Screening strains for microbial biosorption technology of cadmium.

Heavy metals contaminate the environment and provide a threat to public health through drinking water and food chain. Microbial biosorption technology provides a more economical and competitive solution for bioremediation of toxicants such as heavy metals, and microbial genetic modification may modify microbes towards optimal sorption. It is very important to screen suitable strains for this purpose. In this study, three different types of microorganisms Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae were isolated and identified, from uncontaminated soils, and compared their sorption differences with respect to cadmium (Cd2+). We evaluated the effects of contact time and initial concentration on Cd2+ uptake, and found pseudo-second-order kinetic models were more suitable to describe biosorption processes. Adsorption isotherms were used to reflect their biosorption capacity. The maximum biosorption capacities of three strains calculated by the Langmuir model were 37.764, 56.497, and 22.437 mg Cd/g biomass, respectively. In bacteria, Cd2+ biosorption mainly occurred on cell wall, while the difference in biosorption between yeast inside and outside the cell was not significant. We found that due to the structural differences, the removal rate of E. coli surface decreased at a high concentration, while S. cerevisiae still had a lower biosorption capacity. FTIR spectroscopy reflected the difference in functional groups involved in biosorption by three strains. SEM-EDS analysis showed the binding of Cd2+ to microorganisms mainly relied on ion exchange mechanism. Based on the above results, we suggested that B. subtilis is more suitable to get genetically modified for heavy metal biosorption.

Mitigative effects of zinc on cadmium-induced reproductive toxicity in the male freshwater crab Sinopotamon henanense.

Cadmium (Cd) is a highly harmful environmental contaminant, which can cause reproductive toxicity. Zinc (Zn) is an essential trance element that may protect the organism from the harmful effects of Cd. However, the mechanism of Zn against Cd-induced reproductive toxicity remained to be elucidated. The aim of this study was to assess the effects of subchronic exposure to Cd on the relative testis weight (RTW), the histopathology, the activity of stress marker antioxidant enzymes, the level of lipid peroxidation of testis, as well as the mitigative effects of Zn on Cd-induced reproductive toxicity in male freshwater crab Sinopotamon henanense. For this purpose, male crabs were divided into 10 groups including a control group (without metals) and metal exposure groups with Cd alone in three concentrations and Cd combined with Zn in six concentrations for 14 days. The results showed that Cd evoked concentration-dependent reproductive toxicity of male Sinopotamon henanense as showed by decreased RTW, appearance of morphological lesions, increased SOD, CAT, GPx activity, and MDA levels. Nevertheless, Zn combined with Cd exposure significantly alleviated Cd-induced reproductive toxicity as proved by increased RTW, reappearance of normal histological morphology, increased SOD activity, recovered CAT and GPx activity, and decreased MDA levels in testis. Our study demonstrated that the application of Zn can mitigate Cd-induced reproductive toxicity by ameliorating the testicular oxidative stress and improving the antioxidant status.

Clone, identification and functional characterization of a novel toll (Shtoll1) from the freshwater crab Sinopotamon henanense in response to cadmium exposure and Aeromonas hydrophila infection.

Toll is essential in innate immune system which is important for defense against bacterial, fungal and viral infections in invertebrates. Our previous study showed that cadmium (Cd) could change the expression pattern of ShToll3 in the epithelium (gills and midgut from the freshwater crab Sinopotamon henanense) infected by Aeromonas hydrophila. To investigate the diverse innate immune roles of crustacean homolog Tolls, in this study, we cloned Shtoll1 from S. henanense. The full-length cDNA of Shtoll1 was 4746 bp, with an ORF of 3033 bp encoding a putative protein of 111 amino acids, a 5'-untranslated region of 255 bp and a 3'-untranslated region of 1713 bp. Phylogenetic analysis showed that ShToll1 was clustered into the group of DmToll1, DmToll 4 and DmToll 5. In addition, the tissue distribution results showed that Shtoll1 was expressed widely in different tissues, with the highest expression in heamocytes. Besides, Shtoll1 expressions were upregulated in heamocytes and hepatopancreas after A. hydrophila infection. At the same time, the increase of Shtoll1 expressions were examined in heamocytes in response to Cd exposure and A. hydrophila infection in combination. Through western blotting and immunohistochemical analysis, the ShToll1 expressions in heamocytes were increased in response to A. hydrophila and Cd independently as well as in combination. Moreover, the mRNA level of three antimicrobial peptides (AMPs) alf5, alf6, and c-lys, which possibly responded to Cd and A. hydrophila stimulation through Shtoll1, were analyzed. Thus, we conclude that Cd expand the susceptibility of ShToll1 to A. hydrophila infection in heamocytes. This suggest that ShToll1 may contribute to the innate immune defense of S. henanense against A. hydrophila and Cd in heamocytes.

