Long-Term Exposure to Environmental Concentrations of Azoxystrobin

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Ecotoxicology and Human Environmental Health

Long-term exposure to environmental concentrations of azoxystrobin delays sexual development and alters reproduction in zebrafish (Danio rerio) Fangjie Cao, Christopher J Martyniuk, Peizhuo Wu, Feng Zhao, Sen Pang, Chengju Wang, and Lihong Qiu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b05829 • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 8, 2019

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Environmental Science & Technology

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Long-term exposure to environmental concentrations of azoxystrobin delays sexual development

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and alters reproduction in zebrafish (Danio rerio)

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Fangjie Cao,† Christopher J. Martyniuk,‡ Peizhuo Wu,† Feng Zhao,† Sen Pang,† Chengju Wang,† and

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Lihong Qiu†*

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† Department

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China

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‡

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University of Florida Genetics Institute, Interdisciplinary Program in Biomedical Sciences Neuroscience,

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College of Veterinary Medicine, University of Florida, Gainesville, FL, 32611, USA

of Applied Chemistry, College of Science, China Agricultural University, Beijing, 100193,

Department of Physiological Sciences and Center for Environmental and Human Toxicology,

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*

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Lihong Qiu,

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E-mail: [email protected]; Tel: +86 (0)10 62733924

Corresponding author:

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ACS Paragon Plus Environment


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ABSTRACT: The strobilurin fungicide azoxystrobin (AZO) can induce adverse effects in aquatic

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organisms, but data are lacking on endpoints associated with sexual development and reproduction

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following a chronic exposure to AZO. In this study, zebrafish embryos (F0) at 2–4 hours post-fertilization

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(hpf) were exposed to 0.2, 2.0 and 20.0 µg/L AZO until 120 days post-fertilization (dpf). Decreased male

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ratio and increased intersex ratio were observed by 20.0 µg/L AZO at 42 and 60 dpf, but this effect

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disappeared at 120 dpf. AZO at 20.0 µg/L inhibited growth, retarded gonadal development, and disrupted

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sex hormone and vitellogenin in females at 60 and 120 dpf, and in males at 42, 60 and 120 dpf. These

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effects were associated with altered expression of cyp19a, cyp19b, hsd3b, hsd17b, vtg1 and vtg2.

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Exposure to 2.0 µg/L AZO altered mRNA levels of these transcripts in females at 120 dpf, and in males

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at 60 and 120 dpf. Reproduction ability was reduced by 20.0 µg/L AZO at 120 dpf. Developmental

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defects were observed after F1 embryos from exposed parents of 20.0 µg/L were reared in AZO-free

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water at 96 hpf. Overall, these data provide new understanding of fish sexual development and

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reproduction following chronic exposures to AZO.

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INTRODUCTION

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Azoxystrobin (AZO), a strobilurin fungicide, remains one of the most widely used fungicides

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around the world (i.e. USA, Australia, UK and China),1 and is worth $1.27 billion in global sales reported

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by Phillips McDougall in 2016.2 AZO is also one of the frequently detected fungicides in aquatic

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environment,3-6 with mean detected concentration of 0.16 µg/L in streams in the US,7 1.43 µg/L in 20

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streams in Lower Saxony, Germany,8 0.15 µg/L in the surface water in Demark,9 and 34 µg/L in river

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water in Shanghai, China.4 AZO is a contaminant of concern because it induces both lethal and sub-lethal

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effects in aquatic organisms.10-17 Previous studies report that AZO disrupts the endocrine system of

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zebrafish.12-14 For example, a 21-day exposure to 200 µg/L AZO decreased 17β-estradiol (E2) and

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vitellogenin (Vtg), increased testosterone (T), rewarded ovarian development, up-regulated hsd3b,

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hsd17b and cyp17, as well as down-regulated cyp19a, cyp19b, vtg1 and vtg2 in female zebrafish;

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moreover, male zebrafish displayed increased E2 and Vtg, decreased T, inhibited testicular development,

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up-regulated hsd3b, hsd17b, cyp17, cyp19a, cyp19b, vtg1 and vtg2, which impaired reproductive

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endpoints.14 Vtg and E2 were increased in zebrafish embryos following 48 and 72 exposure to 0.1 and

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10.0 µg/L AZO, and an increase in vtg and a decrease in cyp19b were induced after 48 and 72 h exposure

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to 1, 10 and 100 µg/L AZO.12 Similarly, a 21-day parental exposure to 200 µg/L up-regulated cyp19a,

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cyp19b, vtg1 and vtg2 in F1 zebrafish embryos.13 However, data are lacking regarding long-term impacts

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of AZO at environmental concentrations on sexual development and reproduction in F0 generation, and

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developmental success in F1 offspring of fish.

