Procambarus Bouvieri Research Articles

Computational analysis of molt-inhibiting hormone from selected crustaceans

Molt-inhibiting hormone (MIH) is a principal endocrine hormone regulating the growth in crustaceans. In total, nine MIH peptide sequences representing members of the family Penaeidae (Penaeus monodon, Litopenaeus vannamei, Marsupenaeus japonicus), Portunidae (Portunus trituberculatus, Charybdis japonica, Charybdis feriata), Cambaridae (Procambarus bouvieri), Parastacidae (Cherax quadricarinatus) and Varunidae (Eriocheir sinensis) were selected for our study. In order to develop a structure based phylogeny, predict functionally important regions and to define stability changes upon single site mutations, the 3D structure of MIH for the crustaceans were built by using homology modeling based on the known structure of MIH from M. japonicus (1J0T). Structure based phylogeny showed a close relationship between P. bouvieri and C. japonica. ConSurf server analysis showed that the residues Cys8, Arg15, Cys25, Asp27, Cys28, Asn30, Arg33, Cys41, Cys45, Phe51, and Cys54 may be functionally significant among the MIH of crustaceans. Single amino acid substitutions ‘Y’ and ‘G’ at the positions 71 and 72 of the MIH C-terminal region showed an alteration in the stability indicating that a change in this region may alter the function of MIH. In conclusion, we proposed a computational approach to analyze the structure, phylogeny and stability of MIH from crustaceans.

A neuropeptide family from the sinus gland of the Mexican crayfish, Procambarus bouvieri (Ortmann)

The main neuroendocrine axis of crustaceans is constituted by the brain, medulla terminalis X-organ (MTXO) -sinus gland (SG) system. A variety of hormonal peptides are synthesized in the MTXO where the peptidergic neuronal somata are situated. The peptides are then transported via axonic flow to the SG, which is a conglomerate of bulbous axonic terminals in close contact with the hemolymph, where these hormones are liberated to the circulation by a process of exocytosis. Four peptides have been isolated from crude aqueous extracts of microdissected SG of the Mexican crayfish, Procambarus bouvieri, by means of a single-step reverse-phase high pressure liquid chromatography (RP-HPLC) on a μBondapak-Phenyl column; these have been characterized physiologically by specific bioassays, and structurally by means of tryptic peptide mapping and sequencing. These procedures have identified (in the order of elution) a gonad-inhibiting hormone (GIH), a molt-inhibiting hormone (MIH), a major isomorph of the crustacean hyperglycaemic hormone (CHH-I) and its minor isomorph (CHH-II), in approximate concentrations of 5, 18, 60, and 20 ng per SG, respectively. Their common characteristics are: (1) acidic pl, (2) hydrophobicity, (3) molecular masses between 8300 and 8400 Da, (4) approximately 72 amino acid residues, (5) blocked N- and C-termini, (6) six cysteines forming three disulfide bridges, (7) lack of histidine, methionine, and tryptophan residues, (8) long tracts of sequence identity. These features clearly establish these four peptides as members of one neuropeptide family. Recently, a factor with gonad-stimulating activity has been isolated from this same crude extract by means of RP-HPLC. This factor stimulated by 300% the specific incorporation of radioactive leucine into yolk proteins in an in vitro bioassay consisting in the incubation of ovary fragments from the adult female shrimp, Penaeus vannamei, and immunoprecipitation of the proteins with a specific antibody to shrimp yolk protein. Thus, the SG extract from the crayfish, P. bouvieri, contains both a gonad-inhibiting hormone and a gonad-stimulating hormone (GSH), capable of acting on ovarian yolk protein synthesis in the shrimp, P. vannamei. The output of shrimp larvae spells the success of a shrimp hatchery. The induction of maturation of shrimp in captivity is done at present via eyestalk ablation of female broodstock, but this method causes increased mortality, disruption of the shrimp's endocrine system, and decreased larval viability with repeated spawnings. GSH could be used in aquaculture to increase the effect of eyestalk ablation or to substitute for this procedure completely.

Amino acid sequence of the minor isomorph of the crustacean hyperglycemic hormone (CHH-II) of the Mexican crayfish Procambarus bouvieri (Ortmann): presence of a D-amino acid.

