Starvation induces hepatopancreas atrophy in Chinese mitten crab (Eriocheir sinensis) by inhibiting angiogenesis.

BACKGROUND: The hepatopancreas of crustaceans serves as a significant organ for both the synthesis and secretion of digestive enzymes, as well as energy storage. In the event of food shortage, the hepatopancreas can provide energy for survival. To investigate the potential regulatory mechanisms of the hepatopancreas in response to starvation in Eriocheir Sinensis, transcriptome analysis, histological study and qRT-PCR were performed. RESULTS: The results showed that starvation caused a decrease in the hepatopancreas index of E. sinensis, which had certain effects on the tissue structure, metabolism and angiogenesis in the hepatopancreas. In addition, WGCNA and linear regression analysis showed that the genes significantly related to the hepatopancreas index were mainly enriched in the angiogenesis pathway, in which AKT signaling played an important role. Starvation may inhibit AKT signaling pathway by reducing the expression of TGFBI, HSP27, HHEX, and EsPVF1, thereby hindering angiogenesis, promoting apoptosis, and leading to hepatopancreas atrophy. CONCLUSION: These results indicate that AKT plays an important role in the angiogenesis pathway and apoptosis of the starvation induced hepatopancreas index reduction, which is beneficial to further understand the effect of starvation stress on hepatopancreas of Chinese mitten crab.

Multifunctional carbonized nanogels to treat lethal acute hepatopancreatic necrosis disease.

BACKGROUND: Shrimp aquaculture has suffered huge economic losses over the past decade due to the outbreak of acute hepatopancreatic necrosis disease (AHPND), which is mainly caused by the bacteria Vibrio parahaemolyticus (V. parahaemolyticus) with the virulence pVA1 plasmid, which encodes a secretory photorhabdus insect-related (Pir) toxin composed of PirA and PirB proteins. The Pir toxin mainly attacks the hepatopancreas, a major metabolic organ in shrimp, thereby causing necrosis and loss of function. The pandemic of antibiotic-resistant strains makes the impact worse. METHODS: Mild pyrolysis of a mixture of polysaccharide dextran 70 and the crosslinker 1,8-diaminooctane at 180 â„ƒ for 3 h to form carbonized nanogels (DAO/DEX-CNGs) through controlled cross-linking and carbonization. The multifunctional therapeutic CNGs inherit nanogel-like structures and functional groups from their precursor molecules. RESULTS: DAO/DEX-CNGs manifest broad-spectrum antibacterial activity against Vibrio parahaemolyticus responsible for AHPND and even multiple drug-resistant strains. The polymer-like structures and functional groups on graphitic-carbon within the CNGs exhibit multiple treatment effects, including disruption of bacterial membranes, elevating bacterial oxidative stress, and neutralization of PirAB toxins. The inhibition of Vibrio in the midgut of infected shrimp, protection of hepatopancreas tissue from Pir toxin, and suppressing overstimulation of the immune system in severe V. parahaemolyticus infection, revealing that CNGs can effectively guard shrimp from Vibrio invasion. Moreover, shrimps fed with DAO/DEX-CNGs were carefully examined, such as the expression of the immune-related genes, hepatopancreas biopsy, and intestinal microbiota. Few adverse effects on shrimps were observed. CONCLUSION: Our work proposes brand-new applications of multifunctional carbon-based nanomaterials as efficient anti-Vibrio agents in the aquatic industry that hold great potential as feed additives to reduce antibiotic overuse in aquaculture.

The effects of ammonia-N stress on immune parameters, antioxidant capacity, digestive function, and intestinal microflora of Chinese mitten crab, Eriocheir sinensis, and the protective effect of dietary supplement of melatonin.

