Photoperiod controls egg laying and caudodorsal cell hormone expression but not gonadal development in the pond snail Lymnaea stagnalis.

Photoperiod is a reliable cue to regulate growth and reproduction for seasonal adaptation. Although photoperiodism has been well studied in Chordata and Arthropoda, less is known about Mollusca. We examined photoperiodic effects on egg laying, body size, gonad-somatic index, oocyte size and relative amounts of caudodorsal cell hormone mRNA in individual rearing conditions in the pond snail Lymnaea stagnalis. Twenty-five weeks after hatching, the percentages of egg-laying snails under a photoperiod of 12 h light and 12 h darkness (12L:12D) were significantly smaller than those under longer days. The total numbers of eggs and egg masses under 12L:12D were significantly smaller than those under longer days. Significant differences between 16L:8D and 12L:12D were not observed in the soft body and ovotestis weight, and the gonad-somatic index. Photoperiodic effects were also not observed in oocyte diameters twenty-two weeks after hatching. Twenty-seven weeks after hatching amounts of caudodorsal cell hormone mRNA were significantly lower in the cerebral ganglia with commissure under 12L:12D than 16L:8D. L. stagnalis exhibited a clear photoperiodic response in egg laying and the amount of caudodorsal cell hormone mRNA, but not in gonadal development. Under 12L:12D suppression of caudodorsal cell hormone expression might suppress egg laying.

D-Amino Acids in Peptides from Animals, Including Human: Occurrence, Structure, Bioactivity and Pharmacology.

All life forms typically possess homochirality, with rare exceptions. In the case of peptides and proteins, only L-amino acids are known to be encoded by genes. Nevertheless, D-amino acids have been identified in a variety of peptides, synthesized by animal cells. They include neuroexcitatory and neuroprotective peptides, cardioexcitatory peptides, hyperglycemic hormones, opioid peptides, antimicrobial peptides, natriuretic and defensin-like peptides, and fibrinopeptides. This article is a review of their occurrence, structure and bioactivity. It further explores the pharmacology and potential medical applications of some of the peptides.

The serotonin or dopamine by cyclic adenosine monophosphate-protein kinase A pathway involved in the agonistic behaviour of Chinese mitten crab, Eriocheir sinensis.

Agonistic behaviour is common in an encounter between two crustaceans. It often causes limb disability and consumes a lot of energy, which is harmful for the growth and survival of commercially important crustaceans. In the present study, we mainly focused on the agonistic behaviour of the Chinese mitten crab, Eriocheir sinensis, which is an important species of the aquaculture industry in China. We recorded agnostic behaviour with a high-definition camera and preliminarily evaluated the role of serotonin (5-HT) or dopamine (DA)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway and eyestalk in the behaviour. The results showed that agonistic behaviour in E. sinensis consisted of three stages: approach, contact and fight. We found that the number of fights and cumulative time of fight were significantly higher in the male vs. male group than in the female vs. female and female vs. male groups (P < 0.05). After 1 h of agonistic behaviour, 5-HT concentration showed a significant increase and DA concentration showed a significant decrease when compared with the control group (no encounter; P < 0.05). 5-HT1B and 5-HT2B mRNA levels showed a significant increase in the eyestalk (P < 0.05). 5-HT7 mRNA levels showed significant downregulation in the thoracic ganglia and DA1A mRNA levels showed upregulation in the intestine (P < 0.05). DA2 mRNA levels showed a significant decrease in the eyestalk (P < 0.05). These changes were accompanied by a significant increase in cAMP level and significant decrease in PKA level in the haemolymph (P < 0.05). In addition, a significant decrease in glucose levels was detected after the agonistic behaviour. Crustacean hyperglycemic hormone (CHH) mRNA levels showed significant upregulation in the eyestalk and significant downregulation in the intestine (P < 0.05). The number of fights and cumulative time of fight in the left eyestalk ablation (L-X vs. L-X) group were more and longer than those in the intact eyestalk (C vs. C), right eyestalk ablation (R-X vs. R-X) and bilateral eyestalk ablation (D-X vs. D-X) groups. In short, E. sinensis shows special agonistic behaviour modulated by 5-HT or DA-cAMP-PKA pathway and eyestalk, especially the left eyestalk.

