Title: De novo transcriptomic analysis predicts the effects of phenolic compounds in Ba
River on the liver of female sharpbelly (Hemiculter lucidus)
Members:Jiahua Guo1, Jiezhang Mo2, Jianglin Peng1, Shan Liu1, Mirella Kanerva3, Hisato Iwata3,
Affiliations
1. Shaanxi Key Laboratory of Earth Surface System and Environmental Carrying Capacity,
College of Urban and Environmental Sciences, Northwest University, Xi’an 710127, China.
2. Department of Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China
3. Center for Marine Environmental Studies, Ehime University, Bunkyo-cho 2-5, Matsuyama,
Ehime prefecture, Japan. 790-8577.

1. Background
Ba River as one of the biggest urban river of Xi’an has been overburdened with theeffluent discharged from 5th wastewater treatment plant (WWTP) in Xi’an, which mainly
receives and treats the municipal and industrial sewage. Wang et al. (2018) have measured
the concentrations and distribution of BPA, NP, and 4-t-OP in three sampling sites, including
a mid-stream site (M) located near the outfall of WWTP, and two sites (up- (U) anddown-stream (D)) situated approximately four kilometers away from the WWTP discharges.
In regards to sites U, M, and D, the mean concentrations for BPA were 297, 1573.1, and334.9 ng L-1, respectively; the exposure levels for NP were 125.2, 634.8, and 185.6 ng L-1;
and concentrations for for 4-t-OP were 85.4, 126, and 56 ng L-1, respectively. In the U, M,and D sites, the mean concentrations in sharpbelly were 16.7, 26.4, 11.5 (ng g-1, dry muscleweight) for 4-t-OP, 15.5, 103.5, 30.8 ng g-1 for NP, and 24, 146.9, 28.7 ng g-1 for BPA,
respectively.
2. Aims
The objectives of the present study were: (1) to assess the potential phenotypicchanges induced by phenolic compounds containing WWTP effluent; (2) to decipher themolecular pathways dysregulated by phenolic compounds in WWTP effluent; and (3) to link
the potential contaminants in the effluent with the dysregulated molecular pathways,
predicting the potential adverse effects.
3. Procedure
3.1 Sampling sites and fish collection
Sharpbelly were collected from the Ba River that includes an outfall of the 5th municipal
wastewater treatment plant (WWTP; Fig. 1). Three sampling sites were selected including asite located up-stream (U) from the WWTP outfal, a mid-stream site (M) located near theoutfall of WWTP, and a site located down-stream (D) from the WWTP discharges. Here,sharpbelly collected from up-, mid- and down-stream of Ba River that were designed to beinterpreted as control, high, and low treatment groups, respectively. While fifteen fish were1 / 5

caught from each site by fishing, more than 90% were females. Thus, four adult female
sharpbelly in each site were randomly selected for further transcriptomic analysis.
After capture, sharpbelly were immediately sacrificed following the animal handing
procedures of Northwest University. Four biometric parameters including body weight (g),Fulton’s condition factor (CF = fish weight/ fish length), gonado-somatic index (GSI = gonadweight/ body weight), and hepatosomatic index (HSI = liver weight/ body weigh) wereemployed to evaluate the phenotypic effects of WWTP effluent on all the captured sharpbelly.
Liver tissues submerged in Trizol (Shanghai Yuanye Bio-Technology Co., Ltd, China) were
subjected to snap freezing by liquid nitrogen and stored at -80 0C prior to RNA extraction.

Fig. 1. Map of the study area and the sampled site location. The study design incorporated threesampling sites, including a site located up-stream from the WWTP outfall, a mid-stream site
located near the outfall of WWTP, and a site located down-stream from the WWTP discharges.

3.2 RNA extraction, library preparation, and illumina sequencing
3.3 De novo assembly and functional annotation

3.4 Differentially expressed genes (DEGs) enrichment analysis
Methods in 3.2-3.4 followed the sophisticated bioinformatics steps so they are not repeated.
4. Results
4.1 Phenotypic assessment
Internal examination of these sharpbelly revealed that CF and body weight in themid-stream fish were remarkably increased by approximate 20% in comparison with theup-stream (Fig. 2). Liver weights characterized by HSI in the sharpbelly dwelling in the mid- anddown-stream were higher than that in the up-stream, but no significant difference was recorded

