Differential expression of CMG peptide and crustacean hyperglycemic hormones (CHHs) in the eyestalk of the giant tiger prawn Penaeus monodon

Mouse antiserum against C-terminal amide of Pem-CMG (a peptide in the family of CHH/MIH/GIH) penta-deca peptide (RPRQRNQYRAALQRLamide=CMG-15) was generated and used for localization of the peptide in tissue and extract of the eyestalk of Penaeus monodon by means of immunohistochemistry and dot-ELISA in comparison with anti-T+ antiserum (T+=YANAVQTVamide : the putative C-terminal amide of crustacean hyperglycemic hormone (CHH) of Macrobrachium rosenbergii). The anti-CMG-15 antiserum did not show cross-reactivity to T+ peptide by dot-ELISA and vice versa for anti-T+ antiserum. In dot-ELISA of eyestalk extract of P. monodon after one step separation by RP-HPLC, anti-CMG-15 antiserum recognized different peptide fractions (F38-39) from those recognized by anti-T+ antiserum (F19, 40-41 and 47-51). Most of the T+ immunoreactive fractions (except F19) show higher hyperglycemic activity than the CMG immunoreactive fractions. In immunohistochemical localization, anti-CMG antiserum recognized only 2-3 neurons in medulla terminalis X-organ complex (MTXO) with long processes terminated in the sinus gland. The CMG-immunoreactive neurons were clearly distinct from CHH containing neurons situated in the same area. This evidence confirms the existing of CMG peptide which may play distinct roles from CHHs in hormonal regulation in P. monodon.     

Identification and characterization of a hyperglycemic hormone from freshwater giant prawn, Macrobrachium rosenbergii

Crustacean hyperglycemic hormone (CHH), a physiologically important neurohormone stored in the sinus gland of eyestalks, primarily regulates carbohydrate metabolism and also plays significant roles in reproduction, molting and other physiological processes. In the freshwater giant prawn, Macrobrachium rosenbergii, an injection of X-organ sinus gland (XOSG) extract evoked a hyperglycemic response, peaked in 1 h. The hyperglycemic effect of the eyestalk extract was maximal at the dose of 0.5 eyestalk equivalent. CHH fractionated by RP-HPLC, in M. rosenbergii was identified by its hyperglycemic activity and partial amino acid sequence, and the molecular weight of 8534 was determined by matrix-assisted laser desorption ionization mass spectrometry--time of flight analysis (MALDI-TOF). The amino acid sequence of the first 25 residues of CHH showed 72% homology with the first 25 residues of CHH A and CHH B of the American lobster Homarus americanus.     

Three more novel FMRFamide-like neuropeptide sequences from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii

In addition to five FMRFamide-like peptides (FLPs) previously isolated from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii (16), three more new FLPs (Mar-FLP6-8) were identified from minor immunoreactive fractions of 5,000 eyestalk extracted in methanol/acetic acid/water: DGGRNFLRFamide, GYGDRNFLRFamide and VSHNNFLRFamide. These three peptides share 5-6 common residues at the C-terminus with Mar-FLP1,2 and 3. This evidence reveals that the structural diversity and complexity of the FLP family in M. rosenbergii are similar to that found in other invertebrate species.     

Purification and characterization of an isoform of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii.

Isolation of the crustacean hyperglycemic hormone (CHH) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii was performed using 5,000 ground eye-stalks extracted in methanol-acetic acid-water (90:1:9). After the extract was partially purified using C18 cartridges, it was further purified by eight steps of RP-HPLC using four kinds of columns: C18, C8, cyano and phenyl, and three solvent systems: acetonitrile (ACN)/trifluoroacetic acid, ACN/heptafluoroacetic acid and ACN/triethylammonium acetate. The bioassay of CHH during purification was done by injection of eluate fractions into eyestalk-ablated prawns and determination of the ability of the fractions to elevate glucose in the haemolymph. A complete amino acid sequence analysis was performed on one isoform of CHH (Mar-CHH-1), consisting of 71 residues. The sequence of Mar-CHH-1 shows considerable similarity (45-68%) to CHHs reported in other crustaceans. It is expected that there might be more than one isoform of CHH in M. rosenbergii.     

