Hemolymph ecdysteroids do not affect vitellogenesis in the lubber grasshopper

The role of hemolymph ecdysteroids in the reproduction of non-dipteran insects is unclear. We examine the role(s) of hemolymph ecdysteroids during egg production in the lubber grasshopper, Romalea microptera. In all individuals, hemolymph ecdysteroids rose to a sharp peak with similar maxima and then fell to undetectable levels. The time from the adult molt to the maximum ecdysteroid titer (E(max) titer) varied in response to food availability, whereas the time from E(max) titer to oviposition was unrelated to food availability. Because both the timing of egg production and the timing of E(max) responded similarly to environmental changes, ecdysteroids may be involved in egg production. We hypothesized that this role is the stimulation of vitellogenesis. Ovariectomized females had vitellogenin but no ecdysteroids, so ecdysteroids are not necessary for vitellogenin production. In addition, treatment of females with ecdysteroids altered neither Vg titers nor ovarian growth. Ovarian ecdysteriods increased at the same age in development as hemolymph ecdysteroids. In contrast to hemolymph ecdysteroids, ovarian ecdysteroids persisted until oviposition. Despite this, [(3)H]ecdysone injected into the hemolymph was detected later only at very low levels in the ovary, suggesting that hemolymph ecdysteroids are not sequestered by the ovary. In summary, our studies indicate that hemolymph ecdysteroids in adult females of the lubber grasshopper are associated with the timing of egg production, but they neither regulate vitellogenesis nor act as a source of ecdysteroids for the ovary.     

Plasticity and canalization in the control of reproduction in the lubber grasshopper

The ability to change reproductive tactics during adult developmentin response to environmental variation is predicted to enhancefitness. Many organisms show phenotypic plasticity early innon-embryonic development, but later exhibit phases of developmentalinflexibility (=canalization). Therefore, we studied reproduction-relatedhormones and proteins and their relationships to plasticityin the Eastern lubber grasshopper. Diet-switching experimentsdemonstrated plasticity early in the egg production cycle, buta switch to canalization late in the cycle. We measured developmentaltiters of 4 hemolymph compounds from single individuals fromadult molt until first oviposition. These 4 compounds were theegg-yolk precursor protein vitellogenin, juvenile hormone (thecentral regulator of insect reproduction), major hemolymph proteins,and ecdysteroids (the arthropod molting hormone that ultimatelyis stored in the egg). Using diet manipulations, we investigatedhow these developmental titers relate to the switch from plasticto canalized egg production. All 4 hemolymph compounds reachedtheir peak levels during the canalized phase, about 12 day beforeoviposition. Diet switches after these peak levels did not affectthe timing to oviposition. Therefore, these peak titers werephysiological events that occurred after the individual committedto laying. We compared these patterns in reproduction to thedevelopment toward adult molt, another major life-history eventin insects. We observed an extended canalized phase before theadult molt. This canalized phase always included a peak of ecdysteroids.The similar patterns in the physiology of these life-historyevents suggested that common limitations may exist in majordevelopmental processes of insects that are directed by hormones.     

Interpopulation variation in developmental titers of vitellogenin, but not storage proteins, in lubber grasshoppers

We examined simultaneous plastic and latitudinal interpopulation variation in the time course of hemolymph protein titers during egg production in the lubber grasshopper. Our goal was to gain insight into possible evolutionary changes in the physiology underlying reproductive plasticity. We used lubbers from three locations in the United States (Florida [FL], Louisiana [LA], and Georgia [GA]), each offered three daily food rations. Previous genetic analysis indicated that grasshoppers from FL (the low-latitude population) and GA (the high-latitude population) were phylogenetically closer to each other than to LA grasshoppers (the intermediate-latitude population). The ages at maximum titers of vitellogenin (Vg(max)) and three storage proteins that were referred to as major hemolymph proteins (MHP(max)) were used as indices of the progress of oocyte development. Age at Vg(max) was affected significantly both by diet and by population. Perhaps most importantly, age at Vg(max) was less for GA grasshoppers than for FL and LA grasshoppers; this pattern differs from the phylogenetic relationships of the populations. Age at MHP(max) was significantly affected only by diet and not by population. Hence, the regulation of these proteins may differ across populations. Finally, we found no evidence that plasticity of reproductive investment in response to food availability differs across populations (as indicated by nonsignificant interactions of population and feeding environment).     

