Long-Term Pyrene Exposure of Grass Shrimp, \u3ci\u3ePalaemonetes pugio\u3c/i\u3e, Affects Molting and Reproduction of Exposed Males and Offspring of Exposed Females

The objective of this study was to investigate the impact of long-term pyrene exposure on molting and reproduction in the model estuarine invertebrate, the grass shrimp (Palaemonetes pugio). Grass shrimp were exposed to measured concentrations of 5.1, 15.0, and 63.4 ppb (mu g/L) pyrene for 6 weeks, during which time we determined molting and survivorship. At the end of the exposure, we immediately sacrificed some of the shrimp for biomarker (CYP1A and vitellin) analyses. The remaining shrimp were used to analyze fecundity and embryo survivorship during an additional 6 weeks after termination of pyrene exposure. Male shrimp at the highest pyrene dose (63 ppb) experienced a significant delay in molting and in time until reproduction, and showed elevated ethoxycoumarin o-deethylase (ECOD) activity immediately after the 6-week exposure period. In contrast, 63 ppb pyrene did not affect these parameters in female shrimp. Females produced the same number of eggs per body weight, with high egg viability (98-100%) at all exposure levels, but with decreased survival for the offspring of the 63-ppb pyrene-exposed females. In addition, vitellin levels were elevated only in females at 63 ppb pyrene after the 6-week exposure. We hypothesize that the elevated vitellin binds pyrene and keeps it biologically unavailable to adult females, resulting in maternal transfer of pyrene to the embryos. This would account for the lack of effect of pyrene exposure on ECOD activity, molting, and reproduction in the adult females, and for reduced survival of their offspring

Biochemical Defense Mechanisms Against Copper-Induced Oxidative Damage in the Blue Crab, \u3ci\u3eCallinectes sapidus\u3c/i\u3e

The blue crab (Callinectes sapidus) has a very dynamic copper metabolism associated with the biosynthesis and degradation of its respiratory pigment hemocyanin. In this study we report on the cellular defense mechanisms used by the crab to protect itself from copper toxicity. Short-term copper-exposure studies, conducted by incubating hepatopancreas tissue explants in copper-containing medium, show that copper taken up by the cells during the first 60 min combines with low-molecular-weight copper complex(es), which include Cu(I)–glutathione. Thereafter, copper binds to newly synthesized metallothionein (MT), with a concomitant decrease in Cu(I)–glutathione. Copper does not displace zinc from the endogenous ZnMT pool. Long-term exposure by means of copper-rich diets results in the synthesis of two MT isoforms in the hepatopancreas: CuMT-I and CuMT-II (D. Schlenk and M. Brouwer, 1991,Aquat. Toxicol.20, 25–34). Transfer of copper from Cu(I)–glutathione to apoMT-I and apoMT-II can be accomplishedin vitro.Cu(I) binding by the two isoforms is very different. Cu(I) binds to apoMT-I in a strictly cooperative manner. No partially filled Cu(I)–thiolate clusters appear to be present. In contrast, the Cu(I)–thiolate clusters in MT-II are formed only after more than four Cu(I) ions are bound. Long-term copper exposure leads to increased activity of two antioxidant enzymes: glutathione peroxidase and manganese superoxide dismutase (SOD). No CuZnSOD is found. Activities of catalase and glutathione reductase and the intracellular levels of glutathione are unaffected by copper. The defense mechanisms are not entirely sufficient for preventing copper-induced oxidative damage. Levels of oxidized lipids are significantly higher in copper-exposed crabs, but oxidized protein levels are nearly the same

Role of a Copper-Specific Metallothionein of the Blue Crab, \u3ci\u3eCallinectes sapidus\u3c/i\u3e, in Copper Metabolism Associated with Degradation and Synthesis of Hemocyanin