Hepatopancreas is the likely organ of vitellogenin synthesis in the freshwater prawn, Macrobrachium rosenbergii

The objective of the present study was to investigate the source of vitellogenin in the freshwater prawn, Macrobrachium rosenbergii. Ovarian development of M. rosenbergii was classified into five stages (stage I-V). Vitellin/vitellogenin was detected in the ovary and the hepatopancreas in different stages by native-PAGE and Western blotting. Two and three subunits of vitellin were observed in the ovary at the early- (I-II), mid- and late- (III-V) stages, respectively. The subunit of vitellogenin was not detected in the hepatopancreas at different stages of prawns. Hepatopancreas had positive immunocytological staining (against vitellin antibody) in different ovarian stages of prawn. Only vitellogenic oocyte but not previtellogenic oocytes and follicle cells had a positive immunocytological staining. Hepatopancreas could synthesize radiolabeled immunoreactive proteins after incubation with radiolabeled glycine on the basis of immunoprecipitation (against vitellin antiserum). Therefore, it is concluded that hepatopancreas is the most likely organ to synthesize vitellogenin in the freshwater prawn, M. rosenbergii.     

Immunohistochemical Identification of Hyperglycemic Hormone- and Molt-Inhibiting Hormone-Producing Cells in the Eyestalk of the Kuruma Prawn, Penaeus japonicus

This study deals with the localization of crustacean hyperglycemic hormone (CHH, Pej-SGPIII) and molt-inhibiting hormone (MIH, Pej-SGP-IV) in the eyestalk of the kuruma prawn Penaeus japonicus using immunohistochemistry. High-titer and highly specific antisera were raised in rabbits against synthetic Pej-SGP-III C-terminal peptide (Glu-Glu-His-Met-Ala-Ala-Met-Gln-Thr-Val-NH2) and Pej-SGP-IV C-terminal peptide (Val-Trp-Ile-Ser-Ile-Leu-Asn-Ala-Gly-Gln-OH), both of which were conjugated with bovine serum albumin by a cross linker. Eyestalks were removed from mature male prawns at the intermolt stage of the molting cycle and fixed in Bouin's solution. Serial sections stained immunohistochemically showed that neurosecretory cells of Pej-SGP-III and Pej-SGP-IV were located in the same cluster of the medulla terminalis ganglionic X-organ (MTGX), and that three kinds of neurosecretory cells, which were stained with anti-PejSGP-III antiserum and/or anti-Pej-SGP-IV antiserum were present. The number of neurosecretory cells which stained with both antisera was much fewer than that of neurosecretory cells which stained with one of the antisera only. The axon and axon terminals in the sinus gland were also stained and the staining density of the sinus gland was always deeper than that of the neurosecretory cells.     

Preparation of two recombinant crustacean hyperglycemic hormones from the giant freshwater prawn, Macrobrachium rosenbergii, and their hyperglycemic activities

Crustacean hyperglycemic hormone (CHH) is released from the X-organ/sinus gland complex located in the eyestalks, and regulates glucose levels in the hemolymph. In the giant freshwater prawn (Macrobrachium rosenbergii), two cDNAs encoding different CHH molecules were previously cloned by other workers. One of these (Mar-CHH-2) was expressed only in the eyestalks, whereas the other (Mar-CHH-L) was expressed in the heart, gills, antennal gland, and thoracic ganglion, but not in the eyestalks. However, their biological activities had not yet been characterized. Therefore, in this study, recombinant Mar-CHH-2 (rMar-CHH-2) and Mar-CHH-L (rMar-CHH-L) were produced using an E. coli expression system, by expression in bacterial cells and recovery in the insoluble fraction. Thereafter, rMar-CHH-2 and rMar-CHH-L were subjected to refolding and were subsequently purified by reversed-phase HPLC. The rMar-CHH-2 and rMar-CHH-L thus obtained exhibited the same disulfide bond arrangements as those of other CHHs reported previously, indicative of natural conformation. In in vivo bioassay, rMar-CHH-2 showed significant hyperglycemic activity, whereas rMar-CHH-L had no effect. These results indicate that Mar-CHH-L does not function as a CHH, but may have some other, unknown function.     

