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.

Vitellogenesis in spiders: first analysis of protein changes in different reproductive stages of Polybetes pythagoricus.

Vitellogenesis represents one of the most vital processes of oviparous species during which various proteins, carbohydrates, and lipids are synthesized and stored inside the developing oocytes. Through analyzing protein changes in the midgut diverticula, hemolymph, and ovaries of females throughout the different vitellogenic stages of the spider Polybetes pythagoricus, we determined the origin of the different proteins involved in the formation of lipovitellins (LVs) along with the existence of a linkage between the hemocyanin and this vital process. An increase in the total protein content of the midgut diverticula, hemolymph, and ovary occurred throughout vitellogenesis followed by a decrease in those levels after laying. The presence of hemocyanin in egg and in LV2, as well as its accumulation in the ovary throughout the vitellogenesis process, was determined. Considering that all biologic processes depend on the correct structure and function of proteins, this study establishes, for the first time for the Order Araneae, the coexistence of three different origins of vitellogenesis-related proteins: one predominantly ovarian involving peptides of 120, 75, 46, and 30 kDa; another extraovarian one originated from the midgut diverticula and represented by a 170 kDa peptide, and a third hemolymphatic one, represented by the 67 kDa peptide.

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.

Ultrastructure and histochemistry of the digestive gland of the giant predator snail Adelomelon beckii (Caenogastropoda: Volutidae) from the SW Atlantic.

This study deals with the structure of the digestive gland of the carnivorous gastropod Adelomelon beckii in Mar del Plata area (Argentina) and discusses the function of its cell types and compare with other gastropods. According to histological and transmission electron microscopy observations the epithelium is composed of two types of cells that are subject to cyclical changes, involving three phases: absorption, digestion and fragmentation. The majority of the cells, called digestive cells, have a basal nucleus with a cytoplasm filled by spherical digestive vesicles in different stages. The apical pole of the cell is covered with microvilli and cilia and shows evidence of endocytotic activity during the absorption phase. The intracellular digestive process passes through: (1) fusion of endocytotic vesicles; (2) transformation in heterolysososmes by fusion with enzyme-rich vesicles; (3) formation of residual bodies after digestion, which are pinched off to the lumen gland during fragmentation phase. The second type of cell in the acini are called vacuolated cells, which occur in clusters, the nucleus is in the mid-basal region and the cytoplasm is basophilic, filled with stacks of RER. During the absorption phase the vacuolated cells secrete neutral mucins that probably serve as lubrication to facilitate transport of food particles. During all phases, a large amount of lipofuscin, an insoluble pigment that is accumulated after lysosome digestion, is accumulated. The functional morphology of the different cell types found in this carnivorous gastropod is very similar to that found in herbivorous and deposit-feeding gastropods, which suggests that the feeding type does not influence the micro-morphology of the digestive gland.