| The increased contamination in the aqutic system has caused detrimental effects on the health of the aquatic organism as well as the balance of the ecology.The current study evalutated the toxicity effects of the two categories of contaminants,ammonia and herbicides,using economical species（grass carp,Ctenopharyngodon idellus）,wildfish（Delta Smelt,Hypomesus transpacificus）and model species（Japeness medaka,Oryzias Latipes）,respectively,to give an intergrated illustration of the toxicity mechanism of pollutants on fish.The industrial wastes,sewage effluents,agricultural run-off and decomposition of biological waste may cause high environmental ammonia in water,and subsequently may lead to detrimental effects and even mass mortality of fish.Grass carp is an intensively cultured fish species for its economic value.Ammonia,as a critical environmental factor,could be a threaten to the grass carp cultural industy.Therefore,grass carp was employed in the present study to obtain the transcriptomic profiles in the liver after high environmental ammonia（HEA）exposure,by applying high-throughput sequencing and bioinformatics analysis.Then the differentially expressed genes（DEGs）,GO and KEGG annotation of the transcriptomic data were analysied.Based on the KEGG analysis,a further study about the mechanism of oxidative stress and apoptosis induced by ammonia were illustrated.This study shed a new light on the detoxification of ammonia in fish liver.The invasive aquatic weeds have disrupted the ecological system of the San Francisco Delta.To address this issue,herbicides penoxsulam,imazamox,fluridone and glyphosate have been intensively applied in the region to control the growth of the aquatic plants.However,little information is available about the effects of these chemicals on non-target organisms in the aquatic environment.Delta Smelt is a native fish species in the Delta and is listed as an endangered species by United States Fish and Wildlife Service（USFWS）due to the continuous population decline caused by the deterioration of aquatic environment.The present study was to assess sub-lethal effects of the herbicides penoxsulam,imazamox,fluridone,and glyphosate on non-target organism using the wildfish species,Delta Smelt,which will provide information to the environmental agencies regarding the effect of herbicide application on non-targeted species such as the endangered Delta Smelt.Then inspired by the foundings in Delta Smelt study,we performed a study using the model species Japeness medaka by incorporated with RNA-seq analysis to further inllustrated the toxicity mechanism of fluridone on the early development stage of medaka.The results are as follows:1、Two c DNA samples from liver in control and HEA treatment group were sequenced,and a total of 911 DEGs were identified in the liver of grass carp after HEA exposure,with 563 genes were up-regulated and 348 genes were down-regulated.631 DEGs were then successfully assigned to GO terms,with the number of the up-and down-regulated genes was 410 and 221,respectively.The RT-qPCR analysis of 10 randomly selected genes validated the results of transcriptomic sequencing.2、After KEGG analysis of the DEGs in the grass carp liver exposed to HEA,a total of 238 DEGs with 168 up-regulated genes and 70 down-regulated genes were assigned to 124 KEGG pathways,in which pathways involved in oxidative stress and apoptosis were indentified.Further analysis on oxidative stress,histopathology and cellular apoptosis in grass carp liver after HEA exposure revealed interesting findings.Increased reactive oxygen species（ROS）content and superoxide dismutase（SOD）activity together with the decreased catalase（CAT）activity were detected,which may be related with the DEGs and related pathways such as FOXO signaling pathway.The histopathology and TUNEL assays results confirmed that apoptosis was induced in liver when fish had suffered HEA.The RT-qPCR results showed that apoptosis related genes such as Bax,c-Myc,casps9 and P53 were up regulated by HEA exposure.Combined with the results of transcriptomic experiments,we concluded that intrinsic mitochondrial pathway could be involved in grass carp liver apoptosis induced by ammonia stress.3、The sub-lethal effects of the herbicides penoxsulam,imazamox,fluridone,and glyphosate on Delta Smelt were evaluated after 6 h exposure,by measuring the expression of the endocrine related genes,17β-estradiol（E2）and glutathione（GSH）concentrations in the liver and acetylcholinesterase（AChE）activity in the brain.The results showed that the induced expression of choriogenin（CHG）or vitellogenin（VTG）gene was observed in the liver of fish exposed to imazamox and glyphosate.E2 concentrations were significantly increased by fluridone exposure in both sexes(0.08 mg L-1)and glyphosate exposure in male fish(0.064,0.64 and 640 mg L-1),whereas penoxsulam and imazamox did not show any effect on the concentration of E2.Significant inhibition of AChE activity was observed in both sexes exposed to penoxsulam,imazamox,and fluridone,with more sensitive in female than that in male.All concentrations of penoxsulam and imazamox tested in this study caused a significant reduction of AChE activity in female while the male showed significant inhibition of AChE activity only at the two highest concentrations of penoxsulam and the lowest and highest concentrations of imazamox.GSH concentration decreased in male exposed to fluridone at 0.83 mg L-1 and higher concentrations and glyphosate at 0.64 mg/L,however,no effect was observed in fish exposed to penoxsulam and imazamox.The results indicate that fluridone and glyphosate disrupt the endocrine system and anti-oxidative process on Delta Smelt,whereas penoxsulam,imazamox and fluridone possess neurotoxic effects on the fish.This study demonstrates the detrimental effects of commonly used herbicides on Delta Smelt.4、The results of toxicity effects of fluridone on the early developmetal stage of medaka showed that the hatching rate of medaka embryo was decreased with the increasing of the fluridone concentration,and dropped to 12.5 and 25% of the female and male embryo exposed to the highest concentration(4 mg L-1),respectively.Suffered from 6 h exposure of fluridone,the male and female larval madaka strat to show the retarded response to the stimulation at the concentration of 20.85 and 104.25 mg L-1,respectively.Then the male larval fish in control and 20.85 mg L-1 of fluridone group were subjected to RNA-seq analysis,and an average of 5.97 and 6.39 million reads were obtained in each group,having the mapping rate of 79.1-81.1% with the reference genome sequence of Japeness medaka.After de novo assembling,totally 59037 unigenes were obtained,among which 799 unigenes were significantly differently expreesed including 555 up-regulated and 244 down-regulated genes.According to the GO enrichment analysis for the DEGs,some biological processes such as oxidative stress,nerves system as well as androgen biosynthetic and metabolic process were affected by fluridone,and some genes related to the above process such as AChE,glutathione reductase（GR）and glutathione S-transferase（GST）were analyzed.The results showed that the change pattern of the gene expression was dose-and sex-dependent.The present study revealed that fluridone could lead to a detrimental toxic effect on Japeness medaka by disrupting the function of neuro system and endocrine system as well as inducing oxidative stress.The intergrated analysis demonstrate that the different aquatic pollutants may have the similar toxic effects on various fish sepceis even though they could induce various responses in different organs.In the current study,both ammonia and herbicides could target the liver of fish and induced oxidative stress,thus led to the disruption of the homeostasis.Highly related to oxidative stress,apoptosis was also an important defense strategy in the fish exposed to chemicals.Moreover,hormone regulation mediated by endocrine system may be also one of a mechanism of the fish responsing to the environmental stress,which could play a role in the fish to cope with the toxic effect of contaminants. |
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