Cadmium bioaccumulation and elimination in tissues of the freshwater mussel Anodonta woodiana.

Experiments were carried out to investigate the bioaccumulation and elimination of cadmium (Cd) in tissues (kidney, gills, digestive gland, mantle, visceral mass, foot, adductor muscle and hemolymph) from the freshwater mussel, Anodonta woodiana. The mussels were exposed to subchronic Cd at concentrations of 0.168 and 0.675 mg L-1 for 28 d of bioaccumulation and 28 d of elimination. During the bioaccumulation phase, Cd bioaccumulations increased in all tissues. The highest bioaccumulation of Cd was found in the kidney. The second-highest and third-highest bioaccumulations of Cd were found in the digestive gland and gills, respectively. The Cd bioaccumulations in the tissues of A. woodiana increased with exposure time and concentration, except for hemolymph, which reached the highest value on d 14. The bioaccumulation factors (BCFs) increased with exposure time, but an inverse relationship was observed between BCFs and exposure concentration. During the elimination phase, the visceral mass showed the highest Cd elimination rate. In the kidney, digestive gland and gills, the elimination rates almost reached 40%, but their concentrations were still higher than in other tissues. Thus, we concluded that the kidney, gills and digestive gland of A. woodiana are target tissues for subchronic Cd toxicity.

Biogenic selenium and its hepatoprotective activity.

Elemental selenium nanoparticles (SeNPs) have multiple biological activities. In this study, we investigated the protective effects of biogenic SeNPs (BioSeNPs) on CCl4-induced liver damage in mice. The results showed that: (i) when compared to sodium selenite (SS), BioSeNPs has a similar tissue distribution after intragastrical administration to mice; (ii) BioSeNPs and SS showed comparable efficacy in increasing the activities of glutathione peroxidase and thioredoxin reductase in liver cell lines, mice blood and liver; (iii) pretreatment with BioSeNPs inhibiting the elevation of activities of various enzymes significantly which included aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase and liver lipid peroxide (p < 0.05 or p < 0.01) in CCl4-treated mice; (iv) activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) were significantly increased (p < 0.05 or p < 0.01) after a pretreatment with BioSeNPs in CCl4-treated mice; (v) histopathological damages in the liver from CCl4-treated mice were ameliorated by a pretreatment with BioSeNPs. In conclusion, these results have shown that BioSeNPs is able to protect the liver from CCl4-induced hepatic damage via increasing the antioxidant capacity and inhibiting oxidative damage. BioSeNPs may have the potential to be used as a trace element food supplement inducing antioxidant bioactivities.

Identification of a novel toll gene (Shtoll3) from the freshwater crab Sinopotamon henanense and its expression pattern changes in response to cadmium followed by Aeromonas hydrophila infection.

Toll signaling is essential for expression of immune genes which are important for defense against bacterial, fungal and viral infections in invertebrates. Although several toll genes have been identified in the crustaceans, none of them has been investigated in freshwater crab Sinopotamon henanense. Moreover, the effect of cadmium (Cd) on toll gene expression has never been examined on the freshwater crabs which live in the sediments and are prone to heavy metal bioaccumulation. Our transcriptomic analysis of hepatopancreas tissue reveals that toll3 gene expression has been decreased when treated with Cd. In this study, we cloned one toll gene (hereby designated Shtoll3) from the crab. The full-length cDNA of Shtoll3 was 4488 bp, with an ORF of 3693 bp encoding a putative protein of 1230 amino acids, a 5'-untranslated region of 414 bp and a 3'-untranslated region of 781 bp. Phylogenetic analysis showed that ShToll3 was clustered into the group of DmToll8. The tissue distribution results showed that Shtoll3 was expressed widely in different tissues, with the highest in gills, and the lowest in hemocytes. Shtoll3 expression was down-regulated only in midguts after Aeromonas hydrophila infection. With Cd presence, Shtoll3 expression in response to A. hydrophila were up-regulated in midguts and gills, which was further confirmed by western blotting analysis. Moreover, the mRNA level of two antimicrobial peptides (AMPs) crustin and c-lys, which possibly responded to Cd and A. hydrophila stimulation through Shtoll3, were analysised. Thus, we conclude that Cd changes the susceptibility of Shtoll3 to A. hydrophila infection in gills and midguts. This suggest that Shtoll3 may contribute to the innate immune defense of S. henanense to A. hydrophila and Cd can modify the immune function in epithelium.

