Effect of starvation and pesticide exposure on neutral lipid composition of the digestive gland of males of the apple snails Pomacea canaliculata.

Pollutants as well as starvation usually modify homeostasis of neutral lipids in aquatic organisms. However, studies on the simultaneous effects of both stressors are scarce. The aim of this study was to evaluate the effect of toxicant exposure under starvation conditions on neutral lipids of the freshwater snail Pomacea canaliculata, selected as the model organism. Starved adult male snails were exposed to sublethal concentration of the pesticide cypermethrin (100 µg/L) during 4 and 10 days. Fed snails were sacrificed at the onset of the experiment (T0), along with starved snails exposed to the pesticide vehicle (ethanol) and another group without solvent served as controls. Total lipid content, neutral lipid classes, fatty acid composition, and pesticide accumulation were determined in the digestive gland of snails. The ethanol concentration used was not an additional stressful agent. As expected, starvation caused a decrease in neutral lipid content in the digestive gland of snails with respect to T0 snails. Pesticide exposure caused, on the other hand, an increase in triacylglycerol content compared to ethanol exposure at day 10 of the bioassay. This increment correlated with the bioconcentration of cypermethrin, which was 47% higher by day 10 than by day 4. The fatty acid profile of triacylglycerols in the digestive gland was significantly altered under starvation and pesticide exposure. Stressed male snails showed the ability to preserve polyunsaturated fatty acids, as evidenced by their significant increase with respect to T0 snails. These results suggest that the alteration of lipid homeostasis could be involved in an adaptive mechanism of aquatic organisms to lipophilic and obesogenic pollutants.

Ecological risk assessment of pesticides in sediments of Pampean streams, Argentina.

After their application in agricultural areas, pesticides are dispersed throughout the environment, causing contamination problems. In Argentina, the main promoter of transgenic biotechnology in the region, the total consumption of agrochemicals has increased significantly in recent years. Most chemicals dumped near surface waters eventually end up in bottom sediments and can be toxic to the organisms that live there. However, published data on the mixing of pesticides in this compartment is still scarce. The objective of this work was to detect and quantify pesticide residues in the sediment of rural streams in the Pampas region and to carry out acute and chronic risk assessment in these aquatic ecosystems. The study area comprises the mountainous system of Tandilia, located in one of the most productive agricultural areas in the country. The concentration of atrazine, acetochlor, chlorpyrifos, cypermethrin, and 2,4-D in the sediment of four rural streams was determined in three different seasons, and the toxic units (TU) and the risk ratios (RQ) were calculated. All the compounds analyzed were detected in most of the sampling seasons and study sites, at concentrations higher than those established in the national and international quality guidelines for the protection of aquatic biota in surface waters and for human consumption. Chlorpyrifos, cypermethrin, and acetochlor were the main pesticides contributing to the TU and RQ values, representing a medium or high ecological risk in most of the sites. Therefore, the evaluation of these pesticides in the bottom sediments could be a decisive factor in assessing the risk to the aquatic environment.

Sensitivity of embryos and larvae of the freshwater prawn Macrobrachium borellii to the latest generation pesticide spirotetramat.

The aim of this study was to evaluate the effects of the last generation insecticide spirotetramat (STM) on embryos and larvae of the freshwater prawn Macrobrachium borellii. Both embryos and larvae were exposed to serial dilutions of STM to determine the LC50 values. After 96-h of exposure, live larvae were fixed for histological analysis. In addition, ovigerous females were exposed to a sublethal concentration of STM (1.7 mg/L) for 96 h to evaluate the activity of the enzymes catalase, glutathione-S-transferase, and superoxide dismutase as well as the lipoperoxidation (LPO) and protein oxidation levels in embryos. The larvae showed a high sensitivity to STM evidenced by the LC50-96 h value (0.011 mg/L). On the contrary, the embryos were highly resistant to STM exposure, and no lethal effect was observed in the treatments with high concentrations of this insecticide (LC50-96 h > 150 mg/L). Among all the biochemical parameters evaluated in the embryos exposed to STM, only LPO showed a significant increase compared to controls. This was probably due to a restricted entry of the insecticide through the embryonic coat. Thus, a preliminary study of the structure and permeability of the embryonic coat was carried out in control embryos. The analysis by electron microscopy revealed that its structure is formed by four embryonic envelopes composed of multiple layers while the assay with a fluorescent probe revealed that the embryonic coat increases its permeability during development. STM caused significant histopathological alterations in the hepatopancreas and gills of larvae. This study showed that although the embryos of M. borellii could be protected by the embryonic coat, the larvae are very vulnerable to the STM toxicity. So, it is necessary to continue evaluating the effects of these new pesticides on non-target organisms, such as aquacultured species, to help predict their ecotoxicological risks derived from the increasing agricultural activity developed worldwide.