Ammonia nitrogen pollution seriously affects the economic benefits of Chinese mitten crab (Eriocheir sinensis) farming. In this study, we first evaluated the protective effects of melatonin (MT) on immune parameters, antioxidant capacity, and digestive enzymes of E. sinensis under acute ammonia nitrogen stress. The results showed that ammonia-N stress significantly decreased the antibacterial ability of crabs, nevertheless MT could significantly improve it under ammonia-N stress (P < 0.05). Ammonia-N group hemolymph antioxidant capacity indicators (T-AOC, T-SOD, GSH-Px) were significantly decreased than control (p < 0.05), while the MT ammonia-N group hemolymph T-SOD activity significantly increased than ammonia-N group (p < 0.05). For hepatopancreas, ammonia-N group GSH-PX activity significantly decreased than control group, but MT ammonia-N group was significant increased than ammonia-N (p < 0.05). Ammonia-N stress has significantly increased the content of MDA in hemolymph and hepatopancreas (p < 0.05), but MT ammonia-N treatment significantly decreased than ammonia-N group (p < 0.05). Compared with the control group, ammonia-N significantly reduced the activities of Trypsin in the intestine and hepatopancreas (p < 0.05), while MT ammonia-N group can significantly improve the intestinal trypsin activity than ammonia-N (p < 0.05). The intestinal microbiota of E. sinensis results showed that ammonia-N stress significantly decreased the relative abundance of Bacteroidetes (p < 0.05). Ammonia-N stress significantly decreased the Dysgonomonas and Rubellimicrobium, and the Citrobacter significantly increased. In summary, melatonin has a protective effect on E. sinensis under ammonia-N stress. Acute ammonia-N stress may lead to the decrease of probiotics and the increase of pathogenic bacteria, which may be closely related to the impairment of digestive function and immune function.

Therapeutic effect and immune mechanism of chitosan-gentamicin conjugate on Pacific white shrimp (Litopenaeus vannamei) infected with Vibrio parahaemolyticus.

To explore the disease resistance mechanism of chitosan conjugates, chitosan-gentamicin conjugate (CS-GT) was synthesized and systematically characterized, the immune mechanism of CS-GT on Litopenaeus vannamei infected with Vibrio parahaemolyticus was further explored. The results showed that imine groups in CS-GT were effectively reduced. Dietary supplementation of CS-GT can significantly increase the survival rate, total hemocyte counts, the antioxidant and immune related enzyme activity levels of shrimps (P < 0.05), which are all dose-dependent under the experimental conditions. In addition, CS-GT can protect the hepatopancreas from invading bacteria and alleviate inflammation. Particularly, CS-GT promotes the expressions of legumain (LGMN), lysosomal acid lipase (LIPA) and Niemann-Pick type C2 (NPC2) up-regulated. It is speculated that CS-GT may stimulate the lysosome to phagocytose pathogens more effectively. In conclusions, shrimps fed with CS-GT can produce immune response via lysosome and greatly improve the disease resistance to Vibrio parahaemolyticus.

Accumulation and histopathological effects of cadmium on the great pond snail Lymnaea stagnalis Linnaeus, 1758 (Gastropoda: Pulmonata).

Toxic metal ions are an important stress factor for a living organism. In this study, accumulation and histopathological changes in foot, mantle and hepatopancreas of great pond snail, Lymnaea stagnalis exposed to different Cadmium (Cd) concentrations in laboratory conditions were investigated. Great pond snails were exposed to sublethal concentrations of 7.92 µg/L, 15.85 µg/L, 31.7 µg/L and 63.4 µg/L Cd. At the end of 7, 14, 21 and 28 days, snail foot, mantle and hepatopancreas were removed to investigate and determine Cd accumulation and histopathological alterations by light microscopy. Cd levels determined in hepatopancreas were higher than those measured from the foot and the mantle of studied specimens. A positive correlation was found between Cd levels in tissues and exposure days. As a result of Cd application, we observed increase in the number of mucosit, pigment and protein cells and desquamation in the epithelium in the foot, atrophy in muscle fibrils, connective tissue cells and increase in the lipid vacuoles in the mantle, increase in the lipid vacuoles and amoebocyte in the hepatopancreas. The severity of the alterations resulting from Cd increased with dose-time dependent.

Astaxanthin Attenuates Fish Oil-Related Hepatotoxicity and Oxidative Insult in Juvenile Pacific White Shrimp (Litopenaeus vannamei).