Toxic effects of Pb2+ entering sperm through Ca2+ channels in the freshwater crab Sinopotamon henanense.

Lead (Pb) is a heavy metal that can damage animal sperm. To study the effects of Pb on calcium homeostasis and calcium channel in the sperm of freshwater crab Sinopotamon henanense, the induction of acrosome reaction (AR) and acrosin activity were investigated when crabs were exposed to different Pb concentrations (0, 3.675, 7.35, 14.7, 29.4 and 58.8mg/L) for 3, 5 and 7 d separately. Fluorescent probe Fluo-3/AM was loaded into the sperm, and [Ca2+] in the sperm was measured by fluorescence microscopy and using microplate reader. The calmodulin (CaM) concentration was measured by ELISA method. Verapamil (VRP), a calcium channel blocker, was used to evaluate whether Pb can enter the sperm through calcium channels leading to sperm damage. After sperm were exposed at 50µg/L VRP, 100µg/L Pb, 50µg/L VRP+100µg/L Pb, 1000µg/L Pb and 50µg/L VRP+1000µg/L Pb for 1h in vitro，sperm quality parameters (sperm survival and sperm DNA integrity) and levels of parameters indicating oxidative stress (protein carbonylation [PCO] and malondialdehyde [MDA]) were measured. Our data showed that Pb reduced the induction of acrosome reaction (AR), down-regulated the acrosin activity, decreased the intracellular concentration of Ca2+ and elevated CaM concentration. Compared to controls, Pb alone induced significant stress, as reflected by decreasing sperm survival and sperm DNA integrity, and increasing PCO and MDA contents. In the presence of VRP, 100µg/L Pb-induced stresses were reduced, all the measured parameters in the sperm exposed at 100µg/L Pb returned to control levels. Our results indicate that Pb enters the sperm of the crab S. henanense through calcium channels, the inhibition of which blocks Pb-induced stresses such as sperm quality decline and oxidative damage.

Oxidative damage, ultrastructural alterations and gene expressions of hemocytes in the freshwater crab Sinopotamon henanense exposed to cadmium.

Toxicity of Cd was tested with the hemocytes of the freshwater crab, Sinopotamon henanense, which were exposed to concentrations of 0, 0.725, 1.450, and 2.900mgL-1 Cd for 7, 14 and 21 d. We investigated the effects of Cd on the total antioxidant capacity (TAC), and oxidative damage of biomarkers, such as malondialdehyde (MDA), protein carbonyl derivates (PCO), and DNA-protein crosslink (DPC). Transmission electron microscopy (TEM) was applied to assess ultrastructural changes of hemocytes. The mRNA expression levels of prophenoloxidase (proPO), lysozyme (LSZ), metallothionein (MT), and the activity of phenoloxidase (PO) were also determined. Our results showed that TAC was inhibited by Cd, resulting in an increase of MDA contents, PCO contents, and DPC levels in hemocytes, respectively. Ultrastructural observations revealed that chromatin condensation, nucleus deformation, mitochondrial dilation, rough endoplasmatic reticulum (rER) degranulation and secondary or tertiary lysosomes were observed in hemocytes of crabs exposed to Cd. Meanwhile, the expression levels of proPO were down-regulated, while the activity of PO was up-regulated in hemocytes. The expression levels of LSZ and MT were up-regulated to some extent. Our findings suggest these parameters could be used as biomarkers in the monitoring of heavy metal pollution and quantitative risk assessments of pollutant exposure.

