Mexican Crayfish Research Articles

ザリガニ類(ウチダザリガニ,メキシコザリガニ,マロン,レッドクロー)の移入に関する考察

ザリガニ類(ウチダザリガニ,メキシコザリガニ,マロン,レッドクロー)の移入に関する考察

Risk assessment for the Mexican freshwater crayfish: the roles of diversity, endemism and conservation status

Abstract The freshwater fauna has been judged to be one of the most threatened biotic components in the world. In many tropical‐temperate freshwater habitats worldwide the largest invertebrates are crayfish, as is the case with the cambarid crayfish in Mexico. With 98% of endemic species, most of them with reduced distribution ranges, the Mexican crayfish have not been analysed to examine how diversity, endemism and threat are distributed. A data set was analysed containing 1419 locality records for the 56 species of crayfish occurring in Mexico arranged in a 251 cell grid. Spatial autocorrelation analyses using Moran's I and G* were conducted; species richness, endemism and threat indices were calculated and mapped. An integrated risk score was derived from the two indices. Spatial autocorrelation analyses revealed a pattern that significantly departs from a random arrangement. Moran's I showed a positive autocorrelation between cells that are less than 800 km apart; while G* analysis identified one hotspot of diversity. Four areas with high endemism and seven areas with intermediate endemism values were recognized. The western portion of the Trans‐Mexican Volcanic Belt, and some areas in the north are of special concern owing to the presence of threatened microendemic species. The areas where more threatened species occur differ from those with high endemism values. This distinction makes evident that for species with low dispersal capabilities the areas with high endemism are the product of historical and geological events, while the areas with high numbers of threatened species are those where human activities have had a major impact. The integrated risk score, however, resulting from the combination of endemism and threat, peaks along the Trans‐Mexican Volcanic Belt and where it joins the Sierra Madre Oriental. The integrated risk score proposed in this study, based on well known and frequently used indices in conservation biology, can be used with existing data to determine areas where crayfish species richness, endemism and threat peak to make conservation efforts more cost‐effective. © 2016 The Authors. Aquatic Conservation: Marine and Freshwater Ecosystems published by John Wiley & Sons, Ltd.

Mitotic karyotype of the tropical freshwater crayfish Procambarus (Austrocambarus) llamasi (Decapoda: Cambaridae)

In Mexico, the biology of Procambarus has been more studied than the biology of other Cambarids because of its diversity and potential use in aquaculture. We determined the karyotype of the Mexican tropical freshwater crayfish Procambarus (Austrocambarus) llamasi from 189 metaphase spreads from gill tissues of 17 adults. They had 98-120 chromosomes (mode 2n=120 chromosomes). There are 60 pairs of monoarm, telocentric chromosomes. Sex chromosomes were not detected and we propose that the P. llamasi karyotype can be used to distinguish this species from other Mexican crayfish. Additionally, we suggest using karyological data in aquaculture and conservation biology.

Hyperglycaemic effects of dopamine in tiger shrimp, Penaeus monodon

The presence and the physiological functions of biogenic amines as neuroregulators mediating the release of neurohormones have been documented in crustaceans. The possible involvement and the stimulatory pathway of dopamine in hyperglycaemia of the tiger shrimp, Penaeus monodon, were investigated. Two isoforms of crustacean hyperglycaemic hormone (CHH), CHH1 and CHH2, were identified. They were equally potent in the diabetic effect, and showed a great similarity in their amino acid composition profile, which is however, distinct from those reported from other crustacean species, such as the shore crab, Mexican crayfish and American lobster. Thus, species variation in the amino acid composition of CHH exists among the crustaceans. Dopamine was found to mimic the action of CHH in inducing hyperglycaemia in intact shrimps, but not in bilaterally eyestalk-ablated individuals. The hyperglycaemic response of shrimps that were treated with the agonist and antagonist to D 1 and D 2 receptors, suggest that dopamine functions as a neuroregulator, which in turn stimulates the release of CHH mainly through D 1 receptors in tiger shrimps.

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 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.

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.