Enzymatic and histopathologic biomarkers as indicators of contaminant exposure and effect in Asian clam (Potamocorbula amurensis)

Enzymatic and histopathologic alterations of the digestive gland, gill, gonad, and kidney were studied in Asian clam (Potamocorbula amurensis) in April, 1997 from each of four United States Geological Survey (USGS) stations in the San Francisco Estuary. Stations were selected based on differing body burdens of metallic contaminants in clams (Stn 4.1> 6.1> 8.1>12.5) observed over 7 years. Because no pristine sites are known within the estuary and because no laboratory-reared stocks of P. amurensis were available, clams from station 12.5 served as reference animals. Histopathologic analysis revealed no lesions in clams collected from station 12.5. Mild digestive gland atrophy and moderate distal kidney tubular vacuolation were seen in clams collected from station 8.1. Mild digestive gland atrophy, moderate kidney tubular atrophy, and moderate gill inflammation were seen in clams collected from station 6.1. Lesions found only in clams from station 4.1 were: (1) severe inflammation and moderate atrophy of primary ducts and diverticula, and decreased numbers of heterophagosomes and heterolysosomes in diverticula of the digestive gland; (2) severe gill inflammation; (3) severe kidney tubular atrophy; (4) severe ovarian and testicular inflammation and necrosis; (5) decreased numbers of mature ova; and (6) decreased number of glycogen storage cells in the ovary and testis. Localization of specific enzymes including adenosine triphosphatase (ATP), acid phosphatase (ACP), alkaline phosphatase (ALKP), gamma-glutamyl transpeptidase (GGT), and glucose-6- phosphate dehydrogenase (G6PDH) was performed and correlated, in serial sections with glycogen (PAS) and haematoxylin and eosin stains. Enzymatic analysis revealed: (1) increased digestive diverticula ATP in stations 6.1 and 4.1; (2) decreased digestive diverticula ACP in stations 6.1 and 4.1 and proximal kidney tubular ACP deficiency in station 4.1; (3) no ALKP differences among stations; (4) increased distal kidney tubular GGT at station 12.5 and decreased distal kidney tubular GGT at station 4.1; (5) decreased digestive diverticula G6PDH G6PDH in all stations except 12.5 and decreased proximal kidney tubular G6PDH in stations 8.1 and 6.1. It is possible that other anthropogenic and natural stressors may have affected the results in this study. However, the prevalence and increased severity of lesions in clams with highest metal body burden suggests a contaminant-associated etiology. Enzymatic and histopathologic biomarker alterations identified in this study were positively correlated with the metal body burden. Clams with the higher prevalence of diseases and enzyme alterations also showed a lower condition index and glycogen content in the month when histopathological assessment was performed. Further study will seek to develop enzymatic and histopathologic biomarkers for use in controlled laboratory conditions to help validatethe field study.     

Cardiac and branchial physiology associated with copper accumulation and detoxication in the mytilid mussel Perna viridis (L.)

Acute, sublethal, copper exposures induced bimodal physiological responses in the mytilid mussel Perna viridis. Whole animal studies revealed that copper increased heart rate (mild tachycardia) whilst clearance rates and hence gill cilia were simultaneously inhibited. The inhibitory bioamine dopamine reversed tachycardia, indicating that elevated heart rates were due to nervous (serotonergic) stimulation of the myocytes. The excitatory bioamine 5-hydroxytryptamine failed to reverse copper-induced inhibition of clearance rates, suggesting that mechanical damage or blockage of the cilia rather than overt neurotoxicity was impairing the function of the gill. The digestive diverticula was the major copper-accumulating organ although rapid metal exocytosis was evident when individuals were allowed to depurate in uncontaminated seawater. Waste products are expelled from the digestive diverticula into the intestine and as the rectum passes through the heart, elevated rates of ventricular contraction may facilitate egestion of metal-rich faeces. Elevated cardiac activity during exposure to copper may serve to increase ventricular pulsatile pressure on the rectum for the expulsion of metal-rich faecal inclusions (coproliths) derived from the digestive diverticula lysosomes. This study describes a relationship between the heart, alimentary canal and digestive diverticula lysosomes as a pathway for metal detoxication in mytilid mussels.     

