The effects of fluorene-9-bisphenol on female zebrafish (Danio rerio) reproductive and exploratory behaviors

Accepted Manuscript The effects of fluorene-9-bisphenol on female zebrafish (Danio rerio) reproductive and exploratory behaviors Ping Mi, Qiu-Ping Zhang, Shu-Hui Zhang, Chao Wang, Shao-Zhi Zhang, Yong-Chun Fang, Jian-Zhao Gao, Dao-Fu Feng, Dong-Yan Chen, Xi-Zeng Feng PII:

S0045-6535(19)30830-6

DOI:

https://doi.org/10.1016/j.chemosphere.2019.04.170

Reference:

CHEM 23678

To appear in:

ECSN

Received Date: 8 January 2019 Revised Date:

20 April 2019

Accepted Date: 22 April 2019

Please cite this article as: Mi, P., Zhang, Q.-P., Zhang, S.-H., Wang, C., Zhang, S.-Z., Fang, Y.-C., Gao, J.-Z., Feng, D.-F., Chen, D.-Y., Feng, X.-Z., The effects of fluorene-9-bisphenol on female zebrafish (Danio rerio) reproductive and exploratory behaviors, Chemosphere (2019), doi: https://doi.org/10.1016/ j.chemosphere.2019.04.170. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT The effects of fluorene-9-bisphenol on female zebrafish (Danio rerio) reproductive

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and exploratory behaviors

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Ping Mi a,1, Qiu-Ping Zhang b,1, Shu-Hui Zhang a,1, Chao Wang c,1, Shao-Zhi Zhang a, Yong-Chun

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Fang c, Jian-Zhao Gao e, Dao-Fu Feng d,*, Dong-Yan Chen b,*, Xi-Zeng Feng a,*

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a

State Key Laboratory of Medicinal Chemical Biology, The Key Laboratory of Bioactive

Materials, Ministry of Education, College of Life Science, Nankai University, Tianjin 300071,

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China.

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Tianjin Key Laboratory of Tumor Microenvironment and Neurovascular Regulation, Department of Histology and Embryology, School of Medicine, Nankai University, Tianjin 300071, China.

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The Institute of Robotics and Automatic Information Systems, Nankai University, Tianjin 300071, China.

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Department of General Surgery, Tianjin Medical University General Hospital, No. 154 Anshan Road, Tianjin 300052, China.

School of Mathematical Sciences and LPMC, Nankai University, Tianjin 300071, China.

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These authors contributed equally to this work.

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* Corresponding author: [email protected] (X. Feng). [email protected] (D. Chen),

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[email protected] (D. Feng).

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ACCEPTED MANUSCRIPT Abstract: Endocrine disruptor chemicals induce adverse effects to animals’

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development, reproduction and behavior in environment. We investigated the effects

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of fluorene-9-bisphenol (BHPF), one substitute of bisphenol A, on courtship behavior

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and exploratory behavior of adult zebrafish. Customized apparatus was used to

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evaluate courtship behavior. The result showed that the male spent less time with

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BHPF and anti-oestrogenic fulvestrant (FULV) treated female in region of

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approaching (ROA). Courtship index between BHPF-exposed female and male

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decreased. The body orientation of BHPF- and FULV-exposed female to male

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decreased. Furthermore, BHPF exposure downregulated the expression of genes

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related to estrogen receptor, steroidogenesis and upregulated oxidative stress related

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genes. It indicated that BHPF exposure interfered the preference of male and female

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in courtship, and induced detrimental effects on reproduction. BHPF treatment

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decreased locomotor activity and time spent in top, increased freezing bouts, and

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induced anxiety/depression-like behavior. The tyrosine hydroxylase in brain

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decreased under BHPF exposure. Here we showed the potential adverse effects of

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BHPF on reproduction and exploratory behaviors.

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Keywords:

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anxiety/depression-like behavior, fluorene-9-bisphenol

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zebrafish,

courtship

behavior,

exploratory

behavior,

1. Introduction

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Endocrine disrupting chemicals (EDCs) are exogenous chemicals that mimic the

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actions of endogenous hormones or interfere with the synthesis, secretion, transport, 2

ACCEPTED MANUSCRIPT metabolism, receptor binding or elimination of natural endogenous hormones (Iguchi,

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1998; Keith, 1998). Bisphenol A (BPA) is a representative of EDCs which had

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adverse effects on development and reproduction (Gould et al., 1998; Laws et al.,

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2000; Della Seta et al., 2006; Li et al., 2017). Over the past decades, studies focusing

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on the estrogenicity of BPA substitutes, such as BPF, BPS, BPAF showed the adverse

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effects of bisphenol compounds (Le Fol et al., 2017; Moreman et al., 2017; Moreman

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et al., 2018; Mu et al., 2018).

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Fluorene-9-bisphenol (CAS NO. 3236-71-3), also known as 9, 9-bis

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(4-hydroxyphenyl)-fluorene (BHPF), is a member of fluorenes which has good

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thermal stability (Wang and Zhang, 2015). Therefore, it is used as one of BPA

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substitutes (Zhang et al., 2017) in the synthesis of polyester polymers such as

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polycarbonates, epoxy resins, polyurethanes, polyesters (Dalebroux and Glanz, 1992;

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Dai et al., 2009). In recent years, materials containing BHPF have been used to

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produce a variety of products, including protective coatings, semiconductor

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encapsulations, composite materials, insulation materials, solder-resistant materials,

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epoxy floor coatings and structural adhesives (Liu et al., 2008; Dai et al., 2009; Zhang

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et al., 2017). However, the research about effects of BHPF on animals is scarce.

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Zhang et al. found BHPF existed in some “BPA-free” plastic bottles and the average

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concentration of BHPF in the drinking water samples filled in plastic bottles was

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19.51±29.55 ng/L, with a maximal concentration of 81.47 ng/L. BHPF was detected

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in the plasma of 7 individuals in 100 human samples, with a mean level of 0.34±0.21

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ng/ml. BHPF caused adverse pregnancy effects in mice by altering the homeostasis of

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ACCEPTED MANUSCRIPT the endocrine system, and it showed BHPF had anti-oestrogenic effects in mice

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(Zhang et al., 2017). Another report showed that acute BHPF exposure (100 -150 µM)

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disrupted mouse oocyte maturation and reduced oocyte quality (Jiao et al., 2019).

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Besides that, BHPF could alter sleep/wake behavior in zebrafish larvae (Mi et al.,

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2019). However, no data was available on the reproductive disrupting effect of BHPF

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in aquatic organisms.

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For aquatic animals, including teleost, the exposure route to environmental

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contaminants is mainly via uptake directly from the water across the skin and/or gill

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surfaces. Zebrafish is similar to mammalian models in toxicity testing and used as a

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common model for detecting the effects of EDCs (Evans, 1987; Van der Oost et al.,

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2003; Handy et al., 2008).

