Effects of ecdysone and juvenile hormone on epidermal cell development in Hyalophora cecropia.

Pupae of Hyalophora cecropia were injected with various doses of beta-ecdysone (molting hormone) or juvenile hormone and the epidermal cell ultrastructure was then studied with the electron microscope. The hormonal effects were rapidly manifested and appeared to be cell specific and dose dependent. The initial response to both hormones was an outward blebbing of the apical plasma membrane. Large doses of beta-ecdysone elicited both precocious cuticle deposition and premature autophagic vacuole formation. Juvenile hormone prevented the appearance of the autophagic vacuoles which normally preceded cell differentation into cells capable of adult synthesis. After prolonged exposure to juvenile hormone, there appeared to be an exaggerated separation of the epidermal cells at the basal region suggesting that the juvenile hormone may act at the membrane level.     

Insecticidal properties of genetically engineered baculoviruses expressing an insect juvenile hormone esterase gene.

Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (jhe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed under the control of a strong, modified viral promoter. This virus, however, naturally encodes an ecdysteroid UDP-glucosyltransferase which inactivates ecdysone, the hormone which initiates molting. Since ecdysteroid UDP-glucosyltransferase could mask the effects of jhe expression by blocking molting entirely, jhe-expressing viruses in which the ecdysteroid UDP-glucosyltransferase gene was deleted or disrupted were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins from infected cells revealed several intracellular proteins and two major secreted proteins which reacted with antibodies to authentic juvenile hormone esterase. Western blot analysis coupled with tunicamycin treatment indicated that differential glycosylation was responsible for the multiple products. Hemolymph of recombinant virus-infected fourth-instar Trichoplusia ni larvae contained levels of juvenile hormone esterase activity 40-fold higher than maximal levels found in uninfected larvae. However, little or no difference in developmental characteristics, weight gain, or time of mortality was observed between insects infected with the jhe-expressing viruses and control viruses.     

Insecticidal properties of genetically engineered baculoviruses expressing an insect juvenile hormone esterase gene.

Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (jhe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed under the control of a strong, modified viral promoter. This virus, however, naturally encodes an ecdysteroid UDP-glucosyltransferase which inactivates ecdysone, the hormone which initiates molting. Since ecdysteroid UDP-glucosyltransferase could mask the effects of jhe expression by blocking molting entirely, jhe-expressing viruses in which the ecdysteroid UDP-glucosyltransferase gene was deleted or disrupted were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins from infected cells revealed several intracellular proteins and two major secreted proteins which reacted with antibodies to authentic juvenile hormone esterase. Western blot analysis coupled with tunicamycin treatment indicated that differential glycosylation was responsible for the multiple products. Hemolymph of recombinant virus-infected fourth-instar Trichoplusia ni larvae contained levels of juvenile hormone esterase activity 40-fold higher than maximal levels found in uninfected larvae. However, little or no difference in developmental characteristics, weight gain, or time of mortality was observed between insects infected with the jhe-expressing viruses and control viruses.     

Expression and functions of dopa decarboxylase in the silkworm, Bombyx mori was regulated by molting hormone

Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. Molting includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis shed and other series of continuous processes. Polyphenol oxidases, dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the dopa decarboxylase (BmDdc) was considered as an enzyme for epidermal layer’s tanning and melanization. This work suggested that dopa decarboxylase is one set of the key enzymes in molting, which closely related with the regulation of ecdysone at the time of biological molting processes. The data showed that the expression peak of dopa decarboxylase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was also observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the dopa decarboxylase expression was significantly elevated compared to the control. BmDdc RNAi induced dopa decarboxylase expression obviously declined in the silkworm larvae, and caused the pupae appeared no pupation or incomplete pupation. BmDdc was mainly expressed and stored in the peripheral plasma area near the nucleus in BmN cells. In larval, BmDdc was mainly located in the brain and epidermis, which is consisted with its function in sclerotization and melanization. Overall, the results described that the dopa decarboxylase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting.     

