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Transcriptomic and physiological changes in western mosquitofish (Gambusia affinis) after exposure to norgestrel
Ecotoxicology and Environmental Safety 171 (2019) 579–586
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Transcriptomic and physiological changes in western mosquitofish (Gambusia affinis) after exposure to norgestrel
T
Liping Houa,b, Shanduo Chena, Juan Liuc, , Jingwen Guod, Zhong Chene, Qiaoling Zhue, ⁎ Wei Zhangf, GuoLiang Xug, Ye Lianga, Rongrong Wua, Xuwen Fanga, Cuiping Zhanga, Ke Xinga, ⁎
a
School of Life Sciences, Guangzhou University, Guangzhou 510655, China Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou 515063, China c Key Laboratory of Water Quality and Conservation in the Pearl River Delta, Ministry of Education, Guangdong Provincial Key Laboratory of Radionuclides Pollution Control and Resources, Institute of Environmental Research at Greater Bay, Guangzhou University, Guangzhou 510006, China d College of Environmental Science and Engineering, Guangzhou University, Guangzhou 510655, China e NanWu Middle School, Guangzhou 510655, China f Guangzhou Tieyi Middle School, Guangzhou 510655, China g Rural Non-point Source Pollution Comprehensive Management Technology Center of Guangdong Province, Guangzhou 510655, China b
ARTICLE INFO
ABSTRACT
Keywords: Endocrine disruption Norgestrel Secondary sexual characteristics transcriptomics Gambusia affinis
Norgestrel (NGT) is a synthetic progestin used in human and veterinary medicine. Adult female mosquitofish were exposed to NGT for 42 d at 377 ng L−1. The fin morphology and the liver transcriptome were assessed. NGT exposure increased ray 4:6 length ratio. As compared to the control, NGT treatment affected the expression of 11,772 annotated transcripts in female mosquitofish. Specifically, we found 5780 were repressed while 5992 were significantly induced. Gene ontology (GO) analysis showed that 53 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and 158 GO terms were significantly over expressed. Genes showing the largest magnitude of expression changes were related to fin development, androgen biosynthesis, and lipid and fatty acid metabolisms, suggesting the involvement of these biological processes in response to NGT exposure in G. affinis. This first comprehensive study on the transcriptomic alterations by NGT in G. affinis not only provides valuable information on the development of molecular markers but also opens new avenues for studies on the molecular mechanisms of effects of NGT in particular and possibly other progestins in G. affinis.
1. Introduction During the past twenty years, endocrine-disrupting chemicals (EDCs) have been increasingly recognized as global aquatic contaminants. More recently, the effects of environmental gestagens (including synthetic and natural progestins) on aquatic organisms have drawn widespread concerns (Fent, 2015). Synthetic progestins are widely used in veterinary medicine and other hormonal therapies (Kejuan et al., 2007; Klijn et al., 2000). These compounds contaminate the environment via wastewater effluents due to their incomplete removal by treatment plants ( Huang et al., 2012). Recent studies have shown that both natural and synthetic progestins may cause endocrine disruption in fish even at low concentrations (Han et al., 2014; Kugathas et al., 2012; Murack et al., 2011). A previous study reported that Norgestrel (NGT) is present in surface waters at concentrations up to 199 ng L−1 ( Buxton and Kolpin,
⁎
2005; Liu et al., 2011). NGT was found in municipal wastewater treatment plant effluents at a concentration of up to 11 ng L−1 in China (Liu et al., 2012a) and in the surface water at concentrations varying between 30.5 and 465 ng L−1 (Liu et al., 2012b). Our previous studies have reported that NGT exposure leads to changes in gonad histopathology and masculinization of female western mosquitofish G. affinis at environmentally relevant concentrations (Hou et al., 2018); it also affects sexual development and leads to transcriptional alterations of genes of the hypothalamic–pituitary–gonadal (HPG) axis in zebrafish Danio rerio (Liang et al., 2015a). Western mosquitofish G. affinis is considered as an important animal model for investigations on endocrine disruption (Kavitha and Venkateswara, 2007, 2008). It is characterized by marked sexual dimorphism and distinct reproductive behaviors (Hou et al., 2017, 2018; Orlando et al., 2007). Previous investigations have shown that exposure to androgens, which include 17b-trenbolone (Angus et al., 2001) and
Corresponding authors. E-mail addresses: [email protected] (J. Liu), [email protected] (K. Xing).
https://doi.org/10.1016/j.ecoenv.2018.12.053 Received 10 September 2018; Received in revised form 8 December 2018; Accepted 18 December 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
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androstenedione (Stanko and Angus, 2010), induces anal fin alterations in females. Although some progestins are known to be androgenic, the available information on the androgenic effects of NGT at the molecular level is very limited. Therefore, the aim of the present study was to conduct transcriptional profiling for examining various acute modulatory pathways in the livers of female mosquitofish (G. affinis) which had been exposed to NGT using next generation sequencing technology. This may provide novel insights into the mechanisms through which NGT modifies the female mosquitofish anal fins and impairs female reproduction. We hypothesized that changes in physiology and gene expression would be indicatives of the androgenic property of NGT.
China). Briefly, mRNA purification using the total RNA samples was performed with polyT oligo-attached magnetic beads (Qiagen, USA), and then cleaved into small fragments by adding a fragmentation buffer. First-strand cDNA synthesis by reverse transcription, using random hexamers as templates, was then performed, which was followed by second-strand cDNA synthesis with DNA polymerase I and RNase H (NEB, USA). Paired-end adapters were attached to the resulting double-stranded cDNA, followed by the selection of suitable fragments and PCR amplification to yield cDNA libraries. The resulting 150-bp paired-end reads were then sequenced on an Illumina HiSeq™ 2500 platform. 2.5. Assembly of transcriptome and gene function annotation
2. Methodology
First, the generated raw reads were filtered by removing the following: reads attached to adapters, reads with unknown nucleotides (Nts) that were > 5%, as well as low-quality reads (> 10% of bases have a Q score ≤20). The remaining clean reads generated from each sample were then assembled using Trinity v2.0.6 software (Heinl et al., 2011) using default settings, and minimum contig length of 200 bp. Functional annotation of all the unigenes was performed using various databases such as the non-redundant protein database (Nr), Swiss-Prot database, Clusters of Orthologous Groups (COG) protein database, Kyoto Encyclopedia of Genes and Genomes (KEGG), as well as Gene Ontology (GO). If a unigene could not be aligned to any database, ESTScan ( Lottaz et al., 2003) was used to predict the coding regions as well as sequence direction. Functional annotation of the unigenes was conducted using WEGO ( Ye et al., 2006).
2.1. Mosquitofish rearing conditions Adult female mosquitofish, Gambusia affinis, were collected in November 2016 from the Liuxi River, which is protected from pollution (Hou et al., 2011). Ten sexually mature female G. affinis per tank were acclimatized in 40-L glass aquaria ( three replicates) containing with 30 L of filtered and aerated tap water. A 12 h:12 h light/dark photoperiod and a controlled temperature (24 ± 1 °C) were utilized. The mosquitofish were fed each day with commercialized red worm flakes ( Haisheng Co., Shanghai, China) during the acclimation period. Food leftovers and fecal matter were siphoned out from the aquaria each day. 2.2. Hormonal treatment Norgestrel (> 99.9% purity) was purchased from Sigma-Aldrich (StLouis, MO, USA). An NGT stock solution (1 mg mL−1) was a diluted to different concentrations and used in the treatments. The female G. affinis were exposed to 0 and 500 ng L−1 of NGT. Three replicate tanks containing 10 females each were used. DMSO was used as a solvent with a final exposure concentration of 0.003% (v/v). The solvent controls consisted of 0.003% DMSO only. The experiment was carried out in a system that was semi-static at a temperature of 25 ± 1 °C. Daily measurement of water chemistry parameters such as conductivity (22 ± 0.1 μS cm−1), water hardness (153 ± 3.7 mg L−1 as CaCO3), dissolved oxygen (DO, 7.7 ± 0.1 mg L−1), and pH (7.6 ± 0.4) were performed daily using a water quality meter with multiple parameters (YSI Model 85 m; Yellow Springs, OH, USA). The medium of exposure was renewed every day.
2.6. Analysis and functional enrichment differentially expressed genes (DEGs) Mapping of clean reads of each sample to the assembled unigenes was performed, followed by estimation of their relative abundances using RSEM software v1.2.12 (Li and Dewey, 2011) using the fragment per kilo bases per million reads (FPKM) method. The detection of DEGs between the NGT and the control was performed using the R package DEGseq ( Wang et al., 2010) using a threshold of | log2 (fold change) | > 1 and a corrected p-value (q-value) < 0.05. To detect overrepresented GO terms as well as KEGG pathways, DEG enrichment analyses were performed using the R package GOseq V1.16.2 (Young et al., 2010) and KOBAS ( Xie et al., 2011). 2.7. Validation by real-time PCR
2.3. Biometrics and fin morphology
Ten candidate genes, which included those with transcripts that are associated with fin development, androgen production, and lipid and fatty acid metabolism, as well as housekeeping genes were employed to design qRT-PCRs (Table S1). The sequences of the detected unigenes were compared with the homologues (blastx) to verify our annotation. These were then used to design qPCR primers. All the primers were validated using end-point PCR. The 10 candidate genes selected were used for determining transcript abundance in mosquitofish liver.
After 42 d of exposure, ten female mosquitofish from each tank (total = 30) were anesthetized (MS-222, 300 mg L−1). Seven fish were randomly selected from each tank (total = 21) and used for measuring anal fin length with a calibrated dissecting microscope (Olympus SZHILLK). Individual fish weight and length were also determined. The width of anal fin ray 3 ( ± 0.1 mm), lengths of rays 4 and 6, and the number of segments in ray 3 were measured. Three mosquitofish from every group were sacrificed by spinal severance. The livers were collected and immersed in Trizol ( Invitrogen, Shanghai), followed by storage in −80 °C until analysis.