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Behavioral and physiological alterations occur to response to changes with the

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ambient. Courtship or mating behavior in zebrafish, consists of male approaching and

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chasing the female (Kalueff and Cachat, 2011). Courtship preference is based on

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visual and olfactory cues (Gerlach and Lysiak, 2006; Kalueff and Cachat, 2011). BPA

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exposure could alter courtship behaviors of male zebrafish (Li et al., 2017).

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Exploratory behavior reflects the propensity to inspect a novel object. When zebrafish

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was exposed to novel environment, a series of robust anxiety responses could be

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evoked as many other species (Blaser and Gerlai, 2006). The novel tank test for

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zebrafish is similar as open field test for rodent. It shows the natural instinct of

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zebrafish to seek protection in an unfamiliar environment through diving and staying

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in the bottom of the tank until they feel safe to explore other place (Levin et al., 2007;

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ACCEPTED MANUSCRIPT Egan et al., 2009). The behavioral parameters are used to evaluate anxiety, such as

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latency to the top, transitions into the top, freezing bouts. Anxiety/depression-like

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behavior in zebrafish showed as a longer latency to the top, reduced time spent in top

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(Barcellos et al., 2007; Levin et al., 2007; Egan et al., 2009) and increased freezing

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bouts and duration (Levin et al., 2007).

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In this study, we investigated the effects of BHPF on female zebrafish courtship and exploratory behavior. Meanwhile,

we observed

the histological and

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morphological structure of the ovary, and detected the expression of genes related to

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estrogen binding, steroidogenesis and oxidative stress in ovary. The level of tyrosine

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hydroxylase in brain was also detected. The results showed the potential effects of

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BHPF to alter preference of courtship behavior and induce anxiety/depression-like

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behavior.

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2. Materials and methods

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2.1. Ethics

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The protocols and procedures of this study involving zebrafish were approved by

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the Committee for Animal Experimentation of the College of Life Science at Nankai

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University (no. 2008) and were performed in accordance with the NIH Guide for the

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Care and Use of Laboratory Animals (no. 8023, revised in 1996).

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2.2. Chemicals exposure BHPF was purchased from Meryer Chemical Technology Co., Ltd. (Shanghai, 5

ACCEPTED MANUSCRIPT China). FULV (purity > 99.0%) was purchased from Meilunbio (Dalian, China). We

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prepared BHPF stock solutions by dissolving 10 mg BHPF in 1 mL dimethyl

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sulfoxide (DMSO), and for FULV stock solutions by dissolving 5 mg BHPF in 1 mL

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DMSO. Then the final BHPF concentration was 300 µg/L and the final FULV

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concentration was 150 µg/L. The final concentration of DMSO was 0.003% in BHPF

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or FULV. The control group was treated with 0. 003% DMSO only. The zebrafish (AB

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strain) were cultured at 28.5℃ during the treatment, the light cycle was 14 h light: 10

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h dark, pH 7.0-7.5, conductivity 500-550 µS. Feed with freshly hatched brine shrimp

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(Artemia salina) twice daily.

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The exposure treatments were shown in Scheme S1A. 36 female zebrafish and 24

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male zebrafish at age of one year old were used in our experiment. Twelve females

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were exposed to control group (0.003% DMSO). Twelve females were exposed to 300

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µg/L BHPF or 150 µg/L FULV. The chemical exposure last for 30 days. The exposure

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solution was updated every day after the second feeding. All the experimental

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condition was consistent with the standard system. One female in FULV treatment

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group jumped out the culture tank and died during the chemical exposure period, so

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the repetition for FULV treated group was 11 in the final test.

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2.3. Courtship behavior apparatus

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The courtship assay apparatus is a chambered polypropylene (PP) box (23 cm

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*15 cm*15 cm) divided into three chambers by two transparent dampers, permitted

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the visual contact among the zebrafish individuals in each chamber. One male 6

ACCEPTED MANUSCRIPT zebrafish was put in one side of the tank, one control female and one BHPF or FULV

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treated female zebrafish were put separately in the two smaller chambers on the other

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side of the tank (Scheme S1B). The outside walls of the PP box were black opaque to

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prevent external visual interference. As well as, the inside walls were white opaque to

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avoid the visual disturbance of mirror reflection of individual itself.

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2.4. Courtship behavior test

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In the laboratory condition, domesticated zebrafish breed all year round (Kalueff

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and Cachat, 2011). Zebrafish have a 24 h reproductive cycle, and light stimulation

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trigger the courtship behavior, the courtship behavior continue for about one hour

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from the first minute of exposure to light following continuous dark (Darrow and

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Harris, 2004). The male chases the female rapidly, and nudges the flank of the female

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with his snout and attempt to lead the female to a spawning site (Kalueff and Cachat,

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2011). Courtship behavior consist of two main steps: chase (approach) and follow (Li

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et al., 2017).

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In this study, the male and female zebrafish were isolated before the test. The

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male and female zebrafish were put to the courtship behavior test tank for 10 min to

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acclimate the test condition at 19:00-21:00 the day before test. The courtship behavior

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test start at 7:00-8:00 a.m. One male zebrafish was put in one side of the test tank, one

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control female and one treated female (BHPF or FULV exposure) were put into the

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two smaller chambers in the other side. For male zebrafish, they could make the

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choice swimming close to the control female or to the treated female. After 2 min

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acclimation, turn on the light and recording the courtship behavior for 5 min. After the

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test, the tank was washed carefully, to make sure no chemical or hormonal was left.

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Other groups repeated this test process.

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2.5. Novel tank test

The novel tank test is usually used for evaluating anxiety/depression behavior.

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The novel tank was made from transparent plexiglass, was a 5 L rectangular box (23

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cm length * 15 cm width * 15 cm depth), with 12 cm depth of water, used to assess

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anxiety/depression behavior of zebrafish. We divided the tank into two equal

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horizontal portions virtually by marking a midline on the outside walls of the tank

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with water body. The region above this midline was indicated as “top” of the novel

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tank, while the area below was indicated as the “bottom” of the novel tank. The novel

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tank was placed over a light source, an LED array, with an acrylic diffuser located

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above. The light source was composed of white light (500 lux) arrays and a

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transparent platform. Two CCD cameras (MV-VS078FM, Microvision, 10 frames/s)

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were fixed to obtain the top (dorsal) view and side (lateral) view of the moving

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zebrafish (Scheme S1C).

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The novel tank behavior started from 10:00 a.m. to 15:00 p.m. after the courtship

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behavior test. Zebrafish behaviors were recorded for 5 min by the two CCD cameras

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synchronously.