In vivo and in vitro inhibitory action of 17ß-estradiol and environmental estrogen 4-nonylphenol on gonad-inhibiting hormone (GIH) expression in the eyestalks of Litopenaeus vannamei.

The gonad-inhibiting hormone (GIH) belongs to a neuropeptide family synthesized and released in an X-organ sinus gland complex of crustacean eyestalks. GIH inhibits crustacean ovarian maturation by suppressing vitellogenin (Vtg) synthesis, whereas estrogen is responsible for the stimulation of vitellogenesis (not established). In this study, the effects of 17ß-estradiol (E2, 10(-6) M), estrogen receptor antagonist tamoxifen (TAM, 10(-6), 10(-7), and 10(-8) M), and the environmental estrogen nonylphenol (NP, 1 µg/L and 100 µg/L) on LvGIH expression in the eyestalks of shrimp were determined by quantitative real-time PCR. Results showed that LvGIH expression decreased significantly during the L. vannamei ovarian maturation cycle. E2 and NP significantly reduced LvGIH transcripts in vivo, but TAM neutralized the inhibitory action of E2 in a dose-dependent manner (P < 0.05). In addition, the LvGIH expression levels decreased significantly in a time-dependent manner (P < 0.05) when ovary fragments were cultured in vitro with E2. The results of this study suggested that estrogen regulates GIH expression in L. vannamei eyestalks. E2 promoted ovarian development not only by directly upregulating vitellogenesis in the hepatopancreas, but it was also capable of downregulating LvGIH expression, which indirectly resulted in the stimulation of L. vannamei vitellogenesis.

How Did Arthropod Sesquiterpenoids and Ecdysteroids Arise? Comparison of Hormonal Pathway Genes in Noninsect Arthropod Genomes.

The phylum Arthropoda contains the largest number of described living animal species, with insects and crustaceans dominating the terrestrial and aquatic environments, respectively. Their successful radiations have long been linked to their rigid exoskeleton in conjunction with their specialized endocrine systems. In order to understand how hormones can contribute to the evolution of these animals, here, we have categorized the sesquiterpenoid and ecdysteroid pathway genes in the noninsect arthropod genomes, which are known to play important roles in the regulation of molting and metamorphosis in insects. In our analyses, the majority of gene homologs involved in the biosynthetic, degradative, and signaling pathways of sesquiterpenoids and ecdysteroids can be identified, implying these two hormonal systems were present in the last common ancestor of arthropods. Moreover, we found that the "Broad-Complex" was specifically gained in the Pancrustacea, and the innovation of juvenile hormone (JH) in the insect linage correlates with the gain of the JH epoxidase (CYP15A1/C1) and the key residue changes in the binding domain of JH receptor ("Methoprene-tolerant"). Furthermore, the gain of "Phantom" differentiates chelicerates from the other arthropods in using ponasterone A rather than 20-hydroxyecdysone as molting hormone. This study establishes a comprehensive framework for interpreting the evolution of these vital hormonal pathways in these most successful animals, the arthropods, for the first time.

Pacific white shrimp (Litopenaeus vannamei) vitellogenesis-inhibiting hormone (VIH) is predominantly expressed in the brain and negatively regulates hepatopancreatic vitellogenin (VTG) gene expression.

Ovarian maturation in crustaceans is temporally orchestrated by two processes: oogenesis and vitellogenesis. The peptide hormone vitellogenesis-inhibiting hormone (VIH), by far the most potent negative regulator of crustacean reproduction known, critically modulates crustacean ovarian maturation by suppressing vitellogenin (VTG) synthesis. In this study, cDNA encoding VIH was cloned from the eyestalk of Pacific white shrimp, Litopenaeus vannamei, a highly significant commercial culture species. Phylogenetic analysis suggests that L. vannamei VIH (lvVIH) can be classified as a member of the type II crustacean hyperglycemic hormone family. Northern blot and RT-PCR results reveal that both the brain and eyestalk were the major sources for lvVIH mRNA expression. In in vitro experiments on primary culture of shrimp hepatopancreatic cells, it was confirmed that some endogenous inhibitory factors existed in L. vannamei hemolymph, brain, and eyestalk that suppressed hepatopancreatic VTG gene expression. Purified recombinant lvVIH protein was effective in inhibiting VTG mRNA expression in both in vitro primary hepatopancreatic cell culture and in vivo injection experiments. Injection of recombinant VIH could also reverse ovarian growth induced by eyestalk ablation. Furthermore, unilateral eyestalk ablation reduced the mRNA level of lvVIH in the brain but not in the remaining contralateral eyestalk. Our study, as a whole, provides new insights on VIH regulation of shrimp reproduction: 1) the brain and eyestalk are both important sites of VIH expression and therefore possible coregulators of hepatopancreatic VTG mRNA expression and 2) eyestalk ablation could increase hepatopancreatic VTG expression by transcriptionally abolishing eyestalk-derived VIH and diminishing brain-derived VIH.