2.8. Measurement of NGT levels in the exposure media Since the exposure media were renewed each day, the actual exposure concentrations of NGT were determined at the initiation of the exposure (0 h, T0) and prior to water renewal (24 h, T 24) in the first day for all aquaria. Approximately 500 mL water samples were taken from each aquarium. NGT concentrations in the exposure medium were measured,as described in our previous study (Xu et al., 2014). NGT extraction from the water samples was performed using CNWBOND LCC18 SPE cartridges (200 mg, 3 mL) (Germany), followed by analysis using RRLC–MS/MS (Xu et al., 2014). The respective NGT limit of detection (LOD) and the limit of quantitation (LOQ) were 0.02–0.25 ng L
2.4. RNA extraction, library construction, and sequencing Total RNA was extracted from the livers of mosquitofish using a TRIzol kit (Promega, USA), followed by DNase I (Takara Bio, Japan) treatment to remove genomic DNA. RNA quality and purity were assessed by agarose gel electrophoresis using a Bioanalyzer 2100 system (Agilent, CA, USA). cDNA libraries construction and Illumina sequencing were carried out by the Total Genomics Solution (TGS) Company (Guangzhou, 580
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and 0.071–0.79 ng L −1. The spiked sample showed a recovery rate ranging from 72% to 107% (Liu et al., 2014).
fin development, androgen biosynthesis, lipid metabolism, and fatty acid metabolism (Table 1). Moreover, KEGG pathway analysis indicated that these DEGs were related to various biological functions, which included metabolic pathways, tryptophan metabolism, protein processing involving the endoplasmic reticulum, insulin resistance, adipocytokine signaling pathway, production of unsaturated fatty acids, the AMPK signaling pathway, PPAR signaling pathway, pyruvate metabolism, biosynthesis of lysine, and the insulin signaling pathway (Table 2).
2.9. Statistical analyses Statistical analyses were conducted using SPSS (version 13.0, IBM, Chicago, IL, USA). Normality and homogeneity of variances were assessed using Kolmogorov–Smirnov and Levene's tests, respectively. Differences between the control and the NGT exposed group were evaluated by one-way ANOVA. The differences were considered statistically significant at a p-value < 0.05.
3.6. RNA sequencing validation using real-time PCR To validate the results of gene expression profiling using RNA-Seq, 10 selected genes, consisting of five upregulated and five downregulated genes, were analyzed by real-time PCR (Table S2). Fig. S1 shows that the expression patterns of these selected genes coincided with the results obtained by RNA-seq. This proves that the gene expression profiles generated by RNASeq were reliable, thereby confirming the observed changes in the expression of genes responsive to NGT stress. (Table 3)
3. Results 3.1. Chemical analysis Table S2 shows the concentrations of the control and the NGT treatments (i.e., 0 ng L−1 and 500 ng L−1). The concentrations of NGT in the solvent control were below the detection limit. Thus, the average NGT concentrations (i.e., 0 ng L−1 and 377 ng L−1) were used for the presentation of data for the SC and the nominal 500 ng L−1 throughout this investigation.
4. Discussion Progestins are potent EDCs in fish as well as in amphibians (Fent, 2015; Kumar et al., 2015). Exposure to progestin results in a variety of adverse effects involving the endocrine systems such as vitellogenin synthesis in male or juvenile fish, disruption of egg maturation, ovotestes/intersex, abnormal reproduction, inhibition of embryonic development, and skewed sex ratios (Hua et al., 2015; Li et al., 2017; Liang et al., 2015b; Runnalls et al., 2013; Zucchi et al., 2012). Our previous study and the present investigation demonstrate that synthetic progestin exposure at environmentally relevant concentrations may induce masculinization of female G. affinis ( Hou et al., 2018; Hou et al., 2017). However, the mechanisms of their masculinization effect remain elusive. The next generation sequencing technique has offered great opportunities for uncovering the molecular pathways related to masculinization effect of progestins. This study used RNA-seq to explore the pathways modulated by NGT in the liver tissues of the adult female mosquitofish to elucidate the molecular mechanisms for masculinization of female G. affinis. We found that the overall hepatic mRNA expression in the female mosquitofish differed substantially between the NGT-treated and the control groups, with altered levels for 11,772 transcripts, including many associated with growth (e.g., fin development), metabolism, or development of mosquitofish. These data could help us better understand the molecular mechanisms of effects of NGT in G. affinis and provide valuable genetic data to develop molecular markers for exposure to progestins in the future.
3.2. Effects of NGT on mosquitofish growth and anal fin morphometrics After 42 d of exposure to NGT, no changes in the length and weight of the adult females were observed (data not shown). However, as compared with the control female mosquitofish, ray 4:6 length ratio was significantly increased by 55.7% (P < 0.001) when exposed to 377 ng L−1 of NGT（Fig. 1）. 3.3. Transcriptome sequencing and assembly After removing redundant sequence from the assembled contigs, approximately 90,304 unigenes were identified. The assembled sequences’ GC content was around 46%. The assembled unigenes showed an average length of 1053 bp, with an N50 value of 2184 bp. Fig. 2 shows that the majority of the unigenes had sizes within the range of 200–500 bp, with 1316 of the unigenes ≥ 3000 bp in length（Fig. 2）. 3.4. Functional annotation of the unigenes The unigenes were subsequently compared with the sequences available in the GO database, and functionally annotated to three major categories, which included biological process, cellular component, and molecular function (Fig. 3). Approximately 18,368 unigenes showed significant hits with the GO database. For the functional category of biological process, the term “cellular process” had the highest number of unigenes, whereas “cell part” and “binding” showed the highest number of transcripts for the respective functional categories of cellular component and molecular function. The KEGG pathway analysis also assigned the unigenes to six different pathways such as Genetic Information Processing, Metabolism, Cellular Processes, Organismal Systems, Human Disease, and Environmental Information Processing (Fig. 4). A total of 37,939 unigenes showed significant hits using the KEGG database, of which 11,706 were related to signal transduction.
4.1. Differential transcriptional expressions by NGT-exposure NGT toxicity occurs via disruption of the normal interaction between E2 that is secreted by gonads and the brain, thereby influencing behavioral and reproductive activities. DE analysis showed that NGT imparts treatment-specific effects on G. affinis. In NGT-exposed females, genes related to the androgen biosynthesis and fin development were upregulated, whereas the genes related to metabolic process, transport, translation, lipid metabolism, carbohydrate metabolism, steroid hormone-mediated signaling pathway, transmembrane transport, biosynthetic process, and proteolysis were downregulated. The impact of NGT on the transcription of genes associated with fin development, biosynthesis of androgens, and lipid and fatty acid metabolism are presented in detail in the next sections.
3.5. Differential gene expression analysis In total, we identified 9190 annotated unigenes with significantly different expression levels after exposure to NGT. Among these, 3712 were significantly induced and 5478 were repressed. To further investigate the functions of these unigenes and their corresponding biological processes, we performed enrichment analysis using the GO and KEGG pathway. GO enrichment analysis showed that many of the DEGs were related to several sexual development biological processes such as,
4.2. Effects of NGT on fin development Poeciliid fish (e.g., G. affinis and G. holbrooki) are suitable models for investigating the effects of endocrine-disrupting compounds based 581
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Fig. 1. Pictures of anal fin morphology of mosquitofish exposed to norgestrel (NGT) for 42 d. (A) Anal fin of a normal adult female with rays 3 labeled; (B) Anal fin of a normal adult male with rays 3 labeled; (C) Anal fin of a masculinized adult female with rays 3 labeled at 377 ng L−1 NGT; (D) Ratios of the basal length and total length of the anal fin of the control and the adult female Gambusia affinis treated by 377 ng L−1 norgestrel for 42-day to that of the solvent control female. Data are expressed as mean ± standard error (SEM) of three replicate samples ( n = 21). *P < 0.05 and * *P < 0.01 indicate statistically significant differences between solvent control (SC) and exposure groups. Error bars represent the standard deviation of the mean values.
showed that NGT exposure increased ray 4:6 length ratio and number of segments in ray 3 ( Hou et al., 2018). These results demonstrated that NGT could masculinize adult female G. affinis. Similarly, exposure to levonorgestrel reportedly led to altered anal fin development in the eastern mosquitofish G. holbrooki (Frankel et al., 2016). In the mosquitofish, the anal fin differentiates into gonopodium via elongation and modification of the anal fin rays 3, 4, and 5 (Turner, 1941). Therefore, the elongation and outgrowth of the anal fin in the female mosquitofish exposed to progestins can be considered as the first indication of masculinization. In this study, fin development is a key process altered by NGT in mosquitofish. In the liver, a down-regulation occurred in several fin development genes including bmp1×1, sofh, hip, and an up-regulation of TRIM33×3, bmp1 and pl2O5dx at 377 ng/L NGT. In the liver, also RT-qPCR revealed a significant down-regulation of bmp1×1 at 377 ng/ L NGT (Fig. S2). These genes mediate the elongation and subsequent gonopodium development in mosquitofish. It is therefore likely that NGT altered the expression of these genes, which in turn activated the bone morphogenetic proteins to determine the fate of the downstream scleroblast (Laforest et al., 1998), resulting in the elongation and outgrowth of the anal fins. Many studies have shown that progestins including NGT and levonorgestrel are androgenic to fish (Hua et al., 2015; Runnalls et al.,
Fig. 2. Gene transcription profiling. Global expression profile of G. affinis exposed to norgestrel (NGT) at 377 ng L−1. Relative to the control, unigenes of 9190 genes were altered with 3712 genes up-regulated and 5478 genes downregulated. Fold-change cutoff was set at ≥ 2 and the FDR was set < 0.01. Green：down-regulated; Red: up-regulated.
on their morphological traits, particularly their external secondary sexual characteristics. In this study, exposure to 377 ng L−1 of NGT for 42 d resulted in increased ray 4:6 length ratio. Our previous study 582
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Fig. 3. GO annotation of the unigenes. The unigenes were assigned to different terms in three major categories of the GO database, namely biological process, cellular component, and molecular function.
2013; Liang et al., 2015b; Li et al., 2017; Zucchi et al., 2012). It has been demonstrated that genes including sonic hedgehog (shh), muscle segment homeobox C (msxC), and fibroblast growth receptor 1 (fgfr1) are also involved in the anal fin growth of the eastern mosquitofish G. holbrooki exposed to 17 β-trenbolone (an androgen) (Brockmeier et al., 2013).
