Juvenile Hormone Levels Research Articles

Developmental profiles of the mRNAs for Manduca arylphorin and two other storage proteins during the final larval instar of Manduca sexta

When fat body mRNA from the tobacco hornworm larva, Manduca sexta, was translated in a rabbit reticulocyte lysate system, three major polypeptides were found, each having a different developmental profile. One mRNA coded for a 74 kilodalton (K) polypeptide doublet precipitated by an antibody to the arylphorin (manducin). This mRNA was present only during the intermolt feeding phase of the penultimate and the final larval instars. Its appearance 16–24 hr after larval ecdysis was dependent upon the incoming nutrient supply and independent of the juvenile hormone (JH) level. Immunoblots of proteins of the fat body, epidermis, and cuticle revealed the presence of arylphorin in all three tissues. Additionally, several small polypeptides that cross-reacted with the arylphorin antibody were found in the fat body during and up to 24 hr after the last larval molt and in the tanning pupal cuticle. The larval epidermis was also found to contain a small amount of arylphorin mRNA. At the time of the JH decline prior to the onset of metamorphosis, a female-specific mRNA coding for a 79 K translation product appeared. In allatectomized larvae this mRNA was detectable earlier, and its appearance in intact larvae was prevented by application of methoprene, indicating that JH regulates its appearance. At wandering a new mRNA that also codes for a 79 K polypeptide appeared in both sexes and was the major messenger present during the prepupal stage. Neither it nor the female-specific mRNA were translatable after pupal ecdysis.

Juvenile hormone modifications of gene expression in the fat body and posterior silk glands of Bombyx mori

The patterns of protein synthetic activities were determined in the fat body and silk glands of Bombyx mori larvae during the fifth instar. Comparisons were made between control and juvenile hormone or methoprene—treated animals. In normal larvae, the relative synthetic activities of a few proteins increase up to the time of spinning. In treated larvae, the presence of high levels of juvenile hormone or methoprene never results in an arrest of the expression of differentiation at the molecular level. The syntheses of the most abundant markers of development in both organs are reduced by the hormone, but total inhibition has never been observed. The transfer of a large amount of proteins (28–29 kd) from the fat body to the haemolymph is decreased.

Detection of [formula omitted] JH III in several life-stages of [formula omitted] and [formula omitted

We have conducted a reinvestigation into both the identification and quantification of juvenile hormone (JH) from several developmental stages of the cockroach, Nauphoeta cinerea , and the firebrat, Thermobia domestica , using a gas chromatography-mass spectrometric (GC/MS) method. We detected only JH III in these animals in contrast to prior studies in which JH I, II, and/or III had been reported using a different scheme relying on HPLC purification and subsequent GC/MS analysis under chemical ionization (CI) conditions. Very high levels (∼800 ng/g) of JH III were found in N. cinerea embryos at stages after dorsal closure whereas first stadium nymphs and female penultimate stadium nymphs contained only low levels (∼1 ng/g and ∼7 ng/ml respectively); in adult females at the stage of rapid oocyte growth ∼150 ng JH III per ml of hemolymph was measured. T. domestica nymphs and egg laying adults contained only low levels (∼1 ng/g) of JH III. The results emphasize the caution which must be used in interpreting results of procedures for analysis of JH at parts-per-billion levels, and also enforce prior observations that the higher JH homologs are not present except in the Lepidoptera .

Studies of esterase 6 in Drosophila melanogaster—XVI : Synthesis occurs in the male reproductive tract (anterior ejaculatory duct) and is modulated by juvenile hormone

Esterase 6, a carboxylesterase (EC 3.1.1.1.) of Drosophila melanogaster, has been unambiguously shown to be synthesized in the anterior ejaculatory duct of the reproductive tract of adult males. Radiolabelled esterase 6 was immunoprecipitated from homogenates prepared from ducts cultured for 5 hr in an in vitro system containing [ 3H]leucine. The anti-allatotropin, precocene, was used to treat adult males to determine if juvenile hormone levels affect the activity of esterase 6. In treated males the level of esterase 6 was 37% of the control levels. This system may allow for further exploration of the hormonal regulation of reproductive processes in Drosophila.