Phenol oxidase is a necessary enzyme for the silkworm molting which is regulated by molting hormone

Insect molting is an important developmental process of metamorphosis, which is initiated by molting hormone. The molting process includes the activation of dermal cells, epidermal cells separation, molting fluid secretion, the formation of new epidermis and old epidermis excoriation etc. Polyphenol oxidases (PPOs), dopa decarboxylase and acetyltransferase are necessary enzymes for this process. Traditionally, the phenol oxidase was considered as an enzyme for epidermal layer’s tanning and melanization. This work suggested that polyphenol oxidases are one set of the key enzymes in molting, which closely related with the role of ecdysone in regulation of molting processes. The data showed that the expression peak of phenol oxidase in silkworm is higher during molting stage, and decreases after molting. The significant increase in the ecdysone levels of haemolymph was observed in the artificially fed silkworm larvae with ecdysone hormone. Consistently, the phenol oxidase expression was significantly elevated compared to the control. PPO1 RNAi induced phenol oxidase expression obviously declined in the silkworm larvae, and caused the pupae incomplete pupation. Overall, the results described that the phenol oxidase expression is regulated by the molting hormone, and is a necessary enzyme for the silkworm molting.     

CHITIN SYNTHASE B: A MIDGUT‐SPECIFIC GENE INDUCED BY INSECT HORMONES AND INVOLVED IN FOOD INTAKE IN Bombyx mori LARVAE

Chitin synthase (CHS) is the key regulatory enzyme in chitin synthesis and excretion in insects, and a specific target of insecticides. We cloned a CHS B gene of Bombyx mori (BmChsB) and showed it to be midgut specific, highly expressed during the feeding process in the larva. Knockdown of BmChsB expression in the third‐instar larvae increased the number of nonmolting and abnormally molting larvae. Exposure to nikkomycin Z, a CHS inhibitor, reduced the amount of chitin in the peritrophic membrane of molted larvae, whereas abnormally elevated BmChsB mRNA levels were readily detected from the end of molting and in the newly molted larvae. Exogenous 20‐hydroxyecdysone (20E) and methoprene, a juvenile hormone analogue, significantly upregulated the expression of BmChsB when the levels of endogenous molting hormone (MH) were low and the levels of endogenous juvenile hormone (JH) were high immediately after molting. When levels of endogenous MH were high and those of endogenous JH were low during the molting stage, exogenous 20E did not upregulate BmChsB expression and exogenous methoprene upregulated it negligibly. When the endogenous hormone levels were low during the mulberry‐leaf intake process, BmChsB expression was upregulated by exogenous methoprene. We conclude that the expression of BmChsB is regulated by insect hormones, and directly affects the chitin‐synthesis‐dependent form of the peritrophic membrane and protects the food intake and molting process of silkworm larvae.     

昆虫蜕皮激素信号转导途径研究进展

蜕皮与变态是全变态昆虫典型的发育特征。调控昆虫蜕皮与变态的激素主要有蜕皮激素和保幼激素。目前已经阐明了蜕皮激素的核受体EcR及部分核信号转导途径,但蜕皮激素是否存在膜受体及膜信号转导途径研究很少。研究证明,蜕皮激素存在细胞质中的信号转导分子和途径,蜕皮激素通过NTF2和Ran调控EcR入核启动基因转录。蜕皮激素使细胞质中的热休克蛋白Hsc70部分入核与USP结合启动基因转录。蜕皮激素通过蛋白激酶PKC使伴侣蛋白calponin磷酸化,参与蜕皮激素信号途径的基因转录。这些研究结果说明蜕皮激素除了有核受体和核受体信号转导途径外,还存在细胞膜受体和细胞膜信号转导途径。     