4.4. Effects of NGT on lipid and fatty acid metabolism In this study, barring the genes related to androgen biosynthesis, the genes associated with lipid and fatty acid metabolism showed the largest magnitude of alterations in transcriptional expressions. Lipids pertain to a class of biomolecules that play various roles in structural and biological activities by interacting with proteins (Bogdanov et al., 2008; Sul and Smith, 2008). The main organic components of fish include lipids and proteins, which play major roles in its life cycle and physiology, which include growth, movement, reproduction, as well as migration (Tocher et al., 2003). The major function of lipids in fish is to store and provide energy in the form of adenosine triphosphate (ATP) via β-oxidation of fatty acids (Totland et al., 2000). Many studies have reported that hydrophobic pollutants accumulate in the lipids of aquatic organisms (Kainz and Fisk, 2009). In the present study, NGTexposure resulted in a decrease in the expression of some genes that are related to the metabolism of lipids and fatty acids in female mosquitofish. An example is the elongase “elongation of very long-chain fatty acids protein 6 (ELOVL6)” gene. ELOVL6 catalyzes the initial and ratelimiting step of the four that comprise the elongation cycle of longchain fatty acids. Another downregulated gene that is involved in the metabolism of lipid and fatty acids fatty acid desaturase 2 (FAD2), which catalyzes the generation of highly unsaturated fatty acids (HUFAs) using the following precursors: polyunsaturated fatty acids (PUFAs), linoleic acid (LA) (18:2, n-6), and alpha-linolenic acid (ALA) (18:3, n-3). In addition, the transcription of glycerophosphodiester phosphodiesterase 1 (GDPDL1), which hydrolyzes glycerolphosphoglycerol, was also downregulated significantly in female mosquitofish. It has been suggested that dysregulation of these genes by NGT exposure may disrupt the metabolism of lipids and fatty acids, thereby reducing the growth and reproduction of female mosquitofish. An earlier study involving NGT effects on zebrafish has shown that
4.3. Effects of NGT on androgen biosynthesis Sex hormones play a major role in the development of external secondary fish sexual characteristics (Devlin and Nagahama, 2002). Disruption of sex hormone biosynthesis may change the hormone levels of fish, thereby affecting their external secondary sexual features. In the present study, the expression of Hsd17β3 increased significantly after exposure to 377 ng L−1 NGT. Hsd17β3 and many other genes encode the enzymes which are involved in the production of 11-ketotestosterone (11-KT). The role of Hsd17β3 in inducing rapid masculinization of females needs further investigation. A recent investigation has shown that treatment of larval zebrafish (20 d post- fertilization) with NGT at concentrations of 34 ng L−1 and 77 ng L−1 for 40 d creates an all-male population, coupled with a significant decrease in the expression of Cyp11a1, Cyp17, Cyp19a1a, and Hsd3b (Liang et al., 2015b). Exposure to levonorgestrel at a concentration of 86.9 ng L−1 for 21 d induces a significant decrease in 3β-Hsd, 20β-Hsd, and Cyp19a expression in fathead minnow (Pimephales promelas) larvae ( Overturf et al., 2014). These findings indicate that NGT and levonorgestrel disrupt steroidal biosynthesis, which in turn affects fish sexual differentiation ( Liang et al., 2015b; Overturf et al., 2014). These studies also suggest that changes at the transcriptional expression of steroid-producing genes may induce masculinization of female mosquitofish after exposure to progestins. 583
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Fig. 4. KEGG annotation of the unigenes. The unigenes were assigned to different pathways in six major categories of the KEGG database, including genetic information processing, metabolism, cellular processes, organismal systems, human disease, and environmental information processing. Table 1 List of enriched gene ontology (GO) terms in the differentially expressed genes (DEGs). Class
GO_ID
Term
DEGs_in_Term
All_Genes_in_Term
Correct_P_Value
BP BP BP BP BP BP BP BP BP BP BP BP BP BP
GO0009058 GO0033333 GO0032259 GO0055085 GO0006629 GO0043009 GO0007165 GO0030433 GO0042157 GO0006702 GO0060059 GO0007519 GO0006631 GO0007417
biosynthetic process fin development methylation transmembrane transport lipid metalolic process vasculature development signal transduction ER-associated ubiquitin-dependent protein catabolic process lipoprotein metabolic process androgen biosynthetic process embryonic retina morphogenesis in camera-type eye skeletal muscle tissue development fatty acid metabolic process central nervous system development
36 12 31 87 50 18 111 6 11 5 8 7 9 11
109 20 103 418 206 47 579 7 22 8 15 12 19 28
3.01306E-05 0.000306 0.000913 0.00112 0.00145 0.00184 0.0026 0.0039 0.00391 0.0111 0.0178 0.0208 0.0213 0.0326
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Table 2 List of enriched KEGG pathways in the differentially expressed genes (DEGs). Pathway
DEGs in term(7994)
All genes in term(37939)
Q-value
Pathway ID
Metabolic pathways Tryptophan metabolism Protein processing in endoplasmic reticulum Insulin resistance Adipocytokine signaling pathway Biosynthesis of unsaturated fatty acids AMPK signaling pathway PPAR signaling pathway Pyruvate metabolism Lysine biosynthesis Insulin signaling pathway
1080 (13.51%) 56 (0.7%) 158 (1.98%) 110 (1.38%) 76 (0.95%) 30 (0.38%) 136 (1.7%) 81 (1.01%) 50 (0.63%) 6 (0.08%) 141 (1.76%)
4142 (10.92%) 163 (0.43%) 569 (1.5%) 380 (1%) 250 (0.66%) 79 (0.21%) 498 (1.31%) 276 (0.73%) 155 (0.41%) 8 (0.02%) 541 (1.43%)
5.10759E-14 0.00254251 0.003021287 0.005003576 0.007597332 0.008891159 0.009632565 0.01260297 0.01273914 0.02381083 0.03588414
ko01100 ko00380 ko04141 ko04931 ko04920 ko01040 ko04152 ko03320 ko00620 ko00300 ko04910
Table 3 Genes assigned to the biological functions response to fin development, androgen biosynthetic process, lipid and fatty acid metabolic metabolic process analyses of the transcripts significantly differentially expressed in liver of mosquitofish exposed to 377 ng L−1 NGT and control (FDR-corrected p values < 0.05). Categories were assigned from the information of GO-terms found in Zfin and MGI. Sequence description Fin development E3 ubiquitin-protein ligase TRIM33 isoform X3 bone morphogenetic protein 1 isoform X1 suppressor of fused homolog hedgehog-interacting protein bone morphogenetic protein 1 procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 Androgen biosynthetic process testosterone 17-beta-dehydrogenase 3 − 1 testosterone 17-beta-dehydrogenase 3–2 testosterone 17-beta-dehydrogenase 3–3 testosterone 17-beta-dehydrogenase 3–4 testosterone 17-beta-dehydrogenase 3–5 testosterone 17-beta-dehydrogenase 3–6 Lipid and fatty acid metabolic process hepatic triacylglycerol lipase glycerophosphodiester phosphodiesterase domain-containing protein 5-like glycerophosphocholine phosphodiesterase GPCPD1 isoform X1 fatty acid desaturase 2-like glycerophosphodiester phosphodiesterase 1 translocator assembly and maintenance protein 41 homolog elongation of very long chain fatty acids protein 6 patatin-like phospholipase domain-containing protein 2 phosphatidylcholine-sterol acyltransferase N-acylethanolamine-hydrolyzing acid amidase-like
qvalue
Top Blast
log2-Fold
0.009858894 8.77e− 15 0.001931321 0.004958137 4.22e− 05 1.31e− 07
Poecilia reticulata Xiphophorus maculatus Poecilia mexicana Xiphophorus maculatus Poecilia mexicana Xiphophorus maculatus
3.3 − 5.19 − 2.06 − 1.53 3.05 1.45
4.05e-06 9.71e-05 3.33E-05 1.73e-07 1.96e-09 6.36e-10
Xiphophorus Xiphophorus Xiphophorus Xiphophorus Xiphophorus Xiphophorus
4.76 4.52 6.49 7.38 8.01 8.18
6.18e-25 0.001938912 1.14e-05 7.94e-07 1.64e-14 0.000628465 4.43e-05 0.001519201 2.70e-16 3.29e-24
Xiphophorus maculatus Xiphophorus maculatus Poecilia reticulata Poecilia latipinna Poecilia formosa Poecilia mexicana Poecilia latipinna Poecilia latipinna Poecilia mexicana Poecilia mexicana
maculatus maculatus maculatus maculatus maculatus maculatus
− 4.96 4.004 6.78 − 5.61 − 4.26 − 4.48 − 4.58 9.89 4.73 − 4.1
the most significantly enriched GO terms were organ and system development, multicellular organismal development, single-organism developmental processes, as well as developmental processes (Shi et al., 2017). Furthermore, NGT significantly increased the expression of most target genes that are involved in the gonadal Notch signaling pathway (Shi et al., 2017). However, the developmental processes and the Notch signaling pathway did not show significant enrichment, which may be caused by species-specific gene expression during NGT stress. However, future research is should test these hypotheses.
Acknowledgements
5. Conclusions
Appendix A. Supplementary material
To the best of our knowledge, this is the first study that has characterized the transcriptome of female mosquitofish after exposure to a progestin. NGT exposure induced both upregulation and downregulation of certain transcripts. RT-qPCR analysis indicated that transcriptional expression of genes related to fin development, biosynthesis of androgens, and metabolism of lipids and fatty acids were significantly influenced by NGT. Our investigation provides valuable genetic data that may be used for elucidating how mosquitofish are affected by NGT at the genomic level. It also offers hints into the mechanisms underlying female masculinization in G. affinis exposed to environmentally realistic levels of NGT.
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2018.12.053.
This research was funded by the National Natural Science Foundation of China (No. 21607032, 41573008, 41873015), Foundation of Guangdong Provincial Key Laboratory of Marine Biotechnology (No. GPKLMB201603), Fishing Port Construction and Fishery Industry Development (A201701B10) and the Guangzhou University's 2017 training program for young top-notch personnel (BJ201709).