The primary structure of the neurohormone crustacean hyperglycemic hormone (CHH-II) was determined by means of enzymatic digestions, manual Edman degradation, and mass spectrometry. CHH-II is a 72 residue peptide (molecular mass 8388 Da), with six cysteines forming three disulfide bridges that connect residues 7-43, 23-39, and 26-52. The peptide has blocked N- and C-termini, and lacks tryptophan, histidine, and methionine. The CHH-I and CHH-II of Procambarus bouvieri have identical sequences and elicit levels of hyperglycemia that are not distinguishable. The difference between the two isomorphs consists in a posttranslational modification of a L-Phe in CHH-I to a D-Phe in CHH-II at the third position from the N-terminus.

Primary structure of the major isomorph of the crustacean hyperglycemic hormone (CHH-I) from the sinus gland of the Mexican crayfish Procambarus bouvieri (Ortmann): Interspecies comparison

The amino acid sequence of this neuropeptide was elucidated by means of a combined approach of enzymatic digestions, manual and automatic Edman degradations, and mass spectrometry. It is a 72 residue peptide (molecular mass 8388 Da), with six cysteines forming three disulfide bridges connecting residues 7–43, 23–39, and 26–52, with blocked N- and C-termini, and lacking the amino acids histidine, methionine, and tryptophan. The CHH-I of Procambarus bouvieri is compared with the other known CHHs from Orconectes limosus (98.6% identity), Homarus americanus isomorph A (83.3% identity), Homarus americanus isomorph B (79.2% identity), and Carcinus maenas (61.1% identity).

Identification, purification and initial characterization of the vitellogenesis-inhibiting hormone from the Mexican crayfish Procambarus bouvieri (Ortmann)

1. 1. By means of a single step of RP-HPLC on μBondapak-Phenyl column, a neuropeptid with vutellogenesis-inhibiting hormone (VIH) activity was isolated from a crude extract of sinus glands (SG) of the crayfish P. bouvieri. The yield was 5.4 ng/SG. 2. 2. The biological activity was tested in a heterologous in vitro bioassay as the depression of vitellogenin biosynthesis by cultured Panaeus vannamei's ovaries. 3. 3. The molecular mass was determined tobe 8388 ± 2 Da by means of mass spectrometry. 4. 4. The N-terminus was shown to be blocked by means of the dansyl-Cl-method. 5. 5. The following amino acid average composition was obtained by means of precolumn derivatization with phenylisothiocyanate: (Asx) 13, (Glx) 8, (Val) 7, (Arg, Cys, Leu) 6, (Tyr) 5, (Ala, Ile) 4, (Lys) 3–4, (Phe) 3, (Thr) 2–3, (Ser, Gly) 23, (Pro) 1. VIH consists of 72–74 amino acid residues. It does not contain Trp, His, or Met. 6. 6. The tryptic peptides of the reduced and carboxymethylated VIH and of the crustacean hyperglycemic hormone (major isoform, CHH-I) were purified on an Ultrasphere-ODS column and the peptide maps compared with respect to retention times and composition. Forty-six percent of the total amount of amino acid residues were accounted for in 5 peptides which coincided in elution time and composition. 7. 7. The remarkable resemblance between VIH and CHH-I confirms clearly that VIH belongs to the neuropeptide hormone family which includes the molt-inhibiting hormone (MIH) and the two isoforms of the crustacean hyperglycemic hormone (CHH-I and CHH-II, formerly designated CHH-B and CHH-C by us).

Secondary structure of a crustacean neuropeptide hormone family by means of CD

Three hormonal neuropeptides have been purified from the sinus gland of the Mexican crayfish Procambarus bouvieri by means of a single-step HPLC method: The molt-inhibiting hormone (MIH) and two isoforms of the crustacean hyperglycemic hormone (CHH-B and CHH-C). Compositional analysis and partial characterization of the three neuropeptides revealed such a high degree of homology that we consider them to be members of a family. Circular dichroic spectra of the three neuropeptides showed that the secondary structure of both isoforms of the CHH are very similar, but that there are important differences in secondary structure between MIH and the CHHs, especially in helix content and in disordered regions.

