Biosynthesis and metabolism of histamine in the central nervous system of Carcinus maenas

The central nervous system of Carcinus maenas synthesizes radioactive histamine when incubated in the presence of [14C] histidine and pyridoxal-5' phosphate. This biosynthesis increases linearly as a function of the amount of enzyme and the incubation time. It is not effected by heart, muscle or hepatopancreas extracts nor by haemolymph. Thus histamine appears to be synthesized mainly in the nervous system. The latter is also the seat of carcinine (beta-alanylhistamine) biosynthesis. Since carcinine seems to be a product of histamine neutralization, histamine metabolism should take place in its entirety in the nervous system. Thus histamine appears to be implicated in the neuronal activity of Carcinus. Different areas of the crustacean central nervous system: brain, eyestalks and thoracic ganglionic mass biosynthesize and metabolize histamine. Thus they all could contain sites of action for histamine. The nervous systems of two other Decapodes, Cancer and Astacus also effect histamine biosynthesis but don't metabolize it into carcinine.     

Biosynthesis of carcinine (beta-alanyl-histamine) in vivo

Carcinine was biosynthesized by Carcinus maenas from [14C]beta-alanine, [14C] histidine and [14C] histamine. Since carnosine (beta-alanyl-histidine) could not be detected in crab tissues, biosynthesis of carcinine could only be effected by direct coupling of beta-alanine and histamine resulting from histidine decarboxylation. Biosynthesis of carcinine was weak when [14C]beta-alanine and [14C] histidine were used as precursors. On the contrary when [14C] histamine was used, synthesis was important. Thus carcinine appears to be a product of histamine catabolism. After injecting [14C] histamine, radioactive carcinine was concentrated mainly in the heart and nervous system; nonmetabolized [14C] histamine was recovered mainly in the latter. The nervous system might therefore be the seat of carcinine biosynthesis and thus the site of action of histamine.     

Mise en Evidence de la Carcinine Synthétase, une Nouvelle Enzyme Catalysant le Métabolisme de l''Histamine dans le Systeme Nerveux Central de Carcinus maenas

Carcinine biosynthesis was induced in vitro from its two components, beta-alanine and histamine. The reaction was catalyzed by muscle, heart, and CNS extracts from Carcinus maenas. The specific activity of the enzyme, carcinine synthetase, was 15 times higher in CNS than in other organs. Only CNS extracts induced biosynthesis of carcinine from histidine, and only in the presence of pyridoxal-5'-phosphate. Hence the seat of carcinine biosynthesis seems to be the CNS. It is highly probable that in the CNS, histidine is transformed into histamine, which is then catabolized into carcinine. The latter would then be transported and accumulated in the cardiac tissue. Thus histamine--the metabolism of which takes place totally within the CNS--would be implicated as a participant in the neuronal activity of Carcinus maenas. Carcinine synthetase is a soluble enzyme that requires the presence of ATP, beta-alanine, and histamine. Mg2+ and dithiothreitol are also essential for activity. Optimum pH is approximately 7.6. Carcinine synthetase differs from carnosine synthetase and gamma-glutamylhistamine synthetase in that it does not catalyze synthesis of beta-alanylhistidine or gamma-glutamylhistamine.     

γ-GLUTAMYLHISTAMINE: A MAJOR PRODUCT OF HISTAMINE METABOLISM IN GANGLIA OF THE MARINE MOLLUSK, APLYSIA CALIFORNICA

The metabolism of histamine in the nervous system of Aplysia, was studied by incubating ganglia for various time periods in a medium containing radiolabelled histamine. After 4 h of incubation, >89% of the ^3MH- or ¹⁴C-label measured in the ganglion extract was converted to a single radioactive product whose electrophoretic and chromatographic mobilities were identical with γ-glutamylhistamine. No evidence was found to indicate that radiolabelled histamine could be deaminated, N-methylated, N-acetylated, or oxidized in this nervous system. Acid hydrolysis of radioactive material prepared from ganglia incubated in either ¹⁴C-histamine alone or in combination with ¹⁴C-glutamate resulted in the liberation of labelled histamine and/or labelled glutamate. These results indicate that γ-glutamyl-ation of histamine may be an important physiological mechanism for histamine catabolism in molluscan nervous tissue.     

Histopathological effects of trypanorhynch metacestodes in the digestive gland of a novel host, Carcinus maenas (Decapoda)

The green crab Carcinus maenas was introduced to Australian temperate waters in the late 1800s, has since become established, and is now considered to be a pest. We undertook an extensive parasite survey to find potential natural enemies of C. maenas and found it to be infected in Australia by 2 species of larval trypanorhynch tapeworm, Trimacracanthus aetobatidis and Dollfusiella martini. We describe the gross pathology and histopathology of the parasites' new host (C. maenas) and note that the plerocercoid larvae are located in the lumen of the digestive gland tubules. The presence of D. martini in C. maenas with low population numbers suggests that either D. martini has an impact (direct or indirect) on the survival of C. maenas, or that the parasite may be an indicator of high predation pressure. If the former were true, this would contribute to the control of this introduced pest species.     

