Assessment of the effects of 2,4-D-dichlorophenoxyacetic acid-based herbicide exposure in eggs and embryos of golden apple snails using OCT.

This study aimed to evaluate the effects of herbicide 2, 4-D-dichlorophenoxy acetic acid on golden apple snail eggs and embryos. Additionally, the study assessed the applicability of optical coherence tomography (OCT), a non-invasive depth cross-sectional microscopic imaging technique, as a novel method, to the best of our knowledge, for studying morphological changes in golden apple snail eggs and embryos, in comparison to the conventional approach of using white light microscopy. The study revealed that the herbicide 2,4-D-dichlorophenoxy acetic acid affected the hatchery rate and morphological changes of the eggs and embryos. The lethal concentration (LC50), representing the concentration of a substance that is expected to cause death in half of the population being studied, of the golden apple eggs and embryos increased with longer exposure time and higher concentrations. The estimated median effective concentration (EC50), which denotes the concentration producing the desired effect in 50% of the exposed golden apple embryos, exhibited a similar trend of change as the LC50. When compared to the microscopic study, it was observed that OCT could be employed to investigate morphological changes of golden apple snail eggs and embryos, enabling evaluation of alterations in both 2D and 3D structures.

Adverse effects of 2,4-D dimethylammonium based-herbicide on Acetylcholinesterase expression in Nile tilapia (Oreochromis niloticus).

In this study, the evaluation of a 2,4-D dimethylammonium based-herbicide impacted on Nile tilapia was done. The effects focus on Acetylcholinesterase (AChE) expression in the brain, gill, muscle, and plasma using antibody techniques. Our findings revealed a decrease in AChE expression with prolonged exposure. For these, AChE was purified using hydroxyapatite column chromatography. Moreover, the isolated protein was characterized as AChE by Polyclonal Ab specific to AChE through the Western blot. For interpretation at the cellular and molecular level, we employed two analytical techniques, histology, and optical coherence tomography (OCT). Alterations in the gill, liver, and muscle were observed to increase with increased exposure time. Field study concludes that AChE could serve as a biomarker to detect herbicide contamination in water and its accumulation in aquatic animals. This study may aid in surveillance and strategy formulation for managing contamination from such substances in various water sources.

Optical coherence tomography as an alternative tool for evaluating the effects of glyphosate on hybrid catfish (Clarias gariepinus × Clarias macrocephalus).

Glyphosate contamination in fresh water is a major problem in agricultural countries. It affects many vital organs in freshwater organisms that are important in the food chain. Hence, the effects of glyphosate on living organism organs are of particular interest. However, several existing techniques for evaluating the effect of glyphosate on aquatic organisms require stained tissue. To study organ tissue with minimal processing, alternative technique is demanded. Here, we investigated the used of optical coherence tomography (OCT) as an alternative tool for ex vivo evaluation of the effect of glyphosate on organ tissues of aquatic organisms, i.e., hybrid catfish (Clarias gariepinus × Clarias macrocephalus). The targeted samples were organ tissues from the brain, gill, and liver of hybrid catfish after glyphosate exposion at concentration of 10 mg L-1 for 24 h. The alteration was then verified by histology, and immunohistochemistry. The study found that all three techniques provide correlated results. We observed that OCT clearly showed damage to the brain and gill tissues of glyphosate-exposed hybrid catfish. However, the alteration in liver tissue was observable but not clear for this low concentration of exposure. The results from histology and immunohistochemistry confirmed the effect of glyphosate on brain, gill, and liver tissues of hybrid catfish. The results suggest that all three techniques could be used to examine the effects of glyphosate exposure in hybrid catfish. However, the choice of a suitable technique depends upon the purpose of the study.

Development of an antibody technique for acetylcholinesterase expression detection in the gill of Nile tilapia (Oreochromis niloticus) as a glyphosate-based herbicide biomarker.