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Zebrafish (Danio rerio) serve as a good model to assess the effects of chemicals on sexual

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development.18 Zebrafish sexual development progresses as follows: (1) initially, juveniles develop

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undifferentiated, ovary-like gonads at approximately 10 days post-fertilization (dpf); (2) sexual 3



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differentiation begins at approximately 21−23 dpf, with about 50% of the ovaries beginning to undergo

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reabsorption and transformation into testes, and ends at about 42 dpf; (3) the development and maturation

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of ovaries and testes are almost completed at ~60 dpf.19,

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influenced by sex steroid hormones,21 sex determination genes (foxl2, brca2, sox9b, sox9a, amh and

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dmrt1, etc.),22, 23 and environmental factors (e.g. chemicals).18, 24, 25

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Zebrafish sexual development can be

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Estrogens (i.e. E2) regulate feminization by binding to the estrogen receptor,26 while androgens (11-

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ketotestosterone and T) regulate masculinization by binding to the androgen receptor.27 Steroidogenic

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enzymes, such as CYP11, CYP17, CYP19, HSD3B, and HSD17B, determine the synthetic rate of

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estrogens and androgens, and can be dysregulated by chemical exposure.28, 29 Estrogens activate the

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expression of vtg to synthesize Vtg protein, the major precursor of egg yolk. Vtg plays an important role

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in the oocytes.28 While vtg gene is present in male fish, its expression is very low, unless it is activated

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by xenoestrogens.30 Thus, Vtg and vtg expression are strong and widely used biomarkers for the

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estrogenic chemicals.

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The objectives of this study were to evaluate the effects of a long-term exposure to environmentally

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relevant concentrations of AZO on sexual development and reproduction in F0 individuals, beginning at

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fertilization and progressing up to 120 days post-fertilization (dpf). Growth, gonadosomatic index (GSI),

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sex steroid hormones, Vtg, sex ratio, gonadal histology, expression levels of genes in the HPG axis, and

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reproductive capacity in F0 generation were measured. Lastly, developmental success in F1 offspring

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was also assessed, as our previous study demonstrated that AZO exhibited transgenerational effects in

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zebrafish.13

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MATERIALS AND METHODS

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Chemicals 4


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Azoxystrobin (AZO, CAS: 131860-33-8, 98% purity) was obtained from Shenyang Chemical

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Industry Research Institute, China. A stock solution of 10,000 mg/L AZO was prepared in analytical-

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grade acetone containing 5% Tween 80,14 and was stored at 4 °C for further dilution. Reconstituted water

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with a pH value of 7.5 ± 0.5 was used to make exposure solutions, which contained 1.27 mM NaHCO3,

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0.33 mM MgSO4, 0.33 mM CaCl2, and 0.17 mM KCl.31

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Zebraﬁsh maintenance and egg production

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Parental zebrafish (wild-type AB-strain; six months old) were purchased from Beijing

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Hongdagaofeng Aquarium Department, China and were housed in a flow-through feeding equipment

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(Esen Corp., China). Normally fertilized embryos at 2−4 hours post-fertilization (hpf) were collected for

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further exposure. The condition for zebrafish maintenance and AZO exposure were maintained with a

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pH value of 7.5 ± 0.5, a conductivity value of 600 ± 100 μS.cm−1, a light/dark cycle of 14:10 h, a

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minimum dissolved oxygen concentration of up to 80% of air saturation, and a temperature of 27 ± 1 °C.

