The folliculostatins of two dipteran insect species, their relation to matrix proteins and prospects for practical applications

The first insect folliculostatic peptide was isolated from vitellogenic ovaries of the mosquitoAedes aegypti. This decapeptide directly inhibits trypsin biosynthesis in the gut, and indirectly ovarian development. The factor was named Trypsin Modulating Oostatic Factor or TMOF by its discoverers. From the fleshfly Neobellieria bullata 2 folliculostatins have been isolated, the hexapeptide Neb-TMOF and the 19-mer Neb-colloostatin. The available data suggest that at least 2 of the 3 folliculostatins originate from matrix (like) proteins present in the ovary, a hitherto unknown source of hormones. Furthermore, one of the folliculostatins (Neb-TMOF) is a potent inhibitor of ecdysone biosynthesis by larval ring glands of fleshflies. The discovery of the dipteran folliculostatins, which do not show any resemblance to inhibins of vertebrates, may significantly contribute to a better understanding of the hormonal control of growth in insects and perhaps, in other animals as well. None of the 3 folliculostatins is blocked at its N- or C-terminus. This, in combination with the pleiotropy of their effects and the narrow species specificity make such peptides prime candidates for, testing their potential in insect pest control by means of molecular biological methods.     

Physiological and biochemical effects of Olea europaea leaf extracts from four phenological growth stages on the oogenesis of female locust Locusta migratoria

The effects of olive leaf extract (OLE) are studied on several reproductive variables and the ovarian biochemical composition of Locusta migratoria (Orthoptera: Acrididae) adult females. The methanolic extracts are prepared from the leaves sampled during four phenological growth stages of olive tree: cluster formation (Cf), swelling inflorescence buds (Sib), full flowering (Ff) and endocarp hardening (Eh). When applied to adult females during the pre‐ovipositional phase, the treatment elicites a significant adverse effect on their reproductive potential. Indeed, OLE significantly reduces both fecundity and fertility and affects oocyte growth during the first gonadotrophic cycle, as indicated by measurements of ovarian weight, length of terminal oocytes and ovarian index. Furthermore, OLE is examined with respect to ovarian biochemical components. Biochemical analyses reveal a significant reduction of ovarian contents of proteins, lipids and carbohydrates, suggesting a disruption in the incorporation of the haemolymph metabolites in the oocytes and an interference of OLE with the vitellogenesis process. The antigonadotrophic effect is confirmed by a histological study of the ovaries, which clearly shows a delay in ovarian development and in yolk accumulation in the basal oocytes of treated females. The most effect is noted with the extract prepared from the leaves collected at the swelling inflorescence buds for all measured parameters, which appears to be related to its high content of polyphenols.     

Effects of a methanolic extract of the plant Haplophyllum tuberculatum and of teflubenzuron on female reproduction in the migratory locust, Locusta migratoria (Orthoptera: Oedipodinae)

The effects of a methanolic extract of the plant Haplophyllum tuberculatum (ME-Ht) and of teflubenzuron (TFB) were compared on several reproductive variables and ecdysteroid titers in the females of Locusta migratoria. The test products were administered orally to newly emerged females at doses of 1500 (ME-Ht) and 10μg/female (TFB). The methanolic extract and TFB had comparable effects on several of the variables examined. Both significantly delayed the first oviposition and reduced fecundity and fertility. ME-Ht and TFB also displayed similar effects on ovarian growth, vitellogenesis and ecdysteroid titers. Both treatments induced a drop in hemolymph protein levels as well as a reduction in vitellogenin uptake by oocytes. This delay in oogenesis was accompanied by a resorption of terminal oocytes. However, whereas TFB completely blocked egg hatch, ME-Ht only had a modest inhibitory effect on this variable. Hemolymph and ovarian ecdysteroid titers, as measured by radioimmunoassay, were similar and low in both control and treated females, except for a peak observed only in control females at the end of vitellogenesis. We discuss the functional significance of the observed effects in the context of the putative modes of action of the methanolic plant extract and TFB.     