Characterization of red pigment concentrating hormone (RPCH) in the female mud crab (Scylla olivacea) and the effect of 5-HT on its expression.

Red pigment concentrating hormone (RPCH) is a member of the chromatophorotropic hormones and, in crustaceans, it is synthesized in the eyestalk. We have isolated a full-length cDNA for a RPCH preprohormone gene (Scyol-RPCH) from the eyestalks of female mud crabs, Scylla olivacea. The open reading frame consists of 642 nucleotides, and encodes a deduced 108 amino acid precursor protein, which includes a signal peptide, the RPCH (pQLNFSPGWamide), and an associated peptide. We show that the mud crab RPCH peptide exhibits 100% identity with 15 other decapods. Expression of Scyol-RPCH within adult mud crab takes place in the eyestalk, brain, and ventral nerve cord, comprising subesophageal ganglion, thoracic ganglion, and abdominal ganglion. In situ hybridization demonstrates specific expression within neuronal clusters 1, 2, 3, and 4 of the eyestalk X-organ, clusters 6, 8, 9, 10, and 17 of the brain, and in neuronal clusters of the ventral nerve cord. We found that administration of 5-HT up-regulates RPCH gene expression in the eyestalk, suggesting that RPCH may play a role as a downstream hormone of 5-HT.

Molecular characterization and expression analysis of an insulin-like gene from the androgenic gland of the oriental river prawn, Macrobrachium nipponense.

The androgenic gland (AG), a male-specific endocrine organ in crustacean, is responsible for the maintenance of male characteristics and gender differentiation. In this study, an AG-specific gene, the Macrobrachium nipponesne insulin-like androgenic gland factor (MnIAG) was isolated from a transcriptome library of M. nipponesne and its full-length cDNA sequences were obtained by RACE method. The cDNA was 1,547 bp in length and encoded a precursor protein of 175 amino acids. The deduced precursor protein consisted of a signal peptide, B chain, C peptide and an A chain, which exhibited the same structural organization as that of previously identified insulin-like androgenic gland in crustacean. The mature peptide of the MnIAG owned two additional conserved cysteine residues, which were also found in the Palaemonidae species reported. Results of the tissue distribution and in situ hybridization showed the MnIAG expressed exclusively in androgenic gland. The quantitative RT-PCR results demonstrated that the MnIAG transcript was present at blastula stage and later developmental stages with low levels, which suggested that the primordial cells of the AG might form at these stages.

Two type I crustacean hyperglycemic hormone (CHH) genes in Morotoge shrimp (Pandalopsis japonica): cloning and expression of eyestalk and pericardial organ isoforms produced by alternative splicing and a novel type I CHH with predicted structure shared with type II CHH peptides.

