K.G. Davey

Cellular and Molecular Actions of Juvenile Hormone. II. Roles of Juvenile Hormone in Adult Insects

Publisher Summary Among animal hormones, juvenile hormone (JH) is distinctive because of its unique structure and the diversity of its effects on insect development and reproduction. This chapter reviews the actions of JH on the fat body, gonads, accessory glands, muscle, and nervous system of adult insects. Whereas the epidermis is a major target of premetamorphic JH action, it has been studied little in adult insects, which generally do not moult. However, since the development of yellow pigmentation that accompanies sexual maturation in adult male locusts is clearly dependent on JH-regulation processes, in which cellular and molecular mechanism are investigated. The rapid recent progress in understanding how ecdysteroids regulate the gene activities has resulted from the opportunities afforded by drosophila melanogaster: mapped and characterized mutants, polytene chromosomes with puffs marking active genes, efficient germ-line transformation. The recognition of two aspects of JH action in the tissues of adult insects is reviewed. A model for understanding some aspects of priming by JH may be found in the action of ecdysteroids, where early genes produce factors needed for the expression of late genes. In structure, thyroxine is very different from JH, but there is considerable resemblance between thyroxine and phenoxyphenoxy carbamate, fenoxycarb. Functionally, there are marked similarities. Thyroxine governs metamorphosis in amphibians, but is remarkably pleiotropic in governing many processes ranging from the maturation of the central nervous system to thermoregulation. The chapter emphasizes the importance of selecting insect systems on the basis of their optimal features for research, rather than historical precedent or economic importance. With selection of appropriate systems and application of the cell and molecular research techniques now available, the elusive problem of JH action should soon yield to enlightenment.

Spruce budworm (Choristoneura fumiferana) juvenile hormone esterase: hormonal regulation, developmental expression and cDNA cloning.

We have used the differential display of mRNAs technique to identify Choristoneura fumiferana genes that are induced by juvenile hormone I (JH I). Of the six PCR products identified, one bound to a 2.8-kb mRNA from CF-203 cells whose abundance increased when the cells were grown in the presence of JH I. The same 2.8-kb mRNA decreased to undetectable levels when the CF-203 cells were grown in the presence of 20-hydroxyecdysone (20E). The PCR fragment probe also detected a 2.8-kb mRNA in the C. fumiferana larval tissues. This 2.8-kb mRNA was present on the first day of the first, third, fourth, fifth and sixth larval and pupal stadia, but was conspicuously absent on the first day of the second larval stadium, as well as during the intermolt periods of the first to fifth instar larval stages. In the sixth instar larvae the 2.8-kb mRNA was detected in the fat body, epidermis and midgut during the intermolt period. The PCR fragment was used as a probe to screen a cDNA library. The deduced amino acid sequence of this 2.8-kb cDNA clone showed similarity with the deduced amino acid sequences of Heliothis virescens juvenile hormone esterases (HvJHE). The deduced amino acid sequence of the cDNA clone contained all five functional motifs that are present in most of esterases, proteases and lipases. The cDNA clone was expressed in the baculovirus expression system, producing a protein that showed JHE activity.

The modes of action of juvenile hormones: some questions we ought to ask

This paper argues that the current dogma that juvenile hormones are structurally unique and constitute a family of derivatives of farnesoic acid which are produced by the corpus allatum (CA), secreted into the hemolymph, frequently transported by binding proteins, enter cells by diffusion across the cell membrane and there the products of the CA interact in some way with the genome, probably via nuclear receptors of the steroid superfamily, may not be tenable. It does so by examining the following questions. How many JHs are there? Are there other sources of JH in insects? Are there non-farnesoids with JH activity in insects? How does JH get into cells? Is the product of the CA the effective hormone? How many modes of action are there? How many receptors are there?

Glutathione S-transferase from the spruce budworm, Choristoneura fumiferana: identification, characterization, localization, cDNA cloning, and expression

A 23-kDa protein that was present at higher levels in diapausing 2nd instar larvae than in feeding 2nd instar larvae of Choristoneura fumiferana was purified, and polyclonal antibodies were raised against this protein. The antibodies were subsequently used to screen a cDNA library that was constructed using RNA from 2nd instar larvae. Eight identical cDNA clones were isolated. The cDNA clone had a 665-bp insert and the longest open reading frame coded for a 203-amino acid protein with a predicted molecular mass of 23.37 kDa. The deduced amino acid sequence showed high similarity to glutathione S-transferases and therefore, the cDNA clone was named C. fumiferana glutathione S-transferase (CfGST). Identity of CfGST was confirmed by using affinity-purification as well as enzyme activity assay. CfGST was closer in similarity to insect GST2 members than GST1 members. The apparent Vmax of the purified CfGST towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. However, the enzyme had a three-fold higher affinity towards CDNB than glutathione. Analyses using Northern blot, immunoblot and immunocytochemistry demonstrated that the fat body was the major tissue where the enzyme was synthesized and stored. Higher levels of CfGST protein were present in diapausing 2nd instar larvae compared to feeding 2nd and 6th instar larvae, suggesting that besides detoxification CfGST may have other roles during insect development that are not readily apparent at present. The CfGST cDNA was expressed in a recombinant baculovirus expression system and an active enzyme was produced.