Ecdysteroid levels associated with epidermal events during worker and soldier differentiation in Macrotermes michaelseni (Isoptera: Macrotermitinae)

Radioimmunoassay of ecdysteroid levels coupled with morphological and histological studies have revealed a close association between hormonal titers and epidermal events during worker and soldier differentiation from lighter and heavier third instars of Macrotermes michaelseni, respectively. Two main peaks of ecdysteroid levels were found during the development of these larvae. The first low one occurred on Day 4 in both cases, although that of lighter larvae was short lived (only 1 day) while that of the heavier larvae continued for another day. Epidermal detachment (apolysis) was shown to start earlier in the heavier larvae (Day 4) than in the lighter ones (Day 6), indicating a differential influence of the first ecdysteroid peak on the epidermis (probably modulated by differing levels of juvenile hormones in the two types of larvae). It was also at this time that morphological differentiation of presoldier mandibles was observed. From this interval onward, hormonal levels remained higher and achieved a second peak in the heavier larvae earlier (Day 9–10) than in the lighter ones (Day 11–12). Epidermal events, i.e., complete apolysis, increased cellular growth, new cuticle deposition, and ecdysis, correlated well with the hormonal level increases, those of the lighter larvae always lagging behind by about 2 days. During presoldier-soldier transformation two distinct ecdysteroid peaks were demonstrated, one occurring on Day 2 (low) and the other on Day 9 (high). The third one was transient and not very distinct from the second peak. The significance of these peaks is discussed in the context of differentiation.

Ecdysteroid and juvenile hormone changes in Bombyx mori eggs, related to the initiation of diapause

We describe a method for the routine determination of changes in juvenile hormone levels in insect eggs. The hormones are first converted into their diol derivatives, then they are purified from other lipids and separated by high-performance liquid chromatography (HPLC). The radioimmunoassay of the fractions was then determined. The method permits the simultaneous assay of ecdysteroids, and it was used for determining the hormonal changes in Bombyx eggs during the pre-diapause development. Our major finding is that the hormonal content of eggs dramatically increased prior to the initiation of diapause. This hormonal rise included ecdysone, 20-OH-ecdysone and 3 juvenile hormones. The HPLC retention time of the latter corresponded to JH 1 JH 2 and JH 3. Subsequently, the embryos entered diapause and the hormonal content of eggs was reduced to traces of ecdysteroids. These dramatic changes in juvenile hormone levels during early embryogenesis raise a number of issues which are developed in the discussion.

Control of corpus allatum activity during the imaginai diapause in females ofLocusta migratoriaL

In the Savio strain of Locusta migratoria an imaginai diapause is induced by long daylength. In diapausing females, the haemolymph level of juvenile hormone (JH) was undetectable during the first 3-wk of imaginai life and later rose only slightly to about 20 ng/JH3IR per ml. Only peripheral cells of the corpora aliata (CA) were active. In nondiapausing animals, or after the termination of diapause, the JH level was high (140–200 ng/ml) and the ultrastructure of the gland exhibited signs of activity. CA severance in 3-wk-old diapausing females terminated diapause as a result of activation of the CA. CA disconnection in the fifth larval instar or at the imaginai moult in long daylength animals did not break diapause and the CA stayed inactive. The lateral cells of the protocerebrum exert a jdual effect: at the end of larval life they bring about CA maturation and render them active, whereas during the imaginai diapause they inhibit CA activity. The median neurosecretory cells of the pars intercerebralis sup...

Changes in ecdysteroid and juvenile hormone levels in developing eggs of Bombyx mori

It is well-known that insect eggs can contain very high concentrations of ecdysteroids, which undergo drastic changes during embryogenesis. We found that this is equally valid for juvenile hormones. Three juvenile hormone-immunoreactive compounds were observed in developing Bombyx mori eggs. They were assumed to be juvenile hormones 1, 2 and 3 according to their retention time in HPLC. These hormones underwent drastic and sudden changes. In the space of one day their concentration was seen to rise rapidly from an undetectable level up to as high as 4 × 10 −6 micromoles per mg of eggs. Their presence was detected as early as the first day of embryonic development, as well as during the blastokinesis period (day 5 to day 9) and in late embryos (day 12 to day 14). Their relative concentrations varied greatly. On two occasions, day 1 and day 8, all three hormones were simultaneously present. Moreover, juvenile hormone 3 was present during the blastokinesis period, either alone or in combination with hormone 2. The latter was the only hormone present in late embryos, before hatching. Thus, with regard to both ecdysteroids (ecdysone and 20-hydroxyecdysone) and juvenile hormones, each day of embryonic development displayed a different hormonal pattern. These patterns undoubtedly constitute a “hormonal code” of embryogenesis control. While 20-hydroxyecdysone can be assumed to trigger cuticulogenesis in embryos as it does in larvae, the effects of the other hormones as well as their possible interactions are questionable.