Vitellogenin and egg production in the moth,Heliothis virescens

The characteristics of vitellogenin (Vg) and the relationship between Vg production and egg production in the tobacco budworm, Heliothis virescens, were studied. The relationship between Vg production and juvenile hormone (JH) and the impact of mating on Vg and egg production were also investigated. Vg appears in the hemolymph of H. virescens about 6 h after moth eclosion. Vg may be separated into two apoproteins (ApoVg-I and ApoVg-II) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights were calculated to be 156,065 ± 800 for ApoVg-I and 39,887 ± 323 for ApoVg-II. SDS-PAGE analysis revealed that the female hemolymph Vg polypeptides appear to be identical to those from eggs but are absent in male hemolymph. Vg concentration was significantly higher in mated females than in virgin females of the same age at 48 h after emergence. Rates of egg production increased as Vg production increased; rates of egg production in mated females were significantly higher than those of virgin females at 48, 72, 96, and 120 h postemergence. Vg production is dependent on JH, because hemolymph from decapitated females lacked Vg while that of decapitated females treated with synthetic JH had Vg at levels comparable to similarly aged, normal H. virescens females. Hemolymph JH titers in mated females were significantly higher compared with those in virgin females at all sampling periods. The high JH level in mated females may explain the high Vg and egg production in mated H. virescens. Arch. Insect Biochem. Physiol. 34:287–300, 1997. © 1997 Wiley-Liss, Inc.     

Quantification of vitellogenin-mRNA during maturation and breeding of a burying beetle

Burying beetles (Nicrophorus orbicollis) are unusual in that to breed they require an unpredictable and valuable resource, a small carcass. Thus the timing of reproduction is unpredictable and beetles' physiological response must be fast. We hypothesized that their pattern of vitellogenin (Vg) synthesis might reflect these requirements. We examined the expression of two Vg genes (sequenced for this study) during sexual maturation and through a reproductive bout. Vg-mRNA, juvenile hormone (JH) titers, ovarian development, and hemolymph concentrations of Vg were quantified in the same individuals. All four variables gradually increased during maturation to peak 15-20 days after eclosion. Twelve hours after the discovery of a carcass, a few hours before oviposition, mRNA was high, hemolymph Vg had decreased, JH and ovarian weight had increased. After oviposition, mRNA was low, hemolymph Vg concentrations and JH were high. This is consistent with our hypothesis that beetles produce and store Vg in the hemolymph prior to the discovery of a breeding resource and replace it quickly. Partial regression of these variables (with the effect of time removed) indicated that JH was not correlated with mRNA, hemolymph Vg, or ovarian weight at any time. Thus the role of JH as a gonadotropin remains unclear.     

Juvenile hormone is a marker of the onset of reproductive canalization in lubber grasshoppers

To meet the challenge of unpredictable environments, many animals are initially developmentally flexible (plastic) but then may become inflexible (canalized) at major developmental events. The control of reproductive output can undergo a switch from flexible to inflexible (Moehrlin, G.S., Juliano, S.A., 1998. Plasticity of insect reproduction: testing models of flexible and fixed development in response to different growth rates. Oecologia 115, 492-500), and juvenile hormone (JH) may control this switch. By manipulating food availability, we tested the hypothesis that JH is involved in the reproductive canalization that appears during oogenesis in lubber grasshoppers. We used four food treatments: (1) high (H); (2) high switched to low (HL); (3) low switched to high (LH); and (4) low (L). We collected hemolymph samples approximately every 4 days and measured the ages at which maximum JH level (JH(max)) and oviposition occurred. Diet significantly affected both age at JH(max) and age at oviposition. In contrast, diet had no significant effect on the time from JH(max) to oviposition nor on the maximum JH level observed. Our data demonstrate that, after JH(max) is reached, the time to oviposition in our grasshoppers was unresponsive to food availability. Hence, reproductive timing appears to be canalized after the JH(max). This is the first demonstration in a phytophagous insect that a particular factor (in this case, JH) can be used to mark the switch from reproductive plasticity to reproductive canalization.     