References Angus, R.A., Mcnatt, H.B., Howell, W.M., et al., 2001. Gonopodium development in normal male and 11-ketotestosterone-treated female mosquitofish (Gambusia affinis): a quantitative study using computer image analysis. General. Comp. Endocrinol. 123, 222–234. Bogdanov, M., Mileykovskaya, E., Dowhan, W., 2008. Lipids in the Assembly of Membrane Proteins and Organization of Protein Supercomplexes: Implications for Lipid-Linked Disorders. Lipids in Health and Disease 2008. Springer, Dordrecht, pp.
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Liang, Y.Q., Huang, G.Y., Ying, G.G., et al., 2015b. The effects of progesterone on transcriptional expression profiles of genes associated with hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes during the early development of zebrafish (Danio rerio). Chemosphere 128, 199–206. Liu, S.S., Ying, G.G., Liu, S., et al., 2014. Analysis of 21 progestagens in various matrices by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) with diverse sample pretreatment. Anal. Bioanal. Chem. 406, 7299–7311. Liu, S., Ying, G.G., Zhang, R.Q., et al., 2012a. Fate and occurrence of steroids in swine and dairy cattle farms with different farming scales and wastes disposal systems. Environ. Pollut. 170, 190–201. Liu, S., Ying, G.G., Zhou, L.J., et al., 2012b. Steroids in a typical swine farm and their release into the environment. Water Res. 46, 3754–3768. Liu, Z.H., Ogejo, J.A., Pruden, A., et al., 2011. Occurrence, fate and removal of synthetic oral contraceptives (SOCs) in the natural environment: a review. Sci. Total Environ. 409, 5149–5161. Lottaz, C., Iseli, C., Jongeneel, C.V., et al., 2003. Modeling sequencing errors by combining hidden Markov models. Bioinformatics 19 (Suppl 2), ii103. Murack, P.J., Parrish, J., Barry, T.P., 2011. Effects of progesterone on sperm motility in fathead minnow (Pimephales promelas). Aquat. Toxicol. 104, 121–125. Orlando, E.F., Bass, D.E., Caltabiano, L.M., et al., 2007. Altered development and reproduction in mosquitofish exposed to pulp and paper mill effluent in the Fenholloway River, Florida USA. Aquat. Toxicol. 84, 399–405. Overturf, M.D., Overturf, C.L., Carty, D.R., et al., 2014. Levonorgestrel exposure to fathead minnows (Pimephales promelas) alters survival, growth, steroidogenic gene expression and hormone production. Aquat. Toxicol. 148, 152–161. Runnalls, T.J., Beresford, N., Losty, E., et al., 2013. Several synthetic progestins with different potencies adversely affect reproduction of fish. Environ. Sci. Technol. 47, 2077–2084. Shi, W.J., Ying, G.G., Huang, G.Y., et al., 2017. Transcriptional and biochemical alterations in zebrafish eleuthero-Embryos (Danio rerio) after exposure to synthetic progestogen dydrogesterone. Bull. Environ. Contam. Toxicol. 99, 1–7. Stanko, J.P., Angus, R.A., 2010. In vivo assessment of the capacity of androstenedione to masculinize female mosquitofish (Gambusia affinis) exposed through dietary and static renewal methods. environmental toxicology. Chemistry 26, 920–926. Sul, H.S., Smith, S., 2008. CHAPTER 6 - Fatty acid synthesis in eukaryotes. In: Vance, D.E., Vance, J.E. (Eds.), Biochemistry of Lipids, Lipoproteins and Membranes, Fifth edition. Elsevier San Diego Office, pp. 155–190. Tocher, D.R., Bell, J.G., Dick, J.R., et al., 2003. Effects of dietary vegetable oil on Atlantic salmon hepatocyte fatty acid desaturation and liver fatty acid compositions. Lipids 38, 723–732. Totland, G.K., Madsen, L., Klementsen, B., et al., 2000. Proliferation of mitochondria and gene expression of carnitine palmitoyltransferase and fatty acyl-CoA oxidase in rat skeletal muscle, heart and liver by hypolipidemic fatty acids. Biol. Cell 92, 317–329. Turner, C.L., 1941. Gonopodial Characteristics Produced in the Anal Fins of Females of Gambusia affinis affinis by Treatment with Ethinyl Testosterone. Biol. Bull. 80, 371–383. Wang, L., Feng, Z., Wang, X., et al., 2010. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics 26, 136–138. Xie, C., Mao, X., Huang, J., et al., 2011. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res. 39, 316–322. Xu, E.G., Liu, S., Ying, G.G., et al., 2014. The occurrence and ecological risks of endocrine disrupting chemicals in sewage effluents from three different sewage treatment plants, and in natural seawater from a marine reserve of Hong Kong. Mar. Pollut. Bull. 85, 352–362. Ye, J., Fang, L., Zheng, H., et al., 2006. WEGO: a web tool for plotting GO annotations. Nucleic Acids Res. 34, W293–W297. Young, M.D., Wakefield, M.J., Smyth, G.K., et al., 2010. Gene ontology analysis for RNAseq: accounting for selection bias. Genome Biol. 11 (R14-R14). Zucchi, S., Castiglioni, S., Fent, K., 2012. Expressional changes by environmental concentrations of progestins and anti-progestin RU486 in zebrafish (Danio rerio) embryos. Comp. Biochem. Physiol. 163, S33–S34.
197–239. Brockmeier, E.K., Ogino, Y., Iguchi, T., et al., 2013. Effects of 17β-trenbolone on Eastern and Western mosquitofish (Gambusia holbrooki and G. affinis) anal fin growth and gene expression patterns. Aquat. Toxicol. 128, 163–170. Buxton, H.T., Kolpin, D.W., 2005. Pharmaceuticals. Hormones, and Other Organic Wastewater Contaminants in U.S. Streams. John Wiley & Sons, Inc, pp. 1202–1211. Devlin, R.H., Nagahama, Y., 2002. Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture 208, 191–364. Fent, K., 2015. Progestins as endocrine disrupters in aquatic ecosystems: concentrations, effects and risk assessment. Environ. Int. 84, 115–130. Frankel, T.E., Meyer, M.T., Orlando, E.F., 2016. Aqueous exposure to the progestin, levonorgestrel, alters anal fin development and reproductive behavior in the eastern mosquitofish (Gambusia holbrooki). General. Comp. Endocrinol. 234, 161–169. Han, J., Wang, Q., Wang, X., et al., 2014. The synthetic progestin megestrol acetate adversely affects zebrafish reproduction. Aquat. Toxicol. 150, 66–72. Heinl, S., Spath, K., Egger, E., et al., 2011. Sequence analysis and characterization of two cryptic plasmids derived from Lactobacillus buchneri CD034. Plasmid 66, 159–168. Hou, L.P., Xie, Y., Ying, G., et al., 2011. Developmental and reproductive characteristics of western mosquitofish (Gambusia affinis) exposed to paper mill effluent in the Dengcun River, Sihui, South China. Aquat. Toxicol. 103, 140–149. Hou, L.P., Xu, H., Ying, G., et al., 2017. Physiological responses and gene expression changes in the western mosquitofish (Gambusia affinis) exposed to progesterone at environmentally relevant concentrations. Aquat. Toxicol. 192, 69. Hou, L.P., Chen, H., Tian, C.E., et al., 2018. Alterations of secondary sex characteristics, reproductive histology and behaviors by norgestrel in the western mosquitofish (Gambusia affinis). Aquat. Toxicol. 198, 224–230. Hua, J., Han, J., Guo, Y., et al., 2015. The progestin levonorgestrel affects sex differentiation in zebrafish at environmentally relevant concentrations. Aquat. Toxicol. 166, 1–9. Huang, G.Y., Ying, G.G., Liu, S., et al., 2012. Regulation of reproduction- and biomarkerrelated gene expression by sex steroids in the livers and ovaries of adult female western mosquitofish (Gambusia affinis). Comp. Biochem. Physiol. 162, 36–43. Kainz, M.J., Fisk, A.T., 2009. Integrating lipids and contaminants in aquatic ecology and ecotoxicology. Lipids Aquat. Ecosyst. 93–114. Kavitha, P., Venkateswara, R.J., 2008. Toxic effects of chlorpyrifos on antioxidant enzymes and target enzyme acetylcholinesterase interaction in mosquitofish. Gambusia Affin., Environ. Toxicol. Pharmacol. 26, 192–198. Kavitha, P., Venkateswara, R.J., 2007. Oxidative stress and locomotor behaviour response as biomarkers for assessing recovery status of mosquito fish, Gambusia affinis after lethal effect of an organophosphate pesticide, monocrotophos. Pestic. Biochem. Physiol. 87, 182–188. Kejuan, F., Meirik, O., Yongang, D., et al., 2007. Once-a-month contraceptive pills in China: a review of available evidence. Contraception 75, 337–343. Klijn, J.G.M., Setyono-Han, B., Foekens, J.A., 2000. Progesterone antagonists and progesterone receptor modulators in the treatment of breast cancer. Steroids 65, 825–830. Kugathas, S., Runnalls, T., Sumpter, J., 2012. Synthetic progestins and glucocorticoids affect fish reproduction and physiology. Toxicol. Lett. 211 (S30-S30). Kumar, V., Johnson, A.C., Trubiroha, A., et al., 2015. The challenge presented by progestins in ecotoxicological research: a critical review. Environ. Sci. Technol. 49, 2625. Laforest, L., Brown, C., G., Geraudie, J., et al., 1998. Involvement of the sonic hedgehog, patched 1 andbmp2 genes in patterning of the zebrafish dermal fin rays. Development 125, 4175–4184. Li, B., Dewey, C.N., 2011. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinforma. 12 (323-323). Li, L., Yang, D., Song, Y., et al., 2017. Effects of bifenthrin exposure in soil on wholeorganism endpoints and biomarkers of earthworm Eisenia fetida. Chemosphere 168, 41–48. Liang, Y.Q., Huang, G.Y., Liu, S.S., et al., 2015a. Long-term exposure to environmentally relevant concentrations of progesterone and norgestrel affects sex differentiation in zebrafish (Danio rerio). Aquat. Toxicol. 160, 172–179.