Distribution of a novel cardioactive neuropeptide (CCAP) in the nervous system of the shore crab Carcinus maenas

A radioimmunoassay (RIA) for the recently discovered crustacean cardioactive peptide (CCAP) has been developed and used to determine contents of CCAP in different parts of the nervous system of the shore crab Carcinus maenas. Immunoreactive material was detected throughout the nervous system. In contrast to the main ganglia which contained low levels of approximately 1.4 pmol CCAP/mg protein (brain and thoracic ganglion), a high concentration was found in a neurohemal structure, the pericardial organs (PO) (868 pmol/mg protein). A predominantly neurohormonal role of CCAP thus suggested is further supported by in vitro release studies. Incubation of POs in high (K+) saline showed that CCAP is secretable in considerable amounts by a Ca++-dependent release mechanism.     

Amino acid sequence of crustacean hyperglycemic hormone (CHH) from the crayfish, Orconectes limosus: emergence of a novel neuropeptide family.

The primary structure of the major form of CHH from sinus glands of the crayfish, Orconectes limosus, was determined by manual Edman microsequencing. It is a 72-residue peptide with a calculated Mr of 8400 Da. In the number of residues, it is identical to the CHH of Carcinus maenas and very similar to MIH (moult inhibiting hormone) of Homarus americanus. All three peptides have pGlu as N-terminus in common, and Val-NH2 is the C-terminal residue in Orconectes and Carcinus CHH. Six Cys residues occupy identical position in the three peptides. There is a 61% sequence identity with Carcinus CHH, and an 81% identity with Homarus MIH.     

Critical salinity, sensitivity, and commitment of salinity-mediated carbonic anhydrase induction in the gills of two euryhaline species of decapod crustaceans

Two euryhaline species of decapod crustaceans, Carcinus maenas and Callinectes sapidus, were subjected to a series of acute low-salinity challenges, and changes in carbonic anhydrase (CA) activity in the gills were monitored in order to characterize the nature of salinity-sensitive CA induction. CA activity is uniformly low in all gills of both species at high salinity, but at a critical salinity of 27 ppt, CA induction occurs in the posterior, ion-transporting gills, with CA activity approximately doubling. This salinity occurs right at, or slightly above, the point at which these species make the transition from osmoconformity to osmoregulation. The regulatory mechanism that controls the levels of CA expression after the initial induction has occurred is also very sensitive. Changes in CA activity occur in response to changes in salinity as small as 20 milliosmoles. CA induction only occurs after a critical minimum amount of time of exposure to low salinity (48-72 hr in C. maenas and 12 hr in C. sapidus), but once induction is begun, it continues regardless of subsequent salinity changes. The timing is most likely due to the time it takes for changes in gene expression and resultant increases in CA mRNA to occur in response to low-salinity exposure, and the delay in CA induction could be an adaptation to avoid making metabolically expensive responses to potentially short-term environmental changes.     

The distribution and orientation of epizoic barnacles on crabs

The distribution and orientation of Balanus crenatus on Carcinus maenas is described. Elminius modestus was also found on C. maenas but it was not common. Both species of barnacle were also found on Cancer pagurus, although neither species of barnacle appeared to be as common on Cancer as on Carcinus. Possible reasons for this are discussed.
Treating the carapaces of similar sized C. maenas as sampling units it is clear that the distribution of B. crenatus over these sampling units is not random but is aggregated. This probably arises because of the gregarious nature of the cyprids when they settle out but other possibilities are considered. B. crenatus on the carapace of C. maenas is found exclusively in the grooves and depressions on the carapace and this is because the cyprids preferentially settle in concavities.
The orientation of B. crenatus also shows a consistent pattern, with the cirral nets facing predominantly backwards. This pattern could arise because the barnacle cyprids orientate to water currents, generated by the exhalant respiratory currents, flowing forwards over the carapace.     

Structure, distribution, and biological activity of novel members of the allatostatin family in the crayfish Orconectes limosus.