Effect of ecdysone agonist RH-0345 on reproduction of mealworm,Tenebrio molitor

RH-0345 belongs to a new group of insect growth regulators (IGRs) with a benzoylhydrazine structure that mimic the action of the natural insect molting hormone 20-hydroxyecdysone. After topical application on female adult beetles of mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae), first oviposition was delayed, the number of eggs per female was reduced by 32%, the follicular epithelium was thinner (-33%) during sexual maturation, the size of deposited eggs was reduced, and egg viability was lost by 68%. Treatment with RH-0345 had also reduced the ovarian protein content and two protein bands were missing in the ovaries. Ultrastructural observations of the ovaries at the end of vitellogenesis in treated females, however, showed no evident differences with the fine structure of both follicular cells and oocytes in controls. In addition, we measured the amount of ecdysteroids in the medium of treated ovary cultures in vitro and in the eggs deposited by treated females. Possible action sites with the reproductive system at different levels in T. molitor are discussed for this novel group of IGRs.     

Ovarian structure and batch fecundity in Lophiomus setigerus

The ovarian structure and batch fecundity of anglerfish Lophiomus setigerus were examined from specimens collected in the East China Sea during March 1991 to September 1995. The right and left ovarian lobes were connected at their posterior ends. Stalk-like ovigerous lamellae protruded from the ovarian wall. During the spawning season, gelatinous material was secreted from the epithelia of both the ovigerous lamellae and ovarian wall, and these epithelia showed morphological changes accompanying the ovarian maturation cycle. Tertiary yolk, migratory nucleus, and mature stage oocytes occurred in the ovaries between May and November, when females with postovulatory follicles and developing vitellogenic oocytes were collected also. These results suggested an extended spawning season during which females undergo repeated spawnings. When the most advanced oocytes attained the secondary yolk stage, they formed a batch that separated from the adjacent group of smaller oocytes. Batch fecundity ( F ) in 20 females with secondary yolk stage ovaries was related to total length (L_T, mm) as F= 556.2 L_T^1.157 (300≤L_T≤396).     

Gonadal development and diandric protogyny in two populations of Dascyllus reticulatus from Madang, Papua New Guinea

Two Dascyllus reticulatus populations from Madang, Papua New Guinea exhibited diandric protogyny. In both populations, gonads began as undifferentiated, and then developed oocytes in the primary growth stage and an ovarian lumen. From this ovarian state or from more developed ovaries containing oocytes beyond the primary‐growth stage, some gonads developed into testes. The first sign of testicular development was degeneration of oocytes, degeneration of oocytes in the primary growth stage in ovarian gonads and degeneration of oocytes of all growth stages present including the primary growth stage in ovaries, which was then followed by development of spermatogenic tissue. In both populations, most of the fish that had gonads with degenerating oocytes were smaller than the smallest mature females, indicating that development towards testes was mostly initiated in immature gonads containing only pre‐vitellogenic oocytes. On some occasions, however, females as large as other mature females also had gonads with degenerating oocytes, suggesting that development towards testes may have occurred in mature ovaries as well. This latter notion is further strengthened by the discovery of a fish having a gonad that contained both degenerating vitellogenic oocytes and developing spermatogenic tissue. Taken together, these results suggest that D. reticulatus can exhibit diandric protogyny, because testes in D. reticulatus developed from juvenile gonads as well as from mature ovaries.     

Effects of flucycloxuron, a chitin synthesis inhibitor, on reproductive events and thickness of chorion in mealworms

Flucycloxuron (FCX), a benzoylphenylurea derivative, was evaluated on Tenebrio molitor. The compound was incorporated into the diet and administrated to newly emerged females at various doses (2, 5 and 10 mg/kg). FCX was found to affect several reproductive events such as the duration of preovipostion and oviposition period, the fecundity, the viability of eggs and the duration of embryonic development, respectively. Morphological study of ovaries showed that FCX reduced both oocytes number, the ovaries weight and the size and the volume of the basal oocyte during the sexual maturation. In addition, it reduced the thickness of chorion from freshly laid eggs. However, electron microscopic study revealed that this compound had no significant effect on the fine structure of chorion. Finally, measurements of ovarian ecdysteroids production by an enzyme immunoassay indicated a reduction in the hormonal amounts recorded.     