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Transcriptomic and physiological changes in western mosquitofish (Gambusia affinis) after exposure to norgestrel
T
Liping Houa,b, Shanduo Chena, Juan Liuc, , Jingwen Guod, Zhong Chene, Qiaoling Zhue, ⁎ Wei Zhangf, GuoLiang Xug, Ye Lianga, Rongrong Wua, Xuwen Fanga, Cuiping Zhanga, Ke Xinga, ⁎
a
School of Life Sciences, Guangzhou University, Guangzhou 510655, China Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou 515063, China c Key Laboratory of Water Quality and Conservation in the Pearl River Delta, Ministry of Education, Guangdong Provincial Key Laboratory of Radionuclides Pollution Control and Resources, Institute of Environmental Research at Greater Bay, Guangzhou University, Guangzhou 510006, China d College of Environmental Science and Engineering, Guangzhou University, Guangzhou 510655, China e NanWu Middle School, Guangzhou 510655, China f Guangzhou Tieyi Middle School, Guangzhou 510655, China g Rural Non-point Source Pollution Comprehensive Management Technology Center of Guangdong Province, Guangzhou 510655, China b
ARTICLE INFO
ABSTRACT
Keywords: Endocrine disruption Norgestrel Secondary sexual characteristics transcriptomics Gambusia affinis
Norgestrel (NGT) is a synthetic progestin used in human and veterinary medicine. Adult female mosquitofish were exposed to NGT for 42 d at 377 ng L−1. The fin morphology and the liver transcriptome were assessed. NGT exposure increased ray 4:6 length ratio. As compared to the control, NGT treatment affected the expression of 11,772 annotated transcripts in female mosquitofish. Specifically, we found 5780 were repressed while 5992 were significantly induced. Gene ontology (GO) analysis showed that 53 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and 158 GO terms were significantly over expressed. Genes showing the largest magnitude of expression changes were related to fin development, androgen biosynthesis, and lipid and fatty acid metabolisms, suggesting the involvement of these biological processes in response to NGT exposure in G. affinis. This first comprehensive study on the transcriptomic alterations by NGT in G. affinis not only provides valuable information on the development of molecular markers but also opens new avenues for studies on the molecular mechanisms of effects of NGT in particular and possibly other progestins in G. affinis.
1. Introduction During the past twenty years, endocrine-disrupting chemicals (EDCs) have been increasingly recognized as global aquatic contaminants. More recently, the effects of environmental gestagens (including synthetic and natural progestins) on aquatic organisms have drawn widespread concerns (Fent, 2015). Synthetic progestins are widely used in veterinary medicine and other hormonal therapies (Kejuan et al., 2007; Klijn et al., 2000). These compounds contaminate the environment via wastewater effluents due to their incomplete removal by treatment plants ( Huang et al., 2012). Recent studies have shown that both natural and synthetic progestins may cause endocrine disruption in fish even at low concentrations (Han et al., 2014; Kugathas et al., 2012; Murack et al., 2011). A previous study reported that Norgestrel (NGT) is present in surface waters at concentrations up to 199 ng L−1 ( Buxton and Kolpin,
⁎
2005; Liu et al., 2011). NGT was found in municipal wastewater treatment plant effluents at a concentration of up to 11 ng L−1 in China (Liu et al., 2012a) and in the surface water at concentrations varying between 30.5 and 465 ng L−1 (Liu et al., 2012b). Our previous studies have reported that NGT exposure leads to changes in gonad histopathology and masculinization of female western mosquitofish G. affinis at environmentally relevant concentrations (Hou et al., 2018); it also affects sexual development and leads to transcriptional alterations of genes of the hypothalamic–pituitary–gonadal (HPG) axis in zebrafish Danio rerio (Liang et al., 2015a). Western mosquitofish G. affinis is considered as an important animal model for investigations on endocrine disruption (Kavitha and Venkateswara, 2007, 2008). It is characterized by marked sexual dimorphism and distinct reproductive behaviors (Hou et al., 2017, 2018; Orlando et al., 2007). Previous investigations have shown that exposure to androgens, which include 17b-trenbolone (Angus et al., 2001) and
Corresponding authors. E-mail addresses: [email protected] (J. Liu), [email protected] (K. Xing).
https://doi.org/10.1016/j.ecoenv.2018.12.053 Received 10 September 2018; Received in revised form 8 December 2018; Accepted 18 December 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
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androstenedione (Stanko and Angus, 2010), induces anal fin alterations in females. Although some progestins are known to be androgenic, the available information on the androgenic effects of NGT at the molecular level is very limited. Therefore, the aim of the present study was to conduct transcriptional profiling for examining various acute modulatory pathways in the livers of female mosquitofish (G. affinis) which had been exposed to NGT using next generation sequencing technology. This may provide novel insights into the mechanisms through which NGT modifies the female mosquitofish anal fins and impairs female reproduction. We hypothesized that changes in physiology and gene expression would be indicatives of the androgenic property of NGT.
China). Briefly, mRNA purification using the total RNA samples was performed with polyT oligo-attached magnetic beads (Qiagen, USA), and then cleaved into small fragments by adding a fragmentation buffer. First-strand cDNA synthesis by reverse transcription, using random hexamers as templates, was then performed, which was followed by second-strand cDNA synthesis with DNA polymerase I and RNase H (NEB, USA). Paired-end adapters were attached to the resulting double-stranded cDNA, followed by the selection of suitable fragments and PCR amplification to yield cDNA libraries. The resulting 150-bp paired-end reads were then sequenced on an Illumina HiSeq™ 2500 platform. 2.5. Assembly of transcriptome and gene function annotation
2. Methodology
First, the generated raw reads were filtered by removing the following: reads attached to adapters, reads with unknown nucleotides (Nts) that were > 5%, as well as low-quality reads (> 10% of bases have a Q score ≤20). The remaining clean reads generated from each sample were then assembled using Trinity v2.0.6 software (Heinl et al., 2011) using default settings, and minimum contig length of 200 bp. Functional annotation of all the unigenes was performed using various databases such as the non-redundant protein database (Nr), Swiss-Prot database, Clusters of Orthologous Groups (COG) protein database, Kyoto Encyclopedia of Genes and Genomes (KEGG), as well as Gene Ontology (GO). If a unigene could not be aligned to any database, ESTScan ( Lottaz et al., 2003) was used to predict the coding regions as well as sequence direction. Functional annotation of the unigenes was conducted using WEGO ( Ye et al., 2006).
2.1. Mosquitofish rearing conditions Adult female mosquitofish, Gambusia affinis, were collected in November 2016 from the Liuxi River, which is protected from pollution (Hou et al., 2011). Ten sexually mature female G. affinis per tank were acclimatized in 40-L glass aquaria ( three replicates) containing with 30 L of filtered and aerated tap water. A 12 h:12 h light/dark photoperiod and a controlled temperature (24 ± 1 °C) were utilized. The mosquitofish were fed each day with commercialized red worm flakes ( Haisheng Co., Shanghai, China) during the acclimation period. Food leftovers and fecal matter were siphoned out from the aquaria each day. 2.2. Hormonal treatment Norgestrel (> 99.9% purity) was purchased from Sigma-Aldrich (StLouis, MO, USA). An NGT stock solution (1 mg mL−1) was a diluted to different concentrations and used in the treatments. The female G. affinis were exposed to 0 and 500 ng L−1 of NGT. Three replicate tanks containing 10 females each were used. DMSO was used as a solvent with a final exposure concentration of 0.003% (v/v). The solvent controls consisted of 0.003% DMSO only. The experiment was carried out in a system that was semi-static at a temperature of 25 ± 1 °C. Daily measurement of water chemistry parameters such as conductivity (22 ± 0.1 μS cm−1), water hardness (153 ± 3.7 mg L−1 as CaCO3), dissolved oxygen (DO, 7.7 ± 0.1 mg L−1), and pH (7.6 ± 0.4) were performed daily using a water quality meter with multiple parameters (YSI Model 85 m; Yellow Springs, OH, USA). The medium of exposure was renewed every day.
2.6. Analysis and functional enrichment differentially expressed genes (DEGs) Mapping of clean reads of each sample to the assembled unigenes was performed, followed by estimation of their relative abundances using RSEM software v1.2.12 (Li and Dewey, 2011) using the fragment per kilo bases per million reads (FPKM) method. The detection of DEGs between the NGT and the control was performed using the R package DEGseq ( Wang et al., 2010) using a threshold of | log2 (fold change) | > 1 and a corrected p-value (q-value) < 0.05. To detect overrepresented GO terms as well as KEGG pathways, DEG enrichment analyses were performed using the R package GOseq V1.16.2 (Young et al., 2010) and KOBAS ( Xie et al., 2011). 2.7. Validation by real-time PCR
2.3. Biometrics and fin morphology
Ten candidate genes, which included those with transcripts that are associated with fin development, androgen production, and lipid and fatty acid metabolism, as well as housekeeping genes were employed to design qRT-PCRs (Table S1). The sequences of the detected unigenes were compared with the homologues (blastx) to verify our annotation. These were then used to design qPCR primers. All the primers were validated using end-point PCR. The 10 candidate genes selected were used for determining transcript abundance in mosquitofish liver.
After 42 d of exposure, ten female mosquitofish from each tank (total = 30) were anesthetized (MS-222, 300 mg L−1). Seven fish were randomly selected from each tank (total = 21) and used for measuring anal fin length with a calibrated dissecting microscope (Olympus SZHILLK). Individual fish weight and length were also determined. The width of anal fin ray 3 ( ± 0.1 mm), lengths of rays 4 and 6, and the number of segments in ray 3 were measured. Three mosquitofish from every group were sacrificed by spinal severance. The livers were collected and immersed in Trizol ( Invitrogen, Shanghai), followed by storage in −80 °C until analysis.
