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Metal-specific induction of metallothionein isoforms in the blue crab Callinectes sapidus in response to single- and mixed-metal exposure.

Metallothioneins (MTs) play an important role in the metabolism of copper and zinc during the molt cycle of the blue crab. In this study we examined the hypothesis that MT expression in crabs is metal specific. Anion-exchange chromatography showed one major ZnCuMT (ZnMT-I) in control crabs, two MT isoforms in cadmium-treated crabs (CdMT-I, CdMT-II), and three forms (CuMT-I, CuMT-II, CuMT-III) in copper-treated animals. Amino acid analysis of the carboxymethylated apo-MTs, purified by reversed-phase HPLC, showed minor differences between ZnMT-I, CdMT-I, CdMT-II, CuMT-I, and CuMT-II, while CuMT-III was unique. After combined exposure to cadmium and copper, four MTs with differing copper/cadmium ratios were observed, equivalent to CdMT-II and the three CuMTs. We conclude that the blue crab has four genes that encode different MTs. Transcription of the CdMT-I gene is induced by cadmium, but inhibited by copper. CuMT-I, CdMT-II, and ZnMT-I may be the products of a single gene responsive to copper, cadmium, and zinc. Expression of the CuMT-II and CuMT-III genes is initiated by copper and not by cadmium. We believe that CdMT-I and CuMT-III are important in detoxification, whereas Zn/CuMT-I and CuMT-II are involved in regulatory functions. These results show the importance of the use of mixed-metal exposures in the study of the molecular mechanisms of metal regulation and function.

Animals↗

Neural asymmetry in male fiddler crabs.

In adult male fiddler crabs, Uca pugnax, there is a marked enlargement of the 1st thoracic ganglion and its nerve root on the side of the major cheliped compared to the side of the minor cheliped. Retrograde uptake of cobalt via the cut ends of the motoneurons revealed a significant hypertrophy of their somata and dendritic fields on the major side of the ganglion compared to the minor side in the male fiddler crabs. (In female fiddler crabs which have two minor chelipeds the motoneurons were similar in size on both sides of the ganglion.) Since the number and distribution of motoneuron somata was relatively constant in the two halves of each ganglion, homologies for individual or groups of neurons could be recognized. The number of axon profiles in a cross-sectional montage of the entire nerve root of the major side in a male fiddler crab was several times greater than that of the minor side in random samples which were appropriately scaled in area. In samples of equal areas the axonal density was similar on the major and minor sides, as was also the range of axon diameters; both signify no difference in size of axons between the contralateral nerve roots. Consequently enlargement of the nerve root on the major side is due to a relative increase in the number of axons. This increase is in sensory fibers since the number of motor fibers are bilaterally constant. Thus neural asymmetry in male fiddler crabs involves hypertrophy of the motoneurons and hyperplasia of the sensory neurons associated with the enlarged condition of the major cheliped.

Animals↗

Formation of ecdysteroids by Y-organs of the crab, Menippe mercenaria. I. Biosynthesis of 7-dehydrocholesterol in vivo.

Y-organs of the xanthid crab, Menippe mercenaria, secrete ecdysteroid hormones in vitro, apparently both 3-dehydroecdysone and 25-deoxyecdysone. Studies were initiated on the biosynthetic path(s), in which cholesterol is converted to these ecdysteroids. Crabs were injected with [3H]cholesterol. Y-organs and hemolymph were removed 12 hr later and extracted directly and the extracts were analyzed by HPLC. Both polar and nonpolar sterols were surveyed. The only metabolite of cholesterol detectable in Y-organs was 7-dehydrocholesterol (identified by mass spectrometry). The total amount of 7-dehydrocholesterol and the amount that was labeled were generally greater than for cholesterol and were higher in Y-organs from de-eyestalked crabs than in those from intact crabs. Subcellular fractionation of the Y-organs showed that over 70% of total radioactivity was in cholesterol and 7-dehydrocholesterol of mitochondria and microsomes, distributed about equally between the two organellar fractions. In hemolymph, the only nonpolar sterols present were cholesterol and 7-dehydrocholesterol; the concentration ratio was 20:1. However, 7-dehydrocholesterol was not significantly labeled. Analyses of polar compounds revealed two prominent, uv-absorbing ecdysteroids which coeluted with the authentic standards, 3-dehydro-20-hydroxyecdysone and 25-deoxy-20-hydroxyecdysone (ponasterone A). The radioactivity profile showed, in addition, a third prominent peak that corresponded in retention time with 3-dehydroecdysone. These results indicate that the Y-organs in vivo form 7-dehydrocholesterol from cholesterol and convert the latter to secretion products without accumulation of other intermediates. At least two ecdysteroids are secreted and appear to be converted peripherally in this crab to their respective 20-hydroxy derivatives.