棉铃虫卵巢形态与卵子发生过程观察

害虫发生高峰期、 发生量的准确预测和田间防治适期的确定与种群雌虫卵巢结构及卵子发生过程密切相关。为了明确棉铃虫Helicoverpa armigera卵巢结构及卵子发生过程, 本研究利用光学体视显微镜和透射电子显微镜, 对棉铃虫成虫卵巢管和卵子的超微结构进行了研究, 并确定了发育级别划分标准。结果表明: 根据卵巢的形状、 卵的产生过程、 卵黄沉积情况等将棉铃虫卵巢发育程度分为6个级别, 即发育初期(0级)、 卵黄沉积前期(Ⅰ级)、 卵黄沉积期(Ⅱ级)、 成熟待产期(Ⅲ级)、 产卵盛期(Ⅳ级)和产卵末期(Ⅴ级)。根据卵子发生过程中滋养细胞、 卵母细胞的变化, 将卵子发生期分为3个阶段： 卵黄发生前期、 卵黄发生期和卵黄成熟期。本研究首次对棉铃虫的卵子发生进行电子显微观察, 并完善了棉铃虫卵巢发育的分级标准, 为进一步研究棉铃虫的生殖发育机理提供了理论参考, 对田间棉铃虫种群发生期和发生量的预测预报也有重要的实践参考价值。     

Reproductive biology of the prawn Melicertus kerathurus (Decapoda: Penaeidae) in Thermaikos Gulf (N. Aegean Sea)

The reproductive biology including insemination frequency, ovarian maturation, gonadosomatic index (GSI), size at first maturity, and fecundity of Melicertus kerathurus were investigated using monthly samples from Thermaikos Gulf. Insemination was recorded by the presence of a spermatophore in the thelycum, and ovarian development was based on macroscopic ovarian staging (ST1–ST5). Inseminated females were found throughout the year with high percentages recorded from April to July. Although all ovarian stages were represented in mated females, insemination increased with size and ovarian maturation. High percentages of vitellogenic or mature ovarian stages were observed from May to July, while immature and developing ovaries were predominant mainly in winter. Spawned ovaries occurred from May to October. Carapace length at first maturity based on the presence of a spermatophore (CL_50sp) was estimated at 39.20 mm, while that based on the presence of vitellogenic and mature ovaries (CL_50ov) at 40.70 mm. The seasonal peak in the proportion of mature females (ST4) varied with size. Inseminated females at ST4 and GSI peaked in June–July. GSI varied in relation to insemination status and ovarian stage. In large females (>50 mm CL), the decline in mature ovaries and GSI increment with size indicates a relative reduction in the reproductive output. The number of oocytes ranged from 62,742 to 602,947 (mean ± SD: 268,000 ± 113,000). As the prawns are targeted during the spawning season, mainly by the artisanal fishery, and female size at first maturity is selected by artisanal net size, managerial measures toward artisanal fishery should be implemented.     

Ovarian maturation of Penaeus subtilis (Decapoda: Penaeidae): A new insight to describe oocyte development and somatic structures

We observed the presence of follicular cells (FC) in the ovaries of Penaeus subtilis (n = 1198), which led us to classify the development of germ cells into six phases: oogonia, previtellogenic oocytes, primary and secondary vitellogenic oocytes, mature oocytes and atretic oocytes. The FC changes their shape according to the development of germ cells and showed a different distribution along the ovary, which allowed differentiating vitellogenic oocytes into primary and secondary. We also observed that the postovulatory follicles (POF) are composed of follicular cells. The presence of POF in penaeids ovaries is rarely reported, but allows the differentiation between spent and resting stages, commonly grouped in reproductive biology research. Furthermore, observation of ovarian lining was useful to differentiate immature females from females that had spawned at least once. Thus, ovarian development was classified into six stages: immature, early developing, advanced developing, ripe, spent and resting. The distribution and shape variations of FC, ovarian lining features and presence of POF were considered crucial for the classification of ovarian maturation stages. The methods developed here may improve estimates of their reproductive cycle, size at first maturity and spawning season, which are important variables in future studies of the reproductive dynamics.     

Changes in biosynthesis and degradation of juvenile hormone during breeding by burying beetles: a reproductive or social role?

Burying beetles, Nicrophorus orbicollis, depend on the location of an unpredictable resource, a small vertebrate carcass, for reproduction. When they discover a carcass, they undergo a correlated rapid rise in titers of juvenile hormone (JH) in the hemolymph and ovarian development. This study investigates the regulation of the changes in JH during breeding in both male and female burying beetles and the role of JH in ovarian development. JH biosynthesis by the corpora allata (CA), measured in vitro, increased in females within an hour of their discovery of a carcass and increased later in males. After returning to low rates as oviposition began, JH biosynthesis rose again 3 days later in females but not in males. Neither the ovaries nor testes synthesized JH. There was a concomitant fall in JH esterase activity within 12 h of discovery of the carcass in both males and females. Although the rise in JH titers and biosynthesis and the fall in JH esterase is correlated with ovarian development, application of methoprene or JH III in the absence of a carcass did not result in vitellogenin uptake by the oocytes. Therefore, we conclude that, in spite of the rapid rise in JH before oviposition, it is not sufficient to regulate vitellogenin synthesis and/or its uptake by the ovaries. We suggest that its role has been preempted to organize social behavior and coordinate parental behavior between mates.     

