The hemolymph protein HP19 of the rice moth, Corcyra cephalonica, mediates the 20-hydroxyecdysone (20E)-dependent acid phosphatase (ACP) activity at a nongenomic level. Affinity-purified polyclonal antibody against HP19 (alphaHP19-IgG) was used in the present study to understand the role of HP19 during the postembryonic development of Corcyra. In the in vitro studies, HP19 action was blocked either by immuno-precipitation using alphaHP19-IgG, prior to its addition to the fat body culture or by the addition of the antibody directly to the culture, along with 20E and hemolymph containing HP19. The alphaHP19-IgG blocked the HP19-mediated 20E-dependent ACP activation. In the in vivo studies, the alphaHP19-IgG was injected into the fully developed last (final/Vth) instar larvae of Corcyra, to complex the HP19 in vivo, in order to block the action of HP19. The injection of alphaHP19-IgG resulted in defective development of larvae, which grew either into non-viable larvae or larval-pupal/pupal-adult intermediates relative to the effect of pre-immune IgG injected controls. The present study shows that HP19 plays an important role in controlling the metamorphosis of Corcyra by regulating the 20E-dependent ACP activity. Coupled with the earlier findings, the ecdysteroid hormone regulates this action at a nongenomic level.

Regulation of energy metabolism is one of the major functions of steroid hormones. In this process, mitochondria, by way of oxidative phosphorylation, play a central role. Depending on the energy needs of the cell, on the tissue, on the developmental stage and on the intensity of the hormonal stimulus, the response can be an activation of pre-existing respiratory chain components, an increased transcription of nuclear-encoded and/or mitochondrial-encoded respiratory chain enzyme (OXPHOS) genes and of biosynthesis of the respective enzyme subunits or, in extreme cases of high energy needs, an increase in the number of mitochondria and mitochondrial DNA content per cell. Some of the hormonally regulated systems involving effects on nuclear and mitochondrial OXPHOS genes are reviewed in this paper. The possible molecular mechanisms of steroid hormone action on nuclear and mitochondrial. gene transcription and possible ways of coordination of transcription in these two separate cell compartments involving direct interaction of steroid receptors with hormone response elements in nuclear OXPHOS genes and in mitochondria and induction/activation of nuclear-encoded regulatory factors affecting mitochondrial gene transcription are presented.

This article concerns the localization of glucocorticoid and thyroid hormone receptors in mitochondria of animal cells. The receptors are discussed in terms of their potential role in the regulation of mitochondrial transcription and energy production by the oxidative phosphorylation pathway, realized both by nuclear-encoded and mitochondrially encoded enzymes. A brief survey of the role of glucocorticoid and thyroid hormones on energy metabolism is presented, followed by a description of the molecular mode of action of these hormones and of the central role of the receptors in regulation of transcription. Subsequently, the structure and characteristics of glucocorticoid and thyroid hormone receptors are described, followed by a section on the effects of glucocorticoid and thyroid hormones on the transcription of mitochondrial and nuclear genes encoding subunits of OXPHOS and by an introduction to the mitochondrial genome and its transcription. A comprehensive description of the data demonstrates the localization of glucocorticoid and thyroid hormone receptors in mitochondria as well as the detection of potential hormone response elements that bind to these receptors. This leads to the conclusion that the receptors potentially play a role in the regulation of transcription of mitochondrial genes. The in organello mitochondrial system, which is capable of sustaining transcription in the absence of nuclear participation, is presented, responding to T3 with increased transcription rates, and the central role of a thyroid receptor isoform in the transcription effect is emphasized. Lastly, possible ways of coordinating nuclear and mitochondrial gene transcription in response to glucocorticoid and thyroid hormones are discussed, the hormones acting directly on the genes of the two compartments by way of common hormone response elements and indirectly on mitochondrial genes by stimulation of nuclear-encoded transcription factors.

In late larvae of the blowfly, Calliphora vicina, arylphorin and LSP-2 proteins, which belong to the class of hexamerins, are selectively taken up by the fat body from the haemolymph. Hexamerin endocytosis is mediated by a specific membrane-bound receptor, the arylphorin-binding protein (ABP). Using the two-hybrid technique, we found that the anterior fat body protein (AFP) interacts with the hexamerin receptor. AFP, a homologue of the mammalian calcium-binding liver protein regucalcin (senescence marker protein-30), exhibits a strong binding affinity for a naturally occurring C-terminal cleavage fragment of the hexamerin receptor precursor (the P30 peptide) and other receptor cleavage products that contain P30. Expression of AFP mRNA and protein is restricted to the anterior part of the fat body tissue and to haemocytes in last-instar larvae. AFP mRNA occurs in all postembryonic developmental stages. Our results suggest that AFP plays a role in the regulation of hexamerin uptake by fat body cells along the anterior-posterior axis.