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between sites (Fig. 2).
4.2 Transcriptomic analysis
4.2.1 Illumina sequencing and de novo assemblyRNA-seq data were obtained from 41.7 million to 52.9 million reads per tissue sample. The
majority of reads were of high quality with Q20 values more than 96.52% across all the samples.
After filtering the sequencing data containing a number of adaptors and low quality reads (< Q20),
the clean data occupying > 92.38% were assembled de novo using the Trinity assembler. De
novo assembly produced 261,296 unigenes with the mean length of 618.8 bp and a total lengthof 161.7 Mb.
4.2.2 Functional annotation
The unigenes were annotated using BLAST searches against NR, GO, KEGG, eggNOG,and Swissprot databases. The annotated RNA-seq genes were distributed in NR (25.18%), GO(7.09%), KEGG (12.66%), and Swissprot (17.41%). In the annotation to NR, sequencing data of
sharpbelly showed high similarity to Sinocyclocheilus rhinocerous (18.85%), Sinocyclocheilus
anshuiensis (15.86%), and Sinocyclocheilus graham (14.51%; Fig. 3A). Based on GO, 18538
unigenes were categorized into 3 main categories, including biological process (BP), molecularfunction (MF), and cellular components (CC), and 67 subcategories. From KEGG, 33,069
unigenes were assigned into 35 pathways that were primarily involved in the process of
metabolism, genetic information processing, environmental information processing, cellular
process, and organismal systems.
4.2.3 Differentially expressed genes and enrichment analyses
In total, we identified 160 (83 up- and 77 down-regulated), 15 (5 up- and 10 down-regulated),
162 (97 up- and 65 down-regulated) DEGs in U-M, U-D, and D-M groups of female sharpbelly,respectively (Fig. 3C, S3). In heatmap, DEG expression of sharpbelly living near WWTP outfall
were clearly departed from three individuals collected from up- and down-stream sites, whereas
the rest samples were indistinguishable from the mid group (Fig. 3B).
GO analysis was implemented to investigate the roles of DEGs in the cellular function.
Given a set of DEGs, the GO analysis is able to figure out the CC, MF, and BP terms that wereinfluenced by WWTP effluent in the female sharpbelly. Top 10 GO terms enriched in CC, MF, andBP in each group were summarized in Supplementary data. Contrast with U-M, and D-M groups,
we merely identified 3 CC and 4 MF terms in U-D group, suggesting there was no significant
difference in sharpbelly living in both sites. The functional KEGG pathway analysis determined
32 and 50 enriched pathways in U-M and D-M groups that may have been affected by WWTP
effluent. Top 20 pathways enriched in each sites were illustrated in Tables 1 & 2. These pathways
were mainly in relation to drug addiction and metabolism (e.g., amphetamine addiction, cocaine
addiction, and drug metabolism – cytochrome P450 for citalopram), endocrine system (e.g.,
endocrine resistance, endocrine and other factor−regulated calcium reabsorption, thyroid
hormone synthesis and signaling), and cellular process and lipid metabolism (e.g.,
mitogen-activated protein kinase (MAPK), adipocytokine signaling, hepatitis C, and theperoxisome proliferator-activated receptors (PPARs) pathway). Nearly no pathway was enrichedin U-D group.

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4.3 Predicted effects of phenolic compounds on sharpbelly
To sum up, our results provided an insight into the molecular mechanism of action of WWTP
effluent incorporating phenolic compounds and predicted the potential adverse outcomes infemale sharpbelly (Fig. 4).

Fig. 2. Condition factor (CF), gonado-somatic index (GSI), hepatosomatic index (HSI) andbody weight (BW) expressed as a percentage of values in sharpbelly collected from the midand down-streams relative to those in the up-stream. Data represent mean ± standard
deviation (n = 15).
A.

B.

C.

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Fig. 3. Transcriptomic profiling of sharpbelly living down-stream from WWTP outfall. (A) Similarityof sharpbelly sequencing data to other fish species; (B) A heatmap of centered and scaled FPKM
values of DEGs in sharpbelly from different sites; (C) Venn diagram of the number of DEGs ineach group.

Fig. 4. Schematic diagram of the pathways activated in the female sharpbelly living in themid-stream. acsl3: Long-chain-fatty-acid--CoA ligase 3; adcy1: adenylate cyclase 1; esr1:
estrogen receptor 1; fgf13: Fibroblast growth factor 13; hspa5: heat shock protein family A(Hsp70) member 5; mao: monoamine oxidase; ppard: Peroxisome proliferator-activated receptor
delta; scd1: stearoyl-CoA desaturase; socs3: suppressor of cytokine signaling 3; tnfrsf1a: tumor
necrosis factor receptor superfamily member 1A. 1. Effects of WWTP effluent on the behavioral
alteration and reproductive function were unclear.

Publication
This paper has been submitted to Environmental pollution and currently under peer-review.
Perspective In Future
As BPA and NP are typical endocrine disrupting chemicals, a further investigation intomolecular changes in other organs (e.g. gonad) is required for a complete understanding of
the adverse effects caused by phenolic compounds in the WWTP effluent.

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