Deleterious effects of two pesticide formulations with different toxicological mechanisms in the hepatopancreas of a freshwater prawn.

The aim of this study was to evaluate the acute effects of the pyrethroid cypermethrin (CYP) and the last generation pesticide spirotetramat (STM) on the prawn Macrobrachium borellii. Initially, the 96-h LC50 was determined in adult prawns. Then, prawns were exposed to sublethal concentrations of pesticides (5% and 20% of the 96-h LC50 values) for four days and hepatopancreas were dissected for biomarkers analyses. Total protein and uric acid content, glutathione S-transferase (GST) activity, levels of lipid peroxidation (LPO), and protein oxidation (PO) were evaluated. Additionally, the presence of histopathological changes, lipofuscins, and neutral lipids accumulation were analyzed. The 96-h LC50 values were 0.12 µg/L and 8.2 mg/L for CYP and STM, respectively. The total proteins and uric acid content were not significantly affected by the treatments (p > 0.05). STM significantly affected the GST activity only at the highest concentration (p < 0.001). However, LPO and OP levels were affected by the lowest concentrations of both pesticides (p < 0.003). CYP and STM caused dose-dependent histological damage as was indicated by the histopathological index. The accumulation of lipofuscins showed a dose-dependent response, while the neutral lipids were significantly accumulated in the prawns exposed to the lowest concentration of both pesticides (p < 0.001). The integrated biomarker index (IBRv2) results indicated that the histological parameters represented the most sensitive biomarkers in M. borellii exposed to CYP and STM. Besides, the pyrethroid showed the highest response at concentration ranges that could be present in its natural environments.

Ecotoxicology and environmental safety toxicity of pyrethroid cypermethrin on the freshwater snail Chilina parchappii: Lethal and sublethal effects.

The aim of the present work was to study the effect of the pyrethroid cypermethrin (CYP) on the non-target freshwater snail Chilina parchappi. Initially, the sensitivity of adult snails to CYP was evaluated via the 96-h LC50 test. Then, snails were exposed to subtethal CYP concentrations (0.1 and 10 mg/l) for 1, 4 and 10 days and the digestive glands were dissected for biomarkers analyses. Enzymatic activity of catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), as well as total glutathione reduced (GSH) levels, were determined. Histological analyses of morphology, intracellular accumulation of lipofucsins and neutral lipids accumulation in the digestive gland were also evaluated. As compared to other molluscs, C. parchappi showed high resistance to CYP exposure evidenced by the 96-h LC50 value (44.59 mg/l). Snails exposed to sublethal CYP concentrations showed a statistically significant increase (p < 0.01) in GST (79-116%) and GPx (45-190%) activities with respect to controls. However, CAT activity showed a tendency to decrease with CYP treatment but was not statistically significantly different compared to control. Only high CYP concentration caused a statistically significant increase (p < 0.01) in GSH content (95-196%). There was evidence of structural changes in the digestive gland of snails exposed to CYP, showing a dose-dependent response. In exposed snails, some of the main symptoms included a reduction in the thickness of the epithelium, vacuolisation of the digestive cells and an increase in the number of excretory cells. Accumulation of lipofuscins (933-1006%) and neutral lipids (403%) were statistically significantly higher (p < 0.05) in snails exposed to CYP compared to control. This study showed that C. parchappii is quite tolerant to CYP exposure and that at sublethal concentrations, GSH metabolism could play a protective role against the pesticide harm in snails. Therefore, it would be interesting to study the response of this organism to other environmental stressors to assess its potential use in monitoring programs.