Animals↗

Formation of ecdysteroids by Y-organs of the crab, Menippe mercenaria. II. Incorporation of cholesterol into 7-dehydrocholesterol and secretion products in vitro.

The conversion in vitro of cholesterol (Ch) to nonpolar metabolites and ecdysteroids was studied in Y-organs of the xanthid crab, Menippe mercenaria. In one set of experiments, Y-organs were prelabeled in vivo by injecting crabs with 100 microCi of [3H]Ch, and halved glands were then incubated for 24 and 48 hr in the presence of unlabeled Ch. In another set, unlabeled Y-organs were incubated in standard medium containing 10 microCi/ml of [3H]Ch. Both polar and nonpolar metabolites were surveyed by HPLC. The early metabolite, 7-dehydrocholesterol (7-dhCh), was the only labeled derivative of Ch detectable in Y-organ tissue after incubation; preincubation amounts of 7-dhCh were higher in glands from de-eyestalked crabs vs glands from intact crabs, and labeling was an order of magnitude higher in glands incubated with labeled Ch vs those prelabeled in vivo. Specific activity calculations indicate highly efficient conversion of 7-dhCh to ecdysteroid secretions. Analyses of the incubation media revealed two polar secretory products, synthesized from Ch in vitro. These coeluted with the authentic standards, 3-dehydroecdysone and 25-deoxyecdysone. Secretion of 3-dehydroecdysone always exceeded that of 25-deoxyecdysone (ratio range, 1.9 to 14.4), in both de-eyestalked and intact crabs.

Animals↗

Resistance of nerves from certain toxic crabs to paralytic shellfish poison and tetrodotoxin.

The inhibitory effect of paralytic shellfish poison and tetrodotoxin on nerves from toxic and nontoxic crabs was examined. The toxins at concentrations of 10(-3) - 10(-4) M partially or completely inhibited the action potential of nerves isolated from the legs of toxic crab species (Zosimus aeneus, Atergatis floridus and Platypodia granulosa), but had no effect at 10(-6) M, the concentration at which the action potential of nerves from a nontoxic crab (Plagusia dentipes) was inhibited completely. A xanthid crab Daira perlata was intermediate in respect to the resistance to toxins. These results agree with the previous results obtained by i.p. administration of both toxins into those crabs.

Action Potentials↗

Occurrence of tetrodotoxin and paralytic shellfish poison in the Taiwanese crab Lophozozymus pictor.

Paralytic toxicity was detected by tetrodotoxin (TTX) bioassay in all 15 specimens of the xanthid crab Lophozozymus pictor collected from northern Taiwan in 1993. The average toxicity of crab specimens was 921 +/- 231 (mean +/- S.E.) mouse units. The toxin of crab was partially purified and then identified. It was found that the crab toxin contained TTX and gonyautoxin. The ratio of TTX to gonyautoxin for crab toxin was about 9:1.

Animals↗

Zinc accumulation in fiddler crabs Uca annulipes latreille and Uca triangularis (Milne Edwards).