Histophysiology of polysaccharide and lipid reserves in various tissues of Littorina littorea exposed to sublethal concentrations of cadmium

1. Sublethal exposure to cadmium causes glycogen depletion in connective tissues of the mantle, kidney folds, and digestive gland-gonad complex. Glycogen levels are lower at higher environmental concentrations of metal and at longer exposure times.2. Simultaneously with glycogen level reduction in reserve tissues, higher levels of glycogen than in control specimens have been detected in the digestive gland of cadmium exposed winkles. Phosphoglucomutase activity has been detected in kidney, connective tissues, and intestine, but not in digestive tubules. This suggests glycogen mobilisation through digestive tubule epithelia.3. Phosphoglucomutase activity in gills is associated with glycogen level increases in blood vessels and in distal portion of gill lamellae after proximal epithelium disruption.4. Lipid contents of the studied organs are only decreased when glycogen levels are largely reduced. Lipase activity has been demonstrated in digestive tubule, kidney and gill epithelia, but not in connective tissues. It is concluded that lipidic store is intracellular while the polysaccharidic one is organismic.5. Sublethal concentrations of cadmium do not cause impairment of phosphoglucomutase and lipase activities: enzymatic activity is well correlated with reserve consumption, demonstrable activity being lost only after substrate (glycogen or lipid) depletion.     

Glutathione enzymes,glutathione content and t-butyl hydroperoxide induced lipid peroxidation in the gill and digestive gland of the estuarine clam,Rangia cuneata

1. Reduced glutathione (GSH), glutathione reductase (GSSG-reductase) and glutathione peroxidase (GSH-peroxidase) activities were measured in the gill and digestive gland of Rangia cuneata.2. Substantial GSH concentrations were found in both gill (820 ± 80 nmole/g tissue) and digestive gland (930 ± 130 nmole/g tissue). The digestive gland exhibited 2.5-fold greater GSSG-reductase activities and 0.5-fold lower GSH-peroxidase activities relative to the gill.3. In vivo exposure to t-butyl hydroperoxide (BHP) elicited a dose-dependent increase (P < 0.05) in lipid peroxidation in both tissues. Lipid peroxidation occurred earlier and to a greater extent in the digestive gland versus the gill. GSH concentrations in both tissues were unaffected by BHP exposure.4. The study results indicate that gill and digestive gland differ in susceptibility to BHP induced oxidative damage, and the difference is accounted for by differences in tissue GSH metabolism.     

Autometallographical localisation of Cu and Zn within target cell compartments of winkles following exposure to Cu&Zn mixtures

This investigation attempts to determine the usefulness of autometallography to localise particular metals in certain key tissues of molluscs exposed to metal mixtures. For this purpose, winkles (Littorina littorea) removed from shell were exposed to very high concentrations of either copper (Cu), zinc (Zn) or a mixture of both metals (Cu&Zn) dissolved in sea-water for short periods of time. Protein-bound metals were detected by autometallography as black silver deposits (BSD) on histological sections of gills, foot, mantle, digestive gland/gonad complex, stomach and kidney. Copper was localised within cytoplasmic granules of gill ciliated cells, nephrocytes and stomach epithelial cells as well as within digestive cell lysosomes. Zinc was essentially found in the basal lamina (histological sense) of gill, stomach, kidney and digestive gland epithelia. BSD were also evidenced in cytoplasmic granules of pore cells present in parenchymal connective tissue of mantle, foot, gill, digestive gland and stomach. Copper and zinc concentrations were additionally calculated for the whole soft body as well as for certain organs by atomic absorption spectrophotometry (AAS). According to AAS, a synergistic phenomenon would contribute to increase the rate of Cu and Zn accumulation in presence of each other. However, after exposure to Cu&Zn autometallography did not evidence any synergistic phenomenon, and Cu and Zn were localised in their respective accumulation sites. In conclusion, autometallography might indicate the presence of certain metals in the environment irrespective of factors, such as "metal-metal interaction-like" phenomena, affecting metal concentrations in soft tissues.     