In cyclorraphan Diptera, two different types of hemolymph proteins exist which belong to the hexamerin family. During the last larval instar, Calliphora vicina synthesizes, besides the major fraction of arylphorin, a second hexameric protein, LSP-2. Here the developmentally regulated biosynthesis of this protein was analyzed. Western blot analyses showed that LSP-2 is not present in eggs, 1st, and 2nd instar larvae, whereas it can be detected in all tissues of last instar larvae. We report the characterization of the complete cDNA sequence that encodes a LSP-2 subunit, a nascent polypeptide of 701 amino acids with a molecular mass of 83.16 kDa. By Northern blotting, a mRNA of about 2.2 kb coding for LSP-2 is identified exclusively in the fat body of 3rd larval instars reflecting the stage and tissue specificity of LSP-2 gene expression. Phylogenetic analysis demonstrates the existence of two distinct groups of hexamerins in Diptera.

The transcripts of sericin-type genes MG1 and MG2 accumulate in Galleria mellonella silk glands during feeding and decline during molting. Three MG1 (1.9, 3.2, and 4.2 kb) and two MG2 (dominant 3.4 kb and minor 5.2 kb) transcripts are detectable from the penultimate instar until the end of cocoon spinning. Experiments with isolated larval abdomens showed that the dramatic change in the ratio of 1.9 and 4.2 kb MG1 transcripts in the last larval instar is due to lack of JH. Transient rise of ecdysteroid titre in the wandering larvae (day 5 of the last instar) is associated with appearance of additional MG1 transcripts (10.0, 7.2, and 5.0 kb), while the molt-inducing ecdysteroid surge on day 7 causes a drop of all transcripts. The drop is prevented and the profile of transcripts is reverted to a larval-like pattern when the last instar larvae are treated with a juvenoid. The profile of MG1 transcripts is affected only when the treatment is applied prior to the start of cocoon spinning (day 6), whereas the decline of the 3.4 kb MG2 transcript is averted with applications up to day 7. Presented results are interpreted as showing that JH causes a “status quo” effect by preventing the disappearance of certain transcripts during molting, exerts a “juvenilizing” effect by restoring the larval pattern of splicing, and affects developmental “programming,” presumably by affecting the future pattern of gene expression. © 1996 Wiley-Liss, Inc.

A 1.4 kb DNA fragment of the 5′-upstream region of a Calliphora arylphorin gene was sequenced. A series of motifs known as cis-regulatory elements in various genes of different species could be identified. Hybridization experiments and comparison of the sequences of 5 members of the arylphorin gene family show a high degree of homology from the TATA-box downstream to the first intron, whereas the upstream regions appear to be divergent.

Four classes of Galleria mellonella silk gland specific cDNAs, designated PG-1, PG-2, MG-1 and MG-2, were cloned, sequenced and used as probes in Northern and Southern blots. Analysis of the results and their comparison with the data available for Bombyx mori reveal that the isolated cDNAs correspond to mRNAs for major silk proteins. Two small transcripts (1.1 and 1.2 kb), detected with PG-1 in the posterior region of Galleria silk glands, represent mRNAs for small proteins related to the light chain fibroin of Bombyx. A large transcript (⩾ 10 kb) homologous to PG-2 seems to be the mRNA for the heavy chain fibroin. Conceptual translation of available PG-2 sequence yields a product with similar amino acid composition as reported for the chemically analyzed fibroin. Hybridization to Southern blots of Galleria genomic DNA showed that PG-1 and PG-2 correspond to separate genes. In the middle silk gland region, multiple transcripts homologous to MG-1 are produced. They include 1.9 and 4.2 kb species, and in certain developmental periods also 3.2, 7.2 and ⩾ 10 kb products. Two different transcripts, one dominant (3.4 kb) and one rare (5.2 kb) were revealed with the MG-2 probe. MG-1 and MG-2 cDNAs contain somewhat similar repeats, and their deduced translation products resemble Bombyx sericins by the dominance of serine and the high content of glycine and asparagine. Southern blots indicated that MG-1 and MG-2 represent different sericin genes apparently corresponding to Ser-1 and Ser-2 genes of Bombyx.