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AZO exposure assay

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To determine the long-term effects of AZO on sexual differentiation and reproduction in F0

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generation, as well as developmental success in F1 offspring, zebrafish embryos (F0) at 2–4 hpf were

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randomly assigned into groups and exposed to water control, solvent control (0.02‱ acetone and 0.001‱

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Tween 80, v/v), 0.2, 2.0 and 20.0 µg/L AZO (equal to 0.496, 4.96 and 49.6 nM, respectively) for 120

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days. The growth, sex ratio, gonadal development, sex hormone and Vtg, mRNA levels of genes in HPG

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axis, and reproductive capacity in F0 generation were assessed. Additionally, development and survival

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in F1 embryos originating from F0 parents were determined at 96 hpf. Exposure concentrations were

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selected to span a wide range of environmentally relevant concentrations reported in the aquatic

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environment.4, 7-9 Moreover, our previous study demonstrated that 2.0 and 20.0 µg/L AZO altered cyp11a, 5



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hsd3b, cyp19a, vtg1 and vtg2 in male zebrafish.14 The experimental design is presented in Figure S1 and

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sample size is shown in Table S1. Abnormal phenotypes and mortality were recorded daily. Dead

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individuals were removed immediately and approximately 90% of exposure solutions were renewed

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daily. Institutional Animal Care and Use Committee (IACUC) of China Agricultural University approved

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the experimental procedures.

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The procedure for F0 exposure was as follows: (1) Initially, 200 fertilized embryos at 2–4 hpf were

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maintained in glass Petri dishes of 20-cm diameter (N = 4 replicates per treatment) containing 200 mL

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of exposure solutions. (2) After hatching (approx. 96 hpf), zebrafish larvae were transferred to 2-L

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beakers containing 1.6-L of exposure solutions. Zebrafish larvae were fed daily starting at 5 dpf. The

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detailed feeding procedure is provided in the SI. (3) After 21 dpf, ~180 individuals (< 10% mortality

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occurred before 21 dpf) from each beaker were transferred to 25-L tanks containing 20-L of exposure

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solutions until 120 dpf.

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At 90 dpf, 10 females and 10 males were selected randomly from each tank and placed into a 10-

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L breeding tank containing 6-L of exposure solution. Each breeding tank was equipped with a removable

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spawning tray of stainless steel mesh at the bottom of the breeding tank so that embryos could fall through.

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One week prior to the end of F0 exposure (about 113 dpf), females and males were separated using

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dividers, continuously exposed to AZO until 120 dpf. At 120 dpf, water in all breeding tanks was changed

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to reconstituted water without AZO and the spawning was stimulated at 8:00 PM in the next morning

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when the light was turned on, followed by egg collection one hour later. F1 embryos of each breeding

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tank were rinsed three times with chemical-free reconstituted water and were examined under a

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stereomicroscope to calculate average egg production per female and fertilization rate. Fifty fertilized

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and normally developed F1 embryos at 2–4 hpf were reared in AZO-free reconstituted water until 96 hpf 6


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to calculate mortality, hatching rate, and malformation rate. The images of morphological abnormity

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were photographed using an Aigo GE-5 digital microscope (Aigo Digital Technology Co. Ltd, China).

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Sample collections in F0 generation at 42, 60 and 120 dpf were conducted as follows: (1) Five

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individuals of each gender from each replicate were randomly selected and euthanized with anesthetic

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buffer (100 mg/L tricaine and 300 mg/L NaHCO3). Wet body weight and total body length were

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measured using an electronic balance and digital caliper (Mitutoyo Corp., Japan), respectively.

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Additionally, weights of female ovaries and male testes in each replicate were measured to calculate GSI.

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(2) Twenty fish were randomly selected from each replicate to assess the phenotype and sex ratio using

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histological observation. (3) Five females and males in each replicate were sampled to gonadal

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development. (4) Blood samples of 2 fish of each gender from each replicate were collected, pooled,

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frozen and stored at −80 °C for sex steroid hormones and Vtg measurement. (5) The brains, gonads, and

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livers of 3 individuals of each gender per replicate were pooled, frozen and stored at −80 °C for RNA

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extraction, respectively.

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Sex ratio and gonadal histological examination

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The sample preparation for gonadal histology was conducted according to a published protocol.14

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Gonadal histology in F0 zebrafish was photographed using an Olympus microscope (Olympus, Japan).

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Two cross-sections of each gonad (five females and five males per replicate) were selected to evaluate

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gonadal development.32

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Sex steroid hormones and Vtg measurement

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Detailed information on the sex steroid hormones (E2 and T) and Vtg measurements in zebrafish can

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be found in SI.