A neuropeptide with molt-inhibiting hormone activity from the sinus gland of the mexican crayfish Procambarus bouvieri (Ortmann)

1. 1. By means of reverse-phase high-performance liquid chromatography (RP-HPLC) on a μBondapak-Phenyl column, a neuropeptide with molt-inhibiting hormone (MIH) activity was isolated from a crude extract of 2000 sinus glands from the crayfish Procambarus bouvieri (Ortmann) with a yield of 25 ng/SG. 2. 2. The activity was tested in a heterology in vitro assay as the depression of ecdysteroid biosynthesis by cultured Orconectes limosus' Y-organs. 3. 3. On SDS-PAGE, the peptide migrated as a single band of approximately 6000 Da, and on IEF, it migrated as a single band to a pI of 5.50. 4. 4. The N-terminus was shown to be blocked by means of the dansyl-Cl method, while the C-terminus was shown to be isoleucine by means of carboxypeptidase-Y digestion. 5. 5. The following amino acid average composition of MIH was obtained: (Asx) 7, (Val) 6, (Glx, Leu, Arg) 5, (Cys, Lys) 4, (Ala, Ile, Tyr, Phe) 3, (Ser) 2, (Thr, Gly) 1–2, (Pro) 1. MIH has 53–55 amino acid residues and its minimal molecular mass is calculated as 6243–6402 Da. It does not contain Trp, His nor Met. 6. 6. 37.7 μg (6.3 nmol) of the molt-inhibiting hormone (MIH) and 70.7 μg (11.8 nmol) of the major peak of the crustacean hyperglycemic hormone (CHH-B) were reduced, carboxymethylated and digested with trypsin. The tryptic peptides of each of the hormones were purified on an Ultrasphere-ODS column and the peptide maps compared with respect to retention times and the composition of each peak. 7. 7. 57.4% of the total amount of amino acid residues were accounted or in five peptides which coincided in elution time and composition. 8. 8. As we have found a clear-cut difference of only two residues between MIH and CHH-B (without taking into account the amides of aspartic and glutamic acids), the present results confirm that the two hormones belong to a family of neuropeptides comprising at least one molt-inhibiting hormone and two hyperglycemic hormones. 9. 9. Evidence is presented for the existence of microheterogeneity in the structure of each of these hormones.

Single-step purification of two hyperglycaemic neurohormones from the sinus gland of procambarus bouvieri: Comparative peptide mapping by means of high-performance liquid chromatography

A crude aqueous extract from 2000 sinus glands of the Mexican crayfish Procambarus bouvieri (Ortmann) was fractionated on a μBondapak-Phenyl column. Two isoforms of the crustacean hyperglycaemic hormone, designated CHH-B and CHH-C in order of elution, were isolated in pure form. Their biochemical characterization showed a remarkable degree of homology. A tryptic digest of each isohormone was fractionated on an Ultrasphere-ODS column. Only one tryptic peptide in CHH-C was eluted later than its homologous peptide in CHH-B. On acid hydrolysis both tryptic peptides had the same composition but, as they contain Asp and Glu, we suspect that the difference resides in a double reciprocal amidation-deamidation of two acidic residues.

A neurosecretory hyperglycemic hormone from the sinus gland of the mexican crayfish Procambarus bouvieri (ortmann)—II. Structural comparison of two isoforms of the hormone

1. 1. The hyperglycemic activity of a crude extract of Procambarus bouvieri (Ortmann) sinus glands was resolved into two UV-absorbing peaks by means of a single step of reverse-phase high performance liquid chromatography (RP-HPLC) on a μ-Bondapak-Phenyl column. These peaks have been designated Crustacean Hyperglycemic Hormones CHH-B and CHH-C in the order of elution. 2. 2. The ratio CHH-B/CHH-C was approximately 3:1, both in area under the curve and in protein content. 3. 3. A structural comparison of the two isoforms of the CHH showed a substantial homology manifested in molecular weight (6000–6200), pI (4.79), number of residues (52–53), number of cysteines (4), number of acid residues, including their amides (12), number of basic residues (8), missing amino acids (methionine, histidine and tryptophane), amino end (blocked) and carboxyl end (isoleucine). 4. 4. The only clear difference between the two isoforms of the CHH is their degree of hydrophobicity which might be due to minor differences in the number of neutral hydrophobic residues and/or postranslational modifications of the type amidation/deamidation of acid residues which cannot be detected in acid hydrolysates.