Crustacean hyperglycemic hormone (CHH) peptide family members play critical roles in growth and reproduction in decapods. Three cDNAs encoding CHH family members (Pj-CHH1ES, Pj-CHH1PO, and Pj-CHH2) were isolated by a combination of bioinformatic analysis and conventional cloning strategies. Pj-CHH1ES and Pj-CHH1PO were products of the same gene that were generated by alternative mRNA splicing, whereas Pj-CHH2 was the product of a second gene. The Pj-CHH1 and Pj-CHH2 genes had four exons and three introns, suggesting the two genes arose from gene duplication. The three cDNAs were classified in the type I CHH subfamily, as the deduced amino acid sequences had a CHH precursor-related peptide sequence positioned between the N-terminal signal sequence and C-terminal mature peptide sequence. The Pj-CHH1ES isoform was expressed at a higher level in the eyestalk X-organ/sinus gland (XO/SG) complex and at a lower level in the gill. The Pj-CHH1PO isoform was expressed at higher levels in the XO/SG complex, brain, abdominal ganglion, and thoracic ganglion and at a lower level in the epidermis. Pj-CHH2 was expressed at a higher level in the thoracic ganglion and at a lower level in the gill. Real-time polymerase chain reaction was used to quantify the effects of eyestalk ablation on the mRNA levels of the three Pj-CHHs in the brain, thoracic ganglion, and gill. Eyestalk ablation reduced expression of Pj-CHH1ES in the brain and Pj-CHH1PO and Pj-CHH2 in the thoracic ganglion. Sequence alignment of the Pj-CHHs with CHHs from other species indicated that Pj-CHH2 had an additional alanine at position #9 of the mature peptide. Molecular modeling showed that the Pj-CHH2 mature peptide had a short alpha helix (α1) in the N-terminal region, which is characteristic of type II CHHs. This suggests that Pj-CHH2 differs in function from other type I CHHs.

Gene expression profiling of the cephalothorax and eyestalk in Penaeus monodon during ovarian maturation.

In crustaceans, a range of physiological processes involved in ovarian maturation occurs in organs of the cephalothorax including the hepatopancrease, mandibular and Y-organ. Additionally, reproduction is regulated by neuropeptide hormones and other proteins released from secretory sites within the eyestalk. Reproductive dysfunction in captive-reared prawns, Penaeus monodon, is believed to be due to deficiencies in these factors. In this study, we investigated the expression of gene transcripts in the cephalothorax and eyestalk from wild-caught and captive-reared animals throughout ovarian maturation using custom oligonucleotide microarray screening. We have isolated numerous transcripts that appear to be differentially expressed throughout ovarian maturation and between wild-caught and captive-reared animals. In the cephalothorax, differentially expressed genes included the 1,3-ß-D-glucan-binding high-density lipoprotein, 2/3-oxoacyl-CoA thiolase and vitellogenin. In the eyestalk, these include gene transcripts that encode a protein that modulates G-protein coupled receptor activity and another that encodes an architectural transcription factor. Each may regulate the expression of reproductive neuropeptides, such as the crustacean hyperglycaemic hormone and molt-inhibiting hormone. We could not identify differentially expressed transcripts encoding known reproductive neuropeptides in the eyestalk of either wild-caught or captive-reared prawns at any ovarian maturation stage, however, this result may be attributed to low relative expression levels of these transcripts. In summary, this study provides a foundation for the study of target genes involved in regulating penaeid reproduction.

GABA and GAD expression in the X-organ sinus gland system of the Procambarus clarkii crayfish: inhibition mediated by GABA between X-organ neurons.

In crustaceans, the X-organ-sinus gland (XO-SG) neurosecretory system is formed of distinct populations of neurons that produce two families of neuropeptides: crustacean hyperglycemic hormone and adipokinetic hormone/red pigment-concentrating hormone. On the basis of electrophysiological evidence, it has been proposed that γ-aminobutyric acid (GABA) regulates both electrical and secretory activity of the XO-SG system. In this work we observed that depolarizing current pulses to neurons located in the external rim of the X-organ induced repetitive firing that suppressed the spontaneous firing of previously active X-organ neurons. Picrotoxin reversibly blocked this inhibitory effect suggesting that the GABA released from the stimulated neuron inhibited neighboring cells. Immunoperoxidase in X-organ serial sections showed co-localization of GABA and glutamic acid decarboxylase (GAD) including the aforementioned neurons. Immunofluorescence in whole mount preparations showed that two subpopulations of crustacean hyperglycemic hormone-containing neurons colocalized with GABA. The expression of GAD mRNA was determined in crayfish tissue and X-organ single cells by RT-PCR. Bioinformatics analysis shows, within the amplified region, 90.4% consensus and 41.9% identity at the amino acid level compared with Drosophila melanogaster and Caenorhabditis elegans. We suggest that crustacean hyperglycemic hormone-GABA-containing neurons can regulate the excitability of other X-organ neurons that produce different neurohormones.

Endogenous thyroid hormone synthesis in facultative planktotrophic larvae of the sand dollar Clypeaster rosaceus: implications for the evolutionary loss of larval feeding.