2.8. Measurement of NGT levels in the exposure media Since the exposure media were renewed each day, the actual exposure concentrations of NGT were determined at the initiation of the exposure (0 h, T0) and prior to water renewal (24 h, T 24) in the first day for all aquaria. Approximately 500 mL water samples were taken from each aquarium. NGT concentrations in the exposure medium were measured,as described in our previous study (Xu et al., 2014). NGT extraction from the water samples was performed using CNWBOND LCC18 SPE cartridges (200 mg, 3 mL) (Germany), followed by analysis using RRLC–MS/MS (Xu et al., 2014). The respective NGT limit of detection (LOD) and the limit of quantitation (LOQ) were 0.02–0.25 ng L
2.4. RNA extraction, library construction, and sequencing Total RNA was extracted from the livers of mosquitofish using a TRIzol kit (Promega, USA), followed by DNase I (Takara Bio, Japan) treatment to remove genomic DNA. RNA quality and purity were assessed by agarose gel electrophoresis using a Bioanalyzer 2100 system (Agilent, CA, USA). cDNA libraries construction and Illumina sequencing were carried out by the Total Genomics Solution (TGS) Company (Guangzhou, 580
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and 0.071–0.79 ng L −1. The spiked sample showed a recovery rate ranging from 72% to 107% (Liu et al., 2014).
fin development, androgen biosynthesis, lipid metabolism, and fatty acid metabolism (Table 1). Moreover, KEGG pathway analysis indicated that these DEGs were related to various biological functions, which included metabolic pathways, tryptophan metabolism, protein processing involving the endoplasmic reticulum, insulin resistance, adipocytokine signaling pathway, production of unsaturated fatty acids, the AMPK signaling pathway, PPAR signaling pathway, pyruvate metabolism, biosynthesis of lysine, and the insulin signaling pathway (Table 2).
2.9. Statistical analyses Statistical analyses were conducted using SPSS (version 13.0, IBM, Chicago, IL, USA). Normality and homogeneity of variances were assessed using Kolmogorov–Smirnov and Levene's tests, respectively. Differences between the control and the NGT exposed group were evaluated by one-way ANOVA. The differences were considered statistically significant at a p-value < 0.05.
3.6. RNA sequencing validation using real-time PCR To validate the results of gene expression profiling using RNA-Seq, 10 selected genes, consisting of five upregulated and five downregulated genes, were analyzed by real-time PCR (Table S2). Fig. S1 shows that the expression patterns of these selected genes coincided with the results obtained by RNA-seq. This proves that the gene expression profiles generated by RNASeq were reliable, thereby confirming the observed changes in the expression of genes responsive to NGT stress. (Table 3)
3. Results 3.1. Chemical analysis Table S2 shows the concentrations of the control and the NGT treatments (i.e., 0 ng L−1 and 500 ng L−1). The concentrations of NGT in the solvent control were below the detection limit. Thus, the average NGT concentrations (i.e., 0 ng L−1 and 377 ng L−1) were used for the presentation of data for the SC and the nominal 500 ng L−1 throughout this investigation.
4. Discussion Progestins are potent EDCs in fish as well as in amphibians (Fent, 2015; Kumar et al., 2015). Exposure to progestin results in a variety of adverse effects involving the endocrine systems such as vitellogenin synthesis in male or juvenile fish, disruption of egg maturation, ovotestes/intersex, abnormal reproduction, inhibition of embryonic development, and skewed sex ratios (Hua et al., 2015; Li et al., 2017; Liang et al., 2015b; Runnalls et al., 2013; Zucchi et al., 2012). Our previous study and the present investigation demonstrate that synthetic progestin exposure at environmentally relevant concentrations may induce masculinization of female G. affinis ( Hou et al., 2018; Hou et al., 2017). However, the mechanisms of their masculinization effect remain elusive. The next generation sequencing technique has offered great opportunities for uncovering the molecular pathways related to masculinization effect of progestins. This study used RNA-seq to explore the pathways modulated by NGT in the liver tissues of the adult female mosquitofish to elucidate the molecular mechanisms for masculinization of female G. affinis. We found that the overall hepatic mRNA expression in the female mosquitofish differed substantially between the NGT-treated and the control groups, with altered levels for 11,772 transcripts, including many associated with growth (e.g., fin development), metabolism, or development of mosquitofish. These data could help us better understand the molecular mechanisms of effects of NGT in G. affinis and provide valuable genetic data to develop molecular markers for exposure to progestins in the future.
3.2. Effects of NGT on mosquitofish growth and anal fin morphometrics After 42 d of exposure to NGT, no changes in the length and weight of the adult females were observed (data not shown). However, as compared with the control female mosquitofish, ray 4:6 length ratio was significantly increased by 55.7% (P < 0.001) when exposed to 377 ng L−1 of NGT（Fig. 1）. 3.3. Transcriptome sequencing and assembly After removing redundant sequence from the assembled contigs, approximately 90,304 unigenes were identified. The assembled sequences’ GC content was around 46%. The assembled unigenes showed an average length of 1053 bp, with an N50 value of 2184 bp. Fig. 2 shows that the majority of the unigenes had sizes within the range of 200–500 bp, with 1316 of the unigenes ≥ 3000 bp in length（Fig. 2）. 3.4. Functional annotation of the unigenes The unigenes were subsequently compared with the sequences available in the GO database, and functionally annotated to three major categories, which included biological process, cellular component, and molecular function (Fig. 3). Approximately 18,368 unigenes showed significant hits with the GO database. For the functional category of biological process, the term “cellular process” had the highest number of unigenes, whereas “cell part” and “binding” showed the highest number of transcripts for the respective functional categories of cellular component and molecular function. The KEGG pathway analysis also assigned the unigenes to six different pathways such as Genetic Information Processing, Metabolism, Cellular Processes, Organismal Systems, Human Disease, and Environmental Information Processing (Fig. 4). A total of 37,939 unigenes showed significant hits using the KEGG database, of which 11,706 were related to signal transduction.
4.1. Differential transcriptional expressions by NGT-exposure NGT toxicity occurs via disruption of the normal interaction between E2 that is secreted by gonads and the brain, thereby influencing behavioral and reproductive activities. DE analysis showed that NGT imparts treatment-specific effects on G. affinis. In NGT-exposed females, genes related to the androgen biosynthesis and fin development were upregulated, whereas the genes related to metabolic process, transport, translation, lipid metabolism, carbohydrate metabolism, steroid hormone-mediated signaling pathway, transmembrane transport, biosynthetic process, and proteolysis were downregulated. The impact of NGT on the transcription of genes associated with fin development, biosynthesis of androgens, and lipid and fatty acid metabolism are presented in detail in the next sections.
3.5. Differential gene expression analysis In total, we identified 9190 annotated unigenes with significantly different expression levels after exposure to NGT. Among these, 3712 were significantly induced and 5478 were repressed. To further investigate the functions of these unigenes and their corresponding biological processes, we performed enrichment analysis using the GO and KEGG pathway. GO enrichment analysis showed that many of the DEGs were related to several sexual development biological processes such as,
4.2. Effects of NGT on fin development Poeciliid fish (e.g., G. affinis and G. holbrooki) are suitable models for investigating the effects of endocrine-disrupting compounds based 581
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Fig. 1. Pictures of anal fin morphology of mosquitofish exposed to norgestrel (NGT) for 42 d. (A) Anal fin of a normal adult female with rays 3 labeled; (B) Anal fin of a normal adult male with rays 3 labeled; (C) Anal fin of a masculinized adult female with rays 3 labeled at 377 ng L−1 NGT; (D) Ratios of the basal length and total length of the anal fin of the control and the adult female Gambusia affinis treated by 377 ng L−1 norgestrel for 42-day to that of the solvent control female. Data are expressed as mean ± standard error (SEM) of three replicate samples ( n = 21). *P < 0.05 and * *P < 0.01 indicate statistically significant differences between solvent control (SC) and exposure groups. Error bars represent the standard deviation of the mean values.
showed that NGT exposure increased ray 4:6 length ratio and number of segments in ray 3 ( Hou et al., 2018). These results demonstrated that NGT could masculinize adult female G. affinis. Similarly, exposure to levonorgestrel reportedly led to altered anal fin development in the eastern mosquitofish G. holbrooki (Frankel et al., 2016). In the mosquitofish, the anal fin differentiates into gonopodium via elongation and modification of the anal fin rays 3, 4, and 5 (Turner, 1941). Therefore, the elongation and outgrowth of the anal fin in the female mosquitofish exposed to progestins can be considered as the first indication of masculinization. In this study, fin development is a key process altered by NGT in mosquitofish. In the liver, a down-regulation occurred in several fin development genes including bmp1×1, sofh, hip, and an up-regulation of TRIM33×3, bmp1 and pl2O5dx at 377 ng/L NGT. In the liver, also RT-qPCR revealed a significant down-regulation of bmp1×1 at 377 ng/ L NGT (Fig. S2). These genes mediate the elongation and subsequent gonopodium development in mosquitofish. It is therefore likely that NGT altered the expression of these genes, which in turn activated the bone morphogenetic proteins to determine the fate of the downstream scleroblast (Laforest et al., 1998), resulting in the elongation and outgrowth of the anal fins. Many studies have shown that progestins including NGT and levonorgestrel are androgenic to fish (Hua et al., 2015; Runnalls et al.,
Fig. 2. Gene transcription profiling. Global expression profile of G. affinis exposed to norgestrel (NGT) at 377 ng L−1. Relative to the control, unigenes of 9190 genes were altered with 3712 genes up-regulated and 5478 genes downregulated. Fold-change cutoff was set at ≥ 2 and the FDR was set < 0.01. Green：down-regulated; Red: up-regulated.
on their morphological traits, particularly their external secondary sexual characteristics. In this study, exposure to 377 ng L−1 of NGT for 42 d resulted in increased ray 4:6 length ratio. Our previous study 582
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Fig. 3. GO annotation of the unigenes. The unigenes were assigned to different terms in three major categories of the GO database, namely biological process, cellular component, and molecular function.
2013; Liang et al., 2015b; Li et al., 2017; Zucchi et al., 2012). It has been demonstrated that genes including sonic hedgehog (shh), muscle segment homeobox C (msxC), and fibroblast growth receptor 1 (fgfr1) are also involved in the anal fin growth of the eastern mosquitofish G. holbrooki exposed to 17 β-trenbolone (an androgen) (Brockmeier et al., 2013).
