| Environmental androgens(EAs)are steroid hormones with stable structure and high-efficiency bioactivity.They mainly come from sewage treatment plants,livestock farms,paper mills and aquaculture drainage.They could seriously affect the endocrine system of organisms even at trace levels in the environment.Corbicula fluminea,as an important part of the aquatic food web,is widely distributed in freshwater environments.It plays a significant role in the nutrient cycle and energy flow of the aquatic ecosystem.However,there are few studies on the effects of environmental androgens on C.fluminea.In order to understand the response of benthic bivalves to EAs,the effects of methyltestosterone(MeTS,the representative of EAs)on sex hormone levels,histopathology and apoptosis in C.fluminea were studied in this paper.We also studied the effects of MeTS on endocrine and immune system pathways in C.fluminea by transcriptome sequencing.Moreover,the expression of genes related to the immune system of C.fluminea after MeTS exposure were analysed by RT-qPCR.This study aims to reveal the toxic effects and the immune damages of MeTS to C.fluminea from the tissue level,the physiological and biochemical level,and molecular level.The main results are as follows:(1)Firstly,MeTS could significantly affect sex hormone levels in C.fluminea.The content of testosterone in the MeTS treatment group were significantly higher than that in the control group,while the content of estradiol and 11-ketotestosterone in the MeTS treatment group were significantly lower than that in the control group.It was showed that there was an opposite variation trend between the content of estradiol and testosterone,and that sex hormone levels were observably associated with the duration and concentration of MeTS exposure.The results showed that MeTS exhibited androgenic effects,which might affect the normal developmental and reproductive processes of C.fluminea.Secondly,histopathological analysis showed that the tissues of C.fluminea could be damaged by MeTS.Specifically,the exposure of C.fluminea in MeTS for 28 d resulted in degeneration of the digestive tubules,infiltration of blood cells,and vacuolation of cells for the digestive glands.And for the gonad,there would be vacuolation of gonad cells,loss of contact between the oocyte membrane and the follicular cell layer.For the gill,it was appaerant that loss of structural integrity,falling out of cilia,severe swelling of the gill lamellae,and massive hyperplasia of the gill epithelium.For the foot,muscle distortion and loss of structural integrity appeared.For the mantle,the loss of structural integrity and rupture of the connective tissue appeared.Among the above organs,the damages to the digestive gland,gonad and gill are more serious.Finally,after the exposure of C.fluminea in MeTS for 28 d,the proportion of apoptotic cells in all organs except the foot in the treatment group was significantly higher compared to the control group.Among all organs,the percentage of apoptosis cells in the digestive gland and the gill were relatively more than other organs.Therefore,MeTS would be more toxic to the digestive gland and gill.(2)Transcriptome analysis was performed on the digestive gland of C.fluminea after MeTS exposure.Illumina sequencing data were assembled into 272110 transcripts and 112948 unigenes,and a total of 1300 differentially expressed genes(DEGs)were detected.Further enrichment analysis showed that the DEGs from C.fluminea exposed to MeTS were mainly enriched in pathways related to the endocrine system,such as the relaxin signalling pathway,the estrogen signalling pathway and the glucagon signalling pathway.As well as DEGs were mainly enriched in pathways related to the immune response,such as the antigen processing and presentation,the leukocyte transendothelial migration and the IL-17 signalling pathway.C.fluminea could respond to MeTS by up-regulating the expression levels of genes(mmp、gs、hsp90、hsp70、krt19、pcreb、gi/o)and down-regulating the expression levels of genes(gabab 和cam)associated with the estrogen signalling pathway in the endocrine system,and by up-regulating the expression levels of genes(mhc I、calr、β2m、hsp90、hsp70 和 ctsb/l/s)associated with the antigen processing and presentation pathway.(3)According to the transcriptome analysis results,five immune-related genes(hsp70、aif-1、ctls、mex3c 和 gpx)were selected to study the effects of MeTS on the expression of immune-related genes in different tissues.The results showed that the expression of all five immune-related genes were relatively high in the digestive gland and gonad.MeTS could regulated the immune system by influencing the expression of immune-related genes in C.fluminea to affect the pathways associated with heat shock response,inflammation,immune recognition,ubiquitination and oxidative stress.MeTS could lead to the overall down-regulating of the five immune-related genes in the digestive gland of C.fluminea,while lead to the overall up-regulating in the gonad.And the expression of five immune genes was strongly associated with both the concentration and duration of MeTS exposure.Therefore,it could be concluded that MeTS could cause immune damages in the digestive gland,but had relatively little effect on gonad.In summary,MeTS could cause changes in sex hormone levels and damages to different tissues in C.fluminea.The immune system of C.fluminea can up-regulating or down-regulating the expression of immune-related genes in response to the damage of MeTS.The results of the present study would provide an important basis for a comprehensive study of the ecological effects of environmental androgens. |
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