Purification and characterization of biliverdin-binding vitellogenin from the hemolymph of the common cutworm,Spodoptera litura

Biliverdin-binding vitellogenin (Vg) was purified from adult female hemolymph of the common cutworm, Spodoptera litura, by using gel filtration and ion exchange chromatographies. The molecular mass of the protein was 490 kDa and it was composed of two 188-kDa subunits. Three internal amino acid sequences obtained by digestion of the protein with lysylendopeptidase showed high similarity to those of Bombyx mori Vg, supporting the purified blue protein to be vitellogenin. latroscan analyses demonstrated the presence of biliverdin in Vg that occupied 2.4% of total lipid components. Among the lipids of Vg (9.5 micrograms total lipids per 100 micrograms protein), diacylglycerol was the most predominant, followed by phospholipid, hydrocarbons, and then triacylglycerol, while in biliverdin-binding proteins (BPs) purified from larval hemolymph (3.1 micrograms total lipids per 100 micrograms protein), phospholipid was the most abundant lipid followed by diacylglycerol; hydrocarbons and triacylglycerol were minor components. Vg was first detected in the hemolymph of female pupae one day before eclosion, but injection of 5 micrograms of methoprene into a 3-day-old pupa induced Vg in the hemolymph 4 days earlier than in the control. Methoprene also induced a faster decline in BP-A and BP-B titers in the hemolymph with a corresponding increase of the Vg titer. These results suggest that juvenile hormone (JH) induces not only vitellogenesis but also the uptake of these proteins by stimulating the metamorphosis of fat body during the pupal stage.     

Regulation of vitellogenesis in the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae)

Studies were undertaken to investigate vitellogenesis and its regulation in female adults of the fall armyworm, Spodoptera frugiperda. A single female-specific protein, likely to be the S. frugiperda vitellogenin (Vg), appeared approximately 5 h after adult eclosion in the hemolymph of virgin females. The concentration of the protein increased with age as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed. A protein with the same relative molecular mass was also present in egg extracts, but absent from hemolymph samples from male moths. The relative molecular mass of the designated S. frugiperda Vg was determined as 164.5+/-2.5 kDa. Vitellogenic oocytes became visible 36-48 h after emergence and egg deposition began on day 3 of adult life. Vg could not be detected in the hemolymph of females decapitated directly after eclosion. When decapitated virgin females were injected with the JH-mimic methoprene (MP), the level of Vg was comparable to that in non-decapitated moths, indicating that vitellogenesis in S. frugiperda depends on juvenile hormone (JH). However, the number of vitellogenic oocytes was somewhat lower than in non-decapitated virgin females. Injection of 20-hydroxyecdysone (20E) promoted Vg production to a similar extent in decapitated female moths, but in contrast to methoprene injection, treatment with 20E never resulted in the production of vitellogenic oocytes. In vitro cultivated ovaries of adult females dissected directly after eclosion produced lower amounts of ecdysteroids than those isolated on day 1 after emergence. Our results suggest a crucial role for 20E in the induction of vitellogenesis in the noctuid S. frugiperda, while JH seems to be essential for the continued uptake of Vg by developing oocytes and may trigger 20E biosynthesis in the ovary.     

Juvenile hormone biosynthesis,oocyte growth and vitellogenin accumulation in Choristoneura fumiferana and C. rosaceana: a comparative study

We assessed the effects of age and mating status on in vitro juvenile hormone (JH) biosynthesis, oocyte growth, egg production and vitellogenin (Vg) accumulation in the tortricid moths, Choristoneura fumiferana and C. rosaceana. To determine whether vitellogenesis is dependent on the presence of JH, we also examined the effects of decapitation and JH analog treatments on egg production. In both species, the corpora allata (CA) of adult females released fmol quantities of JH, with JH II being the major homolog produced. The CA began producing detectable quantities of JH around the time of emergence. Full activation of the CA was observed a few hours sooner in C. fumiferana than in C. rosaceana. In pharate adults and young virgin females of both species, growth of the basal oocyte reflected changes in CA activity. Decapitation of newly emerged females significantly reduced egg production, but treatment of decapitated females with the JH analog methoprene resulted in egg production that was similar to (C. fumiferana) or greater than (C. rosaceana) that of controls, indicating that JH is required for oocyte maturation. Vg was first observed in the hemolymph before the presumptive time of CA activation, suggesting that the synthesis of this protein is not dependent on JH. The presence of normal quantities of Vg in the hemolymph of pupae decapitated before CA activation confirmed this hypothesis. The Vg titer underwent a transient decline following CA activation and was significantly lower in mated than in virgin females of both species 3 and 5 days after copulation. Since CA activation at emergence and mating are both expected to cause a rise in the JH titer, we suggest that the declines in the levels of Vg result from JH-enhanced Vg uptake by the developing oocytes. Mating induced a significant increase in egg production but had no measurable impact on rates of JH biosynthesis in vitro.     