Quality decline and oxidative damage in sperm of freshwater crab Sinopotamon henanense exposed to lead.

Lead (Pb) induces male infertility in vertebrates. Whether lead is related to reproductive abnormalities in aquatic invertebrates remains uncertain. In this work, effects of Pb on the sperm quality and oxidative stress of the freshwater crab Sinopotamon henanense were investigated after 3, 5 and 7d exposure to different Pb concentrations (0, 3.675, 7.35, 14.7, 29.4 and 58.8mg/L). Sperm quality indices including sperm plasma-membrane integrity and acrosomal-membrane integrity were measured by flow cytometry. DNA integrity was measured by fluorescence microscopy. The results showed that Pb levels in sperm increased significantly upon Pb exposure in most treated groups, sperm plasma-membrane integrity, acrosomal-membrane integrity, and DNA integrity were reduced at higher concentrations after 5 d and 7d. Oxidative stress of sperm induced by Pb was reflected in significant up-regulation of reactive oxygen species (ROS) levels after 3, 5 and 7d. A significant reduction of the total antioxidant capacity levels occurred after exposure to 14.7mg/L Pb and above at 7d compared to the control. The results of oxidative damage to lipids, proteins and DNA of sperm showed that malondialdehyde, protein carbonylation and DNA-protein crosslinks were increased in a concentration- and time-dependent manner. Our findings document that Pb can induce harmful effects on several reproductive endpoints in a freshwater crab.

Lead accumulation, oxidative damage and histopathological alteration in testes and accessory glands of freshwater crab, Sinopotamon henanense, induced by acute lead exposure.

Lead (Pb) is one of the most toxic environmental pollutants and known to exert multiple toxic effects including gonadotoxic and spermiotoxic effects. In order to understand toxic mechanisms of lead (Pb) on the testes and the accessory glands of crabs, we investigated Pb accumulation in testes and accessory glands and the survival rate of sperms of freshwater crab, Sinopotamon henanense. The tissue damaging effects of Pb was also investigated by histopathological examination and analyses of antioxidant enzymes as well as lipid peroxidation. Crabs were exposed to different Pb concentrations (0, 3.675, 7.35, 14.7, 29.4 and 58.8 mg/L) for 3, 5 and 7 days. The results showed that Pb levels in testes and accessory glands increased significantly following Pb exposure for 5 and 7 days in almost all treated groups, and survival rate of sperm decreased with increasing Pb concentrations at 5 and 7 days. Morphological changes identified histologically were discovered in testes, including a disordered arrangement of germ cells, a decreased number of sperm in the lumina of the seminiferous tubules, extensive necrosis in the germinal layer of the seminiferous tubules, etc. At the same time, histological abnormalities were discovered in accessory glands, the wall cells were separated from the basement membrane, and wall cells were missing partly. The activities of SOD, GPx and CAT in testes showed no statistically significant changes compared to the control for 3 days, and initially increased and subsequently decreased with increasing Pb concentrations at 5 and 7 days. The antioxidant enzyme activities in accessory glands initially increased and subsequently decreased with increasing Pb concentrations and Pb exposure. This was accompanied with an increase in malondialdehyde (MDA) content in a concentration-dependent manner. These results showed that acute Pb exposure led to a reduction of survival rate of sperm and harmful effects at the cellular level of crab testes and accessory glands, which are most likely linked to Pb-induced oxidative stress.

Subcellular distribution of Cd and Zn and MT mRNA expression in the hepatopancreas of Sinopotamon henanense after single and co-exposure to Cd and Zn.

The freshwater crab Sinopotamon henanense was exposed to Cd and Zn either on their own or in combination. At 14 and 28 days the hepatopancreas was taken and total metal and metallothionein MT) in transcript abundance were assessed. In addition, the subcellular contents of Cd and Zn also were examined following a differential centrifugation. The results showed that concentration of Cd was more responsive to waterborne metal exposures than the concentration of Zn; Zn was better regulated and exhibited only 2-3 fold increases relative to the control. Of the subcellular fractions, the heat stable protein (HSP) fraction was the predominant metal-binding compartment for Cd. The proportion and accumulation of Cd in this fraction increased with the single Cd exposures, which suggest that metallothionein-like proteins play a key role in metal detoxification in the hepatopancreas of S. henanense. Despite the increases in Cd in the HSP fraction during single Cd exposures, some accumulation of Cd was observed in metal sensitive fraction (MSF), which showed that metal detoxification was incomplete. The results demonstrated that the Cd content decreased in metal mixture groups especially when mixed with higher Zn, while the Cd accumulation in MSF was also reduced when Zn was added. MT mRNA expression was also determined in our report, the results showed that both Cd and Zn had the ability of inducing MT mRNA expression. Additionally, the MT mRNA transcription level was enhanced when Cd was mixed with Zn.

