Effects Of Octylphenol Exposure On Gene Expression And Flora Structure Of Tadpoles In Rana Chensinensis

Octylphenol(OP),one of degradation product of alkylphenol ethoxylates(APEs),is widely used as adhesives,non-ionic surfactants and plasticizers in the manufacture of several consumer products and in industrial processing,and has caused a large area of aquatic pollution.The embryo and larval stages of most amphibians take place in water,and adult stages take place on land.Aquatic chemical pollutant is one of the main factors that contribute to the decline of amphibian population.Numerous studies have shown that OP exposure has many negative effects on intestine,liver and kidney and other internal organs of animals.The digestive tract,an important organ involved in the metabolism of the organism,play a key roal in the digestion of dietary components and nutrient transporters,digestion and absorption.Intestine is the main reservoir for the trillions of commensal bacteria which inhabit the intestine and which are essential for health.However,the composition of the gut microbiome is highly diverse,and this diversity can be readily affected by environment,diet,bacterial/viral infection,and antibiotics.This raises the possibility that gut microbiome dysbiosis induces a serious threat to the host health.Accumulating evidence indicates that dysregulated gut microflora contributes in a significant way to a variety of diseases,including diabetes,obesity,cardiovascular diseases,allergies,inflammatory bowel disease,and others.Aquatic chemical pollutants may invade the organism of aquatic animals through digestive tract,skin and gills,particularly for the digestive tract.Therefore,it is necessary to study the effect of chemical pollutants on the structure and function of digestive tract and the structure and abundance of microflora,thereby further exploring the mechanism by which pollutants interfere with the growth and development of the organism.In the present study,Rana chensinensis larva was selected as model organism to investigate the effect of OP on intestinal structure and gut microbiota of amphibians.Firstly,R.chensinensis were exposed to different OP concentrations(0,10-8,10-7 and 10-6 mol/L OP)from Gosner stage(Gs)25 to 38.Snout-vent length,intestinal length,body weight and intestinal weight of tadpoles at Gs 38 in each group were measured,and the microstructures of intestines were observed through pathological section.Secondly,the transcriptome of intestine of R.chensinensis were conducted using RNA-seq approach,and six differentially expressed genes(DEGs)related to the fat digestion and absorption were also validated by qRT-PCR technology.Finally,effects of OP exposure on intestinal microbial structure and composition in R.chensinensis tadpoles were evaluated via high-throughput sequencing of V3-V4 hypervariable region of the bacterial 16S ribosomal RNA gene.The main results and conclusions are as follows:1.Morphological measurement results show that OP increased the weight increase velocity of R.chensinensis tadpoles.Tadpoles exposed to 10-8 mol/L OP had significantly higher intestinal weight than that of the control.Furthermore,significant increases in body weight,intestinal length and intestinal weight relative to controls were observed at 10-7 mol/L OP.In high dose OP treated group(10-6 mol/L),tadpoles had significantly longer SVL and intestinal length and significantly higher total body weight and intestinal weight than the control tadpoles.We conclude that OP are responsible for the tadpole obesity by interfering the lipid metabolism.Further studies are needed to explain the phenomenon.2.Histological examinations for intestine sections stained with hematoxylin-eosin(H&E)showed that the enterocytes were arranged closely and homogeneously,all cells displayed clear cytoplasm and well-delineated cytoplasmic borders in the control tadpoles.The enterocyte morphology of tadpoles in any OP exposure group was almost the same as that in the control group.The results indicated that OP had no significant effect on intestinal microstructure of tadpole.3.15 genes related to the fat digestion and absorption were found in the cDNA library of intestine by using RNA-Seq technology,and the expression profiles of these genes in intestine of tadpoles at Gs 38 from control and 10-6 mol/L OP treated groups were analyzed.In addition,six of these genes were validated by qRT-PCR.The qRT-PCR results showed 10-6 mol/L OP treatment could down-regulate CLPS,LIPASE and I-FABP mRNA levels,while up-regulate APOA-1,APOB-48 and CD36 mRNA levels in the intestine of R.chensinensis.Transcript level changes of CLPS,LIPASE,I-FABP,APOA-1 and APOB-48 were consistent with the results of qRT-PCR.It indicates that the results of the present study are reliable.In addition,based on the analysis of mRNA levels of these DEGs,we assumed that OP exposure may interfere with the fat digestion and absorption pathways in the digestive tract of tadpoles,thereby causing potential interference to their health.4.Through 16S rRNA gene sequencing,168 OTUs were obtained from the intestines of R.at control group and different OP treated groups(10-8,10-7 and 10-6 mol/L).The distinct OUT assignments and alpha-diversity analysis of intestinal microbiota revealed that 10-7 mol/L OP exposure caused decrease in the diversity of the intestinal microbiota compared to the control group,which may be harmful to gut homeostasis and health of R.chensinensis tadpoles.Exceptionally,no significant changes were found in gut microbial diversity at 10-6 mol/L OP exposure group.Further studies are needed to explain the phenomenon.5.Beta-diversity of the intestinal microbial communities in intestines of R.chensinensis from control and OP exposure groups were analyzed.At the phylum level,10-6 mol/L OP could significantly increase the proportion of Bacteroidetes,while the abundance of Proteobacteria and Firmicutes were significantly decreased in 10-6mol/L OP treated group than that of the control group.At the genus level,the abundance of Aeromonas was significantly increased at 10-8 mol/L OP exposure group.The results showed that OP exposure altered the intestinal microbiota composition of R.chensinensis.6.The PCoA plot base on UPGMA clustering and the hierarchical clustering tree confirmed that the control and 10-8 mol/L OP exposure groups OTUs clustered together into one group by phylogenetic composition,the 10-7 mol/L OP exposure group independent into a group and clustered together with the first two groups,and 10-6 mol/L OP group independent into another group.7.On the basis of function analysis using 16S rRNA profile,the relative abundance of Energy Metabolism,Metabolism of Cofactors and Vitamins,Metabolism of Terpenoids and Polyketides,and Transport and Catabolism were significantly increased,whereas Amino Acid Metabolism,Carbohydrate Metabolism,Environmental Adaptation,and Metabolism of Other Amino Acids were significantly decreased in 10 6 mol/L OP exposure group compared to the control.For disease,we found that 10-6mol/L OP can significantly elevated the risk of Cancers,Infectious Diseases and Metabolic Diseases on larvae compared to the control.In summary,the morphological parameters presented herein demonstrated that exposure to OP could promote the growth of tadpoles.In addition,we found that OP treatment could affect the expression level of genes related to the fat digestion and absorption with RNA-Seq and qRT-PCR technology.Besides,according to 16S-rRNA sequencing analysis,exposure to OP obviously altered the intestinal microbial diversity,structure and composition,affected metabolism level and increased risk of disease.The present study thus will greatly contribute to our understanding of the effects of phenolic compounds on the growth and development of amphibian larvae.Moreover,the present study will provide a valuable genomic resource and background for the future study of fat digestion and absorption response to phenolic compounds in amphibian.