A highly conserved red pigment-concentrating hormone precursor in the blue crab Callinectes sapidus.

A cDNA library was established from the eyestalk ganglia of the blue crab Callinectes sapidus. One clone was isolated (644 bp excluding the poly(A) tail) which encodes the red pigment-concentrating hormone (RPCH)-precursor, consisting of the 25 amino acid residue signal peptide, the RPCH, and a 73 amino acid residue RPCH-precursor related peptide. This clone displays high sequence similarity with a clone isolated from an eyestalk cDNA library of the shore crab Carcinus maenas, in accordance with the close phylogenetic relationship between these species. Northern blot experiments indicated the presence of two different mRNA transcripts which hybridized with a specific RPCH-cDNA probe pointing to the possibility of multiple RPCH isoforms in the blue crab. Although crustacean RPCH and the insect adipokinetic hormones (AKH) are structurally related, their precursors show little similarity.

Molecular cloning of two pigment-dispersing hormone (PDH) precursors in the blue crab Callinectes sapidus reveals a novel member of the PDH neuropeptide family.

A cDNA library was established from the eyestalk ganglia of the blue crab Callinectes sapidus. Screening resulted in the isolation of a clone (497 bp excluding poly(A) tail) which encodes a beta-PDH previously found in several crustacean species. It displays high sequence similarity with a clone isolated from an eyestalk cDNA library of the shore crab Carcinus maenas, indicating the close phylogenetic relationship of both species. A second clone (414 bp exclusive of the poly(A) tail) encodes a novel beta-PDH analog which displays 400-fold less potency in crab bioassays. Both cDNAs encode open reading frames of 234 bp for the prepropeptides, consisting of signal peptides, PDH-precursor-related peptides, and PDH sequences.

Pigment-dispersing hormone-like peptide in the nervous system of the flies Phormia and Drosophila: immunocytochemistry and partial characterization.

beta-pigment-dispersing hormone (beta-PDH) isolated from the fiddler crab (Rao et al., '85) is a member of an octadecapeptide family of neuropeptides common to arthropods. Whereas earlier studies of these peptides in insects were limited to orthopterans, this investigation focuses on dipteran flies. Extracts of heads from the blowfly Phormia terraenovae were assessed in a fiddler crab bioassay for PDH activity. Immunocytochemistry, dose-response curves, gel filtration chromatography and reversed-phase HPLC, combined with bioassay and enzyme-linked immunosorbent assay (ELISA), indicate the presence of PDH-like peptide in the blowfly. Immunocytochemical mapping of PDH-like immunoreactive (PDHLI) neurons was performed for the entire nervous systems of Phormia and the fruitfly Drosophila with a beta-PDH antiserum. In the cephalic ganglion (brain, optic lobe and subesophageal ganglion) PDHLI cell bodies could be detected (34 in Phormia and 16 in Drosophila). In both species, each hemisphere contains 8 PDHLI cell bodies in the optic lobes. These innervate the optic lobe neuropils bilaterally. In Phormia, another set of 8 cell bodies are located in each of the lateral neurosecretory cell groups in the superior protocerebrum. These neurons send axons to the corpora cardiaca-hypocerebral ganglion complex and to portions of the foregut. In contrast, only the optic lobe neurons display immunoreactivity in Drosophila. Except for the optic lobes, PDHLI processes are distributed only in nonglomerular neurophils of the brain of both species. In the fused thoracico-abdominal ganglia of Phormia, 28 PDHLI cell bodies were found (only six were found in Drosophila). In both species, six abdominal PDHLI neurons are efferents with axons innervating the hindgut. We also found that some of the PDHLI neurons in the Phormia brain and abdominal ganglion contain colocalized FMRFamide-like immunoreactivity. Since the flies studied here do not display hormonally controlled, fast pigment migrations, the PDH-like peptide may have a role as neurotransmitter or neuromodulator in the central nervous system, especially in the visual system, and a regulatory role in the stomatogastric system and the hind-gut.

Primary structure and relative potency of an analog of beta-PDH (pigment-dispersing hormone) from the crayfish Procambarus clarkii.

A pigment-dispersing hormone (PDH) from eyestalks of the crayfish Procambarus clarkii was purified by gel filtration, cation-exchange chromatography, partition chromatography, and reversed-phase HPLC. Based on automated sequencing and by the identical chromatographic behavior of the native PDH and the synthetic amidated form of the deduced sequence, the primary structure of Procambarus PDH has been established as: Asn-Ser-Glu-Leu-Ile-Asn-Ser-Ile-Leu-Gly-Leu-Pro-Lys-Val-Met-Asn-Glu-Ala- NH2. This peptide differs from beta-PDH of the fiddler crab Uca pugilator at a single position, Glu17 in place of Asp17. Because of this substitution, Procambarus PDH was 4 to 7-fold less potent than beta-PDH in causing pigment dispersion in the erythrophores, leucophores, and melanophores of Uca. In contrast, Procambarus PDH was 4-fold more potent than beta-PDH in eliciting pigment dispersion in the erythrophores of Procambarus. These peptides displayed less marked differences in potency in triggering leucophore pigment dispersion and light-adaptational distal eye pigment movement in Procambarus. These findings indicate that the structural requirements for PDH-receptor interactions vary with the species and with the target cell type within a given species.

Primary structure of an analog of crustacean pigment-dispersing hormone from the lubber grasshopper Romalea microptera.

An octadecapeptide capable of inducing pigment dispersion in the chromatophores of the fiddler crab Uca pugilator has been isolated from lyophilized heads of the lubber grasshopper Romalea microptera. This pigment-dispersing factor (PDF) was purified by gel filtration, ion-exchange chromatography, partition chromatography, and reversed-phase high performance liquid chromatography. Automated gas-phase sequencing, followed by the identification of the carboxyl-terminal amide, established the primary structure of this PDF as Asn-Ser-Glu-Ile-Ile-Asn-Ser-Leu-Leu-Gly-Leu-Pro-Lys-Leu-Leu-Asn-Asp-Ala- NH2. This structure was confirmed by chemical synthesis and by demonstrating that the synthetic and native PDF displayed identical chromatographic behavior and biological activity. The Romalea PDF is structurally related to the crustacean pigment-dispersing hormones (PDHs), which are also octadecapeptides. The sequence of grasshopper PDF shows 78% homology with beta-PDH (from the crabs U. pugilator and Cancer magister) and 50% homology with alpha-PDH (from the prawn Pandalus borealis). This study provides the first direct chemical evidence for the structural relatedness of insect PDF to the crustacean PDHs, thus identifying them as an authentic family of arthropod peptides.