Arylphorin was purified from larvae of the blowfly Calliphora vicina and studied in its oligomeric form and after dissociation at pH 9.6 into native subunits. In accordance with earlier literature, it was electrophoretically shown to be a 500 kDa hexamer (1 x 6) consisting of 78 kDa polypeptides (= subunits). Electron micrographs of negatively stained hexamers show a characteristic curvilinear, equilateral triangle of 12 nm in diameter (top view) and a rectangle measuring 10 x 12 nm (side view). Alternatively, particles in the top view orientation exhibit a roughly circular shape 12 nm in diameter. Crossed immunoelectrophoresis revealed the presence of a major subunit type; the nature of a very minor and a third immunologically separated component remains unclear. A novel 2 x 6 arylphorin particle was detected and isolated. It comprises less than 10% of the total arylphorin material and shows a long, narrow interhexamer bridge in the electron microscope. An arylphorin dissociation intermediate identified as a trimer (1/2 x 6) was isolated; its possible quaternary structure is discussed on the basis of electron micrographs. The epitope of monoclonal antibody Ec-7 directed against tarantula (Eurypelma californicum) hemocyanin subunit d and also reactive to Calliphora arylphorin was traced to a highly conserved peptide of 27 amino acids localized in the center of the protein. The primary structure of Calliphora arylphorin as published in our preceding paper (Naumann and Scheller 1991) is compared in detail to the sequences of spider and spiny lobster hemocyanin. This revealed a basic framework of 103 strictly conserved amino acids. Isofunctional exchanges are proposed for another 76 positions. On the basis of these similarities, and the published three-dimensional model of spiny lobster hemocyanin, a detailed model of the quaternary structure of Calliphora arylphorin is presented. A second larval storage protein previously termed protein II was purified from Calliphora hemolymph. It was demonstrated to be a 500 kDa hexamer of 83 kDa subunits. In the electron microscope it shows a cubic view 9 nm in length with a large central hole and a rectangular view (9 x 10 nm) with a large central cavity. A morphologically very similar hemolymph protein was detected in Drosophila melanogaster larvae. From its structural appearance it is uncertain whether protein II belongs to the hemocyanin superfamily or not.

Abstract Incubation of deproteinized larval cuticle (chitin flakes) with purified arylphorin (calliphorin) or larval haemolymph of Calliphora vicina resulted in the formation of a chitin-protein complex. Enzymatic oxidation of N-β-alanyldopam ine (NDAB) in the presence of chitin flakes or the chitin-protein complex, resulted in various degrees of cross-linking of NBAD-quinone formed with chitin. This study confirms the involvement of arylphorin in the process of quinone tanning of insect cuticle.

Nuclei from fat body of different developmental stages of Calliphora vicina were isolated. They appear to be polyploid and show polytene chromosomes. The isolated nuclei were incubated with [ 32 P]GTP and the RNA transcribed in vitro was hybridized with a DNA fragment encoding a polypeptide subunit of calliphorin. The isolated nuclei transcribe the calliphorin-mRNA correctly and with the same stage specificity as observed in vivo .

The stage- and tissue-specific biosynthesis of calliphorin was analysed during the development of the blowfly, Calliphora vicina. Western blot analyses show that the protein is not present in eggs, whereas it can be detected in fat body, brain, imaginai disk, salivary gland and epidermis throughout all postembryonic stages, including the adult one. By Northern analysis a unique 2.6 kb mol.wt. mRNA coding for calliphorin is identified exclusively in the fat body tissue of larvae, pupae and adults. Hybridization experiments of in vivo labelled poly(A)+ RNA with filter-bound calliphorin genes indicate that the genes are transcribed until pupariation. However, the translation of the calliphorin mRNA stops at the end of the feeding stage, as shown by [35S]-methionine incorporation.

The stage-specific appearance of calliphorin in cuticles of Calliphora vicina was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The fate of the protein, injected into last instar larvae, was pursued by autoradiography of histological sections. Fractionation of sclerotized pupal cuticle in buffer-soluble, urea-soluble and NaOH-soluble fractions shows that calliphorin forms covalent and non-covalent links with other cuticle components. Calliphorin traverses the epidermal cells and enters the cuticle in an undegraded state and appears to be an important constituent of the sclerotizing system.

A library of Calliphora vicina genomic DNA was constructed in the λEMBL3 vector and screened for recombinant phages containing chromosomal segments encoding calliphorin, the major larval serum protein (LSP) of Calliphora. A large series of recombinants hybridizing with in vitro labelled poly(A)+ RNA from Calliphora larval fat bodies and with specific probes derived from the LSP‐1 genes of Drosophila melanogaster was isolated. Five of these phages, chosen at random, were shown by hybrid selection to retain calliphorin mRNA specifically. Eleven calliphorin mRNA‐homologous regions were located on restriction maps of these phages by hybridization with 5′ end‐labelled poly(A)+ RNA from Calliphora larval fat bodies. Each phage contains at least two calliphorin genes arranged in direct repeat orientation and seperated by 3.5–5 kb intergenic regions. The genes display similar but not identical restriction patterns. Filter hybridization and heteroduplex analysis indicate that they share a detectable homology with the LSP‐1β gene of D. melanogaster. Whole genome Southern analysis showed that these genes belong to a large family of closely related calliphorin genes which were found by in situ hybridization to polytene chromosomes of trichogen cells to be clustered in region 4a of chromosome 2 of Calliphora vicina.