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2.6. Behavioral analysis 8

ACCEPTED MANUSCRIPT The videos of courtship behavior and novel tank behavior were imported into the

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custom video-tracking software which was designed by Microsoft Visual and Open

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CV 2.3.1. Matlab 2010 was used to transform raw data of pixel coordinated of each

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fish into behavioral parameters. In the courtship test, the locomotion behavior, social

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interacting behavior and courtship behavior was analyzed. In the novel tank test, the

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detailed locomotion behaviors were analyzed. The locomotion and courtship

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behavioral parameters in courtship test were defined as in prior studies and literature

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(Li et al., 2017).

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Some parameters were introduced show the behavior alternation. We introduced

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attracting index (AI) which was defined as the horizontal velocity of zebrafish (close

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to the opposite sex). In addition, selecting index (SI) which was defined as the vertical

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velocity of zebrafish was introduced to assess the extent of the motivation of female

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choosing male for courtship or interacting with the other female, and SI of the male

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could show which female was preferred for mating. The method for calculating these

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parameters was shown in Supplementary Information Text.

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2.7. Fecundity and gonadosomatic index Spawning events were conducted in all the female groups with untreated males

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every six days from the beginning of the exposure until the 30 exposed day. The

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number of collected eggs was used to show fecundity. The ovaries were collected to

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calculate the gonadosomatic index (GSI) of the females at the end of the exposure.

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The GSI =gonad mass/body mass×100%. 9

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2.8. Histological analysis After the courtship behavior and novel tank behavior test, the ovaries of the

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female zebrafish were collected and fixed in 4% paraformalydehyde overnight at 4℃.

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The fixed ovaries were used for paraffin embedding, 8 µm sections were cut and used

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for hematoxylin-eosin (HE) staining. The sections were observed and photographed

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using light microscope.

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2.9. qRT-PCR

The total RNA was extracted by Trizol (Invitrogen, USA) from the ovaries

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collected from the female zebrafish after the behavior test, and normalized to the same

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concentration before the reserve transcription by M-MLV reverse transcriptase

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(Promega, USA). The cDNA was synthesized using specific primer of each gene. The

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genes related to estrogen binding, steroidogenesis and oxidative stress, containing of

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estrogen receptor 1 (esr1) and estrogen receptor 2a (esr2a), 3β-hydroxysteroid

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dehydrogenase (hsd3b), cytochrome P450 family 11 subfamily A member 1 (cyp11a1)

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and cytochrome P450 17 A1 (cyp17a1), cytochrome P450 19A1 (cyp19a1), superoxide

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dismutase 1 (sod1), catalase gene (cat) and glutathione peroxidase 1a (gpx1a) which

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encode enzyme to defense against oxidative stress were detected. SYBR Green PCR

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kit (Biorad, USA) was used for qRT-PCR, the procedure was following as: denaturing

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at 95

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extension at 72

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for 2 min; 95

for 30 s, 62

for 30s, 72

for 30s, 40 cycles; finally

for 5 min. Standard curve was made using the different 10

ACCEPTED MANUSCRIPT concentration of the cDNA serially diluted from10-4 to 10-11 µg/µL. Serially diluted

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cDNA were run in parallel to the samples in the rt-PCR to obtain the concentration for

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each sample from the corresponding cycle threshold value. The change in mRNA

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level of each gene was calculated from the concentrations of BHPF or FULV treated

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group relatively to the mean value of the control group (Baudiffier et al., 2013).

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Primer sequence was showed in Table S2.

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2.10. Western blot

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The procedure for western blot was followed as previous studies (Zhang et al.,

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2018) with some modification. The brain of the female zebrafish was collected and

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extracted total protein with radioimmunoprecipitation assay (RIPA) (CWBIO, Beijing,

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China) buffer containing phenylmethylsulfonyl fluride (PMSF) (Sigma-Aldrich,

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USA). Total proteins were separated by 10% sodium dodecyl sulphate polyacrylamide

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(SDS-PAGE) gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF)

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membrane, then the membrane was blocked with 5% skim milk dissolved in

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Tris-buffer saline (TBS) for 1 hour at room temperature. PVDF membrane was

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incubated with first mouse anti-tyrosine hydroxylase antibody (anti-HT) (1:1000;

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Chemicon MAB318, CA, USA) and mouse β-actin monoclonal antibody (1:5000;

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Proteintech, IL, USA) overnight at 4 °C. After washing with PBS + 0.05% Tween-20

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(TBST), the membrane was incubated with secondary anti-mouse HRP-conjugated

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secondary antibody (1:3000; CWBIO, Beijing, China). The membrane was then

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washed with TBST. The image of target protein was detected by Super Signal West

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Pico Chemiuminescent substrate (ThermoFisher Scientific, CA, USA).

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2.11. Data analysis Levene test was used to test the data normality, and One-way ANOVA was used

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to evaluate differences among variances, followed by Turkey’s post-hoc test using

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SPSS 21 (IBM Corporation). Prism 6.0 (GraphPad Software) and MATLAB

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(MathWorks, Inc.) were used for charts producing. The data was shown as mean ±

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SEM. Significance was set as P<0.05 (*), P<0.01 (**), P<0.001 (***).

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More details of experimental treatments and analysis was in Supplementary information.

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3. Results

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3.1. The effects of BHPF on locomotor activity in courtship behavior

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The trajectory and locomotor parameters of control female, BHPF treated female,

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fulvestrant (FULV) treated female and male in courtship behavior was assessed. The

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result showed that: the control female mainly swam along the edges close the male

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and the other female; the BHPF or FULV treated female also swam along the edges

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close the male and the control female; the male mainly swam along the edge close to

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the females (Fig. S1, S2). BHPF and FULV did not change the travel distance in

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courtship, but FULV decreased the turn angle and increased the angle velocity slightly

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compared with control female, the travel distance of the male was longer than the

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control female, while the absolute turn angle and angle velocity had no difference

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ACCEPTED MANUSCRIPT compared with the control female (Fig. S3). The locomotion activity of male was

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stronger than the female in this test, but the locomotor activity of BHPF and FULV

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treated female was similar with control female. In order to eliminate the effects of

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zebrafish size on preference, we measured the body length of each zebrafish in the

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video, the result showed there was no significant difference in body length among all

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the groups (Fig. S3).

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AI was used to investigate the extent of one zebrafish attracted by the opposite

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sex individual during the 300 s courtship test, the result showed that BHPF and FULV

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treated female did not have a significant change in AI compared with control female,

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but the AI of females were higher than the AI of male (Fig. 1A). The result showed SI

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had no difference between the control female and treated females, while the SI of

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male was obviously higher than the three types of female (Fig. 1B). This result

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showed there was no difference of BHPF and FULV treated females in attracting the

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male, the female was predominantly swam in the horizontal direction close to the

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region of the male, while the male was mainly swam in the vertical direction for

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selecting females.

