G.R. Wyatt

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.

Lipophorins, a major class of lipoproteins of insect haemolymph

Abstract The name Lipophorin (Gk. lipos , fat; phoros , bearing) is proposed as a generic term for the class of insect haemolymph lipoproteins that serve to transport lipids between organs of absorption, storage and utilisation.

Regulation of glycogen phosphorylase in fat body ofCecropia silkmoth pupae

SummaryGlycogen phosphorylase of pupal fat body of the silkmoth,Hyalophora cecropia, and its activation by different stimuli have been studied. Spectrophotometric assay in the direction of glycogenolysis, used in most of the experiments, indicated higher amounts of phosphorylasea than assay by release of Pi from glucose-1-phosphate; both assays, however, estimated changes in proportion of phosphorylasea equally. TheKms for Pi were estimated as 5 mM for phosphorylasea in the absence of AMP and 18 mM for phosphorylaseb with 2 mM AMP.When diapausing pupae were held at 4°C, fat body phosphorylase was quickly activated by conversion to thea form up to about 50% of the total, and then declined again after 30 days, when glycerol had accumulated in the hemolymph. Cold activation in vivo was quickly reversed at 25°C. Removal of the brain did not prevent cold activation. After storage at 15°C, sensitivity to cold activation was diminished. Locusts and crickets also showed activation of phosphorylase after chilling.Exposure of fat body to air, transfer to Ringer solution, or physical agitation, caused activation of phosphorylase which is classed as “shock” activation. After about 1 h incubation in Ringer at 25°C, this effect reversed spontaneously. Activation also occurred in fat body in vitro after transfer to 0°C (“cold” activation), and was reversed at 25°C. The previously reported inhibition of activation by glycerol, however, could not be consistently reproduced.In fat body homogenates, phosphorylaseb was converted to phosphorylasea by incubation with ATP and Mg2+, which indicates activity of phosphorylase kinase. In preparations treated with Sephadex G-25 and then incubated, the reverse conversion took place, which was inhibited by fluoride, and indicates activity of phosphorylase phosphatase.Cyclic AMP added to fat body in vitro, or theophylline either in vivo or in vitro, stimulated the activation of phosphorylase. In fat body in vitro, shock activation was paralleled by elevation of tissue cyclic AMP, whereas cold activation was not. Cyclic GMP did not stimulate activation, and showed no significant changes in tissue levels.It is concluded that the conversion of silkmoth pupal fat body phosphorylaseb to phosphorylasea can be stimulated by a shock-initiated mechanism involving cyclic AMP and a distinct cold-initiated mechanism independent of cyclic AMP.

Juvenile hormone-controlled vitellogenin synthesis in Locusta migratoria fat body. Hormonal induction in vivo.

Biosynthetic processes related to the production of vitellogenin (yolk precursor protein) have been examined in the fat body of adult female Locusta migratoria. Vitellogenin-producing capacity was assayed by incubation of fat body with [3H]leucine, followed by precipitation from the medium with specific antiserum. In normal development, vitellogenin synthesis began at about Day 7 after emergence and became maximal at about Day 13, when this protein accounted for 60% of the total fat body protein output. The production of other proteins increased to a lesser extent, becoming maximal at about Day 6. The incorporation of uridine into fat body RNA rose to a maximum at Day 8, which coincided with a marked increase in tissue RNA content. The DNA content in adult female fat body approximately doubled between Days 3 and 8. Vitellogenin synthesis, and the increases in RNA and DNA, were prevented by removal of the corpora allata (the source of juvenile hormone). In allatectomized locusts, vitellogenin synthesis was induced by JH or an analog, ZR-515. Applied topically in acetone, these gave steep dose-response curves, half-maximal at 75 and 150 μg, respectively. After a single treatment with ZR-515, fat body vitellogenin production rose slowly during 48 hr, then steeply to a maximum at 72 hr, but after decay of this effect during 10 days, a second application of ZR-515 induced renewed synthesis with little initial lag. Hormone treatment produced a smaller increase in the output of other proteins, and an increase in incorporation into RNA which preceded the major rise in vitellogenin synthesis. Male fat body produced little or no vitellogenin. These results are consistent with action of JH at the gene level.