Four novel PYFs: members of NPY/PP peptide superfamily from the eyestalk of the giant tiger prawn Penaeus monodon

An immunocytochemical method was used for localization of pancreatic polypeptide (PP) immunoreactive substances in the eyestalk of Penaeus monodon using anti-C-terminal hexapeptide of PP (anti-PP6) antiserum. Approximately 200 neuronal cell bodies were recognized in the ganglia between the medulla interna (MI) and medulla terminalis (MT) and surrounding MT in conjunction with the neuronal processes in medulla externa (ME), MI, MT and sinus gland. About half of the PP immunoreactive neurons were also recognized by a combination of three monoclonal antibodies raised against FMRFamide-like peptides. Isolation of the PP immunoreactive substances from the eyestalk was performed using 7500 eyestalks extracted in methanol/acetic acid/water (90/1/9) followed by five to six steps of RP-HPLC separation. Dot-ELISA with anti-PP6 antiserum was used to monitor PP-like substances in various fractions during the purification processes. Four new sequences of one hexapeptide; RARPRFamide, and three nonapeptides; YSQVSRPRFamide, YAIAGRPRFamide and YSLRARPRFamide were identified, and named as Pem-PYF1-4 due to their structural similarity to the PYF found in squid Loligo vulgaris. Each of the new peptides shares four to seven common residues with the C-terminus of the squid PYF and with the NPFs found in other invertebrates. The NPY/PP superfamily as well as the FMRFamide peptide family may be present throughout vertebrates and invertebrates.     

A comparative immunocytochemical investigation of the crustacean hyperglycemic hormone (CHH) in the eyestalks of some decapod crustacea

The eyestalk of the astacideans Orconects limosus, Nephrops norvegicus, and Homarus gammarus, and the palinuran Palinurus vulgaris, was examined with an antiserum raised against purified crustacean hyperglycemic hormone (CHH) of the astacidean species Astacus leptodactylus. A distinct immunopositive reaction occurs in a group of neurosecretory cells in the medulla terminalis ganglionic X-organ (MTGX), in the MTGX-sinus gland tractus, and in a considerable part of the sinus gland. The immunoreactive sites in the eyestalk of the investigated species correspond to the site of production, storage, and release of the CHH. Preliminary investigations with this antiserum also indicate that a positive immunoreaction can be obtained in the eyestalk of other decapod crustaceans, for example, of the brachyuran Macropipus puber and the caridean Palaemon serratus.     

Dopamine depresses the immune ability and increases susceptibility to Lactococcus garvieae in the freshwater giant prawn, Macrobrachium rosenbergii

The total haemocyte count (THC), phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase activity, and phagocytic activity and clearance efficiency to the pathogen Lactococcus garvieae were measured when freshwater giant prawns Macrobrachium rosenbergii (16.2 +/- 2.1 g) were individually injected with saline, or dopamine at 0.5, 5.0, or 50.0 pmol prawn(-1). The results show that a transient period of immunosuppression occurred between 2 and 8 h after injection of dopamine for all immune parameters except circulating haemocytes and all immune parameters returned to control values within 8-16 h after receiving dopamine. Injection of dopamine also significantly increased the mortality of M. rosenbergii challenged with the pathogen L. garvieae. These results suggest that stress-inducing dopamine suppresses the immune system, which in turn promotes the susceptibility to L. garvieae in M. rosenbergii.     

Five Crustacean Hyperglycemic Family Hormones of Penaeus monodon: Complementary DNA Sequence and Identification in Single Sinus Glands by Electrospray Ionization-Fourier Transform Mass Spectrometry

Five novel neuropeptides, designated Pm-sgp-I to -V, of the crustacean hyperglycemic hormone (CHH) family have been identified from the giant tiger prawn Penaeus monodon by isolation of the preprohormone genes from an eyestalk complementary DNA library. On the basis of sequence similarity, the encoded peptides have been classified as CHH-like type I hormones, which include all known CHHs and the molt-inhibiting hormone (MIH) of the lobster Homarus americanus. Consistent with CHH type I preprohormones, the Pm-sgp precursors include a signal peptide, a CHH precursor-related peptide (CPRP), and the CHH-like hormone. Analysis by electrospray ionization-Fourier transform mass spectrometry enabled the neuropeptide complement of individual sinus glands to be resolved. It also confirmed the presence of the five Pm-sgp neuropeptides within the sinus gland of an individual animal, in that the masses observed were consistent with those predicted from the gene sequence of the Pm-sgps after posttranslational modification. These modifications included cleavage of the signal peptide and precursor protein, carboxy-terminal amidation, and formation of three disulfide bridges. Analysis of crude extracts of single sinus glands from different animals revealed variation in neuropeptide content and will provide a tool for determining whether the content varies as a function of the physiological state of the animal. Received March 26, 1999; accepted September 10, 1999.     