Localization and developmental expression of the activin signal transduction proteins Smads 2, 3, and 4 in the baboon fetal ovary

We recently demonstrated that the reduction in the number of primordial follicles in ovaries of near-term baboon fetuses deprived of estrogen in utero was associated with increased expression of alpha-inhibin, but not activin betaA and betaB or the activin receptors. Therefore, we proposed that estrogen regulates fetal ovarian follicular development by controlling the intraovarian inhibin:activin ratio. As a prelude to conducting experiments to test this hypothesis, in the current study we determined whether the primate fetal ovary expressed Smads 2/3 and 4 and whether expression of these activin-signaling proteins was altered in fetal ovaries of baboons in which estrogen production was suppressed. Western blot analyses demonstrated that the 59 kDa Smad 2, 54 kDa Smad 3, and 64 kDa Smad 4 proteins were expressed in fetal ovaries of untreated baboons at both mid and late gestation and that the level of expression was not significantly altered in late gestation by in vivo treatment with CGS 20267 or CGS 20267 and estrogen. Immunocytochemistry localized Smads 2/3 and 4 to cytoplasm of oocytes and pregranulosa cells at midgestation and oocytes and granulosa cells of primordial follicles in late gestation. Smad 4 was also detected in granulosa cell nuclei in late gestation, and nuclear expression appeared to be decreased in fetal ovaries of baboons deprived of estrogen. The site of localization of Smads correlated with localization of the activin receptors IA and IIB, which we previously showed were abundantly expressed in oocytes and (pre)granulosa cells at both mid and late gestation and unaltered by estrogen deprivation. In summary, the results of the current study are the first to show that the intracellular signaling molecules required to transduce an activin signal are expressed in the baboon fetal ovary and that expression was not altered by estrogen deprivation in utero. These findings, coupled with our previous observations showing that estrogen deprivation reduced follicle numbers and upregulated/induced expression of inhibin but not activin or the activin receptors, lend further support to the hypothesis that estrogen regulates fetal ovarian folliculogenesis by controlling the intraovarian activin:inhibin ratio.     

Factors affecting number of surface ovarian follicles and oocytes yield and quality in Egyptian buffaloes

Three experiments were conducted to evaluate factors affecting number of surface ovarian follicles and oocytes yield and quality in buffalo. In Experiment 1, ovaries (n = 126) were collected in pairs from slaughtered anoestrus, early pregnant and cyclic buffaloes. Ovarian follicles (1-3, 4-9 and > or = 10 mm diameter) were counted, aspirated and oocytes were recovered and evaluated. In Experiment 2, ovaries were divided into 2 groups. Group 1, ovaries bearing a CL (n = 74) and Group 2 non-bearing CL (n = 74), ovarian follicles (2-8 mm) were counted, aspirated and oocytes evaluated. In Experiment 3, oocytes were recovered using aspiration or slicing methods. In all experiments, oocytes were classified into good, fair, poor and denuded. Results showed that the development of small and total ovarian follicles are continuous and independent in early pregnant or cyclic buffalo cows, however, it significantly decreased (P < 0.01) in the ovaries of anoestrus buffaloes. Number of medium and large size follicles was significantly increased (P < 0.01) in cyclic buffaloes on Days 10-16 and 17-22 of oestrous cycle, while large follicles was significantly decreased (P < 0.01) in the ovaries of pregnant buffaloes. A significantly higher (P < 0.01) percentage of poor and denuded oocytes were recovered from ovaries of anoestrus and pregnant buffalo. While, the highest (P < 0.01) percentage of good quality oocytes were recovered from ovaries of cyclic buffaloes on Days 1-3 and 10-16 of oestrous cycle, eliciting that the stage of oestrous cycle is affecting the quality of buffalo oocytes. In addition, the presence of a CL stimulates the development of a significantly higher (P < 0.01) number ovarian follicles which produced a significantly higher (P < 0.05) number of good quality oocytes. Slicing of buffalo ovaries produced a significantly higher number of fair, poor and denuded oocytes. In conclusion, number of ovarian follicles and yield and quality of oocytes were affected by the reproductive status, stage of the oestrous cycle, presence of a CL and the method of oocytes retrieval.     