Sequence of the Hexameric Juvenile Hormone-binding Protein from the Hemolymph of Locusta migratoria

The cDNA for the hexameric hemolymph juvenile hormone-binding protein (JHBP) from the migratory locust has been cloned and sequenced. Antiserum raised against purified JHBP was used to identify clones in an expression library. The 4.3-kilobase JHBP mRNA codes for 668 amino acids (74.4 kDa) and contains 2 kilobases of 3′-untranslated region. The derived amino acid sequence reveals that locust JHBP represents a new group within the hexamerin family of arthropod proteins. JHBP appears to be more closely related to arthropod hemocyanins, the believed ancestors of the family, than to the other known insect hexamerins. The mRNA shows a high (89%) bias to codons ending in G or C and the codons ending in A or T are clustered and concentrated toward the 5′ end, suggesting a mosaic gene structure. The recombinant bacterially expressed protein bound [3H]JH III with the same affinity as the protein from hemolymph. A truncated version of JHBP lacking 53 amino acids from the N terminus did not bind JH III. Hybridization analysis of fat body JHBP mRNA in locusts that had been treated with precocene and a JH analog did not give clear evidence for regulation by JH.

Purification and properties of vitellogenin and vitellin from Locusta migratoria

Abstract The vitellogenin (VG) and vitellin (VN) of Locusta migratoria have been purified from ovariectomized female haemolymph and from oocytes, respectively, by a new procedure. The products are free of lipoprotein I, which was present as a contaminant in previous preparations. The isoelectric point of VG and VN is pH 6.3. The amino acid compositions of VG and VN do not differ significantly. The lipid moiety (8.2% by weight of VG and 6.7% by weight of VN) includes diacylglycerol, a trace of triacylglycerol, cholesterol, phosphatidylcholine and phosphatidylethanolamine. The content of diacylglycerol is much lower in VN than in VG, which suggests the release of lipid within the oocyte.

THE FAT BODY AS A PROTEIN FACTORY

Publisher Summary This chapter discusses the problems and opportunities that arise from the biochemical study of the synthesis and storage of specific proteins in the insect fat body. The fat body cells are responsible for a wide range of roles. These include the metabolism of carbohydrates, lipids, and nitrogenous compounds; synthesis and regulation of blood sugar; storage of glycogen, fat, and protein; and synthesis of the major blood proteins— a diversity that may be unequalled in any other metazoan cell. The fat body cell switches its activity pattern in response to nutritional, hormonal, and developmental signals to provide the successive needs of the growing, metamorphosing, migrating, and reproducing insect. Thus, the fat body cell plays not one but an orderly succession of roles. The rapid increase in knowledge in the area of fat body depends on the application of new radioisotope, electrophoretic, and immunochemical techniques and on improved understanding of insect endocrinology and cell biology.

Juvenile hormone-dependent vitellogenin synthesis in Locusta migratoria fat body: Inducibility related to sex and stage☆