Cadmium inhibits molting of the freshwater crab Sinopotamon henanense by reducing the hemolymph ecdysteroid content and the activities of chitinase and N-acetyl-ß-glucosaminidase in the epidermis.

Molting is an essential process during the growth of crustaceans, which is coordinated by ecdysteroids secreted by the Y-organ, molting inhibiting hormone secreted by the X-organ sinus-gland complex, as well as chitinase and N-acetyl-ß-glucosaminidase synthesized by the epidermis. Cadmium is one of the toxic metals in the aquatic environment. However, the endocrine effects of cadmium on the molting of freshwater crabs and the underlying mechanisms are unknown. To investigate these, freshwater crabs (Sinopotamon henanense) were acutely exposed to 0, 7.25, 14.5 and 29 mg/l Cd for 3, 4, 5 days or in some experiments for 4 days after eyestalk-ablation. The concentration of hemolymph ecdysone and the activities of the molting enzymes chitinase and NAG were measured. Histological changes in the epidermal tissues were documented. Our results showed that eyestalk ablation increased the ecdysteroid content as well as the activities of chitinase and NAG, which were inhibited by cadmium in a concentration-dependent manner; histological examinations demonstrated that eyestalk ablation produced storage particles in the epidermal tissues, which was also reduced by cadmium in a concentration-dependent manner. Our data suggest that cadmium disrupts endocrine function through inhibiting the secretion of ecdysteroids by the Y-organ and altering with the regulation of chitinase and NAG activity in the epidermis. This work provides new insights into the mechanisms underlying the molting inhibition effect of cadmium on the crabs.

Reduction of selenite to red elemental selenium by Rhodopseudomonas palustris strain N.

The trace metal selenium is in demand for health supplements to human and animal nutrition. We studied the reduction of selenite (SeO3⁻²) to red elemental selenium by Rhodopseudomonas palustris strain N. This strain was cultured in a medium containing SeO3⁻² and the particles obtained from cultures were analyzed using transmission electron microscopy (TEM), energy dispersive microanalysis (EDX) and X ray diffraction analysis (XRD). Our results showed the strain N could reduce SeO3⁻² to red elemental selenium. The diameters of particles were 80-200 nm. The bacteria exhibited significant tolerance to SeO3⁻² up to 8.0 m mol/L concentration with an EC50 value of 2.4 m mol/L. After 9 d of cultivation, the presence of SeO3²â» up to 1.0 m mol/L resulted in 99.9% reduction of selenite, whereas 82.0% (p<0.05), 31.7% (p<0.05) and 2.4% (p<0.05) reduction of SeO3⁻² was observed at 2.0, 4.0 and 8.0 m mol/L SeO3²â» concentrations, respectively. This study indicated that red elemental selenium was synthesized by green technology using Rhodopseudomonas palustris strain N. This strain also indicated a high tolerance to SeO3⁻². The finding of this work will contribute to the application of selenium to human health.

Expression of metallothionein of freshwater crab (Sinopotamon henanense) in Escherichia coli enhances tolerance and accumulation of zinc, copper and cadmium.

