Reproductive Toxicity Of Ethyl P - Hydroxybenzoate To Male Drosophila And Its DGE Analysis Based On RNA - Seq

Parabens have been widely used as antiseptics in food, medicine, cosmetics and children’s products. Because of its broad antibacterial spectrum, low cost, stable pH characteristic, etc., parabens are considered to be safe and effective. However, studies show that such substances have some estrogenic effects, and the damage to the reproductive system is in a dose-dependent manner. Previous studies found metabolic pathways of such substances are mainly blood and urine with little accumulation phenomenon. But they are found in breast cancer samples in recent studies, and can induce breast cancer cells, which indicate that there is cumulative effect and pose a threat to human health.RNA-seq connects genomic information and its functional protein group. Compared with the whole genome information, RNA-seq sequences obtained by this technology do not contain introns and other non-coding ones, which bringsmore efficient information and advantage in the gene sequence analysis.Short growing cycle, easy feeding, strong propagate ability, multi mutant traits, few chromosomes and homologous with humans gene sequences; these make Drosophila an important model organism in biology and genetics research. This study selects the male Drosophila(Drosophila melanogaster Canton-S strain) as experimental materials. At physiological level, we tested three different EP concentrations (0.03%,0.07%,0.10%) after treatment on male flies, takenstatistics of the egg laying amount and maximum egg laying amount of10d parental generation (Fo) and first filial generation (F1), the emergence rate and emergence amount of F1and second finial generation (F1), the duration time of eggs-arvae, larvae-pupa, and pupa-adult flies, and the total duration time of emergence. At molecular level, we conducted DGE analysis on male fruit flies between0.07%Ethylparaben (EP) treated group and control group based on RNA-seq technique. We then screened differentially expressed genes between the two groups of Drosophila, and performed clustering analysis, GO function annotation, KEEG metabolic pathway analysis, followed by real-time quantitative PCR analysis to verify the DGE on thetwo down-regulated genes up-regulated genesThe results are as follows:1. After adding the concentrationof EP at0.03%,0.07%and0.01%to culture medium, the physiological indexes of drosophila are significant different from compared groups. The results show that fecundity, feather amount and rate of treatment group Fo decrease compared with the control groups, with a significant difference (P<0.01). Total eclosion days of F1also increases with EP concentration.2. Different concentrations of EP are made to Fo male Drosophila. We find fecundity of Fl and feathering amount of F2in EP group (0.10%) fecundity have a very significant difference (P<0.01). Total eclosion days of F2is also shortened (P<0.05), while0.03%and0.07%groups are not significantly different.3. After DEG analysis of0.07%EP deal to male Drosophila and control group,18significantly differentially expressed genes are selected. GO functions of differentially expressed genes are classified into three groups, these arebiological processes, molecular functions, and cellular components categories. The results show that all of the three major categories, carbohydrate metabolic process, cation binding and hydrolase activity and ion binding enriched more differentially expressed genes. Furthermore, in the biological processes category, there is a mannose metabolic process significant enrich term (p<0.05), while there are two lytic vacuole and lysosome in cellular components categories and four alpha-mannosidase activity; mannosidase activity; hydrolase activity, hydrolyzing O-glycosyl compounds and hydrolase activity, acting on glycosyl bonds in categories of molecular function.4. GO analysis on up-regulated and down-regulated genes show that genes in the cellular categories are all down-regulated, while up-and down-regulated genes exist within biological processes and molecular functions. In the biological process, there are a lot of down-regulated genes associated with carbohydrate metabolism and a large number of up-regulated genes associated with oxidative stress. While in the molecular functions group, the functions of hydrolytic activity, α-mannosidase activity, glycosylation, mannosidase activity and carbohydrate binding are mainly associated with down-regulated genes. Peptide and carboxypeptidase activity are related to up-regulated genes.Others such as zinc ions, cations, transition metal ions, metal ions and hydrolytic activity are correlated with both up-regulated and down-regulated genes.5. The paper focuses on the pathway of the differently expressed genes through KEEG and finds that these genes were enriched in8pathways, in which starch and sucrose metabolism, phosphatidylinositol signaling system, metabolic pathways, lysosome, inositol phosphate metabolism, glutathione metabolism and galactose metabolism7pathways were significantly enriched.6. Four genes out of the18differentially expressed genes, including two up-regulated and two down-regulated, are screened and validated by real-time quantitative PCR. The results show that the expression trends of these four genes are consistent with the results based on RNA-Seq analysis of DGE.Comprehensive analysis shows that the EP has certain reproductive toxicity on FO male fruit flies, and the effects of high concentrations of EP on FO are sustainable to the offsprings. Here we speculate that EP can cause disorders in metabolism, hormone secretion and gene expression, as well as alterations in function related genes of different enzymes. These effects will influence the reproduction of male Drosophila, which has certain toxicity to the body.