The present study investigated the effect of dietary astaxanthin (AX) on the growth performance, antioxidant parameters, and repair of hepatopancreas damage in Pacific white shrimp (Litopenaeus vannamei). To evaluate the hepatopancreas protective function of AX in shrimps, we compared the effect of five isonitrogenous and isoenergetic diets under oxidized fish oil conditions with varying AX levels during the 50-day experimental period. The formulated diets were as follows: (i) OFO (oxidized fish oil); (ii) OFO/AX150 (oxidized fish oil + AX150 mg/kg); (iii) OFO/AX250 (oxidized fish oil + AX250 mg/kg); (iv) OFO/AX450 (oxidized fish oil + AX450 mg/kg); and, (v) control group (fresh fish oil). Results showed that the oxidized fish oil with 275.2 meq/kg peroxide value (POV) resulted in a substantial decrease in the final body weight of L. vannamei (P > 0.05) and induced some visible histopathological alterations in the hepatopancreas. Growth performance was significantly higher in shrimps fed with the OFO/AX450 diet than those fed with the OFO diet (p < 0.05). However, no significant difference was observed when the OFO/AX450 diet was compared to the control diet containing fresh fish oil (p > 0.05). Moreover, shrimps under the OFO/AX450 diet displayed a significant improvement in hepatopancreatic health and showed a reduction of malondialdehyde (MDA) compared to those under the OFO diet (p < 0.05). Dietary AX improved the antioxidant capacity of L. vannamei by increasing the catalase (CAT) activity in the hemolymph. Acute salinity change test showed a higher shrimp survival rate under OFO/AX450 diet than the OFO diet (p < 0.05), suggesting that AX can contribute to enhanced stress tolerance. In conclusion, our data suggest that AX confers dose-dependent protection against OFO-induced oxidative insults and hepatopancreatic damage in shrimp.

Oral immunization with recombinant Lactobacillus casei displayed AHA1-CK6 and VP2 induces protection against infectious pancreatic necrosis in rainbow trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis virus (IPNV) primarily infects larvae and young salmonid with serious economic losses, which causes haemorrhage and putrescence of hepatopancreas. To develop a more effective oral vaccine against IPNV infection, the aeromonas hydrophila adhesion (AHA1) gene was used as a targeting molecule for intestinal epithelial cells. A genetically engineered Lactobacillus casei (pPG-612-AHA1-CK6-VP2/L. casei 393) was constructed to express the AHA1-CK6-VP2 fusion protein. The expression of interest protein was confirmed by western blotting and the immunogenicity of pPG-612-AHA1-CK6-VP2/L. casei 393 was evaluated. And the results showed that more pPG-612-AHA1-CK6-VP2/L. casei 393 were found in the intestinal mucosal surface of the immunized group. The Lactobacillus-derived AHA1-CK6-VP2 fusion protein could induce the production of serum IgM and skin mucus IgT specific for IPNV with neutralizing activity in rainbow trouts. The levels of IL-1ß, IL-8 and TNF-α isolated from the lymphocytes stimulated by AHA1-CK6-EGFP produced were significantly higher than EGFP group. For transcription levels of IL-1ß, IL-8, CK6, MHC-II, Mx and TNF-1α in the spleen, the result indicated that the adhesion and target chemokine recruit more immune cells to induce cellular immunity. The level of IPNV in the immunized group of pPG-612-AHA1-CK6-VP2/L. casei 393 was significantly lower than that in the control groups. These data indicated that the adhesion and target chemokine could enhance antigen delivery efficiency, which provides a valuable strategy for the development of IPNV recombination Lactobacillus casei oral vaccine in the future.

Transcriptome analysis of hepatopancreas of Eriocheir sinensis with hepatopancreatic necrosis disease (HPND).

Hepatopancreatic necrosis disease (HPND) is a newly emerging disease in the Chinese mitten crab, Eriocheir sinensis, which has resulted in large economic losses. However, the underlying cause of this disease remains unclear. To better understand the pathogenesis and pathogenic mechanism of HPND, we compared the transcriptome differences of the hepatopancreas of E. sinensis with and without HPND. The analysis yielded > 30 million reads for each sample of three test (with HPND) and three control groups (without HPND). We observed 978 downregulated genes and 644 upregulated genes. Among the gene ontology categories "biological process," "cellular component," and "molecular function", the subcategories cellular process, single-organism process, biological regulation, metabolic process, cell part, organelle, organelle part, binding, and catalytic were enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that "metabolism of xenobiotics by cytochrome P450," "drug metabolism-cytochrome P450," "chemical carcinogenesis," and "material metabolism" were the "five" most significantly enriched pathways in the hepatopancreas of E. sinensis with HPND. The results revealed that material metabolic abnormalities and drug effects from the external environment might be associated with HPND in the Chinese mitten crab. Considering the wide use of pyrethroids for pond cleaning in Xinghua city, we speculated that pyrethroids might cause HPND in the Chinese mitten crab. Our study provided useful information about the cause and pathogenetic mechanisms of HPND and could help to prevent this disease in production practice.