The effect of exposure time and concentration on the tissue-specific accumulation of Zn in two fiddler crabs, Uca annulipes and Uca triangularis, obtained from polluted (Visakhapatnam Harbor) and unpolluted (Bhimilipatnam) areas was studied. Hepatopancreas registered major accumulation of Zn when the crabs were exposed to sublethal concentrations (6.38 to 15.39 ppm) for 30 days. However, when the crabs were exposed to different concentrations of Zn (LCO, LC25, LC50, LC75) ranging from 12.93 to 99.38 ppm, higher accumulation was recorded in gills than in hepatopancreas. There was a progressive increase in the percentage body burden of Zn in hepatopancreas, but that in gills showed a decreasing trend. The standardized body burdens for both species of crabs were calculated to evaluate their sensitivity to exposure time as well as to concentration. The fractional retention coefficients for exposure time estimated for exposed crabs indicate that U. triangularis from Bhimilipatnam, having the highest value, is sensitive and U. annulipes from Visakhapatnam Harbor, showing the lowest value, is resistant.

Animals↗

Gastrin/cholecystokinin-like immunoreactive peptides in the Dungeness crab, Cancer magister (Dana): immunochemical and biological characterization.

The purpose of this investigation was to characterize a gastrin/cholecystokinin-like immunoreactant (G/CCK-LI) extractable from the crab, Cancer magister. G/CCK-LI was extracted best in boiling water and was found mainly in the stomach, hemolymph and carapace. A relatively large immunoreactive peptide in the stomach and apparently smaller forms in the hemolymph and carapace were separated by Sephadex G-50 fractionation. Anion-exchange chromatography further fractionated the stomach form into three major peaks. The crab material cross-reacted with three antisera specific for the common C-terminus of gastrin/CCK, but cross-reacted much less with three antisera directed against other portions of the gastrin molecule. Partially purified crab stomach G/CCK-LI inhibited the binding of labeled CCK to mouse brain G/CCK receptors but not to rat pancreatic CCK receptors. The crab peptide did not stimulate rat gastric acid or rat pancreatic amylase secretion. These results indicate that the crab peptides are structurally similar to, but distinguishable from, the bioactive C-terminal amino acid sequence common to gastrins and CCKs.

Amylases↗

Involvement of cAMP and cGMP in the mode of action of molt-inhibiting hormone (MIH) a neuropeptide which inhibits steroidogenesis in a crab.

In crustaceans, production of molting hormones (or ecdysteroids) by the molting glands (Y-organs; YO), is under negative control exerted by a neuropeptide, the molt-inhibiting hormone (MIH). MIH of the crab Carcinus maenas inhibits in vitro steroidogenesis of basal (intermolt crab) or activated (premolt crab) YO. MIH inhibits secretion of the two ecdysteroids synthesized by crab YO, ecdysone (E) secreted throughout the molting cycle, and 25-deoxyecdysone (25dE), secreted during the premolt period. At a MIH concentration of 10(-8) M, E is reduced about 50% and 25dE 94%. Regardless of the molting stage, this inhibition of steroidogenesis is reversible, dose dependent and measurable after 5 min. On intermolt YO, MIH induced cGMP increase and 8BrcGMP mimics the effect of MIH: at this stage cGMP seems to be involved with MIH inhibition of steroidogenesis. On premolt YO MIH induced a transient increase of cAMP (2-fold) and a long-lasting enhancement of cGMP (60-fold). On active YO, we demonstrated that a low concentration (10(-5) M) of dbcAMP, 8BrcAMP, 8BrcGMP, or agents increasing intracellular cAMP, mimic MIH effects and inhibit steroidogenesis. From these observations it is concluded that both cyclic nucleotides are involved in the mode of action of MIH on activated YO. At this premolt period, MIH/cAMP may act cooperatively with MIH/cGMP in the inhibitory control of steroidogenesis by crab YO.

1-Methyl-3-isobutylxanthine↗

Regulation of neurohormone release in the fiddler crab, Uca pugilator: effects of gamma-aminobutyric acid, octopamine, Met-enkephalin, and beta-endorphin.

Gamma-aminobutyric acid (GABA) blocked concentration of the pigments in melanophores and erythrophores of intact crabs. GABA blocked the release of pigment concentrating hormones from the isolated eyestalk. Octopamine (OA) blocked black pigment dispersion in intact crabs, but did not affect red pigment dispersion or concentration. OA blocked the release of black pigment dispersing hormone from isolated eyestalks. Met-enkephalin, but not Leu-enkephalin, stimulated black and red pigment concentration in intact crabs. Met-enkephalin, but not Leu-enkephalin, stimulated the release of pigment concentrating hormones from isolated eyestalks. Naloxone blocked the effects of Met-enkephalin in intact crabs and on isolated eyestalks. Beta-endorphin induced black pigment dispersion in intact crabs and in isolated legs.