A neurosecretoru hyperglycemic hormone from the sinus gland of the mexican crayfish Procambarus bouvieri (Ortmann)—I. Purification and biochemical characterization of the most abundant form of the hormone

1. 1. The hyperglycemic activity of a crude extract (CE) from the sinus gland (SG) of Procambarus bouvieri (Ortmann) was recovered as a single asymmetric peak by means of reverse phase high performance liquid chromatography (RP-HPLC) on a Nova-Pak C 18 column. This peak was resolved into three symmetric peaks on rechromatography on a μBondapak-Phenyl column. Peaks were designated A, B, and C in the order of elution. Peak A had no hyperglycemic activity, while Peak B was as active as Peak C. 2. 2. Protein determination showed the following results (in ng/SG): CE—907.5, Peak A—14.5, Peak B—30.0 and Peak C—16.5. The protein content of Peaks A + B + C was 61 ng/SG, which is 6.7% of the CE protein. 3. 3. The bioassay was conducted on destalked Procambarus bouvieri. One unit of hyperglycemic activity was found to correspond to 9.4 ng of CE, 860 pg of B and 750 pg of C. 4. 4. Peak B, the most abundant form, was further characterized. On SDS-PAGE it migrated as a single band of approximately 6000 M.W. 5. 5. On I.E.F., peptide B migrated as a single band to a p I of 4.79. 6. 6. By means of the DNS-Cl method, it was shown that peptide B is N-terminally blocked. 7. 7. The carboxyl end of peptide B was found to be isoleucine by carboxypeptidase Y digestion and dansylation. 8. 8. The following amino acid average composition of peptide B was obtained from the acid hydrolysate of 2 nmoles for 20, 48 and 72 hr: (ASX) 7, (VAL) 6, (GLX) 5, (LEU, TYR, CYS, ARG) 4, (ALA, ILE, LYS) 3, (THR, SER, PRO, PHE) 2, (GLY) 1. 9. 9. Peptide B has 52 amino acid residues and its minimal M.W. was calculated as 6042 Da. It does not contain TRP, HIS or MET. 10. 10. While this work was in process, a simplified procedure utilizing only one RP-HPLC step on a μBondapak-Phenyl column was introduced. The same results as above were obtained.

Visual circadian rhythmicity in splitbrain crayfish: A plastic behavioral expression of symmetric circadian pacemakers

Electroretinographic evoked potentials (ERG's) were continuously recorded in dark-adapted, splitbrain crayfishes Procambarus bouvieri. Pulses of light (0.95 cd/m 2) illuminating the left or the right eyes were alternatively applied every 15 or 30 min. As compared to intact crayfish, uni or bilateral damping or suppression of circadian retinal sensitivity rhythm could be caused by surgical bisection of cerebral ganglion in these crustaceans. The damped ERG circadian rhythm was rapidly reversed by reduction or elimination of the test light stimulus to the contralateral eye. Given the redundant processing of pacemaking information coming from bilaterally positioned cephalic circadian pacemakers to the central nervous system in splitbrain crayfish, photodependent damping of ERG rhythm revealed a plastic potential of central circadian pacemakers. The possibility that a strong but reversible inhibitory influence acts simultaneously upon the left and right protocerebral circadian pacemakers while receiving bilateral photic stimulation is considered.

Ionic dependence of screening pigment migrations in crayfish retinal photoreceptors

The ionic dependence of the screening pigment migrations in crayfish (Procambarus bouvieri) retinula cells was studied by incubation of isolated eyes in solutions of different compositions. The pigment aggregation that normally occurs in the dark was inhibited by high potassium in a concentration dependent manner. Inhibition of aggregation was observed also with high external sodium or when sodium was replaced by lithium. Pigment aggregation is favored in the presence of high calcium, cobalt ions or in sucrose solution. The pigment dispersion induced by light is partly inhibited by lithium and in sucrose solution. Neither movement was affected by high magnesium, EDTA or EGTA. Agents expected to increase the intracellular activity of sodium or calcium, as ouabain, caffeine, ruthenium red and the ionophore A23187, all inhibited pigment aggregation. These observations assert the view that the crustacean retinula cells are independent pigmentary effectors, and agree with the notion of an engagement between membrane events and pigment position. It is suggested that sodium influx induced by light results in calcium release from internal stores, which in turn triggers dispersion of the pigment granules within the retinula cells.

Correlation between motor and electroretinographic circadian rhythms in the crayfish Procambarus bouvieri (ortmann)

1. 1. The characteristics of both, motor and electroretinographic circadian rhythms in the crayfish Procambarus bouvieri, were examined. 2. 2. The correlation between both rhythms in intact and brainless crayfish, was obtained. 3. 3. The presence of at least two different but coupled oscillators responsible for the circadian variations in crayfish, is proposed.

