Crustacean hyperglycaemic hormone (CHH)-like peptides and CHH-precursor-related peptides from pericardial organ neurosecretory cells in the shore crab, Carcinus maenas, are putatively spliced and modified products of multiple genes.

About 24 intrinsic neurosecretory neurons within the pericardial organs (POs) of the crab Carcinus maenas produce a novel crustacean hyperglycaemic hormone (CHH)-like peptide (PO-CHH) and two CHH-precursor-related peptides (PO-CPRP I and II) as identified immunochemically and by peptide chemistry. Edman sequencing and MS revealed PO-CHH as a 73 amino acid peptide (8630 Da) with a free C-terminus. PO-CHH and sinus gland CHH (SG-CHH) share an identical N-terminal sequence, positions 1-40, but the remaining sequence, positions 41-73 or 41-72, differs considerably. PO-CHH may have different precursors, as cDNA cloning of PO-derived mRNAs has revealed several similar forms, one exactly encoding the peptide. All PO-CHH cDNAs contain a nucleotide stretch coding for the SG-CHH(41-76) sequence in the 3'-untranslated region (UTR). Cloning of crab testis genomic DNA revealed at least four CHH genes, the structure of which suggest that PO-CHH and SG-CHH arise by alternative splicing of precursors and possibly post-transcriptional modification of PO-CHH. The genes encode four exons, separated by three variable introns, encoding part of a signal peptide (exon I), the remaining signal peptide residues, a CPRP, the PO-CHH(1-40)/SG-CHH(1-40) sequences (exon II), the remaining PO-CHH residues (exon III) and the remaining SG-CHH residues and a 3'-UTR (exon IV). Precursor and gene structures are more closely related to those encoding related insect ion-transport peptides than to penaeid shrimp CHH genes. PO-CHH neither exhibits hyperglycaemic activity in vivo, nor does it inhibit Y-organ ecdysteroid synthesis in vitro. From the morphology of the neurons it seems likely that novel functions remain to be discovered.

Cloning of a novel cytochrome P450 (CYP4C15) differentially expressed in the steroidogenic glands of an arthropod.

Biosynthesis of ecdysteroids, arthropod steroid molting hormones, proceeds from dietary cholesterol through a complex and still incompletely elucidated pathway. Most of the known steps are catalyzed by cytochrome P450 enzymes (CYPs) but none of their genes has yet been identified. We have established a cDNA library of crayfish steroidogenic glands (Y organs). A full length CYP-cDNA was characterized containing a 1539 bp open reading frame encoding a predicted protein of 513 amino acid residues. This novel CYP was assigned to the CYP4 family and designated CYP4C15. Northern blots demonstrated predominant expression of this gene in the active molting glands, suggesting a role in ecdysteroid biosynthesis rather than detoxification.

Ecdysteroid biosynthesis in crayfish Y-organs: feedback regulation by circulating ecdysteroids

In crustaceans, ecdysteroid synthesis in the Y-organs is negatively regulated by the molt-inhibiting hormone (MIH). Reduction or cessation of MIH release from the sinus gland in the eyestalk, probably due to environmental cues, is one of possibly several signals for an increase of edysteroid production and subsequently enhancement of 20-hydroxyecdysone (20E) levels in the hemolymph. The present study asks the question whether the 20E peak in premoult stages D2/D3 is explained solely bythe cessation of MIH release or whether positive feedback mechanisms are also involved. Ecdysteroid production by the Y-organ of the crayfish Orconectes limosus was found to be under negative feedback control by circulating ecdysteroids. Exogenous 20-hydroxyecdysone (20E) as well as RH-5849, a non-steroidal ecdysteroid agonist, reduced ecdysteroid synthesis significantly when injected into intermoult animals. A direct, short loop inhibitory feedback effect was demonstrated by in vitro incubations of Y-organs with RH-5849. Thus, the results presented here do not point to a stimulatory effect of 20E on Y-organ activity but suggest that during intermolt a negative feedback by ecdysteroids plays a role in addition to MIH. Arch. Copyright 1999 Wiley-Liss, Inc.

Cloning and characterization of the L15 ribosomal protein gene homologue from the crayfish Orconectes limosus.

A cDNA homologue of the large unit rat ribosomal protein L15 was cloned from an epidermal cDNA library of the crayfish Orconectes limosus, being the first crustacean ribosomal protein gene cloned to date. It contains 204 amino acids, deduced from the nucleotide sequence, and reveals 70-76% sequence identity with other arthropod and vertebrate ribosomal L15s. A single, abundant 0.7-kb mRNA transcript was constitutively expressed and revealed similar expression levels, among various adult crayfish tissues.

