Long-term feeding with Euglena gracilis cells modulates immune responses, oxidative balance and metabolic condition in Diplodon chilensis (Mollusca, Bivalvia, Hyriidae) exposed to living Escherichia coli.

We evaluated the modulating effect of long-term feeding with lyophilized Euglena gracilis cells on immune response, oxidative balance and metabolic condition of the freshwater mussel Diplodon chilensis. Mussels, previously fed with Scenedesmus vacuolatus (SV) or E. gracilis (EG) for 90 days, were challenged with an environmentally relevant concentration of Escherichia coli in water for 5 days, under feeding or starvation conditions. EG diet increased overall phagocytic activity and tissue hemocyte accumulation (gill and mantle), and favored hemocyte viability upon E. coli challenge. Tissular hemocyte accumulation, and humoral bacteriolytic activity and protein content were similarly stimulated by EG and E. coli, with no further effect when both stimuli were combined. Both, E. coli challenge and EG diet reduced gill bacteriolytic activity with respect to nonchallenged SV mussels, while no effect was observed in challenged EG mussels. Gill and digestive gland protein contents, along with digestive gland bacteriolytic activity were higher in EG than in SV mussels. Both SV and EG mussels showed increased gill mass upon E. coli challenge, while digestive gland mass was increased by bacterial challenge only in SV mussels. Bacterial challenge produced no effect on humoral reactive oxygen species levels of both groups. Total oxyradical scavenging capacity levels was reduced in challenged SV mussels but remained unaffected in EG ones. In general, EG diet decreased glutathione S-transferase and catalase activities in gill and digestive gland, compared with SV diet; but increased enzyme activity was evident in challenged mussels of both groups. Gill and digestive gland lipid peroxidation levels were higher in EG than in SV mussels but E. coli challenge had stronger effect on SV mussels. Adductor muscle RNA:DNA ratio was higher in EG mussels than in SV ones, and increased upon E. coli challenge in mussels of both groups. E. gracilis can be suggested as a nutritional and protective diet complement suitable for filtering bivalves. However, our results obtained from starved mussels show that starvation periods after supplying this diet should be avoided, since these could revert part of the acquired benefits and/or exacerbate detrimental effects.

Resistance in cholinesterase activity after an acute and subchronic exposure to azinphos-methyl in the freshwater gastropod Biomphalaria straminea.

Organophosphorous and carbamates insecticides are ones of the most popular classes of pesticides used in agriculture. Its success relies on their high acute toxicity and rapid environmental degradation. These insecticides inhibit cholinesterase and cause severe effects on aquatic non-target species, particularly in invertebrates. Since the properties of cholinesterases may differ between species, it is necessary to characterize them before their use as biomarkers. Also organophosphorous and carbamates inhibit carboxylesterases and the use of both enzymes for biomonitoring is suggested. Azinphos-methyl is an organophosphorous insecticide used in several parts of the word. In Argentina, it is the most applied insecticide in fruit production in the north Patagonian region. It was detected with the highest frequency in superficial and groundwater of the region. This work aims to evaluate the sensitivity of B. straminea cholinesterases and carboxylesterases to the OP azinphos-methyl including estimations of 48 h NOEC and IC50 of the pesticide and subchronic effects at environmentally relevant concentrations. These will allow us to evaluate the possibility of using cholinesterase and carboxylesterase of B. straminea as sensitive biomarkers. Previously a partial characterization of these enzymes will be performed. As in most invertebrates, acetylthiocholine was the preferred hydrolyzed substrate of B. straminea ChE, followed by propionylthiocholine and being butyrylthiocholine hydrolysis very low. Cholinesterase activity of B. straminea was significantly inhibited by the selective cholinesterases inhibitor (eserine) and by the selective inhibitor of mammalian acethylcholinesterase (BW284c51). In contrast, iso-OMPA, a specific inhibitor of butyrylcholinesterase, did not inhibit cholinesterase activity. These results suggest that cholinesterase activity in total soft tissue of B. straminea corresponds to acethylcholinesterase. Carboxylesterases activity was one order of magnitude higher than cholinesterase. A greater efficiency (Vmax/Km) was obtained using acetylthiocholine and p-nitrophenyl butyrate. Acute exposure to azinphos-methyl did not cause inhibition of cholinesterase activity until 10 mg L(-1) used. Carboxylesterases towards p-nitrophenyl butyrate was inhibited by azinphos-methyl being the IC502.20±0.75 mg L(-1) of azinphos-methyl. Subchronic exposure to environmental concentrations of azinphos-methyl (0.02 and 0.2 mg L(-1)) produced a decrease in survival, protein content and carboxylesterases activity despite no inhibition of cholinesterase activity was observed. B. straminea cholinesterase is not a sensible biomarker. On the contrary, carboxylesterases activity was inhibited by azinphos-methyl. Carboxylesterases could be protecting cholinesterase activity and therefore, protecting the organism from neurotoxicity. This work confirms the advantages of measuring cholinesterases and carboxylesterases jointly in aquatic biomonitoring of pesticide contamination. This becomes relevant in order to find more sensitive biomarkers and new strategies to protect non-target aquatic organisms from pesticide contamination.

Oxidative stress and histological alterations produced by dietary copper in the fresh water bivalve Diplodon chilensis.