Endocrine disruption and health effects of caged mussels, Elliptio complanata, placed downstream from a primary-treated municipal effluent plume for 1 year

Freshwater mussels, Elliptio complanata, were caged in special benthic pens and were immersed at one upstream (Ups) site and two downstream sites (8 and 11 km) of a primary-treated municipal effluent plume for 1 year. The levels of metallothionein-like proteins (MT), lipid peroxidation, protein-free DNA strands and glutathione S-transferase (GST) activity were assayed in digestive gland, gill and gonad tissues to evaluate biological effects and damage. The levels of monoamines (serotonin and dopamine) in nerve ganglia, ATP-dependent transport activity and monoamine oxidase (MAO) activity were also investigated in the homogenates, synaptosomes and mitochondria, respectively. Results showed that significant amounts of sediment accumulated in cages and 82% of mussels survived the yearlong exposure period at the downstream sites. MT-like proteins were induced in all tissues with the following response intensity: gill (3-fold), digestive gland (1.4-fold) and gonad tissues (1.3-fold). Lipid peroxidation decreased (2.5-fold) in digestive gland but increased in gill (1.6-fold) and in gonad tissues (1.5-fold). GST activity was readily increased in digestive gland (2.5-fold), suggesting the presence of organic contaminants in the plume. Levels of protein-free DNA strands did not vary significantly in digestive gland and gill tissues but were significantly reduced in gonad tissues (2.5-fold) relative to the upstream site. In visceral nerve ganglia, both serotonin and ATP-dependent serotonin transport decreased 1.7-fold with a 4-fold increase of 5-hydroxyindole acetate (5-HIAA, a serotonin metabolite) level relative to the upstream site. However, MAO activity was somewhat reduced at downstream sites (0.7- to 0.9-fold of the activity at the upstream site). Dopamine levels were found to be decreased (1.5-fold), but dopamine ATP-dependent transport activity was increased 1.8-fold, suggesting reduced dopaminergic activity. These results indicate that estrogenic chemicals are likely at play, and the increased dopamine and decreased serotonin ATP-dependent transport suggest that the municipal plume was serotonergic for mussels located at the downstream sites. Mussels exposed for 1 year display a complex but characteristic pattern of responses that could lead to harmful health effects including neuroendocrine disruption of reproduction.     

Correlative histochemical studies on preneoplastic and neoplastic lesions in the kidney of rats treated with nitrosamines

Renal tubular lesions induced in male rats by two different carcinogens, N-nitrosomorpholine (NNM) and N-ethyl-N-hydroxyethylnitrosamine (EHEN), using a limited exposure "stop" protocol were investigated histochemically to demonstrate phenotypic cellular changes. The parameters measured included basophilia, glycogen content and the activity of the enzymes glucose-6-phosphatase (G6PASE), glycogen synthetase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase (SDH), alkaline phosphatase (ALP), acid phosphatase (ACP) and gamma-glutamyl transpeptidase (gamma-GT). The lesions observed were predominantly of either basophilic or oncocytic types. In each case, tubular lesions (altered tubules) appeared to give rise to epithelial tumors (epitheliomas) with the same cellular phenotype. Basophilic tubules and epitheliomas proved to be strongly positive for GAPDH and G6PDH while demonstrating a reduction or loss of G6PASE, ALP, ACP, gamma-GT, and SDH compared with controls and the surrounding proximal or distal tubules. In addition, large basophilic epitheliomas demonstrated an increase in both SYN and PHO activities. In contrast, most oncocytic tubules and oncocytomas characterized by abundant densely granular cytoplasm showed a reduction in the activity of G6PDH, but were intensely positive for SDH. However, a few oncocytic lesions demonstrated a decrease in both SDH and G6PDH activity. Rarely, decreased SDH and elevated G6PDH activities were observed in altered tubules resembling oncocytic tubules. It remains to be clarified whether these tubules represent a variation of the oncocytic lesions or, perhaps, another type of tubular lesion. The results indicate that basophilic and oncocytic epithelial tumors differ in their cytochemical pattern and histogenesis. In line with earlier suggestions, the basophilic tumors apparently originate from the proximal renal tubules, while the oncocytomas develop from the distal parts of the nephron. The basophilic tumors are characterized by an increased pentose phosphate pathway and glycolysis, with a corresponding reduction in mitochondrial respiration. However, the majority of the oncocytomas show an increased activity of the mitochondrial enzyme SDH, and a marked decrease in the activity of the key enzyme of the pentose phosphate pathway.     