Animals↗

Association between cadmium and calcium uptake and distribution during the moult cycle of female shore crabs, Carcinus maenas: an in vivo study.

Net influxes into the haemolymph and tissue distribution of 45Ca and 109Cd were studied in vivo in female Carcinus maenas at different moult stages. Net influxes of 45Ca and 109Cd from water were higher in postmoult (A and B) C. maenas than in C3- and C4-intermoult crabs and the net influx of calcium was higher in C3-intermoult crabs than in C4-intermoult crabs. The net influxes of 45Ca and 109Cd increased in postmoult C. maenas with decreasing external calcium concentrations at constant salinity. At all external calcium concentrations a significant correlation existed between 45Ca and 109Cd accumulated in the haemolymph of individual animals. In vivo exposure of postmoult C. maenas to external lanthanum decreased the 45Ca and 109Cd uptake rates to 30 and 10%, respectively, of the control values. About 30% of injected 109Cd were found in the midgut gland, 10-20% in the gills and only a few (1-2) percent was lost to the seawater 24 h after injection. No major variations in tissue distribution of 109Cd were observed between moult stages in these tissues. Premoult crabs retained more cadmium in the haemolymph 24 h after injection than other moult stages, and postmoult crabs retained more in muscle. Between 20 and 40% of the injected 45Ca were excreted to the water, while only a few percent of the injected 45Ca were found in the soft tissues 24 h after injection. Large moult stage variations, however, were observed in the tissue distribution of internalised 45Ca. This study demonstrates that cadmium and calcium uptakes are elevated in postmoult C. maenas. The results indicate that cadmium and calcium in this stage are taken up via Ca2+-channels located in the apical membrane of gill epithelium cells. When internalised, however, cadmium and calcium are metabolised in fundamentally different ways, determined by the chemical properties and biological significance of the two metals.

Analysis of Variance↗

Tissue distribution, subcellular localization and endocrine disruption patterns induced by Cr and Mn in the crab Ucides cordatus.

The essential trace elements Cr and Mn are toxic at high concentrations and information about low concentration is insufficient in the literature. In polluted mangroves, the crab Ucides cordatus can represent a useful tool to assess information on the potential impact of trace elements like Cr and Mn on the environment, since this species is comestible and thus, commercially negotiated. Therefore, U. cordatus crabs were exposed in vivo to different concentrations of Cr and Mn solved in seawater and had their tissue distribution and subcellular deposits evaluated. The gill, hepatopancreas and muscle concentrations were determined by atomic absorption spectroscopy and the results showed that Cr and Mn presented the highest values in the gills rather than in the hepatopancreas and muscular tissue. Electron microscopy and analytical X-ray microanalysis revealed Cr precipitates on the gill surface, co-localized with epiphyte bacteria. In addition, since Cr and Mn did not equally accumulate in most of the tissues studied, glycemic rate of animals, which received injections of extracts of eyestalks of the contaminated crabs, were measured in order to evaluate whether the studied concentrations of Cr and Mn could produce any metabolic alteration. The results indicated that extracts of the eyestalks of crabs submitted to Cr and Mn salts and injected into normal crabs markedly influenced crustacean hyperglycemic hormone synthesis and/or release. The results are discussed with respect to sensitivity of the employed methods and the possible significance of the concentrations of Cr and Mn in the organisms.

Analysis of Variance↗

Osmoregulation and tissue water regulation in the freshwater red crab Dilocarcinus pagei (Crustacea, Decapoda), and the effect of waterborne inorganic lead.