Widely used glyphosate-based herbicides can remain in agricultural fields or be spread into the surrounding environment. This study aimed to develop an antibody technique for assessing acetylcholinesterase (AChE) expression in Nile tilapia (Oreochromis niloticus) after glyphosate exposure in the assessed tissues consisting of plasma, muscle, gills, and liver. Results showed that the cumulative mortality of fish exposed to glyphosate increased with exposure time and glyphosate concentration. The LC50 was evaluated using probit analysis. A sub-lethal concentration of 2 µL L-1 glyphosate-based herbicide altered behavioural and physiological appearances. AChE expression decreased compared to that in the control group with increasing glyphosate exposure time. The 71 kDa AChE was consecutively expressed in plasma, muscle, gill, and liver under laboratory and field conditions, as detected by dot blot and Western blot analysis. Furthermore, laboratory and field studies of the gills showed positive immunohistochemical results. Although this study could detect AChE expression in many tissues, using gills to assess AChE exposure allowed the fish not to be sacrificed compared with other organ studies, and this technique can be used in both laboratory and field conditions. In conclusion, the antibody technique can be applied to measure AChE expression in the gill tissue to assess glyphosate-based herbicide exposure.

Acetylcholinesterase (AChE) monoclonal antibody generation and validation for use as a biomarker of glyphosate-based herbicide exposure in commercial freshwater fish.

Monoclonal antibody specific to acetylcholinesterase (AChE) was extracted from the brain of hybrid catfish after exposure to glyphosate-based herbicide for 24 h. AChE was partially purified using hydroxyapatite and chromatography columns. The specific characteristics of AChE were studied by western blot using commercial polyclonal antibody (Rabbit anti-Fish AChE). It was found that the protein band had a molecular weight of 71 kDa. After mice were injected with AChE 4 times, the spleen showed a response to the induction. Polyclonal B cells from the mouse's spleen were taken and fused with myeloma cells to produce hybrid cells. After two fusions were performed, the clones specific to AChE were selected by dot blot, ELISA, immunohistochemistry and western blot techniques. Two clones, ACHE 33 and ACHE 99, which had the isotype of IgM were found. These two produced monoclonal antibodies specific to AChE in both denatured and native forms. The ACHE 33 monoclonal antibody clone from hybrid catfish could be cross-react with two commercial freshwater fishes, Nile tilapia and climbing perch, based on dot blot, immunohistochemistry, and western blot techniques. Moreover, AChE in Nile tilapia and climbing perch with glyphosate- based herbicide exposure gave a positive result with ACHE 33 as protein with molecular weight of 66 kDa. Based on our results, the produced monoclonal antibody showed specificity and could be applied to test AChE expression to assess glyphosate-based herbicide contamination in hybrid catfish, Nile tilapia and climbing perch. It could be also be a useful tool in indicating the quality of water resources.

Acetylcholinesterase (AChE) polyclonal antibody from hybrid catfish (C. macrocephalus × C. gariepinus): Specification, sensitivity and cross reactivity.

AChE (acetylcholinesterase) is generally classified as a specific biomarker of pesticide exposure. The aim of this study was to produce AChE polyclonal antibody from hybrid catfish that were exposed to commercial glyphosate. The hybrid catfish was exposed to glyphosate (0.75 mL/L) for 24 h. After that, the fish brain was dissected, AChE was extracted and purified by hydroxyapatite column chromatography and eluted with 0.2 M potassium phosphate buffer pH 6.8. This protocol gave 70% yield. Then, the brain extract was characterized using 10% SDS-PAGE and Western blot probed with commercial polyclonal antibody specific to AChE (PAb-AChE). The protein, 71 kDa, was then used as an antigen to immunize mice for antibody production. The polyclonal antibody (PAb) was characterized using dot blot, Western blot and immunohistochemistry for immunolocalization of AChE in hybrid catfish exposed to glyphosate. We found that the appropriate dilution of antibody for both dot blot and Western blot was 1:3500, and 1:2500 for immunohistochemistry. Cross reactivity testing showed that PAb-AChE can be used with AChE from striped snakehead fish at the same dilution as used with AChE from hybrid catfish. It was concluded that PAb specific to hybrid catfish AChE from this work was highly specific and sensitive, and can cross-react with striped snakehead fish AChE. Thus, this polyclonal antibody may be used in monitoring glyphosate exposure in hybrid catfish and striped snakehead fish.