Effects of three compounds with anti-juvenile hormone activity and a juvenile hormone analogue on endogenous juvenile hormone levels in the tobacco hornworm, Manduca sexta

The ability of three anti-juvenile hormones and one juvenile hormone analogue to reduce in vivo juvenile hormone levels in Manduca sexta has been investigated. Two compounds. FMev (tetrahydro-4-fluoromethyl-4-hydroxy-2 H-pyran-2-one) and ETB (ethyl-4-[2-( tert-butylcarbonyloxy)butoxy]-benzoate) reduced the titres of juvenile hormones I and II to near the levels of detection in topically treated larvae. Precocene III (7-ethoxy-6-methoxy-2,2-dimethylchromene) was inactive but the juvenile hormone analogue hydroprene was as effective as the two anti-juvenile hormones in reducing endogenous juvenile hormone titres in larvae. FMev was also shown to reduce the level of juvenile hormones II and III in pharate adults.

Precocene causes male determination in the aphid Myzus persicae

When adult apterous viviparous females of Myzus persicae, reared in short night conditions at 21–23°C, are treated with the precocene analogue 6-methoxy-7-ethoxy-2,2-dimethylchromene, they deposit males towards the end of their reproductive lives. The first-born normal daughters of the treated females also deposit some males at various times in their reproductive lives. Karyotypic analysis was used to investigate the sequence of male and female embryos in the ovarioles of precociously metamorphosed aphids. The experiments support the hypothesis (Mittler et al., 1979) that juvenile hormone level controls the sex determination process in aphids. Since male aphids have an XO sex chromosome constitution, this implies that juvenile hormone level influences the behaviour of the X-chromosomes at or before the single maturation division of the egg. At this division one X-chromosome is eliminated from eggs which will develop as males. Aphids provide the first example of a specific endocrine influence on chromosome behaviour in sex determination.

Seasonal variation in hemolymph juvenile hormone of adult monarchs (Danaus p. plexippus: Lepidoptera)

The Galleria wax test was used to quantify juvenile hormone (JH) activity in the hemolymph of adult monarch butterflies captured in the field at monthly intervals. In animals of both sexes captured in June and July, high levels of hemolymph JH activity were found. By contrast, both sexes had generally lower JH titers from August through January. These results are in general agreement with the state of the JH-sensitive reproductive tract components during these months. Nevertheless, there were notable exceptions, including males in February and September and females in October. During these months relatively high levels of JH were obtained for some hemolymph samples, while in general, reproductive tract development during these periods was variable. This result suggested that temperature, photoperiod, nutrition, flight, JH sensitivity, or some other variable may be important in the triggering of JH-induced reproductive tract growth during these periods. Verification procedures for the assay itself and for the biological material included construction of dose responses for JHI, JHII, JHIII, and Ayerst JH (AJH) (i.e. 16 isomers of JHI), tests for hemolymph JH activity inhibition, effect of allatectomy on JH titer, and comparison of the JH activity of field- and laboratory-collected hemolymph.

Juvenile hormone esterase activity during the last larval and pupal stages of Tenebrio molitor

In vitro analysis of juvenile hormone esterase activity of haemolymph of T. molitor was performed during the end of post-embryonic development. Weak activity was found in penultimate stage larvae as in the major part (except the last day) of last-larval instar, while very high activity was monitored in the early pupae (female or male). This pupal peak was the only one detected during development in the insect, coinciding with the pupal juvenile hormone sensitive period. The first juvenile hormone sensitive period, during the lastlarval instar, does not seem to be protected by any juvenile hormone esterase activity in contrast to other species. These results suggest a central control for the drop in juvenile hormone level ceasing synthesis by the corpora allata after integration of external stimuli. This hypothesis could explain the natural occurrence of prothetelic larvae, the absence of pupal adult intermediates and the variable number of instars in Tenebrio.