Critical roles of hormones in metamorphic life history transitions are well documented in amphibians, lampreys, insects, and many plant species. Recent evidence suggests that thyroid hormones (TH) or TH-like compounds can regulate development to metamorphosis in echinoids (sea urchins, sand dollars, and their relatives). Moreover, previous research has provided evidence for endogenous hormone synthesis in both feeding and nonfeeding echinoderm larvae. However, the mechanisms for endogenous synthesis remain largely unknown. Here, we show that facultatively planktotrophic larvae (larvae that reach metamorphosis in the absence of food but have the ability to feed) from the subtropical sea biscuit Clypeaster rosaceus can synthesize thyroxine endogenously from incorporated iodine (I(125)). When treated with the goitrogen thiourea (a peroxidase inhibitor), iodine incorporation, thyroxine synthesis, and metamorphosis are all blocked in a dose-dependent manner. The inhibitory effect on metamorphosis can be rescued by administration of exogenous thyroxine. Finally, we demonstrate that thiourea induces morphological changes in feeding structures comparable to the phenotypic plastic response of larval structures to low food conditions, further supporting a signaling role of thyroxine in regulating larval morphogenesis and phenotypic plasticity. We conclude that upregulation of endogenous hormone synthesis might have been associated with the evolution of nonfeeding development, subsequently leading to morphological changes characteristic of nonfeeding development.

Production, purification and characterisation of recombinant Fahsin, a novel antistasin-type proteinase inhibitor.

Serine proteinases from inflammatory cells, including polymorphonuclear neutrophils, are involved in various inflammatory disorders, like pulmonary emphysema and rheumatoid arthritis. Inhibitors of these serine proteinases are potential drug candidates for the treatment of these disorders, since they prevent the unrestricted proteolysis. This study describes a novel specific antistasin-type inhibitor of neutrophil serine proteinases, we called Fahsin. This inhibitor was purified from the Nile leech Limnatis nilotica, sequenced and heterologously expressed using a synthetic gene in the methylotrophic yeast Pichia pastoris, yielding 0.5 g(-l) of the protein in the culture medium. Recombinant Fahsin was purified to homogeneity and characterised by N-terminal amino acid sequencing and mass spectrometry. Inhibition-kinetic analysis showed that recombinant Fahsin is a fast, tight-binding inhibitor of human neutrophil elastase with inhibition constant in the nanomolar range. Furthermore, recombinant Fahsin was, in contrast to various other neutrophil elastase inhibitors, insensitive to chemical oxidation and biological oxidation via myeloperoxidase-generated free oxygen radicals. Thus, Fahsin constitutes a novel member of a still expanding family of naturally occurring inhibitors of serine proteinases with potential therapeutic use for treatment of human diseases.

Post-afterdischarge depolarization does not stimulate prolonged neurohormone secretion but is required for activity-dependent stimulation of neurohormone biosynthesis from peptidergic neurons.

Membrane depolarization plays a critical role in stimulating secretion of neuropeptides and can also be important in regulating transcriptional and translational events that control peptide biosynthesis. The purpose of this study was to test the hypothesis that persistent membrane depolarization after the end of an electrical afterdischarge plays an important role in stimulating both prolonged secretion of egg-laying hormone (ELH) and ELH synthesis from peptidergic bag cell neurons of Aplysia. Experimental preparations were treated with a low Na(+) solution to rapidly repolarize membrane potential (Vm). Compared with control preparations, the low Na(+) solution caused rapid membrane repolarization to resting levels, significant shortening of the duration of the afterdischarge, and significant decrease in the decay time constant of cytosolic Ca(2+) ([Ca(2+)](i)) concentrations, but no effect on peak [Ca(2+)](i), total [Ca(2+)](i) above baseline, or duration of elevated [Ca(2+)](i). Contrary to both theoretical expectations and findings in other cell types, low Na(+) treatment and the resulting premature repolarization of Vm did not inhibit ELH secretion. On the other hand, low Na(+) treatment that blocked prolonged depolarization, as well as inhibition of Ca(2+) influx, prevented the afterdischarge-induced stimulation of ELH synthesis. These findings provide support for membrane depolarization acting as a trigger mechanism, rather than a sustained driving force, for cellular events that control ELH secretion. It also demonstrates, for the first time, a critical role for postdischarge Vm in regulating an important aspect of neuroendocrine cell function-that of hormone synthesis.