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Furthermore, we used the locomotion profile, attracting index and selecting index

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for hierarchical clustering, the four groups were divided into three clusters: BHPF and

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FULV treated female gathered into one cluster; the male and the control female gather

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separately (Fig. 1C). This result demonstrated that BHPF and FULV exposure caused

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the similar effect on female in the courtship behavior.

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3.2. The effects of BHPF on time spent in region of approaching and contact in

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courtship behavior There was no difference about time in region of approaching (ROA) among

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females. The female exposed to BHPF spent 37.9% time in ROA, and FULV treated

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female spent 37.6% time in ROA. While the control female spent 43.7% time

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remained in ROA, slightly longer than BHPF and FULV treated females without

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statistical significance. Besides, there was no difference about the time the males

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spent in ROA between BHPF and FULV group, and the male spent average 76.0%

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time in ROA during the test time (Fig. 1D). The male spent 52.0% time in ROA close

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to control female, while the male spent 18.0% time in ROA close to BHPF treated

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female; the male spent 54.0% time in ROA close to control female, while spent 24.0%

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time in ROA close to FULV treated female in the test with FULV group (Fig. 1E).

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However, the velocity of BHPF and FULV treated females in ROA increased

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significantly compared to the control and the male, and the velocity in ROA had no

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difference between BHPF and FULV treated females (Fig. 1F).

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The courtship index was defined as the time the female/male zebrafish

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approached the other male/female in the region of contact (ROC). Courtship index

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was used to evaluate the preference of male and female in courtship. The courtship

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index of male to female (♂→♀) showed that male spent similar time in chasing

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control female and FULV treated female, while the time for chasing BHPF treated

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female was less than the control, but there was no statistical significance (Fig. 1G).

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The courtship index of female to male (♀→♂) showed that BHPF treated female

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ACCEPTED MANUSCRIPT spent less time contact to male compared to the control female (Fig. 1H). This result

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indicated that BHPF exposure disturbed the courtship behavior: (1) BHPF exposure

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led the female less focus on the male; (2) BHPF decreased the attraction of the female

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to the male. The time spent in ROC between control female and BHPF or FULV

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treated female was also compared, the time spent in ROC between the control female

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to BHPF treated female (ctrl♀- BHPF♀) and BHPF treated female to control female

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(BHPF♀-ctrl♀) had no difference, but the time spent in ROC decreased in FULV

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treated female to the control female (FULV♀-ctrl♀) compared with the control female

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to FULV treated female (ctrl♀- FULV♀) (Fig. 1I). It showed that FULV exposure

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disturbed the courtship behavior and influenced the selection of the control female in

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courtship. FULV treated female trended to keep away from the control female, while

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the control female tended to close to FULV treated female. The interaction between

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the control female and treated female was frequent in general.

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3.3. The change of body orientation and Markov chain model of status transform

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during courtship

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The average orientation distribution of every zebrafish holding the body position

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at certain angle (0° to 360°) in ROA was showed in the polar directional histograms

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(Fig. 2A, B). The orientation of resultant pose vector was similar (300° to 360°) for

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the control female when tested together with BHPF or FULV treated female, and the

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pose orientation was mainly towards the male and the other female (Fig. 2A, B). The

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resultant pose vector of BHPF treated female was in 0° to 45° and 330° to 360°, the 15

ACCEPTED MANUSCRIPT orientation was also mainly towards the male and the control female, with the

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direction departed from the other female (330° to 360°) increased and the length of

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resultant pose vector towards the male shorter compared with the control (Fig. 2A,

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Fig. S4). The composite pose vector of FULV treated female was in 0° to 60° mainly

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towards the male and the control female, with shorter length towards the male

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compared with the control (Fig. 2B and Fig. S5). While the resultant pose vector of

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the male was from 120° to 210°, and the pose direction was along the vertical mainly

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towards the control female and BHPF or FULV treated female, there was no obvious

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preferable direction for a certain female, but only leaned to the control female slightly

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(Fig. 2A, B; Fig. S4, S5). The average direction and length of resultant pose vector

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did not show difference between control female, BHPF or FULV treated female (Fig.

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S6). There was no obvious effects of BHPF and FULV on body orientation

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distribution.

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Markov chain model was introduced to explain the patterns during the courtship.

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We virtually divide the residing box of each female and the male zebrafish into two

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equal parts: front and back. We introduce a three-dimensional vectors S = (SC, ST, SM)

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to characterize the status of the three fish. SC is the status of control female, SC =1

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when it was in the front, and SC =0 in the back; ST is the status of treated (BHPF or

347

FULV exposure) female, ST =1 when it was in the front, and ST =0 in the back; SM is

348

the state of the male, SM =1 when it was in the same side of treated female, and SM =0

349

when it was in the side of control female. There are altogether eight different status of

350

the microscopic configurations (Fig. 2C, D). We built a status transition matrix form

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ACCEPTED MANUSCRIPT one of the eight status S = (SC, ST, SM) to the other status S’= (S’C, S’T, S’M). The

352

simple Markov transition matrix from S to S’ was denoted as P S→S’. The video was

353

recorded at 15 frame per second, in two adjacent images with a 1/15 second time

354

interval, in most cases the latter status would maintain the former status, if the latter

355

status change, only one status of the three configurations would shift. From the 5 min

356

trajectories of the three fish we can obtain the probability of the microscopic

357

configuration maintaining or jumping from one status to another. This transition

358

matrix can show the preference of the male and female. In the BHPF treated group

359

(Fig. 2C), the result showed that the probability of status S = (1, 1, 0) was 0.3283,

360

which both the females were in the front and the male in the side of control female;

361

the probability was 0.2730 in status S = (1, 1, 1). The result showed that the control

362

female and treated female was more likely to be in the state of S= (1, 1, 0) and S = (1,

363

1, 1), and the male prefer to attach the control female. The transform probability

364

indicated both the control female and BHPF treated female was more likely to close to

365

the male. The male had more possibility to chase the control female than the BHPF

366

treated female when the females kept away from the male. In the FULV treated group

367

(Fig. 2D), the probability of status S = (1, 1, 0) was 0.2905, and status S = (1, 1, 1)

368

was 0.2987. The probability P(1, 1, 0)→(1, 1, 1) = 0.0149, the probability of reverse process

369

P(1, 1, 1)→(1, 1, 0) = 0.0139. It showed that the control female and treated female was

370

more likely to be in the status of S = (1, 1, 0) and S = (1, 1, 1), but the male showed

371

same preference to either female. The transform probability indicated both the control

372

female and FULV treated female was more likely to close to the male. The status

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probability of S = (0, 0, 0) and S = (0, 0, 1) was lower than other status. It showed the

374

same distribution as BHPF treated group. In the simple transition matrix, we could construct that the control and treated

376

female showed preference to the male, in the BHPF treated group, the male preferred

377

to approach the control female than BHPF treated female, while in the FULV treated

378

group, the male showed similar probability with the control female and FULV treated

379

female.