Development of the accessory reproductive glands and genital ducts in female Schistocerca gregaria

The accessory reproductive glands of female S. gregaria are tubular extensions of the paired genital ducts, which in the mature female contain large amounts of a proteinaceous secretion used in the formation of the egg pod. In the 4th and 5th-instar female the glands are indistinguishable from the remainder of the mesodermally derived genital ducts. Towards the end of the 5th stadium, however, the accessory gland region only acquires characteristic convolutions which persist throughout the adult stages. At this time the epithelium of the entire ducts becomes reorganized into a unicellular epithelium. Only one cell type occurs throughout the length of the glands, and also in the egg calyces and lateral oviducts. The cells are inactive immediately after final ecdysis and remain in this state until the level of juvenile hormone in the haemolymph rises. The hormone acts directly on the cells triggering a rapid proliferation of organelles associated with protein secretion, and thereby increasing the volume of apical cytoplasm. Microvilli develop at the luminar plasma membrane, while irregular infoldings form at the base of the cells. As the gland matures the major organelle, the rough endoplasmic reticulum, changes from the lamellar to the vesicular form. Secretion is released into the lumen by the ‘microapocrine’ method.

Juvenile hormone esterases in diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis

Haemolymph levels of juvenile hormone esterase, 1-naphthyl acetate esterase, and juvenile hormone were measured in synchronously staged diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis. Juvenile hormone esterase levels were monitored using juvenile hormone I as a substrate while juvenile hormone titres were measured with the Galleria bioassay. Haemolymph of nondiapause larvae showed two peaks of juvenile hormone hydrolytic activity: one near the end of the feeding phase and a smaller one just prior to pupal ecdysis. These peaks of enzyme activity correlated well with the low levels of haemolymph juvenile hormone. Juvenile hormone titres were high early in the stadium then showed a second peak during the prepupal stage coinciding with low esterase activity. Diapause haemolymph had peak juvenile hormone esterase activity nearly 4 times the nondiapause level, reaching a peak near the end of the feeding phase. Diapause-destined larvae retained high juvenile hormone titres even during the rise of the high esterase levels. 1-naphthyl acetate esterase levels did not correlate with the juvenile hormone esterase levels in either the diapause or nondiapause haemolymph. High levels of 1-naphthyl acetate esterase activity were associated with moulting periods.

The nature and titre of juvenile hormone in the Colorado potato beetle, Leptinotarsa decemlineata

This paper describes the nature and titre of juvenile hormone at different developmental stages of the Colorado potato beetle, Leptinotarsa decemlineata Say, determined by a selective mass-spectroscopic detection technique, High levels of juvenile hormone III were observed in long-day beetles, whereas low titres occurred in pre-diapause and diapause adults. The level of juvenile hormone III in larvae was low compared with reproductive adults, whereas hardly any juvenile hormone could be detected in pupae. We were not able to detect juvenile hormones I or II. The results agree well with previously reported data using the Galleria bioassay.

Regulation of ommochrome biosynthesis in the tobacco hornworm,Manduca sexta, by juvenile hormone

The absence of juvenile hormone (JH) at the time of head capsule slippage during the molt to the fifth (final) instar of the tobacco hornworm was found to cause ommochrome (primarily dihydroxanthommatin) synthesis in the epidermis during the first two days after ecdysis. Then synthesis decreased until its transient reappearance during the wandering stage. Either JH-I (ED50=8x10−4 μg) or methoprene (ED50=1.4x10−2 μg) applied at this critical time during the molt prevented the first synthesis. A comparison of developmental profiles of tryptophan and its metabolites, kynurenine and 3-hydroxykynurenine, in normal and allatectomized wild type larvae showed that JH at this critical time prevented both the conversion of kynurenine to 3-hydroxykynurenine and 3-hydroxykynurenine to ommochromes. A similar study in normal and methoprene-treatedblack mutant larvae showed that only the latter conversion was inhibited by JH. The accumulation of 3-hydroxykynurenine in the epidermis of the JH-treatedblack mutant is thought to be due to the altered tryptophan metabolism in these mutants in previous instars due to lower JH levels. Neither starvation of theblack mutant nor injection of 3-hydroxykynurenine significantly affected ommochrome synthesis by the epidermis. Preliminary studies of the enzymes involved showed that JH at the critical period suppressed the later activity and/or production of kynurenine 3-hydroxylase in the wild type larva, but had little effect on the particulate ommochrome synthetase activity of the epidermis.

Developmental and teratogenic effects of precocene 2 on the bed bug, Cimex Lectularius L.