4.4. Effects of NGT on lipid and fatty acid metabolism In this study, barring the genes related to androgen biosynthesis, the genes associated with lipid and fatty acid metabolism showed the largest magnitude of alterations in transcriptional expressions. Lipids pertain to a class of biomolecules that play various roles in structural and biological activities by interacting with proteins (Bogdanov et al., 2008; Sul and Smith, 2008). The main organic components of fish include lipids and proteins, which play major roles in its life cycle and physiology, which include growth, movement, reproduction, as well as migration (Tocher et al., 2003). The major function of lipids in fish is to store and provide energy in the form of adenosine triphosphate (ATP) via β-oxidation of fatty acids (Totland et al., 2000). Many studies have reported that hydrophobic pollutants accumulate in the lipids of aquatic organisms (Kainz and Fisk, 2009). In the present study, NGTexposure resulted in a decrease in the expression of some genes that are related to the metabolism of lipids and fatty acids in female mosquitofish. An example is the elongase “elongation of very long-chain fatty acids protein 6 (ELOVL6)” gene. ELOVL6 catalyzes the initial and ratelimiting step of the four that comprise the elongation cycle of longchain fatty acids. Another downregulated gene that is involved in the metabolism of lipid and fatty acids fatty acid desaturase 2 (FAD2), which catalyzes the generation of highly unsaturated fatty acids (HUFAs) using the following precursors: polyunsaturated fatty acids (PUFAs), linoleic acid (LA) (18:2, n-6), and alpha-linolenic acid (ALA) (18:3, n-3). In addition, the transcription of glycerophosphodiester phosphodiesterase 1 (GDPDL1), which hydrolyzes glycerolphosphoglycerol, was also downregulated significantly in female mosquitofish. It has been suggested that dysregulation of these genes by NGT exposure may disrupt the metabolism of lipids and fatty acids, thereby reducing the growth and reproduction of female mosquitofish. An earlier study involving NGT effects on zebrafish has shown that
4.3. Effects of NGT on androgen biosynthesis Sex hormones play a major role in the development of external secondary fish sexual characteristics (Devlin and Nagahama, 2002). Disruption of sex hormone biosynthesis may change the hormone levels of fish, thereby affecting their external secondary sexual features. In the present study, the expression of Hsd17β3 increased significantly after exposure to 377 ng L−1 NGT. Hsd17β3 and many other genes encode the enzymes which are involved in the production of 11-ketotestosterone (11-KT). The role of Hsd17β3 in inducing rapid masculinization of females needs further investigation. A recent investigation has shown that treatment of larval zebrafish (20 d post- fertilization) with NGT at concentrations of 34 ng L−1 and 77 ng L−1 for 40 d creates an all-male population, coupled with a significant decrease in the expression of Cyp11a1, Cyp17, Cyp19a1a, and Hsd3b (Liang et al., 2015b). Exposure to levonorgestrel at a concentration of 86.9 ng L−1 for 21 d induces a significant decrease in 3β-Hsd, 20β-Hsd, and Cyp19a expression in fathead minnow (Pimephales promelas) larvae ( Overturf et al., 2014). These findings indicate that NGT and levonorgestrel disrupt steroidal biosynthesis, which in turn affects fish sexual differentiation ( Liang et al., 2015b; Overturf et al., 2014). These studies also suggest that changes at the transcriptional expression of steroid-producing genes may induce masculinization of female mosquitofish after exposure to progestins. 583
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Fig. 4. KEGG annotation of the unigenes. The unigenes were assigned to different pathways in six major categories of the KEGG database, including genetic information processing, metabolism, cellular processes, organismal systems, human disease, and environmental information processing. Table 1 List of enriched gene ontology (GO) terms in the differentially expressed genes (DEGs). Class
GO_ID
Term
DEGs_in_Term
All_Genes_in_Term
Correct_P_Value
BP BP BP BP BP BP BP BP BP BP BP BP BP BP
GO0009058 GO0033333 GO0032259 GO0055085 GO0006629 GO0043009 GO0007165 GO0030433 GO0042157 GO0006702 GO0060059 GO0007519 GO0006631 GO0007417
biosynthetic process fin development methylation transmembrane transport lipid metalolic process vasculature development signal transduction ER-associated ubiquitin-dependent protein catabolic process lipoprotein metabolic process androgen biosynthetic process embryonic retina morphogenesis in camera-type eye skeletal muscle tissue development fatty acid metabolic process central nervous system development
36 12 31 87 50 18 111 6 11 5 8 7 9 11
109 20 103 418 206 47 579 7 22 8 15 12 19 28
3.01306E-05 0.000306 0.000913 0.00112 0.00145 0.00184 0.0026 0.0039 0.00391 0.0111 0.0178 0.0208 0.0213 0.0326
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Table 2 List of enriched KEGG pathways in the differentially expressed genes (DEGs). Pathway
DEGs in term(7994)
All genes in term(37939)
Q-value
Pathway ID
Metabolic pathways Tryptophan metabolism Protein processing in endoplasmic reticulum Insulin resistance Adipocytokine signaling pathway Biosynthesis of unsaturated fatty acids AMPK signaling pathway PPAR signaling pathway Pyruvate metabolism Lysine biosynthesis Insulin signaling pathway
1080 (13.51%) 56 (0.7%) 158 (1.98%) 110 (1.38%) 76 (0.95%) 30 (0.38%) 136 (1.7%) 81 (1.01%) 50 (0.63%) 6 (0.08%) 141 (1.76%)
4142 (10.92%) 163 (0.43%) 569 (1.5%) 380 (1%) 250 (0.66%) 79 (0.21%) 498 (1.31%) 276 (0.73%) 155 (0.41%) 8 (0.02%) 541 (1.43%)
5.10759E-14 0.00254251 0.003021287 0.005003576 0.007597332 0.008891159 0.009632565 0.01260297 0.01273914 0.02381083 0.03588414
ko01100 ko00380 ko04141 ko04931 ko04920 ko01040 ko04152 ko03320 ko00620 ko00300 ko04910
Table 3 Genes assigned to the biological functions response to fin development, androgen biosynthetic process, lipid and fatty acid metabolic metabolic process analyses of the transcripts significantly differentially expressed in liver of mosquitofish exposed to 377 ng L−1 NGT and control (FDR-corrected p values < 0.05). Categories were assigned from the information of GO-terms found in Zfin and MGI. Sequence description Fin development E3 ubiquitin-protein ligase TRIM33 isoform X3 bone morphogenetic protein 1 isoform X1 suppressor of fused homolog hedgehog-interacting protein bone morphogenetic protein 1 procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 Androgen biosynthetic process testosterone 17-beta-dehydrogenase 3 − 1 testosterone 17-beta-dehydrogenase 3–2 testosterone 17-beta-dehydrogenase 3–3 testosterone 17-beta-dehydrogenase 3–4 testosterone 17-beta-dehydrogenase 3–5 testosterone 17-beta-dehydrogenase 3–6 Lipid and fatty acid metabolic process hepatic triacylglycerol lipase glycerophosphodiester phosphodiesterase domain-containing protein 5-like glycerophosphocholine phosphodiesterase GPCPD1 isoform X1 fatty acid desaturase 2-like glycerophosphodiester phosphodiesterase 1 translocator assembly and maintenance protein 41 homolog elongation of very long chain fatty acids protein 6 patatin-like phospholipase domain-containing protein 2 phosphatidylcholine-sterol acyltransferase N-acylethanolamine-hydrolyzing acid amidase-like
qvalue
Top Blast
log2-Fold
0.009858894 8.77e− 15 0.001931321 0.004958137 4.22e− 05 1.31e− 07
Poecilia reticulata Xiphophorus maculatus Poecilia mexicana Xiphophorus maculatus Poecilia mexicana Xiphophorus maculatus
3.3 − 5.19 − 2.06 − 1.53 3.05 1.45
4.05e-06 9.71e-05 3.33E-05 1.73e-07 1.96e-09 6.36e-10
Xiphophorus Xiphophorus Xiphophorus Xiphophorus Xiphophorus Xiphophorus
4.76 4.52 6.49 7.38 8.01 8.18
6.18e-25 0.001938912 1.14e-05 7.94e-07 1.64e-14 0.000628465 4.43e-05 0.001519201 2.70e-16 3.29e-24
Xiphophorus maculatus Xiphophorus maculatus Poecilia reticulata Poecilia latipinna Poecilia formosa Poecilia mexicana Poecilia latipinna Poecilia latipinna Poecilia mexicana Poecilia mexicana
maculatus maculatus maculatus maculatus maculatus maculatus
− 4.96 4.004 6.78 − 5.61 − 4.26 − 4.48 − 4.58 9.89 4.73 − 4.1
the most significantly enriched GO terms were organ and system development, multicellular organismal development, single-organism developmental processes, as well as developmental processes (Shi et al., 2017). Furthermore, NGT significantly increased the expression of most target genes that are involved in the gonadal Notch signaling pathway (Shi et al., 2017). However, the developmental processes and the Notch signaling pathway did not show significant enrichment, which may be caused by species-specific gene expression during NGT stress. However, future research is should test these hypotheses.
Acknowledgements
5. Conclusions
Appendix A. Supplementary material
To the best of our knowledge, this is the first study that has characterized the transcriptome of female mosquitofish after exposure to a progestin. NGT exposure induced both upregulation and downregulation of certain transcripts. RT-qPCR analysis indicated that transcriptional expression of genes related to fin development, biosynthesis of androgens, and metabolism of lipids and fatty acids were significantly influenced by NGT. Our investigation provides valuable genetic data that may be used for elucidating how mosquitofish are affected by NGT at the genomic level. It also offers hints into the mechanisms underlying female masculinization in G. affinis exposed to environmentally realistic levels of NGT.
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2018.12.053.
This research was funded by the National Natural Science Foundation of China (No. 21607032, 41573008, 41873015), Foundation of Guangdong Provincial Key Laboratory of Marine Biotechnology (No. GPKLMB201603), Fishing Port Construction and Fishery Industry Development (A201701B10) and the Guangzhou University's 2017 training program for young top-notch personnel (BJ201709).