罗氏沼虾胸神经节中促肌肉蛋白质合成激素的初步分离

罗氏沼虾(Macrobrachium rosenbergii)成虾的胸神经节匀浆液,经Sephadex G-50柱层析所得的第2个峰,再经HPLC分离得到9个组分。把第2个组分的溶液注入罗氏沼虾幼虾体内,其肌肉蛋白质合成明显增加(P<0.01),这种促进蛋白质合成的能力随该溶液的蛋白质浓度的增加而增强。结果表明罗氏沼虾胸神经节能分泌促进虾肌肉生长的激素。该激素的分子量在3.4?kDa以下,可能是虾的生长激素。     

Effect of potassium permanganate stress on immune resistance and susceptibility to Lactococcus garvieae in the giant freshwater prawn Macrobrachium rosenbergii

This work is part of a continuing series of investigations on the effect of commonly used aquaculture chemicals on the immune resistance and susceptibility of the giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae. The methodology has been described in earlier publications of the series. Potassium permanganate at 1.0 mg l(-1) in tryptic soy broth (TSB) had no effect on the growth rate of L. garvieae. The mortality of M. rosenbergii challenged with 4 x 10(6) colony-forming units (cfu) prawn(-1) of TSB-grown L. garvieae was significantly greater than that of challenged controls. Addition of potassium permanganate at 1.0 mg l(-1) in TSB significantly increased the virulence of L. garvieae to M. rosenbergii. Exposure of M. rosenbergii to potassium permanganate prior to challenge with TSB-grown L. garvieae at 4 x 10(6) and 3 x 10(6) cfu prawn(-1) revealed that 96 h mortality was significantly lower for prawns held in water containing 0.3 mg l(-1) of the chemical than for prawns in water containing 1.0 mg l(-1) or no chemical. Potassium permanganate caused no significant changes in total hemocyte counts and differential hemocyte counts, compared to the control treatments. However, a concentration of 1.0 mg l(-1) or more for 96 h resulted in decreased phenoloxidase activity, phagocytic activity and clearance efficiency. Respiratory burst increased with exposure to 0.3 mg l(-1). In conclusion, treatment with potassium permanganate at 0.3 mg 1(-1) was effective in reducing M. rosenbergii mortality from L. garvieae infection, but higher concentrations had a negative effect, probably due to reduced prawn defenses.     

Cloning and characterization of a molt-inhibiting hormone-like peptide from the prawn Marsupenaeus japonicus

Recently, it was demonstrated by PCR amplification that an additional molt-inhibiting hormone (MIH)-like peptide was present in the kuruma prawn Marsupenaeus japonicus. In this study, a cDNA encoding this peptide designated Pej-MIH-B was cloned. The Pej-MIH-B gene was expressed strongly in the nerve cord, and weakly in the eyestalk. It was possible to isolate Pej-MIH-B from the sinus glands in the eyestalks. The recombinant Pej-MIH-B expressed in Escherichia coli showed low molt-inhibiting activity, but did not exhibit hyperglycemic activity. These results suggest that Pej-MIH-B does not function as MIH or CHH intrinsically, but may have some unknown functions.     

Secretion of Pem-CMG,a peptide in the CHH/MIH/GIH family of Penaeus monodon,in Pichia pastoris is directed by secretion signal of the alpha-mating factor from Saccharomyces cerevisiae

The CHH/MIH/GIH peptide family of black tiger prawn (Paneaus monodon) is important in shrimp reproduction and growth enhancement. In this study, the cDNA that encodes the complete peptide that is related to the CHH/MIH/GIH family (so-called, Pem-CMG) in the eyestalk of P. monodon was successfully expressed in a methylotrophic yeast Pichia pastoris under the control of an alcohol oxidase promoter. In order to obtain the secreted Pem-CMG, a secretion signal of either the Saccharomyces cerevisiae alpha-factor or Pem-CMG was employed. The results demonstrated that alphaPem-CMG, either with (alpha2EACMG) or without (alphaCMG) the Glu-Ala repeats, was secreted into the medium, while Pem-CMG with its own secretion signal failed to be secreted. The total protein amount that was secreted from the transformant that contained either alpha2EACMG or alphaMG was approximately 60 mg/l and 150 mg/l, respectively. The N-terminus of the Pem-CMG peptide of both alpha2EACMG and alphaCMG was correctly processed. This produced the mature Pem-CMG peptide.