Effects of metazachlor and its major metabolite metazachlor OA on early life stages of marbled crayfish.

The effects of the herbicide metazachlor and its major metabolite metazachlor OA at two concentrations, including environmentally relevant concentrations of metazachlor (0.0115 µmol/l and 0.0790 µmol/l) and metazachlor OA (0.0117 µmol/l and 0.0805 µmol/l), respectively, were evaluated on early ontogeny, growth, behaviour, oxidative stress, antioxidant enzyme levels, histology, and mortality of marbled crayfish Procambarus virginalis. Both tested concentrations of metazachlor and metazachlor OA were associated with significantly lower growth and delayed ontogenetic development compared to controls. Exposure of metazachlor at 0.0115 µmol/l and metazachlor OA at 0.0117 µmol/l and 0.0805 µmol/l resulted in significantly lower activity of total superoxide dismutase (SOD), catalase (CAT), glutathione s-transferase (GST), glutathione reductase (GR), and reduced glutathione (GSH) compared with control and resulted in gill anomalies ranging from wall thinning to focal disintegration of branchial structure. Metazachlor at the environmentally relevant concentration of 0.0790 µmol/l was associated with significant alterations of crayfish distance moved and walking speed. The potential risk associated with metazachlor use in agriculture related to effects on non-target aquatic organisms.

Dietary glutathione supplementation enhances antioxidant activity and protects against lipopolysaccharide-induced acute hepatopancreatic injury and cell apoptosis in Chinese mitten crab, Eriocheir sinensis.

Eriocheir sinensis (E. sinensis) is an important aquaculture species in China. However, deteriorating water environments lead to oxidative stress in these crabs, which subsequently reduces their quality and yield. Glutathione (GSH) is an endogenous antioxidant that is used to mitigate oxidative stress. However, whether dietary GSH can enhance the resistance of E. sinensis to oxidative stress remains unclear. Herein, crabs were fed dietary GSH (the basal diet was supplemented with 0, 300, 600, 900, and 1200 mg/kg diet weight of GSH) for up to 3 weeks and, then, challenged with lipopolysaccharide (LPS; 400 µg/kg body weight). After 6 h, their hepatopancreas were sampled. Diet supplementation with 600 and 900 mg/kg diet weight GSH not only increased the content of GSH in the hepatopancreas, but also enhanced the activities and mRNA expressions of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione-S-transferase (GST) (P < 0.05), compared to that in control crabs challenged with LPS alone. Diet supplementation with 600 or 900 mg/kg GSH also significantly increased the enzyme activities of GSH reductase and γ-glutamyl cysteine synthetase (γ-GCS) in LPS-treated crabs. Haematoxylin-eosin (HE) staining, electron microscopy, and flow cytometry were used to examine the structure and subcellular structure of and apoptosis in the hepatopancreas. The histopathology and sub-microstructure analysis results also showed that diet supplementation with 600 or 900 mg/kg GSH significantly alleviated damage in crabs challenged with LPS and decreased reactive oxygen species (ROS) levels and cell apoptosis ratios in the hepatopancreas, compared to the LPS-treated crabs. To further understand the effect of dietary GSH on LPS-induced apoptosis, the activities and gene or protein expressions of apoptosis-related factors were evaluated. As a result, diet supplementation with 600 or 900 mg/kg GSH significantly decreased the activities of caspases-3, -8, and -9 and inhibited the relative expression of caspase-3 and -8 but increased the expression of B-cell lymphoma-2 (bcl-2) and B-cell lymphoma-2-associated X inhibitor (bax inhibitor) in crabs challenged with LPS. This treatment further significantly downregulated the relative protein levels of caspase-3, -8, -9 and Bax and upregulated those of Bcl-2 in crabs challenged with LPS. However, treatment with 1200 mg/kg GSH caused the opposite effects. Overall, our results reveal that appropriate diets supplemented with 600 or 900 mg/kg GSH could enhance the antioxidant capacity and anti-apoptotic mechanisms in crabs after LPS injection, thereby providing a theoretical basis for the application of dietary GSH in E. sinensis.

Waterborne zinc induced lobe-dependent effect on oxidative stress and energy metabolism in hepatopancreas of Larimichthys crocea.