Effects of starvation on the vitellogenesis,ovarian development and fecundity in the ectoparasitoid,Nasonia vitripennis (Hymenoptera: Pteromalidae)

A female‐specific protein, vitellogenin (Vg), and its corresponding egg vitellin (Vt) are identified in the ectoparasitic wasp Nasonia vitripennis. Both native Vt and Vg have a molecular mass of about 350 kDa, which is composed of two subunits of approximately 190 kDa and 165 kDa under reducing and denaturing conditions (sodium dodecyl sulfate—polyacrylamide gel electrophoresis). An indirect sandwich enzyme‐linked immunosorbent assay developed with both monoclonal and polyclonal antibodies against N. vitripennis Vt. Vg was first detected in the hemolymph on the 10th day after parasitism, and was first observed in oocytes on the 12th day. In adults deprived of food, the highest hemolymph Vg level occurred at the time of adult eclosion and the highest level of Vt in ovaries was found at 30 h after eclosion. In contrast, feeding adults with 20% sucrose resulted in the reduction of Vt uptake by ovaries and the extension of life span, but had little effect on Vg production. Deprived of hosts, starvation of female wasps had no significant effects on ovariole growth and oocyte maturation before the wasps died. However, starvation of female wasps supplied with hosts accelerated the wasps laying progeny into hosts, but resulted in a decrease of total progeny production by comparison with wasps fed with 20% sucrose.     

Tebufenozide disrupts ovarian development and function in silkmoths

Adult development and production of up to 400 eggs within the pupal case of female silkmoths are both dependent on 20-hydroxyecdysone (20E), the steroid hormone of insects. When adult development was initiated with tebufenozide, the non-steroidal ecdysteroid agonist, instead of 20E, full development of all epidermal tissues like the wing was witnessed, but ovarian growth and egg formation was minimal. Administration of tebufenozide to female pharate adults caused disruption of the follicular epithelium, produced nurse cell damage, and inhibited oogenesis. Reduced ability to synthesize RNA and protein accompanied these tebufenozide induced morphological disturbances of the follicles. In vivo accumulation of vitellogenin (Vg) from the hemolymph was reduced in tebufenozide treated female ovaries as well as their ability to accumulate Vg in vitro. Determination of protein staining intensity and antibody reactivity of Vg pointed out that hemolymph Vg level remained fairly constant all through adult development whether induced by 20E or tebufenozide. Measurement of hemolymph volumes and hemolymph Vg levels of control and experimental animals allowed us to conclude that egg development involves the uptake of all the hemolymph proteins and not Vg alone. The loss of hemolymph that accompanies egg maturation was considerably reduced in tebufenozide initiated female pharate adults. 20E could not overcome ovarian growth inhibitory effects of tebufenozide. Dual mechanisms, one involving ecdysteroid antagonist action at the beginning of development, and the other unrelated to that function during heightened egg formation, are needed explain the biphasic inhibitory actions of tebufenozide on silkmoth ovaries.     

JH III production, titers and degradation in relation to reproduction in male and female Anthonomus grandis

Juvenile hormone (JH) is necessary for the production of vitellogenin (Vg) in the boll weevil, Anthonomus grandis. Occurrence of Vg in this species is typically restricted to reproductively competent females, and is not detected in untreated males. However, the JH analog, methoprene stimulates Vg production in intact males and in the isolated abdomens of both male and female boll weevils (where in each case no Vg is detected without treatment), suggesting that males are competent to produce Vg but are normally not stimulated to do so. Preliminary work indicating that male boll weevil corpora allata (CA) produced little or no JH in vitro suggested that failure of males to produce Vg might be due to very low JH levels compared to females. This study re-examines the question of JH in male boll weevils by determining in vitro production of JH III by male CA during the first 10 days after adult emergence, determining hemolymph JH esterase activity during this same time period and hemolymph JH III titers in adults of both sexes. We also re-examine the ability of isolated male abdomens to produce Vg in response to hormonal stimulation, analyzing the effect of a wide range of methoprene and JH III dosages. Results indicate that male A. grandis have circulating JH titers and JH production similar to females. JH esterase activity is slightly but significantly higher in males than females. Vg production by isolated abdomens of both sexes after stimulation with methoprene or JH III was confirmed. Dose response studies indicated that high levels of methoprene were less effective than intermediate doses in stimulating Vg synthesis in both sexes. We conclude that the sexually dimorphic effect of JH on Vg synthesis is not due to differences in JH production or differences in JH titer between the sexes.     