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Gene expression analysis 7



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Total RNA was extracted using RNApre Pure Tissue Kit with DNase digestion (Tiangen Biotech,

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Beijing, China). The quality of all RNA samples was verified using gel electrophoresis. RNA

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concentration was determined using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc.,

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USA). Total RNA (1.5 μg) per sample was reverse-transcribed into cDNA using FastQuant cDNA

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Synthesis Kit (Tiangen Biotech, Beijing, China) and stored at −20 °C for further analysis. Real-time

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quantitative polymerase chain reaction (RT-qPCR) was performed with an ABI 7500 PCR Detection

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System (ABI, USA) using SYBR Green PCR Master Mix Reagent Kit (Tiangen Biotech, Beijing, China).

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No reverse transcriptase (NRT) controls were prepared to assess genomic DNA contamination and there

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was no genomic DNA contamination. The RT-qPCR procedure was conducted as follows: 95 °C for 15

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min and 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 32 s, followed by a melt curve analysis.

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Specific primers of target genes were obtained from published sequences of zebrafish (Table S2).

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The amplification efficiency and linear coefficient of the standard curve of all genes are provided in SI

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(Table S2). The gene of β-actin was selected as a housekeeping gene to normalize mRNA levels of target

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genes due to its stable expression among different tissues and individuals of zebrafish.33 The stable

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expression of β-actin in tissue categories, genders, and ages among different treatments is shown in

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Figure S2. Melt curves of all tested genes are presented in Figure S3-S6. Each treatment contained four

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biological replicates run in triplicate and relative mRNA levels of target genes were analyzed using the

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2 −∆∆Ct method.34

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Chemical analysis

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Exposure solutions (10 mL) in each replicate were sampled at 0, 42, 60 and 120 dpf, and transferred

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into 50-mL centrifuge tubes for chemical analysis. Detailed information of the analytical method of AZO

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is presented in SI. 8


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Data analysis

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Data analyses were conducted using Graph-Pad Prism version 6.0 (GraphPad Software Inc., USA).

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There was no significant difference between water control and solvent control for all investigated

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endpoints and thus the solvent control was used as the control in the following analysis. One-way analysis

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of variance (one-way ANOVA) was conducted to analyze significant differences between the control

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and treatment groups. Clustering maps were constructed using the MultiExperimental Viewer v4.9

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(Dana-Farber Cancer Institute, Boston, MA, USA). Values are shown as the mean ± standard deviation

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(SD). A significant difference was noted when p < 0.05.

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RESULTS

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Chemical analysis

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Chemical analysis of AZO in the water is provided in the SI. The deviations between the actual

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concentrations and nominal concentrations of AZO at 0, 42, 60 and 120 dpf were maintained within ±

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20% (Table S3). Therefore, the nominal concentrations were used to represent the actual concentrations

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of AZO.35

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Growth and GSI in F0 zebrafish

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AZO at 0.2 and 2.0 µg/L did not affect growth or GSI in F0 female and male zebrafish following

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120 days exposure (Table S4 and S5). However, significant reductions in wet body weight, total body

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length, and GSI were induced in F0 females exposed to 20.0 µg/L AZO at 60 and 120 dpf compared with

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the control group (Table S4). In F0 males, 20.0 µg/L AZO significantly decreased wet body weight, total

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body length and GSI at 42, 60 and 120 dpf (Table S5). No significant alteration in CF was observed in

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either gender of zebrafish at the tested doses (Table S4 and S5).

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Sex ratio in F0 zebrafish 9



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Figure 1. Representative images of undifferentiated and intersex gonads in F0 zebrafish exposed to 20.0

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µg/L AZO at 42 (A and B) and 60 (C and D) dpf, and the relative percentages of F0 female, male, intersex

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and undifferentiated zebrafish (E) at 42, 60 and 120 dpf. Blue arrows indicate the undifferentiated gonads

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in zebrafish. PO indicates perinucleolar oocytes, and Sg, Sc, St and Sz represent spermatogonia,

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spermatocytes, spermatids and spermatozoa, respectively (400× magnification in A, B, C and D). Sex

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ratio was calculated as the number of individuals of one phenotype × 100/the total number of individuals

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that were selected for all endpoints at 42, 60 and 120 dpf, respectively. Values are shown as the mean ±

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standard deviation (SD) (N = 4 replicates per treatment, n = 50 fish per replicate at 42 and 60 dpf, and n

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= 80 at 120 dpf). Asterisks (in red) denote significant differences between the control and treatments (*p

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< 0.05 and **p < 0.01 by Dunnett’s post-hoc test).