Inorganic lead has been measured in high concentrations in certain streams in Brazil. This study has evaluated the osmoregulatory effects of lead on the native freshwater red crab Dilocarcinus pagei. In order to probe its osmoregulatory and tissue volume regulatory capabilities and how it would be affected by lead, the crab has been submitted to individual and combined salt and chemical stresses (Pb2+). Male crabs were exposed for 10 days to either: (1) control (freshwater, FW), (2) brackish water of salinity 15 (BW), (3) inorganic lead in freshwater (FWPb), and (4) inorganic lead in brackish water (BWPb), 2.7 mgPb/L. In vivo, whole crabs lost weight transiently when exposed to Pb2+, both in FW and in BW. Haemolymph osmolality and ion concentrations increased and remained elevated upon exposure of crabs to BW, with or without Pb2+, showing a trend to hyper-conformation. In vitro, muscle weight decreased in isosmotic conditions upon exposure to Pb2+. Na+, Cl-, and ninhydrin positive substances (NPS) were increased in muscle exposed to hyperosmotic saline, well above what would be expected from simple efflux of water, suggesting a partial regulatory volume increase (RVI) capacity. This partial RVI involves the Na+, K+, 2Cl- -cotransporter and the Na+/H+ exchanger on Na+ and Cl- uptake, as judged from further decreases in muscle weight in the presence of the respective inhibitors. A breakdown of proteins into NPS seems to follow the uptake of inorganic ions. Pb2+ has affected water and ion movements in D. pagei both in the whole animal and in the isolated tissue. This study has highlighted the relevance of evaluating tissue volume regulation in aquatic animals confronted with metal polluted waters.

Animals↗

CYP330A1 and CYP4C39 enzymes in the shore crab Carcinus maenas: sequence and expression regulation by ecdysteroids and xenobiotics.

Cytochrome P450 enzymes (CYP enzymes) catalyse important metabolic reactions of exogenous and endogenous substrates, including steroid hormones. Here, we report the first two CYP sequences from the shore crab, Carcinus maenas. Two complete cDNAs isolated from crab hepatopancreas encode CYP enzymes named CYP330A1, the first member of a new family, and CYP4C39. CYP330A1 is closest related to members of the CYP2 family (37.3% identical to mouse CYP2J6) and CYP4C39 is most identical to crayfish CYP4C15 (59.5%). CYP330A1 gene expression was induced in hepatopancreas of male green intermoult crabs by ecdysone and ponasterone A, but also by benzo(a)pyrene and phenobarbital. CYP330A1 induction was not observed in red crabs. The present results indicate that the CYP330A1 enzyme may be involved in ecdysteroid metabolism, presumably catabolism, and in the detoxification of environmental pollutants. Ecdysteroids or xenobiotics did not affect CYP4C39 gene expression. The fact that both ecdysteroids and xenobiotics affect CYP330A1 gene expression indicates that mutual interactions between chemical exposures and endocrine functions may exist in the shore crab.

Amino Acid Sequence↗

Laboratory investigations of the effects of predator sex and size on prey selection by the Asian crab, Hemigrapsus sanguineus.

Laboratory studies have shown that the nonindigenous Asian shore crab, Hemigrapsus sanguineus, readily consumes three species of commercial bivalves: blue mussels, Mytilus edulis, soft-shell clams, Mya arenaria, and oysters, Crassostrea virginica. Although crabs can eat bivalves of a wide size range, they preferred the smaller prey (</=10 mm SL). Prey critical size limits exist for M. edulis and C. virginica, but not M. arenaria, possibly because of differences in shell characteristics among the three species. Crabs preferred M. arenaria over both M. edulis and C. virginica, and M. edulis was strongly preferred over C. virginica in pairwise comparison tests. Experiments to determine feeding rates on mussels showed that H. sanguineus can consume large numbers of mussels daily (12.7+/-11.6 mussels day(-1); sexes pooled; N=59). Mussel consumption rates increased with size of the predator and male crabs consumed more mussels than did similarly sized female crabs. The high densities of Hemigrapsus that occur in the wild, their effectiveness as predators of juvenile bivalves and their large appetites suggest an important role for these predators in restructuring the prey communities in habitats into which they have been introduced.

Journal Article↗

Trophic accumulation and depuration of mercury by blue crabs (Callinectes sapidus) and pink shrimp (Penaeus duorarum).