We have identified three MT encoding genes in the blue crab: MT-I. inducible by cadmium, zinc and copper; MT-II, inducible by cadmium and zinc: and MT-III, inducible by copper only [Syring et al., Comp. Biochem. Physiol. C, 125 (2000) 325-332]. To examine the role of the CuMT-I and CuMT-III isoforms in copper metabolism associated with the synthesis and degradation of the oxygen-binding copper protein, hemocyanin, we (l) cloned and sequenced hemocyanin cDNA, (2) examined interaction of the CuMTs with endoplasmic reticulum (ER) vesicles and (3) measured changes in levels of hemocyanin. MT-I. MT-III protein and mRNA that occur in crabs during different stages of the molt cycle. The cDNA-derived hemocyanin amino-acid sequence revealed the presence of a leader peptide indicating that hemocyanin is a secretory protein that is synthesized on the ER. Copper uptake studies show that ER vesicles take up both Cu1+ and Cu2+ in an ATP-independent process. The copper transporter has a K-m of 10.8 +/- 2.4 muM copper and a V-max of 6.1 +/- 0.5 nmol Cu/mg protein/10 min. ER vesicles contain hemocyanin, and bind CuMT-I and, preferentially, CuMT-III. However, binding does not result in copper transfer to the ER. There are statistically significant changes in hepatopancreas MT-III and hemocyanin mRNA, and in hemolymph hemocyanin concentrations during the molt cycle. MT-I mRNA remains constant. Changes in MT-III mRNA are positively correlated with changes in hemocyanin mRNA and hemocyanin protein, which points to coordinate control of MT-III and hemocyanin transcription. No CuMT-III protein is observed in hepatopancreas of intermolt crabs when levels of both MT-III and hemocyanin mRNA are high, suggesting rapid utilization of copper bound to MT-III when cells are actively synthesizing hemocyanin. CuMT-III is present in premolt and softshell crabs, and its emergence appears to coincide with a decrease in hemocyanin synthesis and increase in hemocyanin degradation, These results support the hypothesis that the copper-specific metallothionein is intricately involved in copper homeostasis associated with both the synthesis and degradation of hemocyanin. (C) 2002 Elsevier Science B.V. All rights reserved

Cloning and Sequencing of cDNAs Encoding for a Novel Copper-Specific Metallothionein and Two Cadmium-Inducible Metallothioneins from the Blue Crab \u3ci\u3eCallinectes sapidus\u3c/i\u3e

Metallothioneins (MTs) are cysteine-rich metal-binding proteins found in micro-organisms, plants and all invertebrate and vertebrate animals. Unicellular eukaryotes such as yeast have a copper-MT whose synthesis is induced by a copper-activated transcription factor. Most higher organisms have two major cadmium/zinc MT isoforms, whose synthesis is controlled by a zinc-activated transcription factor. The blue crab, Callinectes sapidus, has two cadmium-inducible isoforms, CdMT-I and CdMT-II, and a third isoform, CuMT-II, which is induced by copper, but not by cadmium. The cDNA sequence of the copper-specific MT, along with those of the two CdMTs, was determined utilizing 3\u27 and 5\u27 rapid amplification of cDNA ends (RACE). CuMT-II cDNA encodes a 63 amino acid protein containing 21 cysteine residues. CdMT-I and CdMT-II cDNA encode a 58 and 57 amino acid protein, respectively, each with 18 cysteines. Molecular phylogeny analysis shows that the CdMT isoforms cluster with other crustacean CdMTs, whereas the copper-specific MT is more closely related to mollusk MTs. CuMT-II shows considerable homology to a copper-specific, non-cadmium inducible, MT from the snail, Helix pomatia. The presence of copper-specific MTs in mollusks and crustaceans, both of which are dependent on hemocyanin for oxygen transport, suggests that CuMT-II is involved in copper homeostasis associated with the synthesis and degradation of hemocyanin. (C) 2000 Elsevier Science Inc. All rights reserved

Replacement of a cytosolic copper/zinc superoxide dismutase by a novel cytosolic manganese superoxide dismutase in crustaceans that use copper (haemocyanin) for oxygen transport.