Embryonic expression of juvenile hormone binding protein and its relationship to the toxic effects of juvenile hormone in Manduca sexta.

The juvenile hormones (JHs) regulate a diverse array of insect developmental and reproductive processes. One molecular target of JH action is its transporter, hemolymph JH binding protein (hJHBP); in the larva of the tobacco hornworm, Manduca sexta, low doses of JH can immediately increase hJHBP gene expression. Less explored are the effects of JH on embryological development, where early hormonal treatment has been shown to affect embryonic development and pupation. This study examines the egg form of JHBP and its gene expression during embryogenesis of M. sexta, as well as the phenotypic effect JH treatment has on embryos and on JHBP gene expression. We here demonstrate that the preponderance of JHBP found in the egg is maternally derived and that the embryonic gene and protein appear identical to those found in the larva. Expression of the JHBP gene begins in both the embryo itself and extra-embryonic tissues 15 h after fertilization, long before emergence of a functional fat body and circulatory system. Topical application of low JH doses to early embryos resulted in larval abnormalities while high doses of the hormone induced embryonic mortality. These effects are not mediated through regulation of the JHBP gene, since embryonic expression appears invariant in response to JH challenge. The toxicity of JH is tightly correlated with the concentration of unbound hormone.

Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification.

Production of recombinant Limulus endotoxin neutralizing protein (rENP) was attained with the GS115 methylotrophic strain of Pichia pastoris transformed with a plasmid, bearing multiple ENP gene copies. The synthetic gene for Limulus ENP was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter. Clones containing a single enp insert were used to construct cassettes bearing 2 and 3 tandem copies of enp. These were then integrated at the HIS locus of P. pastoris GS115 (his4). Clones were chosen for their ability to produce rENP upon methanol induction in shaker flasks, and then the 1x, 2x, and 3x-enp strains were analyzed by Southern blot for the presence of the ENP gene(s). Isolate 3 x 5q, containing a 3x-enp cassette, was the best producer of rENP. Under optimal conditions this strain grown in a fed-batch mode produced yields of >500 mg rENP/L with an average of 5.46 mg rENP/g DCW. Purification of rENP from the clarified broth resulted in a yield of 35% and a purity of >86%. Glycosylated rENP, the main contaminant, was removed with a concanavalin-A column and characterized. The pure rENP neutralized lipopolysaccharide and had the mass, amino-acid composition and N-terminal sequence expected from the cloned gene.

A new case of neuropeptide coexpression (RGamide and LWamides) in Hydra, found by whole-mount, two-color double-labeling in situ hybridization.

The freshwater polyp Hydra has a primitive nervous system that expresses at least six different neuropeptide genes: (1) three genes, coding for the preprohormones-A, -B, and -C that each gives rise to a variety of peptides with the C-terminal sequence Arg-Phe-NH(2) (the Hydra-RFamides); (2) one gene, coding for a preprohormone that gives rise to five peptides with the C-terminal sequence Leu-Trp-NH(2) (the Hydra-LWamides); (3) one gene, coding for a preprohormone that produces a peptide with the C-terminal sequence Lys-Val-NH(2) (Hydra-KVamide, also called Hym-176); and (4) one gene, coding for a preprohormone that gives rise to a peptide with the C-terminal sequence Arg-Gly NH(2) (Hydra-RGamide, also called Hym-355). In a previous paper, we described that a population of neurons in the peduncle (a region just above the foot) of Hydra coexpresses the preprohormone-A and KVamide genes, whereas neurons in the other regions only express either the preprohormone-A, -B, -C, LWamide, or the KVamide genes. Here, we investigated the RGamide gene expression, using whole-mount, two-color double-labeling in situ hybridization, and found that neurons in the basal disk (foot), gastric region, hypostome (a region around the mouth), and tentacles coexpress this gene together with the LWamide gene. A small population of neurons in the hypostome and upper gastric region expresses only the LWamide gene. No coexpression of the RGamide gene with any of the other neuropeptide genes was observed. This is the second example of coexpression of two neuropeptide genes in cnidarians. It demonstrates that many neurons in the primitive nervous systems of cnidarians use combinations ("cocktails") of neuropeptides for their signaling. It also shows that Hydra has at least seven neurochemically different populations of neurons.