棉铃虫蜕皮时期同工酶表达模式

同工酶广泛存在于不同种属生物的组织细胞中,在生物发育的不同阶段有着特定的表达模式和重要的生理功能。昆虫蜕皮是在促前胸腺激素（PTTH）、蜕皮激素和保幼激素共同控制下由一系列基因表达和调控的级联反应。阐明蜕皮发育过程中同工酶的表达模式,可以为研究蜕皮的分子机理提供新的分子靶标,为研制生长调节剂类杀虫剂提供检测的分子标记。该研究分析了棉铃虫Helicoverpa armigera蜕皮时期不同组织中过氧化物酶、乙醇脱氢酶和酯酶的表达模式,找到了3种蜕皮差异表达的过氧化物酶, 2种蜕皮或变态差异表达的乙醇脱氢酶,3种蜕皮差异表达的酯酶。生长调节剂类化学杀虫剂非甾醇类蜕皮激素竞争物RH24-85可以诱导3种酯酶表达上调,可能与蜕皮有关。这些结果为进一步研究棉铃虫蜕皮的分子机理和检测促蜕皮生长调节剂类化学杀虫剂提供了新的分子靶标。     

Developmental arrest induced by juvenile hormone in larvae of Bombyx mori

Injection of the juvenile hormone analog (JHA) methoprene into day 3, fifthinstar larvae of Bombyx mori induced developmental arrest. Feeding activity declined, and the larvae remained as larvae for more than 2 weeks, after which they died. After JHA injection, the hemolymph ecdysteroid titer was low, and the prothoracic glands were almost inactive for 7 days. During this period, prothoracic glands were stimulated by prothoracicotropic hormone (PTTH) in vitro, indicating that JHA did not inhibit the competence of the glands to respond to PTTH. When brain-corpora cardiaca-corpora allata complexes were removed from intact fifth-instar larvae on day 4, the prothoracic glands became autonomously active and produced enough ecdysone for pupation. When PTTH injections were given to larvae previously injected with JHA (7 days before), the larvae recovered feeding activity, purged their guts, and pupated. Injections of 20-hydroxyecdysone into larvae that had been injected with JHA 7 days earlier induced larval molting. These results suggest that JHA affects both the brain and the prothoracic gland.     

Juvenile hormone titers and caste differentiation in the damp-wood termite Hodotermopsis sjostedti (Isoptera, Termopsidae)

Termites are social insects, presenting morphologically distinct castes, performing specific tasks in the colony. The developmental processes underlying caste differentiation are mainly controlled by juvenile hormone (JH). Although many fragmentary data support this fact, there was no comparative work on JH titers during the caste differentiation processes. In this study, JH titer variation was investigated using a liquid chromatography-mass spectrometry (LC-MS) quantification method in all castes of the Japanese damp-wood termite Hodotermopsis sjostedti, especially focusing on the soldier caste differentiation pathway, which was induced by treatment with a JH analog. Hemolymph JH titers fluctuated between 20 and 720pg/microl. A peak of JH was observed during molting events for the pseudergate stationary molt and presoldier differentiation, but this peak was absent prior to the imaginal molt. Soldier caste differentiation was generally associated with high JH titers and nymph to alate differentiation with low JH titers. However, JH titer rose in females during alate maturation, probably in relation to vitellogenesis. In comparison, JH titer was surprisingly low in neotenics. On the basis of these results in both natural and artificial conditions, the current model for JH action on termite caste differentiation is discussed and re-appraised.     

Effects of juvenile hormone analogue on ecdysis prevention induced by precocene in Rhodnius prolixus (Hemiptera:Reduviidae)

Precocene II, added to the meal of fourth-instar larvae of Rhodnius prolixus (25 micrograms/ml of blood), induced an increase in the duration of the molting cycle. This effect was related to the decrease of both the nuclear area of the prothoracic gland cells and the mitotic activity in epidermal cells. Juvenile hormone analogue applied topically (60 micrograms/insect) together with Precocene II treatment avoided atrophy of the prothoracic glands and induced a higher number of epidermal mitosis accelerating the time of subsequent ecdysis. A possible relationship between juvenile hormone and production of ecdysone is discussed.     