Clams (Corbicula fluminea) as bioindicators of fecal contamination with Cryptosporidium and Giardia spp. in freshwater ecosystems in California

This study evaluated clams as bioindicators of fecal protozoan contamination using three approaches: (i) clam tissue spiking experiments to compare several detection techniques; (ii) clam tank exposure experiments to evaluate clams that had filtered Cryptosporidium oocysts from inoculated water under a range of simulated environmental conditions; (iii) sentinel clam outplanting to assess the distribution and magnitude of fecal contamination in three riverine systems in California. Our spiking and tank experiments showed that direct fluorescent antibody (DFA), immunomagnetic separation (IMS) in combination with DFA, and PCR techniques could be used to detect Cryptosporidium in clam tissues. The most analytically sensitive technique was IMS concentration with DFA detection of oocysts in clam digestive gland tissues, which detected 10 oocysts spiked into a clam digestive gland 83% of the time. In the tank experiment, oocyst dose and clam collection time were significant predictors for detecting Cryptosporidium parvum oocysts in clams. In the wild clam study, Cryptosporidium and Giardia were detected in clams from all three study regions by IMS-DFA analysis of clam digestive glands, with significant variation by sampling year and season. The presence of C. parvum DNA in clams from riverine ecosystems was confirmed with PCR and DNA sequence analysis.     

Antioxidant and lipid peroxidation responses in Mytilus galloprovincialis exposed to mixtures of benzo(a)pyrene and copper

This study aimed to assess the antioxidant system potential and lipid peroxidative effects, in the gill and digestive gland of Mytilus galloprovincialis exposed to individual and binary mixtures of benzo(a)pyrene (BaP) and Cu for 7 days. Data demonstrated that in mussels exposed to BaP antioxidant enzymes (catalase--CAT, total glutathione peroxidase--tGPx, glutathione S-transferase--GST and glutathione reductase--GR) and lipid peroxidation (LPO) increased in the gill. On the contrary, in the digestive gland inhibitory antioxidant effects (superoxide dismutase-SOD, GR, metallothioneins-MT) and no changes in LPO levels were detected. Cu was also a potent oxidant agent since MT and LPO levels increased in mussel gill, despite no LPO effect in the digestive gland. For both single contaminants the organ specificity and distinct physiologic/metabolism roles were evident in terms of antioxidant capacity. Gill SOD inhibition, MT and GST unchanged was a result of "simple independent action" of exposure to BaP and Cu. "Interactions" in the binary mixtures, led to absence of changes in LPO effects. In the digestive gland, BaP and Cu interactions were also responsible for the GST and LPO enhancement (antagonistic effects). The current findings demonstrate the differences in antioxidant responses where the organ dependency highlights each contaminant particular mode of action. Generally, in the gill "non-interactive" effects occurred with the lowest Cu concentration while "interactions" exist for the mixture with the highest Cu concentrations. In the digestive gland, "interactions" and "no interaction" effects occurred in all the binary mixtures. Complex contaminant mixtures interact differently based on target tissue which may lead to an imbalance in the mussels health status.     