Juvenile hormone (JH)-dependent vitellogenin (Vg) synthesis in the fat body of Locusta migratoria is normally limited to sexually mature adult females. As a step toward examining the basis of this limitation, we have tested female and male locusts in a series of stages after the third larval molt for inducibility of Vg synthesis by the synthetic JH analog, methoprene. We find that in the fourth and fifth larval instars fat body of both sexes can be induced to produce Vg, but in the adult stage females respond strongly while no more than trace amounts can be induced in males. Quantitative assays show relative responsiveness in the order: adult female greater than fifth instar female greater than fifth instar male much greater than adult male. During the fifth instar of both sexes, maximal vitellogenic response was obtained in midinstar. After the larval-adult ecdysis, female fat body was unresponsive during the first 4 days, then responsiveness increased and by Day 8 after ecdysis fat bodies were fully as competent to produce Vg as at Day 14, the usual maximum of the first vitellogenic cycle due to endogenous JH. Larval and adult female fat bodies implanted into male larvae are competent for Vg synthesis after metamorphosis, so that the differences between adult male and female cannot be imposed by the male milieu intérieur during the larval-adult molt. In male and female precocious adults, produced by treatment of fourth instars with precocene, fat body responded to methoprene as in normal adults. We conclude that factors intrinsic to the fat body cells, determined early in development, are responsible for differential gene programing in males and females, which is partially expressed by the fifth instar but fully manifest only after a molt in the absence of JH.

Locust retinoid X receptors: 9-Cis-retinoic acid in embryos from a primitive insect.

The retinoid X receptor (RXR) is activated by its often elusive cognate ligand, 9-cis-retinoic acid (9-cis-RA). In flies and moths, molting is mediated by a heterodimer ecdysone receptor consisting of the ecdysone monomer (EcR) and an RXR homolog, ultraspiracle (USP); the latter is believed to have diverged from its RXR origin. In the more primitive insect, Locusta migratoria (Lm), RXR is more similar to human RXRs than to USPs. LmRXR was detected in early embryos when EcR transcripts were absent, suggesting another role apart from ecdysone signaling. Recombinant LmRXRs bound 9-cis-RA and all-trans-RA with high affinity (IC50 = 61.2–107.7 nM; Kd = 3 nM), similar to human RXR. To determine whether specific binding had functional significance, the presence of endogenous retinoids was assessed. Embryos were extracted by using modified Bligh and Dyer and solid-phase protocols to avoid the oily precipitate that makes this material unsuitable for assay. These extracts contained retinoids (5.4 nM) as assessed by RA-inducible Cyp26A1-promoter luciferase reporter cell lines. Furthermore, the use of HPLC and MS confirmed the presence of retinoids and identified in any embryo, 9-cis-RA, in addition to all-trans-RA. We estimate that whole embryos contain 3 nM RA, including 9-cis-RA at a concentration of 1.6 nM. These findings strongly argue for a functional role for retinoids in primitive insects and favor a model where signaling through the binding of 9-cis-RA to its RXR is established relatively early in evolution and embryonic development.

Vitellogenin mRNA in locust fat body: Coordinate induction of two genes by a juvenile hormone analog

Levels of vitellogenin (Vg) mRNA in Locusta migratoria fat body were determined as indicators of gene expression induced by the juvenile hormone analog methoprene. After injection of methoprene into juvenile hormone-deprived locusts, excised fat bodies were cultured with [3H]leucine for immunochemical assay of Vg synthesis, and RNA was assayed for Vg mRNA content by hybridization with probes from the previously cloned locust Vg genes A and B. In general, the rise in Vg mRNA paralleled the rise in Vg synthesis. During the primary response to methoprene (in female locusts in which the corpora allata had been destroyed immediately after emergence), Vg mRNA was first detected after 18-24 hr and accumulated rapidly between 36 and 48 hr. The secondary response (in locusts allatectomized during vitellogenesis and kept until Vg disappeared) was accelerated, as Vg mRNA was detectable at 12 hr and titers rose steeply after 18 hr. When Vg synthesis was prematurely induced by injection of methoprene into fifth-stage female larvae, the kinetics of mRNA accumulation were similar to those of primary stimulation in the adult. After allatectomy of vitellogenic females, fat body Vg mRNA decayed with a half-life of about 24 hr, roughly paralleling the decline in Vg synthesis. Assays with the two Vg probes showed coordinate accumulation of gene A and gene B messages under all conditions tested: during primary and secondary stimulation in adult females and in the low-level response obtained by treating male larvae with methoprene.