The study aimed to compare differences in oxidative stress and energy metabolism between the left and right lobe of hepatopancreas in large yellow croaker Larimichthys crocea exposed to 0 (control), 20, and 100 µM Zn for 96 h. Tipical biomarkers were examined including the proportion of white hepatopancreas, lipid content, malondialdehyde (MDA) level, glutathione (GSH) content, activity levels of enzymes (Cu/Zn-superoxide dismutase, Cu/Zn-SOD; catalase, CAT; glutathione peroxidase, GPx; glutathione reductase, GR; mitochondrial ATP synthase, F-ATPase; malate dehydrogenase, MDH; succinate dehydrogenase, SDH; hepatic lipase, HTGL; lipoprotein lipase, LPL), mRNA levels of genes encoding these enzymes (sod1, cat, gpx1a, gr, atp5b, mdh, sdh, htgl, and lpl), and gene expression of signaling molecules the NF-E2-related nuclear factor 2 (nrf2) and Kelch-like ECH-associated protein 1 (keap1). A whitish color in the left lobe of hepatopancreas was observed in the control and Zn-exposed fish. Contrarily, the right lobe of hepatopancreas tended towards red with increasing Zn levels. The phenomenon was further confirmed by that lipid content was reduced in the right lobe and was not significantly affected in the left lobe by Zn. The right lobe showed higher energy consumption than the left lobe as reflected by the up-regulation of activity levels of HTGL, LPL, F-ATPase, MDH, and SDH. Lipid peroxidation declined by 20 µM Zn and was unchanged by 100 µM Zn in both lobes, which could be explained by increased activity levels of Cu/Zn-SOD and GPx. However, the magnitude of increase in Cu/Zn-SOD activity was greater in the right lobe than that in the left one. The difference in enzyme activity between two lobes may be involved in changes in mRNA levels of sod1, gr, atp5b, sdh, htgl, lpl, and nrf2, which was further confirmed by positive relationships between enzyme activity and gene expression. Our data also showed positive correlations between nrf2 expression and mRNA levels of its target genes, suggesting that Nrf2 was required for the protracted induction of these genes. Our results demonstrated the potential molecular mechanism of Zn-induced differences between lobes of hepatopancreas, suggesting that the sampling part of hepatopancreas should be considered with caution when assessing metal contamination.

Changes in growth performance, haematological parameters, hepatopancreas histopathology and antioxidant status of pacific white shrimp (Litopenaeus vannamei) fed oxidized fish oil: Regulation by dietary myo-inositol.

A 58-day feeding trial was conducted to evaluate the effects of dietary myo-inositol (MI) supplementation on growth performance, haematological parameters, hepatopancreas histopathology and antioxidant status of Litopenaeus vannamei fed with oxidized fish oil (OFO). Control diet contained fresh fish oil (FFO) without MI supplementation. The other four diets contained two oxidation levels of OFO (peroxide value: 133.2 and 268.7 meq kg-1) with or without 200 mg MI kg-1 diets (MI0+L, MI0+H, MI200 + L and MI200 + H). Results showed that OFO-supplemented groups (without MI supplementation) showed better growth performance and lower whole-body inositol content when opposed to control group. MI supplementation significantly improved whole-body inositol content in high-oxidized fish oil (HOFO) groups, and also reduced whole-body lipid in low-oxidized fish oil (LOFO) groups. Moreover, Supplementation of OFO and MI markedly hit the fatty acid profile of muscle. HOFO caused severe histopathological changes in hepatopancreas of shrimp, which slightly alleviated by MI supplementation. MI supplementation also grew the total protein (TP) content and alkaline phosphatase (AKP) activity and decreased the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of serum in OFO-supplemented groups. Ingestion of OFO increased levels of lipid peroxidation and protein oxidation in serum or hepatopancreas, which partly ameliorated by MI supplementation. Activities of antioxidant enzymes exhibited different expression patterns because of OFO and MI. In addition, HOFO markedly increased mRNA expression levels of antioxidant genes including ferritin (FT), thioredoxin (Trx), GPX, glutathione S-transferase (GST) and catalase (CAT) and decreased peroxiredoxin (Prx) expression, in which expression of GPX and Prx were increased owing to MI supplementation. Therefore, it suggested that dietary OFO stimulated growth performance, but also induced oxidative stress and caused impairment to hepatopancreas in L. vannamei. The negative impact brought about by OFO was partially mitigated by dietary MI supplementation.

Detoxification and immunoprotection of Zn(II)-curcumin in juvenile Pacific white shrimp (Litopenaeus vannamei) feed with aflatoxin B1.

