Effects Of Bis(2, Ethylhexyl) Phthalate And 4-nonylphenol On The Growth And Development Of Adults Of Pseudomonas Black Spiny Ants

Di(2-ethylhexyl)phthalate(DEHP)and 4-nonylphenol(4-NP)are common endocrine disruptors in the environment.The toxic effects of the two substances on endocrine system,reproduction,development,and carcinogenesis are serious.Once releasing into the soil environment,they can exist for a long time.Studies have shown that DEHP can cause growth retardation and affect survival in midges,drosophila and mice.4-NP can lead to chironomus and daphnia development deformities,decrease their survival rate and influence their reproduction.At present,there are few studies on these two endocrine disruptors effecting to social insects.Molting hormone receptor(Ecdysteroid receptor,EcR)is a unique nuclear receptor in insects.This receptor and the ultra valve protein(Ultraspiracle protein,USP)can form the active complex which activates the molting hormone signaling pathways by starting Broad Complex(BR-C)and response factors expression.The molting hormone signaling pathway can regulate the growth and reproduction of insects.Dopa Decarboxylase(DDC),the important enzyme in the catecholamine biosynthesis pathway.It plays an important role in various biological mechanisms and plays an extremely important role in insect epidermal growth and development.Heat shock protein(hsps)is a kind of stress protein produced in the body in a short time when organisms encounter some external stimuli or bad environment.The expression level of hsc70(Heat shock Cognate protein 70)is stable under normal physiological state,which is mainly closely related to the processing of newly formed peptide chain and the transport of protein precursors.hsc70 belongs to the constituent hsp70.When the external environment becomes harsh,the expression of hsc70 will also be induced,and then participates in maintaining the homeostasis of the body's internal environment.Estrogen-related receptor(ERR)belongs to the nuclear receptor superfamily.It plays a crucial role in metabolism regulation and growth and development.Vitellogenin(Vg)is mainly synthesized from the fat body of sexually mature female,which provides nutrients and functional substances for embryonic development and plays an important role in the reproductive regulation of animals.A large number of microorganisms inhabit the insect digestive tract.These microorganisms play an important role in thedevelopment and reproduction in the insect.At the same time,different physical and chemical environments such as insect digestive tract structure and morphology and feeding conditions will also affect the community structure of microorganisms in the digestive tract.Therefore,the interaction between intestinal microbe and host insect is a good research model.Polyrhachis vicina Roger is a social insect which has a typical caste differentiation.It has strong adapting ability,a short life history and reproductive capacity.There are mainly four stages including egg,larva,pupa and adult.Adult ants can be divided into the worker ants,males and females.This ant is the only medicinal and edible ant which is a good material for studying the growth and development of social insects.In recent years,there have been few reports on the effects of endocrine disruptors on social insects.In this study,DEHP and 4-NP were used to interfere with the social insect P.vicina,and the results were compared and analyzed at the physiological and molecular levels before and after the interference,so as to provide reference for future studies and to assess the effects of environmental pollution on soil organisms and other aspects.This experiment mainly studied the effects of two kinds of environmental endocrine disruptors on the growth and development of different castes of P.vicina.This study was mainly divided into two parts:in the first part,adults of different castes of P.vicina were exposed to different concentrations of DEHP and 4-NP by abdominal administration,and the changes of body weights and mortality of adults of P.vicina before and after treatment were statistically compared.The changes in external morphology of different castes of P.vicina were observed.We investigated the effects of DEHP and 4-NP on the growth and development of different grades of P.vicina from physiological level.Real-time fluorescent quantitative PCR technology was used to detect the change in the expression of PvEcR,PvUSP,PvBR-C,PvDDC,Pvhsc70,PvERR,PvVg at the mRNA level after treatment with two endocrine disruptors.The molecular effects of DEHP and 4-NP on the growth and development of different castes of P.vicina were investigated and the relationship between the gene expressions and