In the central and peripheral nervous system of the crayfish, Orconectes limosus, neuropeptides immunoreactive to an antiserum against allatostatin I (= Dipstatin 7) of the cockroach Diploptera punctata have been detected by immunocytochemistry and a sensitive enzyme immunoassay. Abundant immunoreactivity occurs throughout the central nervous system in distinct interneurons and neurosecretory cells. The latter have terminals in well-known neurohemal organs, such as the sinus gland, the pericardial organs, and the perineural sheath of the ventral nerve cord. Nervous tissue extracts were separated by reverse-phase high-performance liquid chromatography and fractions were monitored in the enzyme immunoassay. Three of several immunopositive fractions have been purified and identified by mass spectroscopy and microsequencing as AGPYAFGL-NH2, SAGPYAFGL-NH2, and PRVYGFGL-NH2. The first peptide is identical to carcinustatin 8 previously identified in the crab Carcinus maenas. The others are novel and are designated orcostatin I and orcostatin II, respectively. All three peptides exert dramatic inhibitory effects on contractions of the crayfish hindgut. Carcinustatin 8 also inhibits induced contractions of the cockroach hindgut. Furthermore, this peptide reduces the cycle frequency of the pyloric rhythms generated by the stomatogastric nervous system of two decapod species in vitro. These crayfish allatostatin-like peptides are the first native crustacean peptides with demonstrated inhibitory actions on hindgut muscles and the pyloric rhythm of the stomatogastric ganglion.     

Biodistribution of N-isopropyl-p-iodoamphetamine in mice

Biodistribution of N-isopropyl-p-iodoamphetamine (IMP) was evaluated in mice. After synthesis of IMP and exchange labeling with radioactive iodine, IMP was injected intravenously in male ddY mice. Activity in the brain reached 8.0 (%dose/g), at 10 min after injection and it was almost constant till 120 min. Activities in the lung and heart were high just after injection, decreased rapidly and was almost constant from 30 to 120 min. Activity in the liver increased slowly compared with other organs, and reached peak level at 60 min. In autoradiography, almost the same activity in the spinal cord as the brain was observed. Uptake and excretion of IMP were shown in kidneys and stomach, which may be the main route of the excretion of IMP and their metabolites. We concluded IMP showed good uptake in the central nervous system.     

Modulatory effects of proctolin on a crab ventilatory muscle

Proctolin enhances nerve-evoked, phasic contractions of a selected respiratory muscle of the shore crab, Carcinus maenas, but has no effect on muscle tonus. Proctolin also increases the work and power output of this muscle. These effects are functionally appropriate in view of previous reports that proctolin stimulates the ventilatory rhythm. They also suggest that proctolin exerts coordinated modulatory control at the central and peripheral levels of the gill ventilatory system. In contrast, serotonin, dopamine and octopamine have no effect on this muscle.     

Calcium-induced calcium release mechanism from the sarcoplasmic reticulum in skinned crab muscle fibres

Mechanically skinned skeletal muscle fibres of the crab Carcinus maenas have been used to investigate the mechanism of calcium release from the sarcoplasmic reticulum. Calcium release has been monitored by the amplitude and kinetics of the tension developed by the fibre. Results show that a very low calcium concentration, insufficient to directly activate contractile proteins, induces a release of calcium from the SR. This release is stimulated by low concentrations of caffeine and inhibited by small amounts of EGTA. Thus, a graded calcium-induced calcium release mechanism dependent on extrareticular calcium concentration has been demonstrated in skinned crab muscle fibre.     

Ultrastructure of the gills of Carcinus maenas

In the present paper it is reported the ultrastructure of the gills from Carcinus maenas, showing the different morphology of the epithelium of the anterior, respect to the posterior gills which can justify, according to some biochemical data, a different function mechanism.     

Positive Darwinian selection on crustacean hyperglycemic hormone (CHH) of the green shore crab, Carcinus maenas

The tissue-specific expression and differential function of the crustacean hyperglycemic hormone (CHH) in Carcinus maenas indicate an interesting evolutionary history. Previous studies have shown that CHH from the sinus gland X-organ (XO-type) has hyperglycemic activity, whereas the CHH from the pericardial organ (PO-type) neither shows hyperglycemic activity nor it inhibits Y-organ ecdysteroid synthesis. Here we examined the types of selective pressures operating on the variants of CHH in Carcinus maenas. Maximum likelihood-based codon substitution analyses revealed that the variants of this neuropeptide in C. maenas have been subjected to positive Darwinian selection indicating adaptive evolution and functional divergence among the CHH variants leading to two unique groups (PO and XO-type). Although the average ratio of nonsynonymous to synonymous substitution (omega) for the entire coding region is 0.5096, few codon sites showed significantly higher omega (10.95). Comparison of models that incorporate positive selection (omega > 1) with models not incorporating positive selection (omega <1) at certain codon sites failed to reject (p=0) evidence of positive Darwinian selection.     