Mercury concentrations in blue crabs (Callinectes sapidus) collected from an area of mercury-contaminated sediments in Lavaca Bay, TX, USA, are more than an order of magnitude greater than concentrations in penaeid shrimp from the same area. Laboratory feeding experiments using mercury-contaminated fish as food showed that both blue crabs and pink shrimp (Penaeus duorarum) could accumulate mercury concentrations comparable to those in their food in 28 days. Calculated mercury assimilation efficiencies averaged 76% for blue crabs and 72% for pink shrimp. Significant depuration of mercury by blue crabs was not observed during a subsequent 28-day period, but pink shrimp lost mercury at a rate of about 0.012 day-1. Model calculations predict biomagnification factors of mercury of about two to three at steady state for both species. The large difference in observed concentrations of mercury in field-collected blue crabs and penaeid shrimp does not result from differences in efficiency of mercury assimilation from their food or from differences in excretion rates. It is more likely the result of differences in residence times in the contaminated area and of differences in feeding habits.

Animals↗

Effects of fenoxycarb exposure on complete larval development of the xanthid crab, Rhithropanopeus harrisii.

Pest control agents, such as juvenile hormone analogues (JHA), have been developed to limit effects on non-target organisms that co-inhabit insect pest habitats. Rhithropanopeus harrisii, an estuarine xanthid crab, was used to observe the impacts of the JHA, fenoxycarb, on the pattern of complete larval development as well as survival of larvae and successful metamorphosis to first crab stage. Significant mortality occurred in the first of four zoeal stages (after 2-3 days of exposure) at the highest treatment of 240 microg fenoxycarb/l and in megalopae exposed to 48 microg fenoxycarb/l. The time required to metamorphose to the first crab stage was significantly increased for megalopae in all treatments 48 microg/l. This delay in development was sufficient to significantly prolong the entire developmental period from zoea to crabs. Unexposed larvae developed to crabs in an average of 16 days; larvae exposed to >/=48 microg/l required 19-20 days. Reduced survival and extended duration of developing larval stages in the life history of a benthic invertebrate may alter the population dynamics of these organisms in the estuary.

Animals↗

PCR cloning and expression of the molt-inhibiting hormone gene for the crab (Charybdis feriatus).

A PCR-based genomic DNA walking technique was used to clone the gene for the molt-inhibiting hormone of the crab, Charybdis feriatus. Several overlapping genomic clones were isolated, and the MIH gene for the crab was reconstructed. DNA sequence determination of the overlapping clone reveals that the MIH gene spans 4.3kb and consists of three exons and two introns. Exons 1 and 2 carry a coding sequence for the signal peptide, and exons 2 and 3 consist of coding sequence for the mature peptide. The exon-intron boundary of the crab MIH gene also follows the 'GT-AG rule' for the splice donor and acceptor. The deduced amino acid sequence of MIH shows the highest overall similarity to those of the crabs, Callinectes sapidus and Carcinus maenas, and the gonad-inhibiting hormone (GIH) of the lobster. The putative polyadenylation signal is approximately 1.0kb 3' downstream of the termination codon (TGA). Genomic Southern blot analysis indicates that few genomic fragments were hybridized to the cDNA probe. The 5' flanking region contains a putative promoter with several putative cis elements similar to some vertebrate neuropeptide genes. The 530-bp flanking region was subcloned separately to two promoterless reporter plasmids carrying either the Green Fluorescent Protein gene (GFP) or the Choramphenicol Acetyltransferase gene (CAT). The DNA constructs were transfected into insect cells (Sf21) and mouse pituitary cells (GH4ZR7), respectively. Green fluorescent protein was detected in some of the transfected insect cells, and expression of the CAT was detected in cells transfected with DNA constructs containing the crab promoter. By RT-PCR, MIH transcripts can be detected in the eyestalk of shrimp in intermolt, early premolt, late premolt stages and females that brood their eggs. It can also be found in the brain, but not in the ovary, hepatopancreas, muscle and epidermis. During early larval development, MIH mRNA can be detected in the pre-hatched and the newly hatched larvae. Unlike the adult, the expression of the MIH in the larvae is exclusively in the brain.

Amino Acid Sequence↗