD are indicated at the left. Arrows indicate the 2 vitellogenin subunits. Arrowheads indicate protein subunits which are found in the male hemolymph and in adult male-incubated ovaries, but which are not found in previtellogenic ovaries. The three male ovary samples contain different quantities of protein because the terminal follicles were of different sizes. std, standards; op, operated (receivedovary transplant); previtell, previtellogenic. ~ ~~ Fig. 7. Scanning electron micrograph ofa chorionated egg from a male-grown adult ovary showing the surface architecture of the chorion. A: Whole view. Arrow 1 indicatesthe micropile region and arrow 2 indicates the honeycomb region. Bar length: 100 pm. 6: Enlargement of the micropile region. Bar length: 10 pm. C: Enlargement of the honeycomb region. Bar length: 10 km. 232 Ballarino et al. ited reduced follicle detachment compared to normal control ovaries (Fig. 2), presumably due to one or more of the following reasons: 1)damage sustained during transplantation; 2) reduced oxygen supply due to severing of the tracheoles; 3) loss of anchorage to the body wall leading to reduced contraction of the ovariole wall. Although the number of follicles that detached from the germarium was similar in transplanted ovaries from both males and female-operated controls, the rate of growth during the vitellogenic period of the pupal stage was reduced in male-grown ovaries compared to both female-operated controls and normal controls (Figs. 3, 4). This reduction in vitellogenic growth may be a result of 1) the smaller size and lower hemolymph resources of males; and/or 2) the absence of vitellogenin in male hemolymph. A possible role for vitellogenin in protein uptake has been suggested by studies in H . cecropia by Kulakosky and Telfer  in which vitellogenin increased the uptake of other proteins into follicles incubated in vitro. The absence of vitellogenin in the male hemolymph may therefore contribute to the reduced vitellogenic growth seen in male-grown ovaries. Although the chorion of adult male-grown eggs appeared normal in surface architecture (Figs. 6, 7), choriogenesis was delayed in male-grown ovaries, and a lower percentage of eggs became chorionated (Fig. 5). The process of chorion formation does not appear to be stimulated by outside hormonal influences since some ovarioles in male-grown ovaries contained chorionated eggs and other ovarioles in the same ovary (and presumably exposed to the same hormonal environment) contained smaller follicles with no evidence of chorion formation. Was the delay in chorion formation in male-grown ovaries due to the longer time it took for male-grown oocytes to reach a prescribed size? Two lines of evidence indicate that oocytes must achieve a prescribed size before chorion formation is initiated: 1) the small variance in the size of chorionated eggs within experimental groups; and 2) the presence in adult male-grown ovaries of ovarioles containing smaller terminal oocytes showing no evidence of chorion formation, while other ovarioles within the same ovary contained chorionated eggs of the prescribed size. The cue for the cessation of uptake and the initiation of chorion formation is unknown and the reason for the difference in the sizes of chorionated eggs in male-grown, normal, and female-operated control ovaries is as yet unclear. SDS-PAGE analysis of hemolymph from males receiving ovaries and of soluble extracts of male-grown ovaries showed that vitellogenin production was not initiated by the ovary transplant and that only some male hemolymph proteins were internalized (Fig. 8, arrowheads). Endocytosis of these male hemolymph proteins thus occurred in the absence of vitellogenin. Zhu et al.  likewise found that male-grown ovaries from B. nzori internalized hemolymph proteins in the absence of vitellogenin. They found that male-grown ovaries were deficient in vitellin, but contained non-sex-linked 30 kD proteins found in the hemolymph as well as an egg-specificprotein. Since endocytosis of male hemolymph proteins occurred in the absence of vitellogenin, this indicates that the onset of the processes of patency and endocytosis are not cued by the prolonged period of high vitellogenin titers which Development of Male-IncubatedOvaries 233 are seen in the hemolymph of last instar gypsy moth larvae. Why is there a prolonged period of high vitellogenin titers in gypsy moths? Perhaps, like storage proteins which are needed during metamorphosis, vitellogenins are produced during the larval feeding stage  and simply stored in the hemolymph in these moths which initiate vitellogenic growth of their oocytes during the pharate adult stage. We have shown that ovarian development proceeds normally in males, but at a slower rate. Why is growth (uptake) slower in males: Is this due to an unknown hormonal difference between male and female pupae? Or to the absence of the vitellogenin ligand? Or is it simply the result of the smaller size and lower nutritive resources of male pupae? Studies by Telfer and Rutberg  in H. cecropia showed that male pupae which received ovary implants and were transfused with hemolymph from female pupae exhibited a greater increase in the size of their oocytes than those which were transfused with male hemolymph. This indicates that some element in the hemolymph of female pupae, possibly vitellogenin, enhances oocyte growth. That the responsible element is vitellogenin is indicated by studies in H. cecropia in which vitellogenin increased the uptake of other proteins into follicles incubated in vitro . We will address this question in the gypsy moth in future studies by injecting purified vitellogenin into male pupae which have received transplanted ovaries, and by incubating follicles in vitro in the presence and absence of vitellogenin and other hemolymph proteins. Since endocytosis occurs in the absence of vitellogenin, this raises the question of whether vitellogenin-receptorproduction and insertion into membranes continues in its absence. Another question of interest is whether the accumulative pathway leading to yolk sphere production is altered in the absence of vitellogenin, as it is in mosquitoes, a species that feeds as an adult . 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