Vitellin of Pteromalus puparum (Hymenoptera: Pteromalidae), a pupal endoparasitoid of Pieris rapae (Lepidoptera: Pieridae): Biochemical characterization, temporal patterns of production and degradation

Vitellin (Vt) and vitellogenin (Vg) profiles were analyzed in Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. Non-denaturing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses indicated that both native Vt and Vg were likely 370 kDa in size, consisting of two subunits of approximate 206 and 165 kDa. An indirect double antibody enzyme-linked immunosorbent assay (ELISA) for monitoring hemolymph Vg and ovarian Vt levels was developed using a monoclonal antibody and a polyclonal antibody made specially against P. puparum Vt. The synthesis and uptake of Vg in this wasp was initiated immediately after adult eclosion. The hemolymph Vg and ovarian Vt reached their highest level of 0.58 and 4.51 microg per female 24 and 48 h after adult eclosion, respectively. Both Vg synthesis and uptake were in parallel with ovarian development. The Vt levels in the developing embryos decreased progressively except 12h after parasitism. Meanwhile, nine new polypeptides with sizes ranging from 59.2 to 151 kDa, possibly resulting from the limited proteolysis of Vt originally accumulated in newly laid eggs, were detected de-novo during embryonic development using Western blotting with the monoclonal antibody against Vt. These studies provide the basis for future investigation into endocrinal regulations of vitellogenesis and understanding the reproductive strategy in this wasp.     

Ecdysteroids in the adults and eggs of Opogona sacchari (Bojer), an invasive alien pest

In order to understand the composition and quantitative variation of ecdysteriods in the adults and eggs of Opogona sacchari (Bojer), an invasive alien pest, we analyzed the ecdysteroid composition and titers in the adult and egg of this pest. On day 4 after eclosion, the titer of ecdysteriods in the male adult was 0.080 ng/adult, much lower than 5.978 ng/adult in the female adult. During the development of ovaries, the titer of ecdysteriods was low on the first two days, and high in the late period, with the peak (10.48 ng/ovary) appearing on day 3. During the development of eggs, the titer of ecdysteriods was about 0.010 ng/egg from day 1 to day 3, and then decreased to 0.006 ng/egg on day 4. In both adults and eggs, three main components of ecdysteriods were found by identification of HPLC/RIA. They were 20-hydroxyecdysone, 26-hydroxyecdysone, and an unidentified component.     

Laying workers in queenless honeybee (Apis mellifera L.) colonies have physiological states similar to that of nurse bees but opposite that of foragers

Honeybee workers shift their labors from nursing their brood to foraging according to their age after eclosion. When the queen is lost from the colony, however, some workers become 'laying workers' whose ovaries develop to lay eggs. Here we investigated whether the physiological state of laying workers is more similar to that of nurse bees or foragers by examining the hypopharyngeal gland (HPG) and hemolymph vitellogenin titers. In a normal colony, nurse bees have well-developed HPGs that synthesize 'major royal jelly proteins' and high hemolymph vitellogenin titers, whereas foragers have shrunken HPGs that synthesize 70-kDa alpha-glucosidase and low hemolymph vitellogenin titers. In queenless colonies, however, workers with developed ovaries (laying workers) tended to have more developed HPGs and to synthesize major royal jelly proteins, whereas workers with shrunken HPGs tended to synthesize alpha-glucosidase and to have undeveloped ovaries. Furthermore, the workers with developed ovaries had higher vitellogenin titers than nurse bees, whereas those with undeveloped ovaries had lower vitellogenin titers. These findings indicate that the physiological state of laying workers is similar to that of nurse bees, but opposite that of foragers.     