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3.4. BHPF decreased the fecundity and GSI, and caused adverse effects in oocyte

382

maturation

The number of collected eggs decreased in BHPF group at the 12, 18, 24 and 30th

384

day of exposure compared with control. At the same time. FULV also decreased the

385

number of collected eggs at 6, 18, 24 and 30th day of exposure compared with control

386

(Fig. 3A). BHPF and FULV exposure also decreased the GSI compared with control

387

(Fig. 3B).

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The hematoxylin-eosin (HE) staining was performed to examine the

389

histopathological effect of BHPF and FULV on ovaries. The oocytes were staged

390

(Selman et al., 1993) : stage ℃, primary growth stage; stage ℃, cortical alveolus stage;

391

stage ℃, early-vitellogenic stage; stage ℃, late-vitellogenic stage. As shown in Fig. 3C,

392

the morphological characteristic of the female zebrafish ovary treated by BHPF and

393

FULV was different to the control. The oocyte maturation level under FULV and

394

BHPF exposure was lower than the control group. The statistical analysis showed that

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ACCEPTED MANUSCRIPT the percentage of primary oocytes (stage ℃) increased obviously in BHPF and FULV

396

groups compared with control (BHPF vs control, P=0.0068; FULV vs control,

397

P=0.0014). However, there was no difference in the percentage of stage ℃ oocytes

398

(BHPF vs control, P=0.9694; FULV vs control, P=0.8725). While the proportion of

399

stage ℃ oocytes decreased in BHPF and FULV group (BHPF vs control, P=0.0340;

400

FULV vs control, P=0.0049). The percentage of late-vitellogenic oocytes (stage ℃)

401

also decreased obviously in BHPF and FULV groups compared with control (BHPF

402

vs control, P=0.0343; FULV vs control, P=0.0083) (Fig. 3D). The oocytes shown

403

morphological abnormality and structural distortion in FULV and BHPF groups. It

404

seemed that FULV and BHPF prevented oocyte maturation and induced abnormal

405

oocytes development.

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3.5. BHPF exposure altered the expression of genes involved in ovarian

408

steroidogenesis and oxidative stress

The courtship behavior was disturbed partly by BHPF and FULV exposure. To

410

further learn the effects of BHPF exposure on reproduction, we extracted RNA from

411

the ovaries and quantified the expression of related genes by qRT-PCR. The

412

expression levels of genes related to estrogen binding and ovarian steroidogenesis

413

changed under BHPF and FULV exposure. The expression of esr1, esr2a and hsd3b

414

in BHPF and FULV group was significantly downregulated compared to the control

415

(Fig. 4A-C). The expression of cyp11a1 was upregulated under BHPF and FULV

416

exposure (Fig. 4D).The expression of other genes encoding steroidogenic enzymes

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ACCEPTED MANUSCRIPT including cyp17a1 and cyp19a1 was significantly downregulated under BHPF or

418

FULV exposure (Fig. 4E-F). It indicated that BHPF and FULV exposure disturbed

419

steroidogenesis and estrogen binding. The expression of sod1, and gpx1a was

420

upregulated in BHPF group but not in FULV group (Fig. 4G, I). The expression of cat

421

did not change under BHPF exposure, but increased under FULV exposure (Fig. 4H).

422

It showed BHPF and FULV exposure potentialy caused oxidative stress and induced

423

adverse damage to the ovary.

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3.6. BHPF inhibited exploratory behavior and caused anxiety/depression-like

426

behavior in female zebrafish

The locomotive behavior of zebrafish was measured by novel tank test, the

428

locomotive parameters of the total distance traveled, turn angle, average velocity and

429

angular velocity was calculated according to the video records of three-dimension (3D)

430

space by dorsal view and lateral view (Fig. 5A, B). Compared with the control, FULV

431

significantly decreased the total distance traveled (P=0.0258), turn angle (P=0.0119),

432

meandering (P=0.0281), average velocity (P=0.0211) and angular velocity (P=0.0100)

433

(Fig. 5C-G). There was no significant change between BHPF treated and control.

434

However, the parameters of the time spent in the top (BHPF group: P=0.0015; FULV

435

group: P=0.0231) (Fig. 5H), time spent ratio of top: bottom (BHPF group: P=0.0022;

436

FULV group: P=0.0218) (Fig. 5J), distance travelled of top: bottom (BHPF group:

437

P=0.0036; FULV group: P=0.0277), latency to enter the top (BHPF group: P=0.0006;

438

FULV group: P=0.0142) and distance travelled in the top (BHPF group: P=0.0037;

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ACCEPTED MANUSCRIPT FULV group: P=0.0129) (Fig. 5L-N) were significantly suppressed in both BHPF and

440

FULV groups. The average entry duration (Fig. 5K) was decreased in both BHPF

441

(P=0.0041) and FULV (P=0.3340) treated group, and entries ratio of top: bottom (Fig.

442

5O) were also decreased in both BHPF (P=0.0026) and FULV (P=0.0410) group. The

443

number of entry into the top was also decreased in BHPF (P=0.281) and FULV

444

(P=0.4290) treated group, but it was not significant (Fig. 5I). Besides, FULV induced

445

the increase of freezing bouts (P=0.0145) and freezing duration (P=0.0163), BHPF

446

also increased the freezing bouts (P=0.0380) and freezing duration (P=0.0430) (Fig.

447

5P, Q). 3D reconstruction of swimming trajectories demonstrated the distinct

448

swimming patterns of zebrafish exposed to BHPF and FULV (Fig. 5R-T). Wild type

449

zebrafish demonstrated normal locomotion patterns, including the wide spatial scope

450

and intensive trajectories of zebrafish (Movie S1). However, both BHPF and FULV

451

exposure disturbed their swimming path patterns, resulting in near bottom trajectories

452

and decreased locomotive activity. Furthermore, BHPF inhibited the exploratory

453

behavior of zebrafish in the novel tank, significantly decreased the swimming path in

454

the top part, and increased the freezing bouts and durations (Movie S2). While FULV

455

also inhibited the exploratory behavior of zebrafish, increased the freezing bouts and

456

duration, decreased the movement in the top (Movie S3). The alternation of

457

locomotive parameters and trajectories showed that BHPF and FULV exposure

458

induced anxiety/depression-like responses.