Contact application of precocene 2 (6,7-dimethoxy-2,2-dimethylchromene) to inseminated adult female bed bugs inhibited egg maturation and affected egg viability. Third-stage nymphs exposed to 100μg/cm 2 showed precocious maturation after the moult to the next stage. Nymphs exposed to lower concentrations of precocene exhibited teratism of the antennae, including the reduction of terminal sensillae. This was thought to be responsible for the bugs not assembling. Reduction in the fecundity in adult bugs, and the morphogenetic and teratogenic activity in nymphs was circumstantially related to reduced levels of juvenile hormone, as a result of exposure to precocene.

Radioimmunoassay of insect juvenile hormones and of their diol derivatives.

We have developed a radioimmunoassay for insect juvenile hormones. The C18 hormone (JH I) was first converted into diol by opening the 10, 11-epoxy ring. Diol was then succinylated and coupled to human serum albumin to make it immunogenic. High titer antisera were obtained from immunized rabbits. Succinyl juvenile hormone was also coupled to the peptide glycyltyrosine and iodinated so as to form a water-soluble 125I-labelled analogue well recognized by antibodies (60% bound by the 1/2000 000 dilution of antiserum routinely used in radioimmunoassay). Standards and biological samples were treated with acidic dioxane in order to convert each hormone into its corresponding diol. In this way, the sensitivity threshold of the radioimmunoassay was under 0.015 pmol. All three diols were equally recognized by the antibodies. Hormones JH I, JH II and JH III could be assayed separately as diols after thin-layer chromatography or high-pressure liquid chromatography purification of the biological samples. This method was used to determine physiological levels of juvenile hormones in the haemolymph of several insects at different development stages including embryos and in corpora allata cultures.

Hormonal control of storage protein synthesis and uptake by the fat body in the silkworm, Bombyx mori

The female silkworm, Bombyx mori, rapidly accumulates two storage proteins, that are synthesized by the fat body, in the haemolymph during the feeding stage of the last-larval instar, and then sequesters them from the haemolymph into fat body during the larval-pupal transformation. The rapid synthesis and uptake of storage proteins by the fat body are shown to be induced by allatectomy in the early-penultimate larval instar. A juvenile hormone analogue, methoprene, is highly effective in inhibiting the allatectomy-induced synthesis, and, in a higher dosage, further blocks the uptake. Allatectomy in the late-penultimate larval instar shortly before moulting does not enhance the storage protein synthesis, but causes the uptake to occur two days earlier in the last-larval instar. Injection of 20-hydroxyecdysone is not stimulatory for synthesis of the proteins, but is effective to induce their uptake. Starvation during the early last-larval instar completely blocks the synthesis. From these results, it is suggested that storage protein synthesis is induced in the absence of juvenile hormone by some supplementary stimulus, possibly the supply of nutrient after feeding, and uptake is induced by ecdysteroids after a decline in the juvenile hormone level.

Juvenile hormone and photoperiodically controlled polymorphism in Aphis fabae: Postnatal effects on presumptive gynoparae

Long days (short nights) (LD 16:8) and high temperatures (> 15°C) have an apterizing effect on the short day (LD 12:12) induced, presumptive gynopara of Aphis fabae. Transfer of presumptive gynoparae to long days (15°C) or to 25°C (short days) at varying times during postnatal development demonstrate that the adult form is determined by the second day of the second instar, i.e. 5 days after birth at 15°C. Transfer on day 1 induces maximum apterization with the proportion of aphids affected decreasing with age at transfer. Apterization induced by long days immediately after birth can, to some extent, be cancelled by return to short days but only up to day 4. Thus long days are morphogenetically more potent than short days at the beginning of larval development. At temperatures above 15°C the proportion of aphids apterized increases almost linearly. Apterized insects can be distinguished from juvenilized insects in the fifth-instar. Topical application of juvenile hormone (JH) induces both apterization and juvenilization of presumptive gynoparae but at different times during larval development, JH treatment during the early-instars promotes apterization but induces little juvenilization, whereas maximum juvenilization, without apterization, is produced by middle-instar treatment. The apterizing effects of JH are, thus, not due to its neotenic action. The response profile of JH-induced apterization is similar to that observed with long days and 25°C. It is suggested that such conditions increase endogenous JH levels in A. fabae. The three naturally occurring JH's differ in activity in the order JH I > JH II > JH III. Both long-day and JH-apterized insects switch from the normal ovipara production of the adult gynopara to vivipara production.