Photoaffinity labeling and characterization of a juvenile hormone binding protein in the membranes of follicle cells of Locusta migratoria

Membrane preparations from vitellogenic follicles of Locusta migratoria incubated with tritium-labeled photoaffinity analogs of juvenile hormones and methoprene in the presence or absence of excess unlabeled hormone or methoprene were irradiated, subsequently solubilized, the peptides separated by SDS-PAGE and the radioactive proteins visualized by fluorography. Both [3H] epoxyhomofarnesyl diazoacetate ([3H]EHDA), the photoaffinity analog of JH II, and [3H] epoxyfarnesyl diazoacetate ([3H]EFDA), the photoaffinity analog of JH III, covalently modified a 35 kDa peptide, and this labeling was displaced by an excess of unlabeled JH II and JH III respectively. No JH 1-displaceable labeling by [3H] epoxybishomofarnesyl diazoacetate ([3H]EBDA), the photoaffinity analog of JH I, was detected. [3H] methoprene diazoacetate ([3H]MDK), the photoaffinity analog of methoprene, exhibited specific binding to a 17 kDa protein. Using (10R)-[3H]JH III as a ligand, membrane preparations were demonstrated to possess specific and saturable binding: Scatchard analysis revealed a single binding site with a Kp of 3.68 ± 0.46 nM and a total binding capacity (Bmax) of 0.375 ± 0.07 pmol per mg protein. The ability of the various JHs to bind reflects previous findings concerning their biological activity on follicle cells in vitro, suggesting that the binding site is a membrane receptor for JH III.

Some properties of Na+K+ ATPase in the follicle cells of Rhodnius prolixus

Abstract The Na + K + -ATPase of follicle cell membrane has been implicated as a likely target by which JH, either directly or indirectly, causes follicle cells to shrink. The characteristics of ATPase activity located in microsomal fractions from vitellogenic follicles are typical for the cation and substrate requirements found for similar ATPases in other systems. JH stimulates the activity of this enzyme in a dose-dependent fashion with optimal stimulation occuring at 4 × 10 −8 M. On the other hand, methyl palmitate, which lacks JH activity, will not stimulate the enzyme.The ATPase from brain is unaffected by JH. This indicates that some, at least, of the ATPase activity found in follicle cells is different from the activity found in other tissues.

Juvenile hormone dependent phosphorylation of a 100 kDa polypeptide is mediated by protein kinase C in the follicle cells of Rhodnius prolixus

Summary The addition of juvenile hormone I (JH I) to membrane preparations of the follicle cells from vitellogenic follicles of the insect Rhodnius prolixus causes a significant increase in the phosphorylation of a 100 kDa polypeptide; and ouabain, a specific inhibitor of Na+K+-ATPase, eliminates this effect. H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine), an inhibitor of protein kinase C (PKC), also eliminates the JH-dependent phosphorylation of this polypeptide. PDBU (phorbol-12, 13-dibutyrate), an activator of PKC, mimics the action of JH in increasing the phosphorylation of the 100 kDa polypeptide. Because these findings parallel the action of JH in causing the patency, the appearance of large spaces between the follicle cells through which vitellogenin gains access to the oocyte surface, they suggest that phosphorylation of one or more membrane proteins is a key event in the development of patency in response to JH. The 100 kDa polypeptide may represent the a-subunit of Na+K+-ATPase.

Hormonal controls on reproduction in female heteroptera

A survey of the literature related to hormonal control of reproduction in female heteropterans reveals many common features. Juvenile hormone secreted by the corpus allatum is the principal hormone governing vitellogenesis. Feeding and nutrition affects egg development via juvenile hormone secreted by the corpus allatum, which is under inhibitory control from the brain. Information about feeding reaches the brain via a humoral route. Similarly, mating increases egg production by altering the degree of inhibition of the corpus allatum by the brain, although the nature of the mating stimulus and the route by which the stimulus reaches the brain remains uncertain. There may be one or more stimulatory factors acting on the corpus allatum. Progress in identifying controls on the corpus allatum is hindered by our ignorance of the identity of the juvenile hormone(s) acting in Heteroptera. Ovulation and oviposition are under the control of a myo-active peptide from the brain, and its release is governed by hormonal inputs from both mating and the ovary. The ovarian input appears to consist of ecdysteroid. A role for ovarian ecdysteroid in controlling the corpus allatum remains uncertain. Arch. Insect Biochem. Physiol. 35:443–453, 1997.

Effects of various compounds on Na/K-ATPase activity, JH I binding capacity and patency response in follicles of Rhodnius prolixus

Abstract This study explores the specificity of the binding of juvenile hormone I to membrane follicle preparations, as revealed by competition studies with juvenile hormone II, juvenile hormone III, and farnesyl methyl ether, and relates their ability to compete to their ability to cause spaces to appear between the cells of the follicular epithelium (patency), and to bring about an increase in Na/K- ATPase activity in microsomal preparations of follicle cells, an important correlate of the ability of JH I to increase patency. None of the compounds tested exhibited significant competitive ability at physiological concentrations, and all them failed to affect patency of ATPase activity. Similar studies were carried out with extracts of the abdominal neurosecretory organs, which owe their antigonadotropic activity to their ability to inhibit the JH I-mediated decrease in cell volume leading to an increase in patency. These extracts failed to affect either the JH I-mediated increase in Na/K- ATPase activity or the binding of JH I to follicle cell membranes.

A possible relationship between ouabain-sensitive (Na+-K+) dependent ATPase and the effect of juvenile hormone on the follicle cells of Rhodnius prolixus

The induction of patency in the follicular epithelium of Rhodnius prolixus by JH is inhibited by ouabain. Ouabain abolishes the reduction in volume of the follicle cells caused by the presence of JH. A JH-sensitive (Na+-K+) ATPase is present in whole ovaries, in isolated follicles and a ‘heavy microsomal’ fraction from vitellogenic follicles.