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459

To compare all the three experimental groups in exploratory behavior, principal

460

component analysis (PCA) based on nine variables (Table S1) was performed. The 21

ACCEPTED MANUSCRIPT three groups in this study and the variables of locomotion parameters in Fig. S7 were

462

showed on two orthogonal axes with two principal components (Fig. 6A-C). The first

463

component in x axis (Fig. 6A) was mainly related to locomotive activity, with most

464

parameters related to locomotion activity in the test. The second component in y axis

465

was primarily related to locomotive characteristics. Average entry duration and entries

466

top: bottom ratio played most contribution in second principal component (Fig. 6A

467

and Fig. S7), which was related to movement between top and bottom. The

468

locomotive activity and movement characteristics between top and bottom were

469

associated with anxiety/depression behavior. The barycenters of three groups were

470

distributed in three quadrants (Fig. 6C). Control group was in one side of the first

471

component, BHPF and FULV groups were in the same side from first component. It

472

showed the same effects of BHPF and FULV for the locomotive activity of female

473

zebrafish, such as total distance traveled, angular velocity, turning angle, meandering.

474

BHPF and FULV groups were divided in the second principal component, it showed

475

the difference of BHPF and FULV can be distinguished from the characteristics

476

between top and bottom. The effect of BHPF induced anxiety/depression-like

477

behavior was stronger than FULV and control group, with the decreasing of average

478

entry duration and entries top: bottom ratio. The effects of FULV had weaker effects

479

on induced anxiety/depression compared to BHPF.

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480 481 482

3.7. BHPF changed the level of tyrosine hydroxylase in brain We investigated the expression of tyrosine hydroxylase (TH) by western blot. The 22

ACCEPTED MANUSCRIPT result showed that TH in brain decreased in BHPF and FULV exposure groups

484

compared to the control (Fig. 6D, E). The decrease of TH may result in the behavior

485

alternation and relate to the anxiety/depression-like behavior under BHPF and FULV

486

exposure zebrafish.

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487 488

4. Discussion

Over the past decades, many studies showed BPA and its analogues had

490

oestrogenic activity (YOKOTA et al., 1999; Nakagawa and Suzuki, 2001; Rastkari

491

et al., 2011; Pisapia et al., 2012; Le Fol et al., 2017; Moreman et al., 2017). However,

492

as one of BPA’s substitutes, BHPF showed anti-estrogenic activity for it could be well

493

fitted into the antagonist pocket of estrogen receptor (ER) α (Zhang et al., 2017).

494

this study, BHPF also showed anti-estrogenic effects in the downregulation of ER

495

genes expression. FULV is an oestrogen antagonist possessing high ER-binding

496

affinity and has multiple effects on ER signaling (Howell, 2006). FULV

497

downregulated the expression of ER (Osborne et al., 2004) and impaired the activity

498

of endogenous oestrogens by binding on the ER (Wakeling et al., 1991; Addo et al.,

499

2002). Therefore, we explored the biological effects of BHPF in zebrafish by

500

comparing with the control and FULV.Courtship behavior plays an important role in

501

reproduction. In this study, we assessed the effects of BHPF on courtship behavior.

502

The locomotor parameters did not change much in BHPF treated female compared to

503

control female, but turn angle and angle velocity in FULV group increased slightly. AI

504

reflected the tendency closing to the opposite sex, SI reflected the trade-off between

In

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ACCEPTED MANUSCRIPT two females (for male to choose which female was preferable). The control, BHPF

506

and FULV treated female showed the similar AI, which was higher than the male, it

507

showed when there was only one male, the females tended to approach the male.

508

However, the SI of male was significantly higher than all the females (Fig. 1). There

509

was no difference among the control, BHPF and FULV treated females in attracting

510

the male, the females were mainly to approach the male, while the male was mainly to

511

select females when there were two females.

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The male had a low courtship index for the BHPF exposure female, and the

513

BHPF exposure female also had low courtship index for the male. This result

514

indicated BHPF exposure interfered the preference of the male in some extent, and

515

reduced the preference of the female to male, in other words, the female showed less

516

focus on male for courting, and had less attraction for the male (Coe et al., 2010).

517

BHPF exposure could disturb the courtship behavior and had potential effects for

518

reproduction. EDCs which mimic sex hormones could interfere courtship,

519

17α-ethinylestradiol (EE2), as one synthetic estrogen, disturbed the courtship behavior,

520

altered aggression and individual social status and productive success in male

521

zebrafish (Colman et al., 2009). EE2 exposure also led to a female-basis sex ratio, the

522

parameters including total paths, average velocity, turn rate, distance swam in

523

spawning area in courtship behavior decreased compared with control male (Larsen et

524

al., 2010). In this study, the interaction between the control female and BHPF or

525

FULV treated female was frequent with high contact between two females (Fig. 1I),

526

besides the effects of BHPF or FULV exposure, it may associate with the aggression

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ACCEPTED MANUSCRIPT between the females for courtship (Coe et al., 2010) or sociality between individuals

528

(Ariyasiri et al., 2019). Hormones played an important role in courtship, female

529

zebrafish attracted the male by a specific steroid glucuronides sex attractant

530

containing oestradiol-17β- and testosterone glucuronide secreted by the ovary (Hurk

531

and Lambert, 1983). Expose to androgenic 17β-trenbolone disturbed normal male

532

courtship behavior (Larsen and Baatrup, 2010). The male spent less time with BHPF

533

or FULV treated female compared to the control in ROA. The courtship behavior was

534

disturbed by BHPF exposure. Under BHPF exposure, females did not focus on the

535

males and the males spent less time with the females, the courting behavior of females

536

toward males diminished, and the preference of male decreased, it showed similar

537

results as EE2 (Coe et al., 2010). The velocity was significantly increased in BHPF

538

and FULV treated female in ROA compared to the control female and the male (Fig.

539

1F), although the average liner velocity in the whole compartment was similar as

540

control and slower than the male. Furthermore, the male spent more time close to the

541

control female and less time close to the BHPF or FULV treated female. Therefore, it

542

could be pointed out that BHPF interfere normal courtship behavior, but in this study

543

it was not as toxic as BPA and other EDCs to affect courtship behavior (Li et al.,

544

2017). FULV did not change courtship behavior obvious in this test. A study showed

545

that FULV (1×10-7 M) did not affect the temporal and spectral features of mating

546

behavior in Xenopus laevis (Hoffmann and Kloas, 2012; Efosa et al., 2017). It showed

547

that BHPF and FULV exposure could affect velocity in courtship and disturb the

548

courtship behavior in a certain extent.

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ACCEPTED MANUSCRIPT The body orientation from tail to head of control female was mainly toward the

550

male and BHPF or FULV treated female; the direction kept away from the control

551

female was increased in BHPF treated female compared to the control female; FULV

552

treated female mainly oriented to the male and control female, with shorter pose

553

vector length towards the male compared with the control. There was no significant

554

difference among the females in pose orientation, while the body orientation of the

555

male main pointed to two different position, one orientated to control female, the

556

other orientated to BHPF or FULV treated female, with a trend towards the control

557

female slightly (Fig. 2A, B; Fig. S4, S5). The three fish in one test mainly oriented to

558

the other two and tended to gathered together, we can see the probability was above

559

50% in the status of S = (1, 1, 0) and S= (1, 1, 1) which the three fishes were all in

560

ROA from the Markov chain model (Fig. 2C, D).