Differential response between histological and biochemical biomarkers in the apple snail Pomacea canaliculata (Gasteropoda: Amullariidae) exposed to cypermethrin.

To develop effective programs to monitor water quality is necessary to identify sensitive biomarkers in indicator species. The aim of this study was to evaluate different biomarkers in the apple snail Pomacea canaliculata exposed to the insecticide Cypermethrin (CYP). Adult male and female snails were exposed to sublethal CYP concentrations (10, 25 and 100µgl-1) for 1, 4, 7 and 14days. The recovery of the exposed snails was also studied by a post-exposure assay. The activities of the enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST), the levels of lipid peroxidation (LPO) and protein oxidation (PC) in digestive gland and gills were studied as biomarkers of exposure. Histopathological changes in target tissues were also evaluated. In digestive gland, CYP caused a significant increase in SOD, CAT and GST activities compared to control (p<0.05) as well as in LPO and PC levels (p<0.05). However, such biochemical effects were neither concentration nor time dependent. Histopatological changes were observed in the exposed groups, such as an increase in the number of basophilic cells, hemocytic infiltration and epithelia atrophy. Additionally, a positive correlation between the surface occupied by pigmented corpuscles and CYP concentrations was observed at all exposure periods. Gills showed greater sensitivity to oxidative damage than digestive gland. CYP caused an acute toxic effect in LPO levels in this respiratory organ. The gill filament of exposed snails, exhibited a reduction or loss of cilia, vacuolization of the columnar cells and an increase in haemocyte content irrespective of the concentration. High concentrations of CYP caused disruptions in the columnar muscle fibers. In general, snails did not show an improvement in their basal state during post-exposure treatment. Apparently, males and females do not have differential sensitivity to the pesticide. The results of this study suggest that histopathological changes are the most sensitive time- and dose-dependent biomarkers of toxicity induced by CYP in P. canaliculata.

Multibiomarker responses in aquatic insect Belostoma elegans (Hemiptera) to organic pollution in freshwater system.

The present study analyzes a battery of biomarkers in the water bug Belostoma elegans from a stream polluted with organic matter (OMS), and another one considered as reference site (RS) during spring-summer season (December to March). Biochemical parameters of glucidic, lipidic and oxidative metabolic pathways were analyzed in males and females of this insect. In general, no significant differences were observed in all biomarkers assayed between both sexes, except lactate concentration which was higher in males than in females (p < 0.0006) in the first three months. About carbohydrate metabolism parameters, only pyruvate-kinase showed significant differences between insects collected in both streams (p < 0.05) during December. However, the total lipid content, saturated fatty acid, and mainly triacylglycerol were higher in insects from RS compared to those from OMS (p < 0.002) in all sampled months. Levels of lipoperoxidation, protein oxidation, reduced glutathione and glutathione-S-transferase activity showed no differences between insects collected from both streams. Nevertheless, the significant increase observed in superoxide dismutase and catalase activities (p < 0.004) could be due to the elevated oxidative metabolism in insects from RS compared to those from OMS with lower dissolved oxygen. Regarding those responding parameters, males accounted for the differences between the two sites during the study period. In conclusion, our results support that lipidic energetic reserves and antioxidant enzyme activities in B. elegans could be used as biomarkers of environmental pollution by organic matter.

Acute toxicity of organophosphate fenitrothion on biomarkers in prawn Palaemonetes argentinus (Crustacea: Palaemonidae).