Juvenile hormone in earwigs: Roles in oogenesis,mating, and maternal behaviors

The role of juvenile hormone (JH) in courtship, mating, maternal behavior, and the ovarian cycle was studied in the ring-legged earwig, Euborellia annulipes (Lucas). The single, median corpus allatum makes and secretes JH III. JH III production was low in newly eclosed adult females, increasing as oocytes developed, maximal at about the time of oviposition, and low again in brooding females. Application of 35 or 122 μg JH III to newly eclosed females hastened the onset of courtship behavior, but had no effect on the age at which females first mated nor on the duration of mating, though the trend is toward advanced onset. Hormone treatment advanced the age of first oviposition and reduced clutch size and the proportion of eggs hatching but did not affect the interval from oviposition of the first clutch to oviposition of the second clutch, nor the size and proportion hatching of the second clutch. Acetone treatment and treatment with 6 μg JH III did not affect these parameters. Application of 50 μg JH III to females on the day of oviposition shortened the duration of maternal care and advanced the onset of the second gonadotropic cycle, compared with that of acetone-treated and precocene II-treated females. The duration of maternal care was positively correlated with the proportion of eggs hatching. JH titer analysis confirmed JH III to be the predominant hormone in this species and clearly demonstrated the absence of other homologues. This work also confirmed our hypothesis that intermediate to high levels of JH are associated with oocyte growth, mating, and cessation of maternal care; low levels of JH are associated with the period of maternal behavior and slow ovarian development. We are currently investigating factors which might regulate corpus allatum activity during the reproductive cycle and the subsequent period of maternal care. Arch. Insect Biochem. Physiol. 35:427–442, 1997. © 1997 Wiley-Liss, Inc.     

Induction of choriogenesis by 20-hydroxyecdysone in the German cockroach

Experiments in vitro have shown that 20-hydroxyecdysone (at a concentration of 0.2 and 2 muM and after 12 and 24 hr of incubation) is able to induce the precocious deposition of chorion materials by the follicular epithelium of young oocytes of the cockroach Blattella germanica. Since previous studies had shown that 20-hydroxyecdysone levels in B. germanica ovaries increase with oocyte maturation to reach a peak just before oviposition, we therefore hypothesize that ovarian ecdysteroids trigger Choriogenesis in this species through an autocrine action.     

Effect of Beauveria bassiana on the ovarian development and reproductive potential of Anastrepha ludens (Diptera: Tephritidae)

We investigated the virulence of a native strain of Beauveria bassiana on the mortality, ovarian development, oogenesis, survival, fecundity and fertility of 4-, 8- and 12-day-old Anastrepha ludens females. In the first study, this strain caused 85% mortality in 8-day-old treated females, with a median lethal concentration (LC₅₀) of 3.1×10⁸ (2.1×10⁸–4.7×10⁸) conidia/mL and a median lethal time (LT₅₀) of 6.5 days. In the second study, the ovarian development and oogenesis of A. ludens females treated with 1×10⁹ conidia/mL were monitored daily in sacrificed flies. The fungus infection did not significantly affect ovarian development of the treated females, although there was a delay in vitellogenesis and a reduction in the quantity of mature basal oocytes and oviposition index in these flies. Moreover, the lowest net fecundity was found in 4-, 8- and 12-day-old treated females, and reduced fertility was observed in 4-day-old treated females. An application to test the potential use of this strain under field conditions is discussed.     

Ovarian cycle in immature and adult stages ofTrichogramma cacoeciae andT. Brassicae (Hym.: Trichogrammatidae)

The ovarian cycle was studied in two species ofTrichogramma, the thelytokousT. cacoeciae and the arrhenotokousT. brassicae. For both species, egg development was recorded (1) in the immature stages (2) in adult females before and after one bout of oviposition. The two species differed in the numbers of immature eggs formed and stored in the ovaries, and in the pattern of egg replenishment after oviposition.     