Is crustacean hyperglycaemic hormone precursor-related peptide a circulating neurohormone in crabs?

Sites of synthesis and release patterns of crustacean hyperglycaemic hormone precursor-related peptide (CPRP) were investigated with those of crustacean hyperglycaemic hormone (cHH), in order to determine whether this precursor-related peptide satisfies certain criteria necessary for its definition as a secretable, circulating hormone. Using the edible crab, Cancer pagurus, sites of CPRP synthesis were determined by immunohistochemistry and release patterns of both peptides were determined in vivo and in vitro by radioimmunoassay of haemolymph and eyestalk superfusates. Both peptides were co-released from sinus glands (SGs) following potassium-evoked depolarization of isolated eyestalk preparations. However, stress-evoked in vivo release resulted in apparent non-stoichiometric circulating peptide profiles. This phenomenon is explained by notable differences in clearance rates of the peptides in haemolymph. In contrast to cHH, CPRP is very slowly degraded in vivo. Although CPRP is clearly a circulating peptide, whose release is concomitant with that of cHH, physiologically pertinent roles for this molecule remain to be discovered.

Expression of crustacean (Callinectes sapidus) molt-inhibiting hormone in insect cells using recombinant baculovirus.

Molt-inhibiting hormone (MIH) negatively regulates the synthesis of ecdysteroid molting hormones by crustacean Y-organs. We report here the expression of blue crab (Callinectes sapidus) MIH in insect cells using recombinant baculovirus. Insect Sf9 cells were transfected with recombinant baculovirus containing a DNA insert encoding the C. sapidus MIH prohormone (signal sequence plus mature hormone). The construct was designed to yield a mature, fully processed recombinant MIH (recMIH). Several baculovirus recombinants showing no contamination with wild-type viral DNA were subsequently analyzed for their ability to direct expression of recMIH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from infected cells revealed time-dependent expression of two proteins of approximately the predicted size for the C. sapidus MIH prohormone and mature hormone. Western blot results (using antiserum against MIH of Carcinus maenas) indicated that the proteins were MIH-immunoreactive. N-Terminal amino acid sequence data and mass spectral analysis indicated the expressed proteins were of the correct sequence and molecular mass. Cell lysates containing the recombinant protein dose-dependently suppressed the synthesis of ecdysteroids by Y-organs in vitro. We anticipate the recombinant peptide will prove useful for studies of the structure and function of MIH.

The shrimp FAMeT cDNA is encoded for a putative enzyme involved in the methylfarnesoate (MF) biosynthetic pathway and is temporally expressed in the eyestalk of different sexes.

Methylfarnesoate (MF), an analogue of the insect juvenile hormone III, has been implicated to play a vital role in the regulation of the growth and reproductive development in crustaceans. Farnesoic acid O-methyltransferase (FAMeT) is the key enzyme involved in catalyzing the final step in the MF biosynthetic pathway. In this study, we report the cloning and characterization of the cDNA encoding the putative FAMeT of the shrimp Metapenaeus ensis. FAMeT comprises 280 amino acid residues with a predicted molecular weight of 32kDa. The predicted putative FAMeT protein reveals a high degree of structural conservation of FAMeT with the lobsters. It shares 79 and 70% sequence identities with the putative FAMeTs of Homarus americanus and Panulirus interruptus, respectively. As revealed by the Southern blot analysis and genomic PCR, only one gene exists in the shrimp genome and the gene is uninterrupted in the coding region. The shrimp FAMeT mRNA is widely distributed in many tissues with the highest expression level observed in the central nervous system. A constant level of FAMeT expression is recorded in the ventral nerve cord of the juveniles and the mature females during the reproductive cycle. Unlike the ventral nerve cord, the eyestalk of the juvenile male, but not the female, expresses FAMeT. Further study shows that the eyestalk of the mature female expresses FAMeT during all stages of ovarian maturation. We speculate that FAMeT may be important for the regulation of eyestalk neuropeptides. This is the first extensive study on the molecular characterization, structural analysis and expression of the crustacean FAMeT.