The effect of the organophosphate fenitrothion (FS) on the non-target freshwater prawn Palaemonetes argentinus was studied. Initially, the 96-h lethal concentration (LC50) of FS was determined in adult prawns. Inhibition of cholinesterase (ChE) in the muscle and hemolymph was assessed. Then, in the hepatopancreas, the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione-S-transferase (GST) were analyzed. Lipid peroxidation (LPO) was also determined in the hepatopancreas. The 96-h LC50 value was 1.12 µg/L. Hemolymph ChE activity showed a significant decrease in exposed prawns to FS compared to the control group, while no significant differences in the muscle were observed between groups (p < 0.05). FS caused a significant increase in the activity of antioxidant enzymes SOD, CAT, and GST compared to the control group (p < 0.02). By contrast, LPO levels were not affected by the pesticide (p < 0.05). These results indicate that P. argentinus is very sensitive to organophosphorus which alter biochemical parameters that are related to antioxidant status. Thus, these parameters could be used as biomarkers for assessing water pollution.

Study of biochemical biomarkers in freshwater prawn Macrobrachium borellii (Crustacea: Palaemonidae) exposed to organophosphate fenitrothion.

Several agrochemicals like organophosphates are extensively used to control pests in agricultural practices but they also adversely affect non-target fauna. The effect of organophosphorous fenitrothion on the prawn Macrobrachium borellii was evaluated. The 96-h LC50 was determined. Activity of superoxide dismutase, catalase, glutathione-S-transferase and lipid oxidation levels, were evaluated in the hepatopancreas from adults exposed to sublethal fenitrothion concentrations for 1, 2, 4 and 7 days. In addition, superoxide dismutase mRNA expression, acetylcholinesterase inhibition and haemocyte DNA damage were determined. The 96-h LC50 was 4.24µg/l of fenitrothion. Prawn exposed to sublethal FS concentrations showed an increase of both catalase and superoxide dismutase activities, mainly after 2 and 4 days exposure and an increase of glutathione-S-transferase activity from day 2 to day 7 while lipid oxidation levels increased mainly on day 1. Superoxide dismutase transcripts were significantly higher in fenitrothion -treated prawns, indicating an induction mechanism. Hemolymph analysis showed that while acetylcholinesterase activity decreased after 2 days, haemocytes displayed most DNA damage after 7-day exposure to fenitrothion. These results indicate that prawn enzymes are highly sensitive to fenitrothion exposure, and these biological responses in M. borellii could be valuable biomarkers to monitor organophosphorous contamination in estuarine environments.

Triacylglycerol catabolism in the prawn Macrobrachium borellii (Crustacea: Palaemoniade).

While invertebrates store neutral lipids as their major energy source, little is known about triacylglycerol (TAG) class composition and their differential catabolism in aquatic arthropods. This study focuses on the composition of the main energy source and its catabolism by lipase from the midgut gland (hepatopancreas) of the crustacean Macrobrachium borellii. Silver-ion thin-layer chromatography of prawn large TAG deposit (80% of total lipids) and its subsequent fatty acid analysis by gas chromatography allowed the identification of 4 major fractions. These are composed of fatty acids of decreasing unsaturation and carbon chain length, the predominant being 18:1n-9. Fraction I, the most unsaturated one, contained mainly 20:5n-3; fraction II 18:2n-6; fraction III 18:1n-9 while the most saturated fraction contained mostly 16:0. Hepatopancreas main lipase (Mr 72 kDa) cross-reacted with polyclonal antibodies against insect lipase, was not dependent on the presence of Ca(2+) and had an optimum activity at 40°C and pH 8.0. Kinetic analysis showed a Michaelis-Menten behavior. A substrate competition assay evidenced lipase specificity following the order: 18:1n-9-TAG>PUFA-enriched-TAG>16:0-TAG different from that in vertebrates. These data indicate there is a reasonable correspondence between the fatty acid composition of TAG and the substrate specificity of lipase, which may be an important factor in determining which fatty acids are mobilized during lipolysis for oxidation in crustaceans.

Antioxidant response and oxidative stress levels in Macrobrachium borellii (Crustacea: Palaemonidae) exposed to the water-soluble fraction of petroleum.