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BHPF exposure further influenced the reproductive function in histochemical

562

morphology and molecular levels. BHPF exposure decreased egg production and GSI

563

of the females. Histopathological section of ovary showed that BHPF and FULV

564

exposure had adverse effects on oocyte maturation and morphology. A study showed

565

that the maturation level of oocytes in ovary of female zebrafish exposure in high

566

temperature induced male-basis sex determination would be delayed compared to the

567

control female in normal temperature (Ribas et al., 2017). BPA as a xenoestrogen

568

could induced the increase of atretic follicles, whole body vitellogenin concentration

569

and altered the expression of cyp19a with an up-regulation at 10 µg/L (Molina et al.,

570

2018). Severe deterioration of ovarian tissue occurred under 3 and 5 mg/L BPA

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ACCEPTED MANUSCRIPT 571

exposure with increased atretic oocytes, structurally distorted and less developed

572

oocytes

573

(2,3,7,8-tetrachlorodibenzo-p-dioxin) and insecticide (deltamethrin) also played

574

negative effects on ovary structure (King Heiden et al., 2005; Koc et al., 2009). In our

575

study, the result was the same with more oocytes in primary stage and less in

576

vitellogenic stage under BHPF and FULV exposure. BHPF and FULV had similar

577

effects to slow down the process of oogenesis in female zebrafish.

and

Akbulut,

2014).

The

toxicant

herbicide

SC

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(Yön

Sex steroids are essential for the regulation of oogenesis and alterations in sex

579

steroid biosynthesis. We investigated the effects of BHPF and FULV on the

580

expression of genes involved in steroid biosynthesis and oogenesis in female

581

zebrafish ovary. The significantly decreased esr1 and esr2a expression showed a

582

remarkable anti-estrogenic effect of BHPF and FULV exposure (Fig. 4). The

583

inhibition of aromatase (cyp19a1) expression level showed that the conversion of

584

testosterone to estradiol in granulosa cells was disrupted (Romano et al., 2012).

585

Cyp11A1 synthesizes pregenolone from cholesterol (Goldstone et al., 2010) and it

586

works on the upstream of 17β-estradiol synthesis pathway. In this study the

587

upregulation of cyp11a1 and the downregulation of hsd3b, cyp17a1 and cyp19a1a

588

would alter the synthesis of sex hormone. The decreasing trends in expression of

589

steroidogenic enzymes (hsd3b, cyp17a1) indicated a possible wider effect on

590

steroidogenic pathways (Richard et al., 2005). The level of sod1 expression elevated

591

in response to various stimulating factors, such as heat shock, heavy metal, hydrogen

592

peroxide (Zelko et al., 2002). SOD activity is an important factor in aging and

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ACCEPTED MANUSCRIPT lifespan determination in Drosophila (Parkes et al., 1998). Oxidative stress such as

594

silver nanoparticles caused the expression of sod1, gpx1 and cat to a greater extent

595

(Kim et al., 2009). The expression of sod1, gpx1 were significantly upregulated in

596

response to reactive oxygen species (ROS) (Patel et al., 2013). The protective agents

597

such as resveratrol upregulated the expression of sod1 and gpx1 in a concentration-

598

and time-dependent way and reduced the oxidative stress (Spanier et al., 2009). In this

599

study, BHPF exposure upregulated expression of sod1 and gpx1a and induced

600

oxidative stress. It showed BHPF had toxicity for the development and reproduction

601

in female zebrafish. Taken together, the alternation of ovarian gene expression

602

suggested that BHPF and FULV might disrupted steroid hormone biosynthesis and

603

also potentially modulated the biological effects of estrogens via modulating the

604

expression of the predominant estrogen receptor esr1 and esr2a in the ovary.

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EDCs can disrupt the hormone balance of vertebrate and invertebrate, not only

606

affect the gonads differentiation, fertility and other reproductive behavior, also cause

607

damage on the hormone secretion and behavior reaction region in brain inducing fear,

608

anxiety and other emotional behavior disorders. Therefore, we tested the effect of

609

BHPF and FULV on the non-reproductive behavior of zebrafish by using the typical

610

anxiety/depression behavior detection method, novel tank test. BHPF and FULV

611

induced anxiogenic effects, with the decrease of locomotor activity, time spent in top,

612

average entry duration. Notably, BHPF induced more serious anxiety/depression-like

613

behavior, with less time spent in top and less numbers entered into the top. The total

614

traveled distance, turning angle, meandering, average velocity, angular velocity, time

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ACCEPTED MANUSCRIPT spent in top, average entry duration decreased in BHPF and FULV exposure females.

616

The locomotion behavior change in novel tank test was not correspond with the

617

change in courtship behavior. As the result showed, the locomotion activity of FULV

618

and BHPF treated female did not trended to decrease (Fig. S3) in courtship behavior,

619

especially in ROA with an obvious increase of velocity (Fig. 1F), while in the novel

620

tank test, the similar parameters were inclined to decrease. These conflicting results

621

could be caused by the effect of other zebrafish individuals disturbing the locomotor

622

behavior, in courtship behavior the locomotor activity was inconsistent with the

623

condition when the zebrafish was isolated into novel tank alone. The reduced

624

exploration, increased freezing behavior and erratic movement was used to assess

625

behavioral induces of anxiety in novel tank test (Cachat et al., 2010). Anxiety in novel

626

test is reflected by reduced exploration and elevated erratic movements and freezing

627

(Stewart et al., 2011; Stewart et al., 2012). Anxiety and depression are major chronic

628

mood disorders and usually caused by exposure to diverse unpredictable stress factors

629

(Chakravarty et al., 2013), BHPF exposure stress induced the mood disorders.

630

Gestational and early life exposure to BPA was associated with anxious and depressed

631

behavior on children (Braun et al., 2011; Harley et al., 2013). BPA exposure increased

632

anxiety/depression-like behavior in mice of both sexes in open field, dark-light

633

transition, elevated plus maze tasks (Xu et al., 2012). Perinatal exposure to

634

environmental dose of BPA caused anxiety/depression-related behaviors in rats and

635

mice (Chen et al., 2015; Xu et al., 2015). In this study, we could find that BHPF could

636

induced anxiety/depression-like behavior as BPA. It showed BHPF disturbed the

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29

ACCEPTED MANUSCRIPT 637

mood and caused stress in zebrafish. Maternal consumption of low levels of dietary BPA decreased attractiveness of

639

male offspring, maternal BPA led to a disadvantage for mating and induced greater

640

anxiety-like behavior in deer mice (Galea and Barha, 2011). BPA exposure also

641

induced sex-specific effects on social and anxiety-like behavior by altering epigenetic

642

programming in juvenile cortex and hypothalamus, the expression alternation in ERα

643

and DNA methyltransferase in males’ cortex and females’ hypothalamus were

644

associated with the alternation of DNA methylation in ERα (Kundakovic et al., 2013).

