| Since amphibians often live,breed,and spent the first phase of their life cycle in areas designed to receive contaminated waters,they are particularly vulnerable to contaminant exposure.Also,due to the permeability of their skin,they are sensitive to a wide range of toxic substances.In particular,the larval stages of amphibians are highly susceptible to pesticides present in water due to their thin and permeable skin and the continuous water flow through their gills.Amphibians stand out as the most imperilled taxa around the world according to the International Union for Conservation of Nature(IUCN).Although climate change and habitat destruction have been invoked as potential contributing factors,evidence is mounting that organophosphate insecticides(OPs)may be primarily responsible for many of these catastrophic declines.The primary mode of action of OPs is the inhibition of the activity of acetylcholinesterase(AChE)and elevating acetylcholine levels in the synaptic cleft of nervous tissues,then leading to subsequent cholinergic neurotoxicity through overstimulation of postsynaptic nicotinic and muscarinic receptors.However,there is now considerable evidence that the above mechanism cannot alone account for the wide range of symptoms and disorders related to a variety of enzymes,proteins,and receptors that can be modified by OPs.As one of the most widely used groups of insecticides,this compounds accounted for about 30%of pesticides used globally.Despite the remarkable benefits for productivity in high-intensity agriculture,application of OPs could also cause many risks for the environment,ecosystem and non-target organisms,especially human health,via volatilization,leaching and indirect inhalation.Furthermore,OPs can contaminate the surface waters in both urban and agricultural landscapes,which can have catastrophic effects on aquatic ecosystems,organisms,populations community and other biological chains who rely upon them.Trichlorfon(O,O-dimethyl(1-hydroxy 2,2,2-trichloroethyl)phosphonate)is an organophosphate insecticide,which has been used in a wide number of systems.A wide range of exposure time and concentrations of trichlorfon(0.25?25 g/L)are used in the treatment of parasitic diseases,and it is applied at 0.1?1.0 mg/L with 0.25 mg/L in 5 applications weekly in aquaculture ponds to eradicate ectoparasites.However,the widespread,long-term and overuse of trichlorfon pose risk for public and environment health.In particular,the varieties form of pesticides leads to persistent pollution of soil,ground and surface waters,which has impact on the entire aquatic ecosystem and threatens the survival of amphibians.In order to evaluate the toxicity of OPs(represented by trichlorfon)on non-target organisms,R.chensinensis at different development stage were selected for the succession researches below.Firstly,on the basis of evaluation of the acute toxicity of trichlorfon on amphibian larvae,the effects of low dosage trichlorfon on the growth and development of R.chensinensis tadpoles were discussed.At the same time,the larval of R.chensinensis were used as a model system to study the behavior responses and social behavior alterations on the developing vertebrate nervous system induced by low doses of trichlorfon.Moreover,after exposure to low level of trichlorfon,the characterization of the content of different neurotransmitter in muscle,brain,and myoloid tissue were clarified.Despite AChE,other neurotransmitter could be the potential specific biomarkers for monitoring of OPs.The results could provide theoretical support for the multiple effects of low level stress of OPs and the secreening of specific biomarkers for monitoring of OPs.In addition,RNA-seq was selected for the construction of cDNA libraries of differentially expressed genes(DEGs)in brain tissue of R.chensinensis,that before and after exposure of low level of trichlorfon.All these are attempt to elucidate how trichlorfon executes toxic effects on aquatic animals under chronic exposure and identify the main components of the pathways related to neurotoxicity.Finally,Western blot was used to detect the protein expression level of DEGs,and Immunohistochemistry were used to localize and analyse the expression of DEGs in brain.The specific results are as follows:1.The acute toxicity of trichlorfon on R.chensinensis tadpoles showed that,the LC50 of trichlorfon for 24,48,72 and 96 h was 142.50±3.23,49.19±1.28,25.68±2.04 and 15.55±1.93 mg/L,respectively.The safe concentration(SC)of trichlorfon in tadpoles was 1.56±0.19 mg/L,which showed the strong acute toxic effects of trichlorfon.The most malformation of death tadpoles during the assay including bent tail,axial flexures,renvers,and oedemas and swollen in appearance.2.In the chronic toxicity test,the effects of trichlorfon on the growth of R.chensinensis tadpoles were evaluated by measuring the weight and the length of tadpoles at different concentration for 21 and 42 d,respectively.The results showed that the tadpoles exposed to low-dosage of trichlorfon for longer time would be inhibited significantly growth and development.Chronic exposure experiment suggests that the mortality of tadpoles increased with the increase of trichlorfon dosage.The cumulative effect of the toxicant dose and exposure time were also showed in this experiment,which was consistent with the results of previous researches.3.The