The aim of the present work was to evaluate the effect of the water soluble fraction of hydrocarbons (WSF) on the antioxidant status of the freshwater prawn Macrobrachium borellii. First, seasonal variations were studied in a non-polluted area. Hepatopancreas and gills showed season-related fluctuations in catalase (CAT), glutathione-S-transferase (GST) activities and in lipid peroxidation levels (LPO), but not in superoxide dismutase (SOD). Then, adults were exposed semi-statically to sublethal doses for 7days. CAT, SOD, GST, and glutathione peroxidase (GPx) activities and LPO, reduced glutathione (GSH) and protein oxidation (PO) levels were determined. Exposed individuals showed significant increases in CAT, SOD, and GST activities in hepatopancreas and CAT activity in gills. GPx activity did not vary in either tissues. While LPO levels increased, GSH levels decreased significantly in hepatopancreas of exposed animals, but PO levels showed no variation. Induction of SOD was also assessed by Real-time PCR mRNA expression in hepatopancreas. The non-enzymatic antioxidant activity was also tested; ABTS 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) was higher in hemolymph of treated-prawns compared to controls, but ferric reducing activity of plasma assay (FRAP) values did not change. Taken together, the present results indicated that the antioxidant defenses of M. borellii, mainly in hepatopancreas, were significantly affected by aquatic hydrocarbon contamination, regardless of the season.

Effect of the water-soluble fraction of petroleum on microsomal lipid metabolism of Macrobrachium borellii (Arthropoda: Crustacea).

The effect of the water-soluble fraction of crude oil (WSF) on lipid metabolism was studied at critical metabolic points, namely fatty acid activation, enzymes of triacylglycerol and phospholipid synthesis, and membrane (lipid packing) properties in the freshwater prawn Macrobrachium borellii. To determine the effect of the contaminant, adults and embryos at different stages of development were exposed to a sublethal concentration of WSF for 7 days. After exposure, microsomal palmitoyl-CoA synthetase (ACS) showed a two-fold increase in adult midgut gland. Embryo's ACS activity was also affected, the increment being correlated with the developing stage. Endoplasmic reticulum acylglycerol synthesis was also increased by WSF exposure in adults and stage 5 embryos, but not at earlier stages of development. Triacylglycerol synthesis was particularly increased (18.5%) in adult midgut gland. The microsomal membrane properties were studied by fluorescent steady-state anisotropy, using the rotational behavior of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Microsomes from midgut gland of WSF-exposed prawn showed no differences in fluidity. Nevertheless, microsomes incubated with WSF in vitro increased their fluidity in a temperature- and WSF concentration-dependent fashion. Both, aliphatic and aromatic hydrocarbons individually tested elicited an increase in membrane fluidity at 10 mg/l, but at 4 mg/l only nC10-C16 aliphatics did. In vivo results indicate that WSF increased the activity of microsomal enzymes that are critical in lipid metabolism, though this change was not due to direct alterations in membrane fluidity, suggesting a synthesis induction, or an enzyme-regulatory mechanism. Nevertheless, hydrocarbons elicited membrane fluidity alterations in in vitro experiments at concentrations that could be found in the environment after an oil spill.

Activation of lipid catabolism by the water-soluble fraction of petroleum in the crustacean Macrobrachium borellii.