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AZO at 20.0 µg/L disrupted sex ratio and sexual differentiation in F0 zebrafish at 42 and 60 dpf.

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Undifferentiated gonads and intersex gonads were observed in zebrafish treated with 20.0 µg/L AZO at

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42 dpf (Figure 1A and B), and intersex phenotypes were still detected at 60 dpf when compared to the

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control group (Figure 1C and D). Exposure to 20.0 µg/L AZO significantly decreased the percentages of

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females and males at 42 dpf, whereas the percentages of intersex and undifferentiated phenotypes were

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significantly increased (Figure 1E). Likewise, a significant decrease in the percentages of males and an 10


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increase in intersex phenotypes were observed in 20.0 µg/L AZO group at 60 dpf (Figure 1E). However,

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no intersex and undifferentiated phenotype in zebrafish treated with 0.2, 2.0 or 20.0 µg/L AZO at 120

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dpf were observed, nor was the sex ratio changed in zebrafish at 120 dpf (Figure 1E).

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Gonadal development in F0 zebrafish

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Figure 2. Representative images of the ovaries in F0 female zebrafish exposed to control (A, D and G),

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2.0 (B, E and H) and 20.0 µg/L (C, F and I) AZO at 42, 60 and 120 dpf, respectively. The oocytes

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included perinucleolar oocytes (PO), cortical alveolar oocytes (CO), early vitellogenic oocytes (EV)

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and late vitellogenic oocytes (LV) (100× magnification).

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Figure 3. Representative images of the testes in F0 male zebrafish exposed to control (A, D and G), 2.0

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(B, E and H) and 20.0 µg/L (C, F and I) AZO at 42, 60 and 120 dpf, respectively. The spermatocytes

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included spermatogonia (Sg), spermatocytes (Sc), spermatids (St) and spermatozoa (Sz) (200×

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magnification).

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Figure 4. The relative percentages of different stages of oocytes in F0 females (A) and spermatocytes in 12


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males (B) exposed to control, 0.2, 2.0 and 20.0 µg/L AZO at 42, 60 and 120 dpf. Values are shown as

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the mean ± standard deviation (SD) (N = 4 replicates per treatment, n = 5 fish per gender of each

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replicate with 10 cross-sections). Asterisks denote significant differences between the control and

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treatments (*p < 0.05 and **p < 0.01 by Dunnett’s post-hoc test).

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Gonadal development in F0 females and males was inhibited by 20.0 µg/L AZO exposure (Figure 2

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and 3). In female ovaries, exposure to 20.0 µg/L AZO significantly increased the relative percentage of

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perinucleolar oocytes (immature oocytes) at 42, 60 and 120 dpf, whereas the relative percentage of late

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vitellogenic oocytes (mature oocytes) were significantly decreased at 60 and 120 dpf compared to the

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control (Figure 4A). In male testes, 20.0 µg/L AZO induced a significant increase in the percentage of

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spermatogonia at 42 dpf (Figure 4B). A significant increase in the relative percentage of spermatogonia

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and spermatocytes (immature spermatocytes) was observed in 20.0 µg/L AZO group at 60 and 120 dpf,

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whereas 20.0 µg/L AZO significantly reduced the relative percentage of spermatozoa (mature

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spermatocytes) at 60 and 120 dpf (Figure 4B).

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Sex steroid hormone and Vtg in F0 zebrafish

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F0 females exhibited no change in E2, T and Vtg, nor the E2/T ratio following 120 days exposure

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to 0.2 and 2.0 µg/L AZO. However, in F0 females, exposure to 20.0 µg/L AZO induced a significant

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decrease in E2 and an increase in T at 60 and 120 dpf compared to the control (Table S6). Moreover, the

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E2/T ratio and Vtg were significantly reduced in females exposed to 20.0 µg/L AZO at 42, 60 and 120

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dpf (Table S6). In F0 males, E2, T and Vtg, or the E2/T ratio were not altered following 120 days