physiological level changes and the morphological changes were analyzed in the ants.In addition,the effects of two endocrine disruptors on the intestinal structure of worker ants were observed by paraffin section and hematoxylin-eosin staining.In the second part,16S high-throughput sequencing technology was used to analyze the effects of DEHP on intestinal microorganisms in the worker ants,and then studied the effects of endocrine disruptors on the growth and development of P.vicina.The results were as follows:1.Different concentrations of DEHP(0.02mg/L,0.2mg/L,2mg/L,20mg/L,200mg/L)were treated through abdomen of P.vicina for 15 days.Compared with the control group,the survival rate of workers treated with DEHP showed dose-dependent changes,the survival rate of males was also reduced,but the survival rate of females was not affected.DEHP treatment increased the body weights of workers and decreased the body weights of females,but had no significant effect on the body weights of males.2.It was observed that the epidermal growth abnormalities were found in the abdominal morphology of the ants of different castes after DEHP treatment.This indicated that DEHP had a direct effect on the normal growth and development of P.vicina.3.The results of qRT-PCR showed that compared with the control group,the mRNA expression levels of PvEcR,PvUSP,PvBR-C,PvDDC,Pvhsc70,PvERR,PvVg in the treatment group were all up-regulated or down-regulated,but there were no significant time-dependent or concentration-dependent changes.DEHP treatment had the greatest influence on PvVg gene of P.vicina,especially on the workers.4.The results of paraffin section and hematoxylin-eosin staining showed that compared with the control group,DEHP mainly affected the midgut of the worker ants.In the DEHP treated group,the muscle layer in the midgut tissue was incomplete,the cell boundary was not clear,and the cell staining was more intense,the nucleus were atrophy.We speculated that DEHP would affect the metabolism of different castes of P.vicina,and thus futherly influence their growth and development.5.Different concentrations of 4-NP solution(0.01mg/L,0.1 mg/L,1mg/L,10 mg/L,100 mg/L)were treated through abdomen of P.vicina for 15 days.Compared with the control group,the survival rate of workers treated with 4-NP decreased by dose-dependent,and the survival rate of both males and females also decreased,among which the survival rate of males was the lowest,followed by that of the workers and the females were highest.Compared with the control group,with the body weights of adults were increased by 4-NP treatment.6.It was found that the abdominal morphology of workers,females and males treated with 4-NP showed abnormal epidermal growth.These results indicated that 4-NP had a direct influence on the normal growth and development of P.vicina.7.The results of qRT-PCR showed that compared with the control group,the expression levels of PvEcR,PvUSP,PvBR-C,PvDDC,Pvhsc70,PvERR,PvVg in the treatment group were all up-regulated or down-regulated,but there were no significant time-dependent or concentration-dependent changes.The 4-NP treatment had the most significant effect on the PvVg gene of P.vicina,among which the workers and the males were the most significant.8.The results of paraffin section and hematoxylin-eosin staining showed that compared with the control group,4-NP mainly affected the midgut of the worker ants.In the 4-NP treated group,the muscle layer of midgut tissue was incomplete,the peritrophic membrane disappeared,the cell secretion was more active,nucleus appeared atrophy.We speculated that 4-NP would affect the metabolism of different castes of P.vicina and would influence their growth and development.9.16S high-throughput sequencing technology was used to analyze the effects of DHEP on the intestinal microorganisms of workers.The results showed that DEHP could change the richness and composition of the intestinal microbiota of workers and affect the metabolism in vivo.To sum up,endocrine disruptors DEHP and 4-NP both had negetive effects on the growth and development of P.vicina.The two disruptors can decrease the survival rate,increase or decrease the body weight of the adults,and cause the abdomen deformity of the adults.These physiological changes may be related to the changes of PvEcR,PvUSP,PvBR-C,PvDDC,Pvhsc70,PvERR,PvVg gene expressions after interference.Different genes differently responded to the two disruptors,and different disruptors had different abilities to change specific gene expressions.In addition,the two endocrine disruptors can damage the intestinal tissue of P.vicina,and then change its intestinal microbiota,affect metabolism,and ultimately harm growth and development of P.vicina.