The aim of this work was to study the oxidative stress effects and histological alterations caused by dietary copper on the filter-feeding freshwater mussel Diplodon chilensis. Bivalves were fed during 6 weeks with the green algae Scenedesmus vacuolatus previously exposed to copper. Metal concentration in algae cultures and bivalve digestive gland was measured by TXRF. A maximum accumulation of 0.49 µg Cu/mg protein was detected at week 6. Also at this week, the hepatosomatic index (HSI) showed the highest decrease (50%) in response to Cu exposure. SOD and GST activities were significantly increased at weeks 4, 5 and 6, reaching an activity on average 50% higher than in controls for GST. CAT activity and GSH increased significantly at weeks 5 and 6. Despite this response, oxidative damage measured as TBARS and carbonyl groups contents increased significantly at weeks 4, 5 and 6, respectively. Digestive tubule and duct atrophy and cell-type replacement in treated mussels were observed by histological studies. The presence of intracellular rhodanine-positive granules, suggests copper accumulation in intracellular vacuoles of digestive cells.

Effect of cadmium, lead and arsenic on the oviposition, hatching and embryonic survival of Biomphalaria glabrata.

Biomphalaria glabrata is a widespread freshwater gastropod mollusc. The easy aquaculture of these organisms allow its use as an accessible tool for contamination bioassays. B. glabrata showed marked metabolic responses when exposed to cadmium, lead and arsenic. Those responses could also affect the reproduction of the snails. Taking into account this hypothesis, B. glabrata were exposed for 96 h (acute laboratory bioassays) to different concentrations of cadmium (0.1, 0.05 and 0 mg/L), lead (0.5, 0.1, 0.05 and 0 mg/L) and arsenic (0.5, 0.1, 0.05 and 0 mg/L). Snails were removed from the aquaria while eggs were left in the same contaminant concentrations. The effect of the assayed toxicants on snail reproduction was registered as the alterations of the total number of laid eggs (TNLE), hatching time and embryonic survival. At 0.10 mg/L cadmium significantly decreased the TNLE (p<0.05) and no embryos survived. The lowest assayed level (0.05 mg/L) of cadmium, delayed the hatching time twice when it was compared with the control group (p<0.01). Lead decreased the TNLE at 0.5 mg/L level (p<0.01). The other assayed doses (0.05 and 0.10 mg/L) also decreased embryonic survival significantly (p<0.05 and p<0.01 respectively) and extended twice the time to hatching (p<0.01). The 0.50 mg/L level killed all embryos. Arsenic at all studied concentrations decreased the TNLE (p<0.05) while the hatching time was increased by 50%. Embryo survival only decreased at the highest level (0.5 mg/L) of arsenic assayed. In summary, the acute exposure (96 h) to cadmium lead and arsenic, altered the reproduction of B. glabrata, modifying the TNLE, hatching time and embryonic survival.

Potential use of glycogen level as biomarker of chemical stress in Biomphalaria glabrata.

Biomphalaria glabrata, a freshwater gastropod mollusc, was tested as biondicator organism to assess cadmium, lead and arsenic exposure using acute laboratory bioassays. Modifications of glycogen levels were measured in different anatomical regions of B. glabrata in order to test the usefulness of this parameter as a general biomarker of chemical stress. The snails were exposed 96 h to different concentrations of the following contaminants: 0.1 and 0.05 mg Cd/L; 0.5, 0.1 and 0.05 mg Pb/L; 0.5, 0.1 and 0.05 mg As/L. Significant decreases in the polysaccharide content were observed in gonadal region for all treated animals. Arsenic and lead at 0.1 and 0.5 mg/L level of exposure were also able to decrease the levels of glycogen in the pulmonary and digestive gland region. Glycogen content in the cephalopedal region of treated animals presented a significant decrease (p<0.05) when compared with control organisms only for arsenic at the highest level of exposure. To establish possible correlations between glycogen and contaminants accumulated by snails, analyses of the elements bioaccumulated in the different anatomical regions of B. glabrata were also performed. Cadmium and lead followed a similar pattern of bioaccumulation with highest values in the digestive gland region. Arsenic bioaccumulation, however, was highest in the gonadal region.

Comparative study on two freshwater invertebrates for monitoring environmental lead exposure.

Two freshwater invertebrate organisms, Biomphalaria glabrata and Lumbriculus variegatus, were tested as potential experimental animal models to assess Pb exposure using acute laboratory bioassays. Since long, the enzyme delta-aminolevulinic acid dehydratase (ALA-D) has been recognised as a useful biomarker of Pb exposure and effect. Therefore, determinations of ALA-D activity were performed in the whole body soft tissues of pigmented and non-pigmented gastropods B. glabrata and in the oligochaete L. variegatus. The organisms were exposed to varying concentrations of Pb for 48 h. Levels of Pb in the invertebrates were also analysed. Highly significant negative correlations were found between the enzymatic activity and the levels of Pb exposure, and also between the enzymatic activity and the metal incorporated by the invertebrates. No significant differences were found in the enzyme activity nor in the resulting metal accumulation based on gastropod pigmentation. The values of Pb concentration that produce 50% of inhibition on the enzyme activity (EIC50) were 0.023 and 0.029 mg Pb/L for pigmented and non-pigmented B. glabrata, respectively. A much higher value was found for L. variegatus (0.703 mg Pb/L). The non-observed effect concentration (NOEC) on enzyme activity for the oligochaetes was 0.05 mg Pb/L, about twice the EIC50 calculated for the gastropods. These data showed that both pigmented and non-pigmented B. glabata were much more sensitive organisms than the oligochaetes. The differences in enzyme inhibition could be attributed, at least partly, to differences in the metal body burden found between the organisms, since levels of Pb accumulated by B. glabrata were approximately three times higher compared to those observed in L. variegatus. Therefore B. glabrata showed to be a more suitable and reliable bioindicator organism for assessing Pb contamination in aquatic ecosystems, especially at low levels of metal exposure.