The blue crab, Callinectes sapidus, which uses the copper-dependent protein haemocyanin for oxygen transport, lacks the ubiquitous cytosolic copper-dependent enzyme copper/zinc superoxide dismutase (Cu,ZnSOD) as evidenced by undetectable levels of Cu,ZnSOD activity, protein and mRNA in the hepatopancreas (the site of haemocyanin synthesis) and gills. Instead, the crab has an unusual cytosolic manganese SOD (cytMnSOD), which is retained in the cytosol, because it lacks a mitochondrial transit peptide. A second familiar MnSOD is present in the mitochondria (mtMnSOD). This unique phenomenon occurs in all Crustacea that use haemocyanin for oxygen transport. Molecular phylogeny analysis suggests the MnSOD gene duplication is as old as the origin of the arthropod phylum. cytMnSOD activity in the hepatopancreas changes during the moulting cycle of the crab. Activity is high in intermoult crabs and non-detectable in postmoult papershell crabs. mtMnSOD is present in all stages of the moulting cycle. Despite the lack of cytCu,ZnSOD, crabs have an extracellular Cu,ZnSOD (ecCu,ZnSOD) that is produced by haemocytes, and is part of a large, approx. 160 kDa, covalently-linked protein complex. ecCu,ZnSOD is absent from the hepatopancreas of intermoult crabs, but appears in this tissue at premoult. However, no ecCu,ZnSOD mRNA can be detected, suggesting that the protein is recruited from the haemolymph. Screening of different taxa of the arthropod phylum for Cu,ZnSOD activity shows that those crustaceans that use haemoglobin for oxygen transport have retained cytCu,ZnSOD. It appears, therefore, that the replacement of cytCu,ZnSOD with cytMnSOD is part of an adaptive response to the dynamic, haemocyanin-linked, fluctuations in copper metabolism that occur during the moulting cycle of the crab

Replacement of a Cytosolic Copper/Zinc Superoxide Dismutase by a Novel Cytosolic Manganese Superoxide Dismutase in Crustaceans That Use Copper (Haemocyanin) for Oxygen Transport

The blue crab, Callinectes sapidus, which uses the copper-dependent protein haemocyanin for oxygen transport, lacks the ubiquitous cytosolic copper-dependent enzyme copper/zinc superoxide dismutase (Cu,ZnSOD) as evidenced by undetectable levels of Cu,ZnSOD activity, protein and mRNA in the hepatopancreas (the site of haemocyanin synthesis) and gills. Instead, the crab has an unusual cytosolic manganese SOD (cytMnSOD), which is retained in the cytosol, because it lacks a mitochondrial transit peptide. A second familiar MnSOD is present in the mitochondria (mtMnSOD). This unique phenomenon occurs in all Crustacea that use haemocyanin for oxygen transport. Molecular phylogeny analysis suggests the MnSOD gene duplication is as old as the origin of the arthropod phylum. cytMnSOD activity in the hepatopancreas changes during the moulting cycle of the crab. Activity is high in intermoult crabs and non-detectable in postmoult papershell crabs. mtMnSOD is present in all stages of the moulting cycle. Despite the lack of cytCu,ZnSOD, crabs have an extracellular Cu,ZnSOD (ecCu,ZnSOD) that is produced by haemocytes, and is part of a large, approx. 160 kDa, covalently-linked protein complex. ecCu,ZnSOD is absent from the hepatopancreas of intermoult crabs, but appears in this tissue at premoult. However, no ecCu,ZnSOD mRNA can be detected, suggesting that the protein is recruited from the haemolymph. Screening of different taxa of the arthropod phylum for Cu,ZnSOD activity shows that those crustaceans that use haemoglobin for oxygen transport have retained cytCu,ZnSOD. It appears, therefore, that the replacement of cytCu,ZnSOD with cytMnSOD is part of an adaptive response to the dynamic, haemocyanin-linked, fluctuations in copper metabolism that occur during the moulting cycle of the crab

Alterations in Prey Capture and Induction of Metallothioneins in Grass Shrimp Fed Cadmium-Contaminated Prey

The aquatic oligochaete Limnodrilus hoffmeisteri from a Cd-contaminated cove on the Hudson River, Foundry Cove, New York, USA, has evolved Cd resistance. Past studies have focused on how the mode of detoxification of Cd by these Cd resistant worms influences Cd trophic transfer to the grass shrimp Palaemonetes pugio. In the present study, we investigate reductions in prey capture in grass shrimp fed Cd-contaminated prey. We also investigate the induction of metal-binding proteins, metallothioneins, in these Cd-exposed shrimp. Grass shrimp were fed held-exposed Cd-contaminated Foundry Cove oligochaetes (for 1 week) or laboratory-exposed Cd-contaminated Artemia salina (for 1 or 2 weeks). Following these exposures, the ability of Cd-dosed and control shrimp to capture live A. salina was compared. Results show that shrimp fed laboratory-exposed Cd-contaminated A. salina for 2 weeks exhibit significant reductions in their ability to successfully capture prey (live A. salina). Reductions in prey capture were also apparent, though not as dramatic in shrimp fed for 1 week on field-exposed Cd-contaminated Foundry Cove oligochaetes. Shrimp were further investigated for their subcellular distribution of Cd to examine if alterations in prey capture could be linked to saturation of Cd-metallothionein. Cd-dosed shrimp produced a low molecular weight (similar to 10,000 daltons) Cd-binding metallothionein protein in a dose- and time-dependent manner. Most importantly, successful prey capture decreased with increased Cd body burdens and increased Cd concentration bound to high molecular weight proteins (i.e., enzymes)