Action of different ecdysteroids on the regulation of mRNAs for the ecdysone receptor, MHR3, dopa decarboxylase, and a larval cuticle protein in the larval epidermis of the tobacco hornworm, Manduca sexta.

To determine which ecdysteroids may be biologically active in the larval epidermis of the tobacco hornworm, Manduca sexta, we studied the action of several known ecdysteroids and metabolites on the expression of the genes encoding the ecdysone receptor (EcR), Manduca hormone receptor 3 (MHR3), dopa decarboxylase (DDC), and a larval cuticle protein (LCP-14). Both Day 2 fourth- and Day 2 fifth-instar larval epidermis contained significant 3 beta-reductase activity which metabolized 3-dehydroecdysone (3DE) and 3-dehydro-20-hydroxyecdysone (3D20E) to ecdysone (E) and 20-hydroxyecdysone (20E), respectively, but had only very low amounts of ecdysone oxidase activity (E to 3DE) and no detectable ecdysone 20-monooxygenase activity (E to 20E). When the expression of the various genes was studied in the epidermis in vitro, 20E and 3D20E had similar effects, whereas E, 3DE, 26-hydroxyecdysone and 20,26-dihydroxyecdysone were ineffective. Exposure of Day 2 fifth-instar epidermis to 500 ng/ml of either 20E or 3D20E for 24 hr caused a rapid, biphasic increase in EcR-B1 mRNA. By contrast, EcR-A mRNA showed a less rapid initial increase followed by a slow steady rise and was less responsive to 3D20E. Ecdysone in a 1:1 mixture with 20E effectively halved the concentration of 20E needed to induce EcR-B1 mRNA but showed no synergism in the induction of EcR-A mRNA. The induction of MHR3 mRNA and of DDC mRNA in Day 2 fourth-instar epidermis as well as the suppression of DDC and LCP-14 gene expression by 3D20E was indistinguishable from that of 20E. Therefore, for Manduca larval epidermis, only 20E and 3D20E are biologically active ecdysteroids. Since the 3D20E can be converted to 20E by the epidermis, its effects are likely mediated by 20E.

Involvement of a 3beta-hydroxysteroid dehydrogenase activity in ecdysteroid biosynthesis.

Ecdysteroid biosynthesis was analyzed in vitro using dissociated Y-organ cells from the shore crab Carcinus maenas. 3-Dehydroecdysone (3DE) was detected as a minor secretory product, in addition to the formerly identified end-products 25-deoxyecdysone and ecdysone (E). In conversion studies, 3DE was formed from tritiated 5beta-ketodiol (2,22,25-trideoxyecdysone), 2,22-deoxyecdysone and 2-deoxyecdysone but not from E. Further experiments were performed in order to understand the interconversions between 3-oxo and 3beta-OH compounds in the crab Y-organ. The enzyme involved in 3beta-dehydrogenation was not ecdysone oxidase, a soluble enzyme found in peripheral tissues of many arthropods but it presented strong similarities with 3beta-hydroxysteroid dehydrogenase enzymes from vertebrates: it was membrane-bound and NAD+-dependent. Moreover, a NADH-dependent 3beta-reduction of several 3-oxo-ecdysteroids was obtained using the same microsomal fraction (100,000 x g pellet) of Y-organs, indicating that the reaction might be reversible. As this activity was specific of molting glands, we hypothesize that there is at least one 3beta-hydroxysteroid dehydrogenase enzyme involved in the biosynthetic pathway of ecdysteroids.

Regulation of steroidogenesis in crayfish molting glands: involvement of protein synthesis.

The involvement of continuous protein synthesis in the mechanisms of crustacean steroidogenesis was investigated using crayfish molting glands (Y-organs). During intermolt, Y-organ steroidogenic activity is low. Eyestalk ablation initiates premolt which is characterized by a rapid increase in the production of ecdysteroids. In vitro incorporation of [14C]leucine into TCA-precipitable proteins was measured in Y-organs. A significant increase of de novo protein synthesis within 2 h and simultaneously led to a strong inhibition of the ecdysteroid synthesis. Sinus gland extracts (containing molt inhibiting hormone) also induced both a limited but reproducible inhibition of Y-organ protein synthesis and a pronounced inhibition of ecdysteroid production within 2 h. The results suggest a functional link between protein synthesis in the Y-organ and sustained ecdysteroid production. The analysis of autoradiographs from one-dimensional gel electrophoreses revealed an overall increase in de novo synthesis of glandular proteins in early premolt but also a more specific effect on distinct proteins (increase of 150, 140, 50-60, 22 and 15-18 kDa proteins) which may be more directly involved in the regulation of ecdysteroidogenesis.