Reproductive biology of female big‐bellied seahorses

In this study, ovarian morphology, reproductive condition and sex steroid levels were investigated in the big‐bellied seahorse Hippocampus abdominalis , collected by snorkel and SCUBA diving in Wellington Harbour, New Zealand. Within the ovary, oocytes were contained between an outer muscular wall and an inner layer of luminal epithelium. Two germinal ridges ran along the entire length of the ovary. In cross‐section, oocytes were arranged in sequential order of development beginning at the germinal ridges and ending at the mature edge. Ovarian lamellae were absent. Vitellogenic and advanced cortical alveoli oocytes were elongated in shape, whereas maturing oocytes were distinctively pear‐shaped. Mature oocytes were large (2·6 – 4·4 mm in length) and aligned with the animal pole towards the muscular wall. Reproductively mature females were found throughout the year indicating a protracted reproductive season. The gonado‐somatic index was significantly different between all ovarian stages, but the hepato‐somatic index was not. Females with previtellogenic ovaries had significantly higher plasma concentrations of testosterone than females with vitellogenic or maturing ovaries. There was no significant difference in plasma concentrations of testosterone between females with vitellogenic or maturing ovaries, or in plasma concentrations of 17β‐oestradiol between females in all ovarian stages. This study contributes to the knowledge on the reproductive biology of female syngnathids.     

Annual reproductive cycle based on histological changes in the ovary of the female mosquitofish,Gambusia affinis, in central Japan

To clarify the annual reproductive cycle of wild female mosquitofish,Gambusia affinis, in Mie Prefecture, central Japan, changes in ovarian histology were investigated. Female mosquitofish kept in aquaria under constant temperature (25°C) and photoperiod (16L: 8D) conditions produced successive broods at intervals of 22.1±0.46 days (n=7). Between days 0–3 following parturition, females began active vitellogenesis. Between days 3–5, fully grown oocytes matured and were fertilized, and embryonic development began in the follicles. By day 10, as fertilized eggs continued embryonic development, some oocytes at the oil-droplet stage had begun to accumulate yolk globules for the next gestation. Thus, vitellogenesis of the succeeding batch of oocytes overlaps with gestation during reproduction in the mosquitofish. A rearing experiment showed the annual reproductive cycle of mosquitofish breeding in Nagashima to be as follows. Although oocytes had not at that point developed to the yolk globule stage, copulation occurred in February. Females began vitellogenesis in early May, the first pregnancy of the year commencing in mid-May. From mid-May to August, females repeated the gestation cycle (vitellogenesis, maturation, fertilization, pregnancy and parturition) at around one month intervals. In September, oocyte recruitment from the oil-droplet to the yolk globule stage ceased. After the final parturition, the ovaries contained only non-vitellogenic oocytes. Spermatozoa in the ovarian cavity were scare from November to January.     

Effect of age,diet, diapause and juvenile hormone on oogenesis and the amount of vitellogenin and vitellin in the twospotted stink bug,Perillus bioculatus (Heteroptera: pentatomidae)

Vitellogenic oocytes from Perillus bioculatus have two native vitellins, Vt1 and Vt2, with molecular masses of 553 and 228 kDa, respectively. The hemolymph contains a major vitellogenin, Vg, with a molecular mass of 528 kDa that consists of three apoproteins with masses of 177, 84 and 59 kDa, respectively. Antibodies to purified Vt2 reacted with ovary extracts, egg extracts and female hemolymph, but not with male hemolymph in immunodiffusion tests. Western blots showed that anti Vt2 reacted with both Vt1, Vt2 and with Vg. Vitellogenesis starts at an ovarian score of 12 at 2.4 days after emergence. The first cycle of egg development is completed in ovaries with a score of 112 at 7.7 days. During this 5.3 day period, the ovaries of a single female incorporated 1833 &mgr;g of protein to form vitellin. Vitellogenin levels start to increase in females 2.5 days after emergence and reached 17.8 &mgr;g/&mgr;l by 5.5 days. After 5.5 days vitellogenin levels fluctuated between 9.7 and 19.9 &mgr;g/&mgr;l. Most diapausing females contained no ovarian follicles in the vitellarium and their hemolymph contained less than 1 &mgr;g/&mgr;l of vitellogenin. Treating diapausing females with 1 &mgr;g of JH III increased vitellogenin levels over 120-fold. Insects maintained on a liver-based artificial diet had lower vitellogenin levels than the controls at all sample times and did not show an increase in vitellogenin concentration until 11.5 days. Treating insects on the artificial diet with 10 &mgr;g of JH III elevated vitellogenin levels to about a fourth of that found in prey-fed insects of a comparable age. This suggests that females fed the artificial diet have low levels of essential materials needed for vitellogenin production.