The Paradigm That All Oxygen-Respiring Eukaryotes Have Cytosolic CuZn-Superoxide Dismutase and That Mn-Superoxide Dismutase is Localized to the Mitochondria Does Not Apply to a Large Group of Marine Arthropods

The enzyme superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide radical, is present in the cytosol and mitochondria of all oxygen-respiring eukaryotes. The cytosolic form contains copper and zinc (CuZnSOD), whereas the mitochondrial form contains manganese (MnSOD). The latter protein is synthesized in the cytosol as a MnSOD precursor, containing an N-terminal mitochondrial-targeting sequence. CuZnSOD is sensitive toward cyanide (CN) and hydrogen peroxide (H2O2), but MnSOD is not. Assays for SOD activity in cytosol from the hepatopancreas of the blue crab, Callinectes sapidus, showed the presence of a CN/H2O2-insensitive form of SOD. No CN/H2O2-sensitive CuZnSOD was found. This unexpected phenomenon was shown to occur in all decapod crustacea (crabs, lobsters, shrimp) examined. The cytosolic and mitochondrial SODs of C. sapidus were purified by means of ion-exchange, size-exclusion, and reverse-phase HPLC. The cytosolic SOD is a homodimeric protein, which exists in a monomer−dimer equilibrium (24 kDa ↔ 48 kDa). The protein contains approximately 1 Mn per subunit. No copper or zinc is present. Amino acid sequence analysis identified the novel cytosolic SOD as a MnSOD precursor with an abnormal mitochondrial-targeting sequence. The mitochondrial SOD of C. sapidus is similar to the MnSOD found in other eukaryotes. N-Terminal amino sequences of mitochondrial and cytosolic blue crab MnSOD differ in several positions. The MnSODs are thus encoded for by two different genes. The paradigm that all eukaryotes contain intracellular CuZnSOD and that MnSOD occurs exclusively in the mitochondria appears not to apply to a large group of marine arthropods

Changes in Mitochondrial Gene and Protein Expression in Grass Shrimp, \u3ci\u3ePalaemonetes pugio\u3c/i\u3e, Exposed to Chronic Hypoxia

Spatial and temporal increases of hypoxia in estuaries are of major environmental concern. Since mitochondria consume most of the oxygen in the cell, we examined the potential role of mitochondrial gene and protein expression in adaptation to chronic hypoxia in the grass shrimp Palaemonetes pugio. Grass shrimp were exposed to DO levels slightly above and below the critical pO(2), 1.8 mg/L, for P. pugio, and hypoxia-induced alterations in gene expression were screened using custom cDNA macroarrays. Mitochondrial gene expression was not affected by exposure to moderate hypoxia (2.5 mg/L DO). However, chronic exposure to severe hypoxia (1.5 mg/L DO) for 7 days resulted in an increase of transcription of genes present in the mitochondrial genome (including 16S rRNA and Ccox 1), together with up-regulation of genes involved in Fe/heme metabolism. This pattern was completely reversed by day 14, when a significant down-regulation of these genes was observed. Separating mitochondrial proteins in two dimensions by IEF and reverse phase chromatography, followed by LC/MS/MS of differentially expressed proteins, showed cytochrome c oxidase subunit 2, encoded by Ccox 2, was down-regulated after 12 d exposure to severe hypoxia. It appears therefore that decreases in mitochondrial Ccox gene transcription result in decreased mitochondrial Ccox protein synthesis. These results suggest that mitochondrial genes and proteins show promise as molecular indicators of exposure to hypoxia. (C) 2008 Elsevier Ltd. All rights reserved