Aflatoxins, which was produced by Aspergillus flavus or Aspergillus parasiticus fungi during grain and feed processing or storage, could cause severe health problems and reduction of yield during shrimp cultures. To evaluate toxic effects of aflatoxin B1 (AFB1) in juvenile Pacific white shrimp (Litopenaeus vannamei) and potential protective effect of Zn(II)-curcumin (Zn-CM), four experimental diets (control, 500 µg/kg AFB1, 500 µg/kg AFB1+100 mg/kg Zn-CM, 500 µg/kg AFB1+200 mg/kg Zn-CM) were formulated in quadruplicate to feed the shrimp for 8 weeks. The results revealed that AFB1 could induce significant decrease in final body weight (FBW), weight gain (WG, %) and visible variations of the hepatopancreas structures in L.vannamei. Compared with AFB1 group, AFB1+100 mg/kg Zn-CM group significantly ameliorated the toxic effects of AFB1 on growth performance, while AFB1+100 mg/kg Zn-CM group had no effect on growth performance. Dietary AFB1+100 mg/kg Zn-CM enhanced phenoloxidase (PO) (P < 0.05) activity. Both dietary AFB1+100 mg/kg Zn-CM and AFB1+200 mg/kg Zn-CM reduced inducible nitric oxide synthase (iNOS) activity and glutathione (GSH) level, decreased the content of malondialdehyde (MDA) (P < 0.05) in hepatopancreas compared with AFB1 group. Transmission electron microscopy (TEM) analysis demonstrated that Zn-CM could relieve the microvilli transformation and mitochondria accumulation reduction caused by AFB1. Consequently, the results demonstrated that suitable Zn-CM could mitigate the AFB1-induced hepatotoxicity and immunotoxicity effects on L.vannamei.

Crude oil toxicity to fiddler crabs (Uca longisignalis and Uca panacea) from the northern Gulf of Mexico: Impacts on bioturbation, oxidative stress, and histology of the hepatopancreas.

The intensive drilling and extraction of fossil fuels in the Gulf of Mexico result in a considerable risk of oil spills impacting its coastal ecosystems. Impacts are more likely to be far-reaching if the oil affects ecosystem engineers like fiddler crabs, whose activities modify biogeochemical processes in the sediment. The present study investigated effects of oil on the fiddler crabs Uca longisignalis and Uca panacea, which are important as ecosystem engineers and as prey for a wide variety of species. The present study used mesocosms and microcosms to investigate the effects of crude oil on fiddler crab burrowing and to assess cellular and tissue damage by the oil. Fiddler crabs were exposed for periods of 5 or 10 d to oil concentrations up to 55 mg/cm2 on the sediment surface. Their burrowing was delayed, their burrows were smaller, and they transported less sediment in the presence of oil. The hepatopancreas had elevated levels of oxidative stress and a higher abundance of blister cells, which play a role in secretory processes. Interspecific differences were observed; most effects were strongest in U. panacea, though burrowing was more strongly affected in U. longisignalis. The present study demonstrates that crude oil is likely to impact fiddler crabs and many species that depend on them for their diet or for the ecological changes that result from their burrowing. Environ Toxicol Chem 2018;37:491-500. © 2017 SETAC.

Effect of cadmium exposure on hepatopancreas and gills of the estuary mud crab (Scylla paramamosain): Histopathological changes and expression characterization of stress response genes.

Cadmium (Cd) is a heavy metal that accumulates easily in organisms and causes several detrimental effects, including tissue damage. Cd contamination from anthropogenic terrestrial sources flows into rivers, and through estuaries to the ocean. To evaluate the toxic effects of Cd on estuary crustaceans, we exposed the mud crab Scylla paramamosain to various Cd concentrations (0, 10.0, 20.0, and 40.0mg/L) for 24h. We also exposed mud crabs to a fixed Cd concentration (20.0mg/L) for various periods of time (0, 6, 12, 24, 48, and 72h). We observed that after exposure to Cd, the surfaces of the gill lamellae were wrinkled, and the morphologies of the nuclei and mitochondria in the hepatopancreas were altered. We analyzed the expression profiles of 36 stress-related genes after Cd exposure, including those encoding metallothioneins, heat shock proteins, apoptosis-related proteins, and antioxidant proteins, with quantitative reverse transcription PCR. We found that exposure to Cd altered gene expression, and that some genes might be suitable bioindicators of Cd stress. Gene expression profiles were organ-, duration-, and concentration-dependent, suggesting that stress-response genes might be involved in an innate defense system for handling heavy metal exposure. To the best of our knowledge, this study is the first one of histopathology and stress-response gene expression pattern of Scylla paramamosain after Cd exposure. Our work could increase our understanding of the effect of environmental toxins on estuary crustaceans.