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exposure to 0.2 and 2.0 µg/L AZO, except for a significant increase in Vtg in the 2.0 µg/L AZO group

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at 120 dpf (Table S7). Moreover, exposure to 20.0 µg/L AZO significantly increased E2 and Vtg, E2/T

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ratio, and decreased T at 42, 60 and 120 dpf (Table S7). 13



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Gene expression alteration in F0 zebrafish

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Figure 5. Clustering map of expression levels of esr1, esr2b, ar and cyp19b in the brains (B), cyp11a,

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cyp17, cyp19a, hsd3b, and hsd17b in the gonads (G), and vtg1 and vtg2 in the livers (L) of female (F)

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and male (M) zebrafish exposed to control (0.0), 0.2, 2.0 and 20.0 µg/L AZO at 42, 60 and 120 dpf. The

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mean values of normalized transcript levels in each treatment were used to draw the clustering map (N

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= 4 replicates per treatment). Asterisks denote significant differences between the control and treatments

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(*p < 0.05 and **p < 0.01 by Dunnett’s post-hoc test). All expression data are presented in SI.

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In F0 female brains, there were no changes in the expression of esr1, esr2b, or ar following 120 days

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exposure to 0.2, 2.0 or 20.0 µg/L AZO, whereas cyp19b were significantly down-regulated in the 20.0

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µg/L AZO group at 60 and 120 dpf compared to the control (Figure 5 and Figure S7A, C and E). In F0

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male brains, cyp19b was significantly up-regulated in the 20.0 µg/L AZO group at 42, 60 and 120 dpf,

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and in the 2.0 µg/L AZO group at 60 and 120 dpf (Figure 5 and Figure S7B, D and F).

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In F0 female ovaries, cyp19a was down-regulated in the 20.0 µg/L AZO group at 42, 60 and 120 dpf,

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and in the 2.0 µg/L AZO group at 120 dpf compared with the control (Figure 5 and Fig. S8A, C and E).

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Expression levels of hsd3b and hsd17b were significantly increased in the 20.0 µg/L AZO group at 42,

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60 and 120 dpf, and in the 2.0 µg/L AZO group at 120 dpf. Likewise, cyp17 was up-regulated in the 2.0

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and 20.0 µg/L AZO groups at 120 dpf (Figure 5 and Figure S8A, C and E). In F0 male testes, 20.0 µg/L 14


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AZO significantly up-regulated cyp17, hsd3b, hsd17b and cyp19a at 42, 60 and 120 dpf, and up-

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regulation of these genes were detected in the 2.0 µg/L AZO group at 120 dpf (Figure 5 and Figure S8B,

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D and F). Conversely, AZO did not alter genes related to sex differentiation (i.e. foxl2, brca2, sox9a,

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sox9b, amh and dmrt1) in female ovaries or male testes at 42 and 60 dpf (Figure S9).

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In F0 female livers, vtg1 and vtg2 were significantly decreased in 20.0 µg/L AZO group at 42, 60

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and 120 dpf, and in the 2.0 µg/L AZO group at 120 dpf compared to the control (Figure 5 and Figure

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S10A, C and E). In F0 male livers, vtg1 and vtg2 were significantly increased in the 20.0 µg/L AZO

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group at 42, 60 and 120 dpf, and in 2.0 µg/L AZO group at 120 dpf (Figure 5 and Figure S10B, D and

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F).

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Reproductive capacity in F0 zebrafish and developmental success in F1 embryos

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AZO impaired reproductive endpoints in F0 zebrafish following 120 days exposure. The average

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number of eggs per female and average fertilization rate were significantly decreased in the 20.0 µg/L

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AZO group (Table S7). Compared to F1 embryos from the non-exposed F0 parents, a significant increase

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in mortality and a significant decrease in hatching rate at 96 hpf were observed in F1 offspring from the

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exposed parents of 20.0 µg/L AZO group, even when F1 embryos were reared in AZO-free water (Table

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S8). Moreover, F1 larvae collecting from F0 group of 20.0 µg/L AZO displayed pericardial edema, yolk

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sac edema and spinal deformation (Figure S11), with a significant increase in malformation rate at 96

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hpf (Table S8).