Critical roles for juvenile hormone and its esterase+ in the prepupa of Trichoplusia ni

In the caterpillar Trichoplusia ni (Lepidoptera: Noctuidae) it has been demonstrated by allatectomy that the appearance of juvenile hormone during the prepupal stage is crucial for the successful larval-pupal ecdysis of most larvae. Application of juvenile hormone or juvenile hormone esterase inhibitors at key times disrupted normal development as well. Thus the subsequent disappearance of juvenile hormone is regulated by degradation by juvenile hormone esterase in addition to a hypothetical reduction in biosynthesis. This reduction in juvenile hormone titer in the prepupa is just as critical for normal development as was its previous appearance. These observations on the critical role of juvenile hormone in the prepupa are in contrast to observations in some other species. For instance, in the case of Manduca sexta (Lepidoptera: Sphingidae), juvenile hormone is considered only supplementary to the action of prothoracicotropic hormone in the postwandering stage and primarily is required for normal pupal development. It thus appears that even within the Lepidoptera the role of juvenile hormone in prepupal development can vary dramatically.     

An ultrastructural and developmental analysis of the corpus allatum of juvenile hormone deficient mutants ofDrosophila melanogaster

Summary The ultrastructure of the corpus allatum of theapterous mutantsap ⁴ andap ^56f ofDrosophila melanogaster during larval-pupal-adult metamorphosis and adult life was correlated with the gland's ability to synthesize juvenile hormone in vitro. During the early wandering period of the third instar of both mutants, a high concentration of smooth endoplasmic reticulum, mitochondria and mitochondrion-scalariform junction complexes are typical features of an active corpus allatum cell. Juvenile hormone biosynthesis by the glands is high at that time and, in fact, only slightly lower than that of wild type glands. In contrast to the wild type gland, the cells of the pupal and pharate adult corpus allatum of both mutants contains highly electron dense mitochondria with tubular cristae but no whorls of smooth endoplasmic reticulum nor glycogen clusters. The frequency and size of the lipid droplets, putatives depots of the juvenile hormone precursors, in cells of theap ^56f gland is a function of the insect's age, but both are lower than in wild type gland cells. Juvenile hormone biosynthesis by both mutant glands remains at the basal level when compared to increased synthesis by the wild type gland. The frequency and density of lipid droplets in cells of theap ⁴ corpus allatum are much lower than in theap ^56f glands. During adult life, the ultrastructural profile of theap ^56f corpus allatum is similar to that of the wild type gland although the in vitro production of juvenile hormone by the former is much lower than that of the wild type gland. The ultrastructural features of the adult corpus allatum ofap ⁴ homozygotes reveal precocious degeneration and support the view that this non-vitellogenic mutant is a juvenile hormone deficient mutation.     

Salmon growth hormone is transported into the circulation of rainbow trout,Oncorhynchus mykiss,after intestinal administration

Summary Transport of recombinant salmon growth hormone (rsGH) into the circulation of rainbow trout following intragastric or rectal administration was investigated. Changes in plasma GH levels were analyzed by radioimmunoassay specific to chum salmon GH. Intragastric administration of rsGH by oral intubation resulted in elevation of GH immunoreactivity in plasma after 11h. The plasma GH increased maximally after 15h, and then declined rapidly to reach a normal level after 19h. On the other hand, oral intubation of rsGH to carp, which has no stomach, caused elevation of plasma GH levels after 1 h which lasted for 21 h. In sharp contrast, rectal administration of rsGH to rainbow trout significantly elevated plasma GH levels after 15 min, which reached a maximum after 30 min. The rsGH was labeled with fluorescein isothiocyanate (FITC) to distinguish exogenous from endogenous GH, and administered into rainbow trout rectally. Subsequent analysis of plasma samples on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed two fluorescent bands at the same molecular weights as those of the monomeric and dimeric rsGHs. Intragastric and rectal administration of rsGH into juvenile trout resulted in significant increases in length and body weight compared to the control fish. This study strongly suggests that the rsGH is transported into the circulation of salmonids via the lower part of the intestine and takes part in growth stimulation.Abbreviations DCA deoxycholic acid - FITC fluorescein isothiocyanate - GH growth hormone - HPLC high-performance liquid chromatography - HRP horseradish peroxidase - RIA radioimmunoassay - rsGH recombinant salmon growth hormone - sGH chum salmon growth hormone - SEM standard error of mean - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TFA trifluoroacetic acid     

Simultaneous determination of molting and juvenile hormone titers of the greater wax moth