Isolation and purification of a neurodepressing hormone from the eyestalk of Procambarus bouvieri (Ortmann).

1. A neurodepressing hormone has been isolated and purified to homogeneity from aqueous extracts of 2000 eyestalks of the Mexican crayfish Procambarus bouvieri (Ortmann). 2. Purification was achieved by gel filtration on Sephadex G-25 and G-15, and preparative paper electrophoresis at four pH valueimately 1200 molecular weight and composed of neutral amino acids. 5. No N-terminal group could be found. From its electrophoretic behavior it is concluded that the C-terminal group is also blocked.

Functional characterization of the neurodepressing hormone in the crayfish.

The effect of the neurodepressing hormone (NDH) was studied on different identified motoneurons in the abdominal ganglia of the crayfish Procambarus bouvieri (Ortmann). Although differences in sensitivity were apparent, all the neurons tested responded to NDH with a reduction in spontaneous firing rate, which lasted as long as NDH was present, and, depending on the concentration and time of action of the hormone, for even longer periods. NDH activity was determined in the various parts of the central nervous system of the crayfish, being highest in the eyestalk, gradually diminishing away from the eyestalk, with a cephalo-caudal gradient, being lowest in the abdominal ganglia. High levels of NDH activity were detected in the blood. After eyestalk ablation, NDH concentration steadily diminishes in the blood and central nervous system, until virtually disappearing after 4 days; from day 5 onwards, the activity is recovered up to its original levels. NDH synthesis takes place with a time constant of approximately 3 hr in cultured isolated segments of central nervous system, being highest in the eyestalk.

Electrophysiological evidences of mutual modulatory influences on the retinal activity of the crayfish Procambarus bouvieri (O)

Electroretinographic evoked potentials (ERGs) were recorded in dark adapted crayfish by the application of pulses of light (0.09 Cd/ft 2) presented every 2.5 min. Heterolateral illumination (HI) for sixty min (0.06−0.3 Cd/ft 2) induced up to 50% decrease in ERG after a latency of 12–25 min. ERG depression was proportional to the intensity of HI and also showed a circadian rhythm. During the a phase the ERG recovery started 3–10 min after HI was turned off. In contrast it started only after 10–20 min during the ρ phase. The time course of the ERG depression, which was abolished in splitbrain animals, strongly suggests that a mutual modulatory influence, probably of neuroendocrine nature, is present in the crayfish visual system.

Neural coupling between left and right electroretinographic circadian oscillations in the crayfish p. bouvieri

1. 1. The effects of light pulse and surgical bisections of the cerebral ganglion upon the coupling of electroretinographic (ERG) circadian rhythm of left and right eyestalks were examined in the crayfish Procambarus bouvieri (Ortmann). 2. 2. The results indicate that the application of light for 60 min (0.15 cd/ft 2) at only one of the eyestalks induces a short-lasting bilateral decrease in the ERG voltage and a simultaneous phase shift in both left and right ERG rhythms in the following oscillations. 3. 3. The relatively long-lasting latency of ERG depression heterolaterally induced (15–25 min) as well as its recovery time (10–20 min) suggest that this contralateral modulation would be of neuroendocrine nature. 4. 4. Four patterns of ERG rhythms were recorded after cerebral ganglion bisection: (1) ERG damped oscillations, (2) bilateral or (3) unilateral ERG rhythms, and (4) lack of ERG oscillations. 5. 5. The above-mentioned changes suggest an important coupling mechanism between both eyestalks, with the result that ERG circadian pacemakers are mutually synchronized.

Habituation of mechanoreceptive interneurons in the crayfish.

The effect of repetition of sensory stimuli was studied on the responses of mechanoreceptive interneurons in the optic tract of the crayfish (Procambarus bouvieri (Ortmann)). The number of spikes recorded from a given unit gradually decreased during a train of stimuli. The decrease showed a negative exponential time course, with a curvature dependent upon the frequency of stimulation, the intensity of stimuli, and the hour of the day. Habituation is selective for the intensity and rate of stimulation, and the particular spot of receptive field stimulated. Locomotor excitation results in a dimminution of the rate of decay. The effect of a single train of stimuli when leading to pronounced habituation may persist for periods longer than 24 hr.

Persistent activity rhythms in the oyster.

Persistent activity rhythms in the oyster.