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DISCUSSION

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In this study, 20.0 µg/L AZO reduced the ratios of female and male zebrafish, and increased the

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proportion of undifferentiated and intersex zebrafish at 42 dpf. Decreased male ratio and increased

293

intersex ratio were also observed in zebrafish exposed to 20.0 µg/L AZO at 60 dpf. However, no 15



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undifferentiated or intersex phenotype was detected in zebrafish exposed to 20.0 µg/L AZO at 120 dpf,

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nor was sex ratio altered. These results suggest that 20.0 µg/L AZO retards sexual differentiation at 42

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and 60 dpf, but may not impact the sex ratio at 120 dpf. Delayed sexual differentiation would induce

297

undifferentiated or the intersex phenotype, which may lead to the alteration of sex ratio in zebrafish,36, 37

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but these impacts may be less pronounced or not present at later stages of life. For example, exposure to

299

0.15 mM methimazole, starting from 3 dpf to 33 dpf, delayed sexual differentiation and induced female-

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biased population in zebrafish at 45 dpf, but had no effect on sex ratio after zebrafish were maintained

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in clean water until 60 dpf.38 The pesticides prochloraz, vinclozolin, and monocrotophos are documented

302

to affect sexual development and sex ratio in zebrafish.39-42 For example, waterborne exposure to 400

303

μg/L vinclozolin, an anti-androgenic fungicide, starting from 21 to 35 dpf permanently shifted sex ratio

304

towards females (55−65%) and impaired gonadal testicular maturation in male zebrafish.39 The

305

organophosphorus pesticide monocrotophos with estrogenic activity increased the proportion of females

306

(71%) at 0.100 mg/L after exposure from fertilization to 40 dpf.40 The fungicide prochloraz, an aromatase

307

inhibitor and androgen receptor antagonist, caused male-bias (77%) population after 60 days exposure

308

to 202 μg/L.40, 42

309

Exposure to 20.0 µg/L AZO increased the percentage of immature oocytes at 42, 60 and 120 dpf,

310

and decreased the percentage of mature oocytes in female zebrafish at 60 and 120 dpf. Likewise, 20.0

311

µg/L AZO increased the percentage of immature spermatocytes and decreased the percentage of mature

312

spermatocytes in male zebrafish at 60 and 120 dpf. These results indicate that 20.0 µg/L AZO retarded

313

gonadal development in female and male zebrafish. The decreased GSI in females and males at 60 and

314

120 dpf further support this hypothesis. These data are in good agreement with that of our previous study,

315

which reported that gonadal development was delayed in female zebrafish exposed to 200 µg/L AZO, 16


Page 16 of 27

Page 17 of 27


316

and in male zebrafish treated with 20 and 200 µg/L AZO following a 21-day exposure.14 Thus, longer

317

exposures to lower doses of AZO also appear to delay gonadal development in both females and males.

318

Here, we found that 20.0 µg/L AZO decreased E2 and E2/T ratio, and increased T in females at 60

319

and 120 dpf. Conversely, an increase in E2 and E2/T ratio, and a decrease in T was observed in males

320

exposed to 20.0 µg/L AZO at 42, 60 and 120 dpf. Our previous study reported that AZO altered E2, T

321

and Vtg levels in females after a 21 day-exposure to 200 µg/L, and in males exposed to 20 and 200

322

µg/L.14 Sex steroid hormones and genes controlling sexual determination regulate gonadal differentiation

323

and sex ratio.43 We propose that an unbalanced E2 and T is a major underlying reason for altered sexual

324

differentiation and retarded gonadal growth in zebrafish treated with 20.0 µg/L AZO as expression levels

325

of foxl2, brca2, sox9b, amh, sox9a and dmrt1 remained unchanged in females and males at 42 and 60

326

dpf. However, there are likely multiple mechanisms that are associated with gonadal differentiation and

327

sex ratios. For instance, norgestrel was reported to affect sexual differentiation and induce male-biased

328

population in zebrafish exposed to 34 and 77 ng/L starting from 20 dpf to 60 dpf. This effect was

329

accompanied by T suppression, increased dmrt1 expression, and decreased figa transcription.21

330

AZO at 20.0 µg/L down-regulated cyp19a and cyp19b, as well as up-regulated hsd3b and hsd17b

331

in female zebrafish at 42, 60 and 120 dpf, whereas up-regulation was observed for cyp19a, cyp19b, cyp17,