Metallothioneins (MTs) are ubiquitous metal-binding, cysteine-rich, small proteins and play a major role in metal homeostasis and/or detoxification in all organisms. In a previous study, a novel full length MT gene was isolated from the freshwater crab (Sinopotamon henanense), a species widely distributed in Shanxi and Henan Provinces, China. In this report, the gene for the crab MT was inserted into a PET-28a-6His-SUMO vector and recombinant soluble MT was over-expressed as fusions with SUMO in Escherichia coli. The recombinant fusion protein was purified by affinity chromatography and its biochemical properties were analyzed. In addition, on the basis of constructing SUMO-MT, two mutants, namely SUMO-MTt1 and SUMO-MTt2, were constructed to change the primary structure of SUMO-MT using site-directed mutagenesis techniques with the amino acid substitutions D3C and S37C in order to increase metal-binding capacity of MT. E. coli cells expressing SUMO-MT and these single-mutant proteins exhibited enhanced metal tolerance and higher accumulation of metal ions than control cells. The results showed that the bioaccumulation and tolerance of Zn(2+), Cu(2+) and Cd(2+) in these strains followed the decreasing order of SUMO-MTt1 > SUMO-MTt2 > SUMO-MT. E. coli cells have low tolerance and high accumulation towards cadmium compared to zinc and copper. These results show that the MT of S. henanense could enhance tolerance and accumulation of metal ions. Moreover, we were able to create a novel protein based on the crab MT to bind metal ions at high density and with high affinity. Therefore, SUMO-MT and its mutants can provide potential candidates for heavy metal bioremediation. This study could help further elucidate the mechanism of how the crab detoxifies heavy metals and provide a scientific basis for environment bioremediation of heavy metal pollution using the over-expression of the crab MT and mutant proteins.

Biochemical, histological and ultrastructural alterations of the alimentary system in the freshwater crab Sinopotamon henanense subchronically exposed to cadmium.

Cadmium (Cd) is one of the toxic metals in the aquatic environment. We investigated the effects of Cd on the digestive enzymes, antioxidant enzymes, malondialdehyde (MDA) content and morphology of the hepatopancreas and intestine in the freshwater crab Sinopotamon henanense. Crabs were exposed to sublethal Cd concentrations of 0, 0.725, 1.450 and 2.900 mg/L for 21 days. After Cd exposure, the activities of maltase, amylase and trypsin of two tissues were lower than the control. The activities of superoxide dismutase, catalase and glutathione peroxidase in the hepatopancreas and intestine were decreased, and the MDA concentration increased in all of the treated groups, over the experimental period. The results of light and transmission electron microscopy showed that 2.900 mg/L of Cd exposure caused profound morphological damages in the hepatopancreas and midgut. After exposure, histological abnormalities of two tissues were discovered, including cellular swelling and necrosis. Additionally, alterations in microvilli, nucleus, mitochondria, rough endoplasmic reticulum as well as Golgi complex in epithelial cells of two tissues were observed. This may be due to antioxidant enzymes activities that were reduced by Cd in the alimentary system of the crabs, and led to membrane lipid peroxidation. The membrane structure was destroyed, and caused further tissue damage, which likely made the alimentary system unable to secrete digestive enzymes, leading to further reduction of digestive enzymes.

The response of metallothionein and malondialdehyde after exclusive and combined Cd/Zn exposure in the crab Sinopotamon henanense.

The purpose of this paper is to show the interactions of Cd and Zn in the freshwater crab Sinopotamon henanense through metallothionein (MT) and malondialdehyde (MDA) level measurements. Laboratory acclimated S.henanense were exposed to Cd (50 µg/L, 100 µg/L, 500 µg/L ), and Zn (100 µg/L, 1000 µg/L) alone and in combined treatments (100 µg/L Zn+50 µg/L Cd, 100 µg/L Zn+100 µg/L Cd, 100 µg/L Zn+500 µg/L Cd, 1000 µg/L Zn+50 µg/L Cd, 1000 µg/L Zn+100 µg/L Cd, 1000 µg/L Zn+500 µg/L Cd) for 7, 14, 21, 28, 35 days. The results demonstrated that the MDA contents increased with exposure time and dose and showed time- and dose-dependence in both gills and hepatopancreas of S.henanense after single Cd exposure, while the changes of MDA levels were not significant with single Zn exposure. The MDA levels decreased when the crabs were exposed to metal mixtures compared to Cd exposure alone, indicating that Zn mediated the cellular toxicity of Cd. MT contents increased after single Cd exposure and also showed a time- and dose-dependence, in a tissue-specific way. Zn showed a limited ability of MT induction both in gills and hepatopancreas of S.henanense. The MT contents represented not a simple addition of single metal exposures but were enhanced at a higher concentration of Zn combined with different Cd concentrations compared to single metal exposure. Whether MT can be used as a biomarker for complex field conditions need to be considered cautiously since different induction patterns of MT were found among single Zn, Cd and combined groups. It is suggested that several biomarkers together as a suite should be used in the monitoring of heavy metal pollution in the aquatic environment.