Alteration of cytochrome C oxidase activity during spermatogenesis in Carcinus maenas

During spermatogenesis in the crab, Carcinus maenas, mitochondria in the developing spermatids degenerate. In close association with mitochondrial fragments in late spermatids, an elaborate lattice-like complex of fused membranes is formed and lies in a position adjacent to the acrosome. Mature sperm possess no mitochondria, but a whorl of membranes is present alongside the acrosome in the diffuse nucleus. To ascertain whether or not cytochrome c oxidase activity is lost as the mitochondria disintegrate, differentiating Carcinus maenas spermatids and mature sperm have been studied cytochemically. Cytochfome c oxidase activity was localised by means of the osmophilic compound 3,3'-diaminobenzidine (DAB). Cytochrome c oxidase activity is confined to the cristae of mitochondria in the testis. As spermatids mature most mitochondria are lost. A few mitochondrial fragments may be caught up within the lamellar complex. While they are recognisalbe as mitochondria they retain cytochrome c oxidase activity. The lamellar complex does not show this enzyme activity. These results therefore suggest that the mature sperm of Carcinus maenas do not contain the enzymes normally incorporated in the mitochondrial membrane, capable of oxidative phosphorylation.     

Angiotensin-converting enzyme-like activity in crab gills and its putative role in degradation of crustacean hyperglycemic hormone

Angiotensin-converting enzyme-like enzyme activity (ACELA) was found in Carcinus maenas using reverse phase high performance liquid chromatography (RP-HPLC) analysis of degradation kinetics of a synthetic substrate (Hippuryl-histidyl-leucine) and a specific inhibitor (captopril). Gills contained the highest ACELA, then brain, muscle, and testis, respectively, while no activity was detected in the following tissues: hepatopancreas, hindgut, hypodermis, heart, and hemolymph. ACELA present in gill membranes exhibited a K(m) of 0.23 mM and V(max) of 7.6 nmol with synthetic substrate. The enzyme activity was dependent on Cl- concentration and was markedly inhibited by captopril, lisinopril, and EDTA. Addition of Zn2+ to membranes previously treated with EDTA restored 89% activity, suggesting that C. maenas ACELA is a Zn2+ metalloenzyme. Gill membranes prepared from premolt crabs showed similar levels of ACELA to those of the intermolt animals. Administration of captopril in vivo lengthened the half life of circulating CHH, while in vitro incubation of gill membranes with captopril reduced CHH. These results suggest that C. maenas ACELA present in gills is likely to be involved in degradation of this neuropeptide.     

Intraspecific morphological variation related to the moult-cycle in colour forms of the shore crab Carcinus maenas

The common shore crab Carcinus maenas exhibits a range of carapace colours from green through orange to red, green forms showing some differences of distribution from red/orange forms. To test the hypothesis that colour differences were moult-related, large numbers of Carcinus were collected intertidally and subtidally in summer when moulting is most prevalent, and their moult stages determined. Red and orange coloration was found only to occur in the larger size classes in crabs in prolonged intermoult, but not solely in the largest crabs in terminal anecdysis. Red crabs were characterized by a heavier load of epibionts and a strong, thicker carapace. In contrast, green crabs were found over the entire size range and appeared to be actively moulting forms; they carried fewer epibionts and had a thinner integument than red forms. The significance of morphological differences between red/orange and green forms of Carcinus maenas is considered in relation to previously reported behavioural, physiological and ecological differences between the colour forms.     

Interspecific hybridization and mitochondrial introgression in invasive carcinus shore crabs

Interspecific hybridization plays an important role in facilitating adaptive evolutionary change. More specifically, recent studies have demonstrated that hybridization may dramatically influence the establishment, spread, and impact of invasive populations. In Japan, previous genetic evidence for the presence of two non-native congeners, the European green crab Carcinus maenas and the Mediterranean green crab C. aestuarii, has raised questions regarding the possibility of hybridization between these sister species. Here I present analysis based on both nuclear microsatellites and the mitochondrial cytochrome C oxidase subunit I (COI) gene which unambiguously argues for a hybrid origin of Japanese Carcinus. Despite the presence of mitochondrial lineages derived from both C. maenas and C. aestuarii, the Japanese population is panmictic at nuclear loci and has achieved cytonuclear equilibrium throughout the sampled range in Japan. Furthermore, analysis of admixture at nuclear loci indicates dramatic introgression of the C. maenas mitochondrial genome into a predominantly C. aestuarii nuclear background. These patterns, along with inferences drawn from the observational record, argue for a hybridization event pre-dating the arrival of Carcinus in Japan. The clarification of both invasion history and evolutionary history afforded by genetic analysis provides information that may be critically important to future studies aimed at assessing risks posed by invasive Carcinus populations to Japan and the surrounding region.