645

BHPF exposure induced alternation of courtship and anxiety/depression-like behavior,

646

further study is needed for learn whether BHPF exposure may had the potential

647

effects on methylation. BPA exposure caused sex-specific influence on reproductive

648

and non-reproductive behaviors, altered characteristics of exploration, anxiety, spatial

649

learning and memory behaviors (Xu et al., 2011). BHPF showed the same tendency to

650

disturb the behaviors and induced anxiety/depression-like behavior like BPA

651

exposure.

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638

TH is a marker enzyme to catalyze amino acid L-tyrosine to L-DOPA (Nagatsu,

653

1995; Meister, 2009). As we know dopamine is one kind of neurotransmitters between

654

nerve cells in the brain, it controls physical movement and emotional behavior.

655

Anxiety is a behavioral consequence under stress, a classic hypothesis considering

656

that the serotonin (5-hydroxytryptamine, 5-HT) in brain can promote anxiety (Fossat

657

et al., 2014). The density of TH immunoreactive fibers in the basolateral complex

658

correlated inversely with anxiety-related behavior in rats, and a reduced anxiety with

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ACCEPTED MANUSCRIPT increased TH immunoreactive density (Yilmazer-Hanke et al., 2004). Formaldehyde

660

exposure increased the levels of anxiety/depression-like behavior and reduced the

661

levels of brain TH (Li et al., 2016). These were corresponded with the result of this

662

experiment. The decrease of TH in BHPF and FULV would cause the reduction of

663

L-DOPA, which was correlated with anxiety/depression-like behavior.

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659

In this study, we can summarize the effects of BHPF on adult female zebrafish.

665

BHPF exposure could not only interfere the courtship behavior but also influence

666

exploratory behavior (Fig. 7): (a) BHPF exposure could reduce courtship index of the

667

male for BHPF treated female or BHPF exposed female for male; BHPF exposure

668

disturb the pose orientation distribution of the female; The male spent less time with

669

BHPF treated female in ROA. Besides that, BHPF exposure decreased fecundity and

670

caused adverse effects on the ovary structure. BHPF exposure also altered the

671

expression of genes related to estrogen binding and steroidogenesis, while

672

upregulated the expression of sod1 and gpx1a and induced oxidative stress. All of

673

these indicated BHPF could induce reproductive interference and had potential

674

harmful effects for reproduction. (b) BHPF exposure decreased locomotor activity,

675

numbers of enter into top, average entry duration, time spent in tops; BHPF induced

676

anxiety/depression-like behavior; the level of tyrosine hydroxylase in brain decreased

677

under

678

anxiety/depression-like behavior. In conclusion, we can see that BHPF has potentially

679

adverse effects on courtship behavior and induces mood disorders. We should be

680

cautious to the application of BHPF.

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Acknowledgments This project was initiated in the State Key Laboratory of Medicinal Chemical

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Biology at Nankai University. This work was supported by the Special Fund for Basic

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Research on Scientific Instruments of the Chinese National Natural Science

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Foundation [grant nos: 61327802, 61127006], and the National Basic Research

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Program (973 program) of China [grant no: 2015CB856500]. The authors declare that

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there are no conflicts of interest.

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Author contributions

X.Z.F and S.H.Z. conceived and designed the experiments. Behavior experiments were conducted by S.H.Z., and the video analysis for behavior was conducted by C.

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W.. The histologic section and qPCR was conducted by Q. P. Z.. Data analysis and

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paper writing were conducted by P. M.. PCA analysis was conducted by S.Z.Z. and J.

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Z.G.. Y.C.F gave important contribution to data analysis. X.F.Z., D.Y.C, D.F.F made

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important contributions to the discussion. All authors discussed the results and

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reviewed the manuscript at all stages.

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References

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Fig. 1. Related parameters in courtship. (A) Attracting index in courtship. (B) Selecting index in

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courtship. (C) Hierarchical clustering of the locomotion parameters. (D) Time in region of approach

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(ROA). (E) The time ratio of male spent in the area close to control or BHPF and FULV treated females

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in ROA. (F) The velocity of the zebrafish in region of approaching (ROA). (G) Courtship index of

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male for females. (H) Courtship index of females for male. (I) Time spent between two females. The

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replication was n=23 for ctrl♀, n=12 for BHPF♀, n=11 for FULV♀, n=23 for male♂ in this test.

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The error bar represents the standard error of the mean (SEM). The statistical significance was set at

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P<0.05 (*), P<0.01 (**), P<0.001 (***).

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Fig. 2. The body orientation and Markov chain model of transform matrix. (A, B) Polar directional 37

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male♂. The angle was the resultant orientation of pose vector of each zebrafish, and the radius was the

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resultant orientation of pose vector. (C, D) Markov chain model of transform matrix in BHPF group (C)

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Fig. 3. The fecundity, GSI and histological structure of ovary. (A) Number of egg collected (n=4). (B)

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The GSI of females (n=4). (C) Histological morphology of oocytes. I, primary growth stage; II, cortical

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alveolus stage, III, early-vitellogenic stage; IV late-vitellogenic stage. Scale bar: 100µm. (D) Stastical

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results of different stage oocytes. The replication was n=3 for each group. The statistical significance

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Fig. 4. Relative mRNA expression of genes in zebrafish ovaries. (A-F) Relative mRNA expression

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related to steroidogenesis. (G-I) Relative mRNA expression of genes related to oxidative stress. The

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replication was n=9 for ctrl , n=9 for BHPF , n=8 for FULV

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standard error of the mean (SEM). The statistical significance was set at P<0.05 (*), P<0.01 (**).

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Fig. 5. The novel tank test of BHPF and FULV exposure. (A) The schematic diagram of overlook view.

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(B) The schematic diagram of lateral view. (C-Q) Locomotor parameters during novel test. (R-T) The

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trajectory charts of different treated zebrafish during the novel test. The replication was n=23 for ctrl

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zebrafish brain. (A) Contribution of the nine variables to the variances of the first and second principal

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component. (B) Projection of barycenters in the control female, BHPF and FULV treated female groups

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into the first and second principal components. (C) The envelopes of the three groups from the same

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principal components analysis were showed. (D) Western blot analysis of TH. (E). Quantification of

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TH content. The replication was n=3 for ctrl

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represents the standard error of the mean (SEM). The statistical significance was set at P<0.05 (*),

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ACCEPTED MANUSCRIPT Highlights • Male spent less time with BHPF treated female in ROA. • BHPF decreased the courtship index between female and male. • BHPF downregulated the expression of genes related to ER and steroidogenesis.

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• BHPF induced anxiety/depression-like behavior and reduced TH level.