Effects of cadmium on lipid storage and metabolism in the freshwater crab Sinopotamon henanense.

Since environmental effects of molecular traits are often questioned we analyze here the molecular effects of cadmium (Cd) on lipid pathways and their effects on tissues development. Lipids are an important energy source for the developing embryo, and accumulate in the ovary and hepatopancreas of decapod crustaceans. The extend of Cd affecting lipid storage and metabolism, is studied here with the freshwater crabs Sinopotamon henanense. Crabs were exposed to water-born Cd at 1.45, 2.9, 5.8 mg/l for 10, 15, and 20 days. With significantly increased Cd accumulation in exposed crabs, lipid content in hepatopancreas and ovary showed a time-dependent and concentration-dependent reduction, being at least one of the reasons for a lower ovarian index (OI) and hepatopancreatic index (HI). After 10-day exposure increased triglyceride (TG) level in hemolymph and up-regulation of pancreatic lipase (PL) activity in the hepatopancreas suggested an increased nutritional lipid uptake. However, two processes led to lower lipid levels upon Cd exposure: an increased utilization of lipids and a down-regulated lipoprotein lipase (LPL) led to insufficient lipid transport. 10-day Cd exposure also triggered the production of ß-nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt hydrate (NADPH), as well as to the synthesis of adenosine triphosphate (ATP) and fatty acids. With increasing exposure time, the crabs at 15 and 20-day exposure contained less lipid and TG, suggesting that more energy was consumed during the exposure time. Meanwhile, the level of NADPH, ATP and the activity of PL, LPL, fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) activity was down-regulated suggesting an impairment of the crab metabolism by Cd in addition to causing a lower lipid level.

Oxidative damages and ultrastructural changes in the sperm of freshwater crab Sinopotamon henanense exposed to cadmium.

The toxicity of several waterborne heavy metals to aquatic organisms is associated with oxidative damages due to the formation of reactive oxygen species (ROS). In the present work, the reproductive toxicity of the heavy metal cadmium was tested with the sperm of freshwater crab. The crabs were exposed to sublethal concentrations of 0, 7.25, 14.5, 29, 58 and 116 mg/L of Cd²âº for 3, 5 and 7 d. Cd²âº accumulation, ROS formation, and the total antioxidant capacity (TAC) in Sinopotamon henanense sperm were measured. Biomarkers of oxidative damage to lipid (Malondialdehyde, MDA), proteins (protein carbonyl derivates, PCO) and DNA (DNA-protein crosslinks, DPC) were investigated to address Cd²âº effects on crucial macromolecules of the S. henanense sperm. Transmission electron microscopy (TEM) was applied to assess ultrastructural changes induced by 29 and 116 mg/L Cd²âº exposure for 7 d. The results showed that sperm Cd²âº levels were significantly increased at 3, 5 and 7 d starting from the 14.5 mg L⁻¹ Cd²âº-treated groups. Meanwhile, ROS levels were significantly increased over the experimental period. In terms of TAC, statistically significant changes were observed only at day 7 with the Cd²âº concentrations of 14.5, 29, 58 and 116 mg/L. This resulted in an increase of MDA content (5 d and 7 d), PCO content (Cd²âº: 58 and 116 mg/L, 7 d), and DPC levels (Cd²âº: 116 mg/L, 3 d and 7 d), by 26.32%, 37.47%, 22.04%, respectively, in the 116 mg/L Cd²âº group at day seven. For ultrastructural observations, the sperm membrane became wrinkled and partly dissolved, the nuclear envelope turned wrinkled and the chromatin condensed, the acrosome was incomplete with a damaged acrosomal membrane in crabs treated with 29 mg/L Cd²âº for 7 d. After treatment with 116 mg/L Cd²âº for 7 d, the sperm membrane was almost dissolved, the chromatin in the nucleus was more heavily condensed, chromatin irregularities and serious acrosome damage were observed.