Cell damage and apoptosis in the hepatopancreas of Eriocheir sinensis induced by cadmium.

Cadmium (Cd) is one of the most common pollutants in the environment and it is known to cause a range of tissue damages and apoptosis in invertebrates. In this study, we investigated the effect of Cd on the hepatopancreas of the crab Eriocheir sinensis, a commercially and ecologically important species of crustacean. The crabs were first exposed to water containing different concentrations of Cd2+ (0, 0.63, 1.26, 2.52, 5.04 and 10.07mg/L) for 6days. Typical morphological characteristics and physiological changes of apoptosis were then observed using various methods, including AO/EB double fluorescence staining, transmission electron microscopy and DNA fragmentation analysis. The results showed that Cd2+ induced cell damage and apoptosis in a concentration-dependent manner. Transmission electron microscopy revealed the presence of cellular swelling and necrosis with reduced number of microvilli on the cell surface and damages to individual organelles. The mitochondria became swollen and vacuolated. The rough endoplasmic reticulum (Rer) was expanded, with membrane rupture and many different sizes of vesicles, suggesting the destruction of protein-synthesizing structures in the hepatopancreatic cells. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidases (GPx) initially increased and subsequently decreased with increasing Cd2+ concentrations. This was accompanied by increases in malondialdehyde (MDA) and H2O2 contents, which led to membrane lipid peroxidation. Crabs exposed to Cd2+ also displayed significant increases in caspase-3, -8, and -9 activities compared to control crabs. Cadmium induced the production and accumulation of ROS in the hepatopancreas, which resulted in oxidative damage and abnormal metabolism. Taken together, the results indicated that Cd2+ could induce oxidative damage as well caspase-dependent apoptosis in E. sinensis hepatopancreas.

Metal uptake, oxidative stress and histopathological alterations in gills and hepatopancreas of Callinectes amnicola exposed to industrial effluent.

Metal uptake by biota due to elevated environmental concentrations elicits oxidative stress and could lead to pathological outcomes. The relationship between the histopathological profile of hepatopancreas and gills and altered biochemical features (antioxidant enzymes i.e. GSH, GPx, CAT, SOD, lipid peroxidation (MDA) and serum protein) in the blue crab, Callinectes amnicola from contaminated parts of the Lagos Lagoon was investigated. Monthly crab, sediment and surface water samples were taken from effluent receiving areas of the Lagos lagoon i.e. Makoko, Okobaba, Iddo, Ikoyi and Mid-lagoon (control site) over an 18-month period and analyzed for metal levels (Pb, Cd, Zn and Cu). Significantly higher levels of GPx and lower levels of Pb, Zn and Cu was recorded in gills and hepatopancreas of crabs from the mid-lagoon compared to crabs from other sites. Reaction patterns of gills across the different sites of the lagoon included regressive (ranging from epithelial lifting, disruption of pilaster cells, detached cuticle to focal necrosis) and circulatory disruptions (oedema); increased activity of GSH and GPx in gills were positively correlated with lesions of lower importance factor. Reaction patterns in hepatopancreas were more regressive including vacuolation/infiltration of fatty lobules, necrosis, granuloma, disintegrated lumen, atrophied tubules and loss of lobular hepatocyte structure; increased activity of GSH, GPx and CAT were positively correlated with lesions of low importance factor in the hepatopancreas. Findings show that lesions in both gills and hepatopancreas of the blue crab could be associated with uptake of metals, depleted antioxidant activity and incidence of lipid peroxidation in tissue.

Single and multi-gene phylogeny of Hepatospora (Microsporidia) - a generalist pathogen of farmed and wild crustacean hosts.