A simple and rapid extraction procedure was developed to determine simultaneously the molting hormone (MH) and juvenile hormone (JH) activity in a single insect tissue sample. From the onset of the last larval stage to adult eclosion of the greater wax moth, Galleria mellonella, three JH peaks were noted: at the time of the sixth larval ecdysis, 1 day before the seventh larval ecdysis, and at the time of adult eclosion. Three MH peaks were recorded for the male: at 1 day before the sixth larval ecdysis, 1 day before the seventh larval ecdysis, and 2 days after pupation. In the female, a fourth peak was shown at the time of adult eclosion. This fourth peak exhibits the highest molting hormone activity of all samples, 1600 Musca units/g of fresh tissue or an equivalent of 5.6 μg/g of ecdysterone. Eighty per cent of this MH accumulated in the ovary. The significance of MH and JH titers as related to the endocrine regulation of development is discussed in the light of this finding.     

Juvenile hormone acid methyltransferase activity in imaginal discs of Manduca sexta prepupae

The occurrence of a peak of juvenile hormone (JH) during the prepupal period has been noted in several lepidopterans. In Manduca sexta and Hyalophora cecropia this peak is known to prevent the precocious onset of adult differentiation in imaginal tissues. However, it has previously been observed in our laboratory that corpora allata (CA) of this age are incapable of making JH owing to a lack of the terminal synthetic enzyme, juvenile hormone acid methyltransferase (JHAMT). Since the CA are required for normal pupation, it is likely that JH acid is the product released by the prepupal CA. Therefore, we analyzed whether JH acid treatment would prevent precocious adultoid differentiation in allatectomized M. sexta larvae. JH acid injections were found to be as effective as JH in normalizing pupation, and acted in a time- and dose-dependent manner. This finding led to a question of whether injected or endogenous JH acid could be methylated to JH. Homogenates of several tissues from prepupae were assayed for the presence of JHAMT. Of the tissues assayed, only imaginal discs possessed significant levels of the enzyme. These results support our previously proposed mechanism for production of the prepupal JH peak in M. sexta.     

Hormonal Control of Molting in Decapod Crustacea

The involvement of the molting hormone, 20-hydroxyecdysone,in the mediation of molting in decapod crustaceans is brieflyreviewed. Aspects of the secretion and metabolism of its precursor,ecdysone, are discussed. Experiments are described that demonstratethe presence of a molt-inhibiting hormone (MIH) in the sinusglands of juvenile lobsters (Homarus americanus). Assays forMIH include measurement of the molt interval and radioimmunoassayof circulating titers of ecdysteroids in eyestalk-ablated lobsters.This latter assay indicates that sinus gland extracts significantlydecrease the concentration of circulating ecdysteroids 24 hrafter injection. Data are also presented on the circulatingtiters of ecdysteroids during multiple molt cycles of lobstersfollowing eyestalk ablation. These data indicate that theremust be another factor that ultimately regulates the circulatinglevels of the molting hormone.     

Embryonic expression of juvenile hormone binding protein and its relationship to the toxic effects of juvenile hormone in Manduca sexta

The juvenile hormones (JHs) regulate a diverse array of insect developmental and reproductive processes. One molecular target of JH action is its transporter, hemolymph JH binding protein (hJHBP); in the larva of the tobacco hornworm, Manduca sexta, low doses of JH can immediately increase hJHBP gene expression. Less explored are the effects of JH on embryological development, where early hormonal treatment has been shown to affect embryonic development and pupation. This study examines the egg form of JHBP and its gene expression during embryogenesis of M. sexta, as well as the phenotypic effect JH treatment has on embryos and on JHBP gene expression. We here demonstrate that the preponderance of JHBP found in the egg is maternally derived and that the embryonic gene and protein appear identical to those found in the larva. Expression of the JHBP gene begins in both the embryo itself and extra-embryonic tissues 15 h after fertilization, long before emergence of a functional fat body and circulatory system. Topical application of low JH doses to early embryos resulted in larval abnormalities while high doses of the hormone induced embryonic mortality. These effects are not mediated through regulation of the JHBP gene, since embryonic expression appears invariant in response to JH challenge. The toxicity of JH is tightly correlated with the concentration of unbound hormone.