A PLANIMETRIC STUDY OF MORPHOLOGICAL VARIABILITY IN THE DIGESTIVE DIVERTICULA OF LITTORINA LITTOREA (LINNAEUS) AND MYTILUS EDULIS LINNAEUS

The inter-and intratubular morphological variability in thedigestive diverticula of Littorina littorea and Mytilus edulishas been investigated by planimetric procedures. Five parametershave been measured: mean epithelial thickness (MET), mean diverticularradius [MDR], mean luminal radius (MLR), MLR/MET ratio and MET/MDRratio. The results indicate that irrespective of the patternof tubule organization within the digestive diverticula (whethertubule types are clustered or not), variability between individualsis greater than that between zones of the digestive gland. Thishas implications for the design of sampling strategies in investigationsof the morphology of digestive diverticula in physiologicaland pathological studies. The variability in the epithelialthickness within diverticular sections is of minor relevancein assessing the overall condition of the digestive gland inthese studies, because variability in epithelial thickness betweentubules is significantly greater than within tubules in bothspecies. (Received 10 March 1989; accepted 16 October 1989)     

Recombinant expression,characterization and expressional analysis of clam <Emphasis Type="Italic">Meretrix meretrix</Emphasis> cathepsin B,an enzyme involved in nutrient digestion

Cathepsin B is one of the most important proteolytic enzymes involved in the nutrient metabolism of clam Meretrix meretrix. The recombinant fusion protein GST-MmeCB (rGST-MmeCB) was obtained at a high level from Escherichia coli and identified using LC-ESI-MS/MS. The GST tag was cleaved from rGST-MmeCB, and the resulting recombinant MmeCB (rMmeCB) was able to degrade the selective substrate carbobenzoxy-l-arginyl-l-arginyl-7-amino-4-trifluoromethylcoumarin (Z-Arg-Arg-AFC) in vitro. The kinetic parameters of the rMmeCB were calculated as follows: K _m, V_max and k _cat are 6.11 μM, 0.0174 μM min⁻¹ and 277.57 s⁻¹, respectively. Rabbit anti-rGST-MmeCB polyclonal antibodies was prepared and used to analyze the tissue distribution of MmeCB protein in M. meretrix. The results showed that the highest level of cathepsin B was found in the digestive gland and moderate levels were found in gill and mantle. Similar expression patterns were found at the mRNA level as detected by real time PCR. Further analysis showed that starvation caused a slight increase in MmeCB protein synthesis in the digestive gland, while refeeding after starvation caused an apparent increase in MmeCB synthesis in digestive gland, gill and mantle. Real time PCR analysis showed that MmeCB mRNA in digestive gland was significantly up-regulated by starvation and returned to normal level after the starved clams were refed. Together, these results indicated that cathepsin B is probably involved in the nutrient digestion of M. meretrix.     

重金属镉对鲫鱼碱性磷酸酶和酸性磷酸酶活性的影响

研究了重金属镉对鲫鱼肠、肝胰脏、鳃组织碱性磷酸酶和酸性磷酸酶活性的影响。结果表明,在0.2、0.4、0.8mg/L镉浓度条件下静态染毒12h、24h、48h、96h后,鲫鱼肠、鳃组织中碱性磷酸酶(AKP)和酸性磷酸酶(ACP)的活性降低,肝和胰脏的碱性磷酸酶活性没有明显变化,其酸性磷酸酶活性则升高。     

Tissue expression of glandular kallikrein and its response to 17 beta-estradiol in the acclimatized carp