Bombyxin secretion in the adult silkmoth Bombyx mori: sex-specificity and its correlation with metabolism

Changes in the hemolymph bombyxin titer of the adult silkmoth Bombyx mori were investigated by time-resolved fluoroimmunoassay. Immediately after eclosion, hemolymph bombyxin titers were low in both males and females, and then increased steeply in males to a very high level and this high titer was maintained for at least 3 h, whereas the titer increment in females was small and transient. The difference in the change of bombyxin titer between males and females suggests that bombyxin is responsible for the regulation of physiological changes underlying sexually different activities of the adult moths. However, no evidence was obtained that bombyxin controls adult metabolism as far as the effects of bombyxin on the concentrations of carbohydrates and lipids in the hemolymph were investigated. The change in the hemolymph trehalose concentration was almost the same between sexes, and between intact and neck-ligated moths. Furthermore, bombyxin injection did not affect the hemolymph trehalose concentration nor trehalase activity in the muscle. Although the hemolymph lipid concentration rose after eclosion in males, it was not influenced by bombyxin. These results exhibit striking contrast to the results of our previous study, in which bombyxin showed hypotrehalosemic activity in the larval stage, thus indicating that the action of bombyxin changes during metamorphosis.     

Temporal profiles of juvenile hormone titers and egg production in virgin and mated females of Heliothis virescens (Noctuidae)

Juvenile hormones (JH) I, II, and III were monitored in hemolymph of virgin and mated females of various ages in Heliothis virescens. JH I was the predominant homologue followed by JH II, but JH II was present at a higher level in young virgin females. JH III was detectable only at a low level. In virgin females, hemolymph JH titers were low at emergence (2.2ng/ml-total amount of JH I and JH II), but increased thereafter and reached a maximum at 24h of age (53.5ng/ml). At 30h and 36h of age, JH titers dropped to a low level, but increased again in older virgin females. After mating, JH titers increased significantly. JH titers at 0h after uncoupling (137.4ng/ml) were nearly 3 times as high as those in 24-h-old virgin females. Within 6h after uncoupling, JH titers decreased slightly, but titers increased with age of mated females and reached a level of 320.2ng/ml hemolymph at 72h after uncoupling. The titer of JH I and JH II was correlated highly with total number of eggs produced (r(2)=0.70, P<0.001). Mating stimulated JH production, resulting in an increase in egg production.     

Age- and density-related oviposition behavior of the European corn borer,Ostrinia nubilalis (Lepidoptera: Pyralidae)

Age-related oviposition patterns ofOstrinia nubilalis were studied at three population densities in the laboratory by releasing newly eclosed adults in wire-screened cages and analyzing their oviposition throughout the adult stage with digital analysis. Oviposition sequences of individual females depositing egg masses were documented on the third and seventh nights after eclosion with a video camcorder. During a sequence, a female produced an egg in an average time of 15 or 26 s on the third and seventh nights, respectively, and completed depositing an egg mass the size of 20–39 eggs in an average time of 316 and 525 s, respectively. Females were not easily disturbed during egg mass deposition and pulsated their abdomen before deposition of each egg. Females produced few egg masses the first night after eclosion. Oviposition increased on nights 2 and 3 but declined steadily thereafter as females matured. Females older than 6 nights produced fewer egg masses; the proportion of egg masses with fewer than 20 eggs increased gradually. By the end of the adults' lifetime, nearly 100% of the egg masses had fewer than 20 eggs. The data are fundamental to our research to define the role of phytochemicals in modifying oviposition behavior of the European corn borer.     

Cypermethrin induction of vitellogenesis and ovarian development in unfed adult female Ornithodoros moubata (Acari: Argasidae)

Summary

Vitellogenesis in ticks is known to be induced by engorgement and mating. In this paper, the synthetic pyrethroid cypermethrin (CyM) is shown to induce production of yolk protein precursor, vitellogenin (Vg), and ovarian development in unengorged mated adult female Ornithodoros moubata. The levels of Vg found in the hemolymph and ovarian development induced by CyM were dose-dependent. i.e., CyM doses of more than 0.2 and 1.0 μg/tick were needed for significant increase of Vg titer in the hemolymph and yolk deposition in oocytes, respectively. Immunological and electrophoretical analyses of Vg and Vitellin (Vn) induced by CyM were identical with those induced by engorgement. Vg titer induced by CyM in unengorged females followed approximately the same time course as that in the normal engorged females. However, Vg titer induced by CyM continued to increase after day 8 and reached a maximum (95 μg/μ1) on day 10 after treatment, while Vg titer induced by engorgement decreased again after reaching a maximum (60 μg/μ1) on day 6, correlated with yolk Vn deposition in oocytes. Ovarian development induced by even high doses (10 or 20 μg/tick) of CyM was slow compared to normal development stimulated by engorgement. Oviposition was not observed in females treated with CyM.