Little is known about the effect of the water-soluble fraction of crude oil (WSF) on lipid metabolism in invertebrates. The effect of the WSF on the triacylglycerol (TAG) mobilization, fatty acid activation and degradation was evaluated in the decapod Macrobrachium borellii, exposing adult and eggs at different stages of development for 7 days to a sublethal concentration of WSF. Using radioactive tracers, mitochondrial palmitoyl-CoA synthetase (ACS), triacylglycerol lipase (TAG-lipase) and fatty acid beta-oxidation system activities were assayed. Before studying the effect of WSF, the kinetic parameters of ACS were determined in purified mitochondria. Its optimal temperature and pH were 32 degrees C and 8.0, respectively, the apparent K(m) 2.48 micromol l(-1), and its V(max) of 1.93 nmol min(-1) mg protein(-1). These kinetic parameters differed significantly from this shrimp's microsomal isoform. After 7 days exposure to a sublethal concentration of WSF (0.6 mg/l), changes were observed in the enzymatic activity of all enzymes or enzymatic system assayed in adult midgut gland as well as in stage 5 eggs, a period of active organogenesis. An increase in the mobilization of energy stores was detected as early as stage 4, where TAG-lipase activity increased by 27% in exposed eggs. The increase was even more marked in exposed eggs at stage 5 where a three-fold rise (154%) was determined. Exposed adult shrimp also showed an augmented lipase activity by 38%. Fatty acid beta-oxidation increased by 51.0 and 35.5% in midgut gland and eggs at stage 5, respectively, but no changes were observed at less-developed stages. Mitochondrial fatty acid activation by ACS also increased in adults and stage 5 eggs by 7.4 and 52.0%, respectively. A similar response of the lipid catabolic pathways to WSF contamination in both adult and eggs, suggests that the exposure to this pollutant causes an increase in the energy needs of this shrimp. When validated by field studies, these catabolic enzymes could be employed as early pollution biomarkers.

Toxicity, uptake, and release of the water-soluble fraction of crude oil in different developing stages of the prawn Macrobrachium borellii.

The effects of a water-soluble fraction of light crude oil dissolved in freshwater (WSF) on Macrobrachium borellii exposed at three life stages was evaluated. Adults, larvae (PL), and embryos were exposed to five levels of WSF for 96 h. At 48 and 72 h PL were significantly more sensitive to WSF than adults, though values for 96-h LC50 were not significantly different (1.56 and 1.41 mg/L, respectively). Mortality was never high enough to allow LC50 calculation in embryos, but chronic exposure to WSF increased the mortality near hatching and significantly decreased PL survival compared to the control group. The uptake, accumulation, and depuration of WSF were evaluated in adult prawns (lipid content 1.5% wet wt) exposed to a sublethal concentration for 96 h followed by a 10-d depuration period. Hydrocarbons were rapidly taken up, and after 24 h most of them reached an equilibrium concentration. Increases in the number and alkylation of the aromatic rings enhanced both their accumulation and their retention. When returned to clean freshwater, levels of lighter aromatics dropped rapidly over 12 h. From these studies we conclude that early life stages of M. borellii are not very vulnerable to WSF toxicity, while adults have a rapid uptake and release of most of the contaminating hydrocarbons, probably due to their low lipid levels.

Synthesis, distribution, and levels of an egg lipoprotein from the apple snail Pomacea canaliculata (Mollusca: Gastropoda).

The site of synthesis of mollusc lipoproteins is hitherto unknown and was investigated for perivitellin 2 (PV2), an egg lipoprotein found in the freshwater snail Pomacea canaliculata. Tissues (albumen gland, gonad-digestive gland complex, and muscle) from vitellogenic females were incubated in vitro with 14C-leucine at 25 degrees C for 12 hr. At the end of incubation, soluble proteins from tissue homogenates and medium were analyzed for de novo protein synthesis by electrophoresis and HPLC, and radiolabeled proteins were quantified by liquid scintillation. Two albumen gland radiolabeled proteins (67 and 31 kDa) co-migrated with the subunits of PV2, and they represented 6.0% of the total labeled protein in that tissue. Western blot analysis confirmed the presence of PV2 only in the albumen gland. In vivo experiments where adult females were injected with 3H-leucine revealed that PV2 was not present in hemolymph. ELISA analysis in all tissues of the snail confirmed the PV2 presence only in the albumen gland and developing eggs with levels of 26 and 98 mg/g protein, respectively. Therefore, the albumen gland is the only site for PV2 synthesis, and no extra-gland synthesis, circulation, or accumulation could be found. PV2 subunits were further characterized analyzing N-terminal sequences which showed no homology with other proteins.