Cyprinus carpio skeletal muscle kallikrein was isolated to apparent homogeneity, and a polyclonal antiserum against the purified protein was generated. Glandular kallikrein expression and tissue distribution were assessed using both Western blots and immunohistochemistry. A 39-kDa protein was detected in skeletal muscle, the gill, kidney, and pituitary gland, where an additional 72-kDa immunoreactive band was observed. Immunohistochemistry revealed immunoreactive kallikrein in the intermuscle tissue, epithelial gill cells, apical portion of distal and proximal tubular cells in the kidney, mucus and epithelial cells of the skin, intestinal tube, and prolactin-producing cells of the pituitary gland. In addition, the effect of 17beta-estradiol on kallikrein expression was analyzed in three different tissues of winter- and summer-acclimatized male carps. A 2.5-fold (p<0.05) increase in kallikrein immunoreactivity due to estrogen treatment was observed in winter-acclimatized carp muscle, but not in summer-acclimatized fish. In contrast, the gill responded differently, since a 2-fold (p<0.05) increase was found only in summer-acclimatized carps. Kallikrein immunoreactivity in the kidney increased both in summer- (2.5 fold) and in winter-acclimatized carps (1.5 fold). The signals obtained demonstrate the existence of tissue-specific variable responses to estrogen treatment in vivo, between winter and summer-acclimatized carp.     

Field validation of hsp70 stress proteins as biomarkers in Asian clam (Potamocorbula amurensis): is downregulation an indicator of stress?

The focus of this paper is to consider the applicability of the hsp70 stress protein response as a biomarker in field studies. Stress proteins (or heat shock proteins, hsp) of the hsp70 family are induced by sublethal concentrations of a variety of environmental pollutants. However, few studies have applied these proteins as biomarkers of environmental stress under field conditions. Our laboratory is investigating hsp70 proteins and other responses of Asian clam (Potamocorbula amurensis) as potential biomarkers in laboratory and field studies. Our efforts include two studies presently being conducted in northern San Francisco Bay: (1) monthly collection of clams from four sites along a cadmium contamination gradient; (2) 7 day in situ exposure of clams at two selected sites at Mare Island Naval Shipyard. Here we present results on hsp70 proteins in P. amurensis in field-collected and outplanted clams. Both field projects are ongoing, therefore the results presented here do not represent completed studies; rather, they illustrate a portion of our experience. For this workshop, we illustrate weaknesses and strengths of these proteins as biomarkers, and we underscore where additional work is needed. In field-collected clams (study no. 1), site-specific differences in levels of two hsp70 proteins, hsp70 and hsp76, were measured in May and June 1997. Although an inverse correlation exists between cadmium tissue concentrations and hsp70 protein levels, differences detected may be reflective of a salinity gradient. Results from recent laboratory exposures to cadmium and a range of salinities are discussed. After in situ exposure for 7 days (study no. 2), both hsp70 and hsp76 levels were significantly reduced in clams from site R. However, given a brief heat-shock in the laboratory, hsp70 protein levels were significantly higher in clams from this site than in controls. Results indicate that downregulation as well as upregulation of hsp70 proteins may be indicators of stress in P. amurensis.     

Cadmium bioaccumulation and detoxification in the gill and digestive gland of the Antarctic bivalve Laternula elliptica

Exposure to a sublethal concentration of cadmium (Cd; 50 microg L(-1)) resulted in significantly increased Cd concentrations in the gill and digestive gland of the Antarctic bivalve Laternula elliptica. Continuous accumulation of Cd in the two organs during the 14-day exposure period was associated with sequestration of Cd to both the soluble cytosolic and insoluble particulate cell fractions. However, the contribution of each cell fraction to Cd sequestration differed between the two organs; in the gill, a larger portion of Cd was associated with the insoluble fraction, while in the digestive gland, both the soluble and insoluble fractions sequestered similar amounts of Cd. Metal-binding components in the insoluble cell fraction were not identified in this study. On the other hand, a metallothionein-like protein (MTLP) was the major Cd-detoxifying component in the soluble cell fraction of the gill and digestive gland. The amount of MTLP increased linearly with exposure time and the amount of Cd accumulated in the tissue, which suggests a potential utility of MTLP as a biomarker for exposure to Cd and possibly other metals.     

Proliferative characteristics of atypical cells in native oysters (Ostrea lurida) from Yaquina Bay, Oregon.