alternations of behaviour manifested the responses of animals to environmental stressors.The behavioral measures of tadpoles recorded in this study showed that the behavior responses of tadpoles showed a dose-dependent reduction in behavior activity after 4 weeks of trichlorfon exposure.The reduction of behavior activity in trichlorfon-treated tadpoles exhibited a dose-and time-dependent relationship.The quantization of activity revealed that the time spent in middur and burdur were increased significantly after 2 weeks' treatment of trichlorfon,then the differences faded away gradually after 2 more weeks of exposure.4.Trichlorfon caused a concentration-related reduction in contact frequency,while the contact duration time presented a positive correlation with the exposure concentration.The decreased locomotor distances and hypoactivity of contaminant tadpoles explored smaller portions of habitats,thus,excluded them from optimal foraging habitats and render them susceptible to predation.5.Though AChE is the molecular target of trichlorfon,studies on neurotransmitter biomarkers of trichlorfon contamination are limited.In order to clarify the effects of low level exposure of trchlorfon on the neurotransmitter changes in R.chensinensis,three main biological sample of muscle,brain,and myoloid tissue were used for the evaluation of acetylcholinesterase(AChE),choline acetyltransferase(ChAT),carboxylesterase(CarE),and acetylcholine(Ach).The result showed that the Trichlorfon produced a reduction of the activity of AChE and CarE with an impaired induction in Ach levels.The effects of trichlorfon on the activity of ChAT depended on the region of tissues.Thus,low level stress of trichlorfon can result in neurotoxicity in amphibian,by disruption of the metabolism and transmission of cholinergic-related neurotransmitter significantly.6.In order to evaluate the interference of trichlorfon on gene transcription patterns in the brain of R.chensinensis with 4 weeks treatment under control conditions and 0.1 mg/L exposure.In total,102,013 unigenes were obtained from the brain tissue of R.chensinensis,and 874 differentially expressed genes(DEGs)were identified.Functional annotation indicated that out of 118,643 unigenes,45,600(44.7%)were annotated in the Nr,Nt,the Swiss-Prot,KEGG,COG,and GO databases.7.The differential expression patterns of 4 genes associated with neural activity were selected and validated by quantitative polymerase chain reaction(qPCR).The results revealed that except for the canonical cholinesterase-based mechanism,trichlorfon could act on other receptors and alter certain types of neuronal ion channels as the major target sites.All of these effects ultimately cause disorders of multifunctional pathways and other neurotransmitter pathways in the host.Furthermore,the results also validated the faithful results of Illumina sequencing and assembles.8.Western blot(WB)and Immunohistochemistry(IHC)were employed to unravel the expression model of JAK2(Janus kinase 2)and GABRD(GABAA receptor)in the brain tissue of R.chensinensis.The result showed that low level of trichlorfon generally decreases the protein expression of JAK2 and GABRD.Furthermore,the reduction expression of JAK2 in IHC assay was related with exposure time.And the GABRD positive cells in brain was reduce for 2 weeks'exposure of trichlorfon,and then returned to normal after exposure for 4 weeks.Thus,despite the canonical mechanism,low level of trichlorfon could also disrupt the GABA system by acting on the GABAA receptor and signal transduction.Thus,low-dosage of trichlorfon for longer time still threatens the survival of tadpoles,and inhibited significantly in growth and development.Trichlorfon caused a time-related reduction in the response to external stimulation,which leaded to sluggish and lose consciousness of tested organisms.The toxic effects exhibited as the reduction in locomotor behavior.The tadpoles exhibited a dose-dependent reduction behavior activity in trichlorfon treatment.A significant increase in middur-time was found after 2 weeks of treatment.The decreased locomotor distances and hypoactivity of contaminant tadpoles explored smaller portions of habitats,thus,excluded them from optimal foraging habitats and render them susceptible to predation.At the same time,low dosage of trichlorfon had a disruptive effect on the social behavior of tadpoles,which decreased in social responses of the individual and induce the delaying behavior.Thus,low level of trichlorfon the interference the metabolism process of the cholinergic transmitter,which then leading to the disorder of cholinergic transmitter,all these induced the neurotoxic effect in organism.The results above confirmed that despite the classical mechanisms of OPs,trichlorfon could act on other receptors and alter certain types of neuronal ion channels as the major target sites,and cause disorders of multifunctional pathways and other neurotransmitter pathways in the host ultimately.The data obtained in this research can provide basis for further study of the toxic mechanism on non-target organisms of OPs,and serve as a fundamental reference for the protection of amphibian species and aquatic ecological risk assessment.Lastly,related neurotransmitters,such as CarE and ChAT can be used as biomarkers in the monitoring of OPs. |
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