Almost half of all known microsporidian taxa infect aquatic animals. Of these, many cause disease in arthropods. Hepatospora, a recently erected genus, infects epithelial cells of the hepatopancreas of wild and farmed decapod crustaceans. We isolated Hepatospora spp. from three different crustacean hosts, inhabiting different habitats and niches; marine edible crab (Cancer pagurus), estuarine and freshwater Chinese mitten crab (Eriocheir sinensis) and the marine mussel symbiont pea crab (Pinnotheres pisum). Isolates were initially compared using histology and electron microscopy revealing variation in size, polar filament arrangement and nuclear development. However, sequence analysis of the partial SSU rDNA gene could not distinguish between the isolates (~99% similarity). In an attempt to resolve the relationship between Hepatospora isolated from E. sinensis and C. pagurus, six additional gene sequences were mined from on-going unpublished genome projects (RNA polymerase, arginyl tRNA synthetase, prolyl tRNA synthetase, chitin synthase, beta tubulin and heat shock protein 70). Primers were designed based on the above gene sequences to analyse Hepatospora isolated from pea crab. Despite application of gene sequences to concatenated phylogenies, we were unable to discriminate Hepatospora isolates obtained from these hosts and concluded that they likely represent a single species or, at least subspecies thereof. In this instance, concatenated phylogenetic analysis supported the SSU-based phylogeny, and further, demonstrated that microsporidian taxonomies based upon morphology alone are unreliable, even at the level of the species. Our data, together with description of H. eriocheir in Asian crab farms, reveal a preponderance for microvariants of this parasite to infect the gut of a wide array of decapods crustacean hosts and the potential for Hepatospora to exist as a cline across wide geographies and habitats.

Histopathological changes in gill and liver of Capoeta capoeta living in the Karasu River, Erzurum.

The contamination of surface waters by different pollutants is an important problem worldwide. In this study, the histopathological effects of water pollution were investigated on freshwater fish species Capoeta capoeta caught from the Karasu River. Fish were caught at three different sites in the Karasu River, namely, Askale, Dumlu, and Serçeme. The histological changes in gill and liver of fish were detected microscopically and evaluated with quantitative analyses. In addition, heavy metals have also been determined in surface water samples from these sites. Results showed that the Askale site was polluted by different kinds of heavy metals. In Askale site, some heavy metals such as Cd, Al, As, Pb, and Mn levels were mostly detected at concentrations above than the accepted values by the Turkish Standards Institute. The presence of gill and liver histological alterations were assessed by the degree of tissue change (DTC). In gill filaments, hypertrophy and hyperplasia of the gill epithelium, lamellar epithelial lifting, lamellae shortening, vasodilatation, lamellar disorganization, blood congestion, fusion, and aneurysm were observed. In the liver, the changes included an increase in the number and size of melanomacrophage aggregates, nonhomogenous parenchyma, proliferation of the hepatopancreas, sinusoidal dilatation, vacuolization, hypertrophy of the hepatocytes, congestion and degeneration of central vein, blood congestion, pyknotic nucleus, focal necrosis, and hepatic granuloma. The histological lesions were comparatively most severe in liver. The DTC means were varied from slight to moderate of gill and moderate to severe of liver tissue in the Askale site, thus the site is considered to be of low quality. Some pathological alterations were observed in the Serçeme site, although their distribution was lower than sites Dumlu and especially Askale. The least DTC means of the Serçeme site demonstrated their good environmental quality. The results suggest that there is a close relationship between amounts of pathological alterations and environmental contamination.

Ecotoxicity of single-wall carbon nanotubes to freshwater snail Lymnaea luteola L.: Impacts on oxidative stress and genotoxicity.

Mammalian studies have raised concerns about the toxicity of carbon nanotubes, but there is very limited data on ecogenotoxicity to aquatic organisms. The aim of this study was to determine eco-geno toxic effects of single walled carbon nanotubes (SWCNTs) in fresh water snail, Lymnea luteola (L. luteola). A static test system was used to expose L. luteola to a freshwater control, 0.05, 0.15, 0.30, 0.46 mg/L SWCNTs for up to 4 days. SWCNTs changed a significant reduction in glutathione, glutathione-S-transferase, and glutathione peroxidase with in hepatopancreas of L. luteola. Lipid peroxidation (LPO) and catalase showed dose- and time-dependent and statistically significant increase in hepatopancreas during SWCNTs exposure compared with control. However, a significant (p < 0.01) induction in DNA damage was observed by the comet assay in hepatopancreas cells treated with SWCNTs. These results demonstrate that SWCNTs are ecogenotoxic to freshwater snail L. luteola. The oxidative stress and comet assay can successfully be used as sensitive tools of aquatic pollution biomonitoring.