Abnormal, large, possibly neoplastic cells from two Ostrea lurida have been successfully labeled with tritiated thymidine, and thus, for the first time, data are available for analyzing a presumptive invertebrate proliferative disorder. Large numbers of labeled atypical cells (AC) were uniformly distributed throughout the connective tissue (CT) underlying the gills, mantle, stomach, gut, digestive diverticula, kidney, and gonad. Two general types of AC are associated with the proliferative disorder. Both are abnormally large with many features characteristic of undifferentiated cells, and both are probably members of a proliferating compartment. The labeling index (LI) for AC ranged from 1.22% in connective tissue surrounding digestive tubules to 14.17% in CT underlying gill epithelium; the LI for all AC was 4.82%. The data strongly suggest that the AC proliferate at a much greater rate than any other oyster cells subjected to similar treatment.     

Cloning and expression of a heat shock protein (HSP) 90 gene in the haemocytes of Crassostrea hongkongensis under osmotic stress and bacterial challenge

Heat shock protein 90 (HSP90) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Here, we report the cloning of the HSP90 homologue in Crassostrea hongkongensis (ChHSP90) through SSH in combination with RACE from cDNA of haemocytes. The full-length cDNA of ChHSP90 is 2459 bp in length, consisting of a 3', 5'-untranslated region (UTR) and an open reading frame of 2169 bp encoding 722 amino acids. The identity analysis of the amino acid sequence of HSP90 revealed that ChHSP90 is highly conserved. Distribution of ChHSP90 mRNA in gonad, heart, adductor muscle, mantle, gill, digestive gland, and haemocytes suggested that ChHSP90 is ubiquitously expressed. The mRNA levels of ChHSP90 under salinity and bacterial challenges were analyzed by real-time PCR. Under hypo-osmotic treatment, ChHSP90 mRNA in gonad, heart and haemocytes were significantly up-regulated on day 2 and onwards; while in gill, digestive gland and adductor muscle it was significantly down-regulated; the expression in mantle was decreased significantly on day 2 and 3 (P < 0.01), and then up-regulated on day 4 (P < 0.05). Under hyper-osmotic treatment, the mRNA level in gonad, heart, adductor muscle was increased on day 2 and onwards; in gill, it was firstly increased, and then gradually decreased, reaching a minimum on day 3. On day 4, the expression level in gill recovered to pre-treatment level; in mantle and digestive gland, the expression levels were decreased, reaching to the minimum on day 3. During Vibrio alginolyticus challenge, the mRNA level of ChHSP90 increased 3-fold at 4 h post-infection, returned to its pre-challenge level at 6 h post-infection, then was further up-regulated from 8 to 36 h post-infection. These experiments demonstrate that ChHSP90 mRNA is constitutively expressed in various tissues and apparently inducible in haemocytes under salinity and bacterial challenges, suggesting its important role in response to both osmotic stress and bacterial invasion.     

Effects of light-emitting diodes on thermally-induced oxidative stress in the bay scallop Argopecten irradians

ABSTRACT

Water temperature is an important stressor that affects the physiological and biochemical responses of scallops. In this study, we investigated the effect of different light-emitting diodes (LEDs; red, green and blue) on oxidative stress in Argopecten irradians. PCR revealed MnSOD mRNA expression in the digestive diverticula, gill, adductor muscle and eye. CAT and HSP70 mRNA were expressed in the digestive diverticula, gill and adductor muscle. Additionally, we measured the changes in the expression of HSP70, MnSOD and CAT as well as H₂O₂ levels during thermal/laboratory stress. In the digestive diverticula, gill and adductor muscle, the mRNA expressions and activities and H₂O₂ levels significantly increased in response to thermal changes. The gene expressions and activities and H₂O₂ levels were significantly lower in scallops that received green LED light than in those that received no mitigating treatment. A comet assay revealed that thermal change groups had increased rates of nuclear DNA damage; however, treatment with green LED reduced the frequency of damage. The results indicated that low or high water temperature conditions induced oxidative stress in A. irradians but that green LED significantly reduced this stress.