References Angus, R.A., Mcnatt, H.B., Howell, W.M., et al., 2001. Gonopodium development in normal male and 11-ketotestosterone-treated female mosquitofish (Gambusia affinis): a quantitative study using computer image analysis. General. Comp. Endocrinol. 123, 222–234. Bogdanov, M., Mileykovskaya, E., Dowhan, W., 2008. Lipids in the Assembly of Membrane Proteins and Organization of Protein Supercomplexes: Implications for Lipid-Linked Disorders. Lipids in Health and Disease 2008. Springer, Dordrecht, pp.
585
Ecotoxicology and Environmental Safety 171 (2019) 579–586
L. Hou et al.
Liang, Y.Q., Huang, G.Y., Ying, G.G., et al., 2015b. The effects of progesterone on transcriptional expression profiles of genes associated with hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes during the early development of zebrafish (Danio rerio). Chemosphere 128, 199–206. Liu, S.S., Ying, G.G., Liu, S., et al., 2014. Analysis of 21 progestagens in various matrices by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) with diverse sample pretreatment. Anal. Bioanal. Chem. 406, 7299–7311. Liu, S., Ying, G.G., Zhang, R.Q., et al., 2012a. Fate and occurrence of steroids in swine and dairy cattle farms with different farming scales and wastes disposal systems. Environ. Pollut. 170, 190–201. Liu, S., Ying, G.G., Zhou, L.J., et al., 2012b. Steroids in a typical swine farm and their release into the environment. Water Res. 46, 3754–3768. Liu, Z.H., Ogejo, J.A., Pruden, A., et al., 2011. Occurrence, fate and removal of synthetic oral contraceptives (SOCs) in the natural environment: a review. Sci. Total Environ. 409, 5149–5161. Lottaz, C., Iseli, C., Jongeneel, C.V., et al., 2003. Modeling sequencing errors by combining hidden Markov models. Bioinformatics 19 (Suppl 2), ii103. Murack, P.J., Parrish, J., Barry, T.P., 2011. Effects of progesterone on sperm motility in fathead minnow (Pimephales promelas). Aquat. Toxicol. 104, 121–125. Orlando, E.F., Bass, D.E., Caltabiano, L.M., et al., 2007. Altered development and reproduction in mosquitofish exposed to pulp and paper mill effluent in the Fenholloway River, Florida USA. Aquat. Toxicol. 84, 399–405. Overturf, M.D., Overturf, C.L., Carty, D.R., et al., 2014. Levonorgestrel exposure to fathead minnows (Pimephales promelas) alters survival, growth, steroidogenic gene expression and hormone production. Aquat. Toxicol. 148, 152–161. Runnalls, T.J., Beresford, N., Losty, E., et al., 2013. Several synthetic progestins with different potencies adversely affect reproduction of fish. Environ. Sci. Technol. 47, 2077–2084. Shi, W.J., Ying, G.G., Huang, G.Y., et al., 2017. Transcriptional and biochemical alterations in zebrafish eleuthero-Embryos (Danio rerio) after exposure to synthetic progestogen dydrogesterone. Bull. Environ. Contam. Toxicol. 99, 1–7. Stanko, J.P., Angus, R.A., 2010. In vivo assessment of the capacity of androstenedione to masculinize female mosquitofish (Gambusia affinis) exposed through dietary and static renewal methods. environmental toxicology. Chemistry 26, 920–926. Sul, H.S., Smith, S., 2008. CHAPTER 6 - Fatty acid synthesis in eukaryotes. In: Vance, D.E., Vance, J.E. (Eds.), Biochemistry of Lipids, Lipoproteins and Membranes, Fifth edition. Elsevier San Diego Office, pp. 155–190. Tocher, D.R., Bell, J.G., Dick, J.R., et al., 2003. Effects of dietary vegetable oil on Atlantic salmon hepatocyte fatty acid desaturation and liver fatty acid compositions. Lipids 38, 723–732. Totland, G.K., Madsen, L., Klementsen, B., et al., 2000. Proliferation of mitochondria and gene expression of carnitine palmitoyltransferase and fatty acyl-CoA oxidase in rat skeletal muscle, heart and liver by hypolipidemic fatty acids. Biol. Cell 92, 317–329. Turner, C.L., 1941. Gonopodial Characteristics Produced in the Anal Fins of Females of Gambusia affinis affinis by Treatment with Ethinyl Testosterone. Biol. Bull. 80, 371–383. Wang, L., Feng, Z., Wang, X., et al., 2010. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics 26, 136–138. Xie, C., Mao, X., Huang, J., et al., 2011. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res. 39, 316–322. Xu, E.G., Liu, S., Ying, G.G., et al., 2014. The occurrence and ecological risks of endocrine disrupting chemicals in sewage effluents from three different sewage treatment plants, and in natural seawater from a marine reserve of Hong Kong. Mar. Pollut. Bull. 85, 352–362. Ye, J., Fang, L., Zheng, H., et al., 2006. WEGO: a web tool for plotting GO annotations. Nucleic Acids Res. 34, W293–W297. Young, M.D., Wakefield, M.J., Smyth, G.K., et al., 2010. Gene ontology analysis for RNAseq: accounting for selection bias. Genome Biol. 11 (R14-R14). Zucchi, S., Castiglioni, S., Fent, K., 2012. Expressional changes by environmental concentrations of progestins and anti-progestin RU486 in zebrafish (Danio rerio) embryos. Comp. Biochem. Physiol. 163, S33–S34.
197–239. Brockmeier, E.K., Ogino, Y., Iguchi, T., et al., 2013. Effects of 17β-trenbolone on Eastern and Western mosquitofish (Gambusia holbrooki and G. affinis) anal fin growth and gene expression patterns. Aquat. Toxicol. 128, 163–170. Buxton, H.T., Kolpin, D.W., 2005. Pharmaceuticals. Hormones, and Other Organic Wastewater Contaminants in U.S. Streams. John Wiley & Sons, Inc, pp. 1202–1211. Devlin, R.H., Nagahama, Y., 2002. Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture 208, 191–364. Fent, K., 2015. Progestins as endocrine disrupters in aquatic ecosystems: concentrations, effects and risk assessment. Environ. Int. 84, 115–130. Frankel, T.E., Meyer, M.T., Orlando, E.F., 2016. Aqueous exposure to the progestin, levonorgestrel, alters anal fin development and reproductive behavior in the eastern mosquitofish (Gambusia holbrooki). General. Comp. Endocrinol. 234, 161–169. Han, J., Wang, Q., Wang, X., et al., 2014. The synthetic progestin megestrol acetate adversely affects zebrafish reproduction. Aquat. Toxicol. 150, 66–72. Heinl, S., Spath, K., Egger, E., et al., 2011. Sequence analysis and characterization of two cryptic plasmids derived from Lactobacillus buchneri CD034. Plasmid 66, 159–168. Hou, L.P., Xie, Y., Ying, G., et al., 2011. Developmental and reproductive characteristics of western mosquitofish (Gambusia affinis) exposed to paper mill effluent in the Dengcun River, Sihui, South China. Aquat. Toxicol. 103, 140–149. Hou, L.P., Xu, H., Ying, G., et al., 2017. Physiological responses and gene expression changes in the western mosquitofish (Gambusia affinis) exposed to progesterone at environmentally relevant concentrations. Aquat. Toxicol. 192, 69. Hou, L.P., Chen, H., Tian, C.E., et al., 2018. Alterations of secondary sex characteristics, reproductive histology and behaviors by norgestrel in the western mosquitofish (Gambusia affinis). Aquat. Toxicol. 198, 224–230. Hua, J., Han, J., Guo, Y., et al., 2015. The progestin levonorgestrel affects sex differentiation in zebrafish at environmentally relevant concentrations. Aquat. Toxicol. 166, 1–9. Huang, G.Y., Ying, G.G., Liu, S., et al., 2012. Regulation of reproduction- and biomarkerrelated gene expression by sex steroids in the livers and ovaries of adult female western mosquitofish (Gambusia affinis). Comp. Biochem. Physiol. 162, 36–43. Kainz, M.J., Fisk, A.T., 2009. Integrating lipids and contaminants in aquatic ecology and ecotoxicology. Lipids Aquat. Ecosyst. 93–114. Kavitha, P., Venkateswara, R.J., 2008. Toxic effects of chlorpyrifos on antioxidant enzymes and target enzyme acetylcholinesterase interaction in mosquitofish. Gambusia Affin., Environ. Toxicol. Pharmacol. 26, 192–198. Kavitha, P., Venkateswara, R.J., 2007. Oxidative stress and locomotor behaviour response as biomarkers for assessing recovery status of mosquito fish, Gambusia affinis after lethal effect of an organophosphate pesticide, monocrotophos. Pestic. Biochem. Physiol. 87, 182–188. Kejuan, F., Meirik, O., Yongang, D., et al., 2007. Once-a-month contraceptive pills in China: a review of available evidence. Contraception 75, 337–343. Klijn, J.G.M., Setyono-Han, B., Foekens, J.A., 2000. Progesterone antagonists and progesterone receptor modulators in the treatment of breast cancer. Steroids 65, 825–830. Kugathas, S., Runnalls, T., Sumpter, J., 2012. Synthetic progestins and glucocorticoids affect fish reproduction and physiology. Toxicol. Lett. 211 (S30-S30). Kumar, V., Johnson, A.C., Trubiroha, A., et al., 2015. The challenge presented by progestins in ecotoxicological research: a critical review. Environ. Sci. Technol. 49, 2625. Laforest, L., Brown, C., G., Geraudie, J., et al., 1998. Involvement of the sonic hedgehog, patched 1 andbmp2 genes in patterning of the zebrafish dermal fin rays. Development 125, 4175–4184. Li, B., Dewey, C.N., 2011. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinforma. 12 (323-323). Li, L., Yang, D., Song, Y., et al., 2017. Effects of bifenthrin exposure in soil on wholeorganism endpoints and biomarkers of earthworm Eisenia fetida. Chemosphere 168, 41–48. Liang, Y.Q., Huang, G.Y., Liu, S.S., et al., 2015a. Long-term exposure to environmentally relevant concentrations of progesterone and norgestrel affects sex differentiation in zebrafish (Danio rerio). Aquat. Toxicol. 160, 172–179.
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