332

hsd3b and hsd17b in male zebrafish treated with 20.0 µg/L AZO at 42, 60 and 120 dpf. AZO at 2.0 µg/L

333

was found to down-regulate cyp19a, and up-regulate cyp17, hsd3b and hsd17b in females at 120 dpf,

334

while exposure to 2.0 µg/L AZO increased cyp19a, cyp19b, cyp17, hsd3b and hsd17b in males at 120

335

dpf. The synthesis of sex steroid hormones is controlled by steroidogenic enzymes of the HPG axis, and

336

transcriptional changes in these key steroidogenic enzymes (e.g. cyp19, cyp17, hsd3b and hsd17b) disrupt

337

the balance between E2 and T.28, 44 Taken together, we hypothesize that the altered expression of genes 17



Page 18 of 27

338

encoding key steroidogenic enzymes affects sex hormones in females exposed to 20.0 µg/L AZO at 60

339

and 120 dpf, and in males treated with 20.0 µg/L AZO at 42, 60 and 120 dpf. This hypothesis is also

340

supported by our previous study demonstrating that the dysregulation of cyp19a, cyp19b, cyp17, hsd3b

341

and hsd17b were associated with altered E2 and T in females and males following a 21-day exposure to

342

AZO.14 In another study, zebrafish larvae displayed an increase in E2, which was associated with an up-

343

regulation of cyp19b and hsd17b after 24, 48 and 72 h exposure to 1 and 100 µg/L AZO.12 However,

344

further studies should be conducted to explore if transcriptional alterations in HPG axis caused by AZO

345

actually led to changes in protein levels and enzyme activity in steroidogenic synthesis pathway.

346

We found that AZO appeared to have more profound effects in males than females and this is

347

consistent to our previous study.14 Difenoconazole, a triazole fungicide, has been shown to exert sex-

348

specific effects in adult zebrafish.45 However, the mode of action of AZO is not yet defined in fish and

349

the mechanisms of sex-dependent sensitivity are complicated. We did not specifically set out to test this

350

hypothesis so we are cautious in our interpretation about severity or sex-specific susceptibility. A novel

351

mechanism related to sex-dependent sensitivity in zebrafish would be interesting to pursue.

352

In this study, exposure to 20.0 µg/L AZO reduced Vtg protein, and down-regulated vtg1 and vtg2

353

in female zebrafish at 42, 60, and 120 dpf. Conversely, Vtg protein and vtg mRNA were increased in

354

male zebrafish treated with 20.0 µg/L AZO at 42, 60 and 120 dpf, and 2.0 µg/L AZO at 120 dpf. Vtg is

355

vital for ovarian development, oocyte maturation and yolk biosynthesis,46,

356

reduced Vtg in females is related to the delayed ovarian development. Vtg in males can be induced by

357

estrogenic or anti-androgenic chemicals, which would inhibit testicular development and maturation.48

358

Our recent study reported that 200 µg/L AZO inhibited ovarian development, decreased Vtg, and down-

359

regulated vtg1 and vtg2 in female zebrafish following 21 days exposure, whereas AZO at 20 and 200 18


47

and it is proposed that

Page 19 of 27


360

µg/L retarded testicular development, increased Vtg, and up-regulated vtg1 and vtg2 in male zebrafish.14

361

Noteworthy was that parental exposure to 200 µg/L AZO up-regulated vtg1 and vtg2 in F1 embryos,

362

even when embryos were reared in clear water.13 Taken together, AZO appears to alter Vtg protein and

363

vtg expression differently in the sexes, and this response is rather consistent over the duration of exposure

364

(> 21d).

365

Long-term exposure to 20.0 µg/L AZO from 2–4 hpf to 120 dpf decreased egg production and

366

fertilization rate in zebrafish. We also observed reduced survival and hatching rate, morphological

367

defects, and increased malformation rate in F1 offspring from exposed parents (20.0 µg/L AZO), despite

368

that F1 embryos were reared in clean water. Reduced fecundity may not be surprising as the number of

369

mature oocytes is influenced by circulating sex steroid levels and Vtg. These, in turn, are directly related

370

to steroidogenic enzyme expression in the gonad and vtg expression in the liver.49 Thus, we propose that

371

impaired reproduction in F0 individuals is a result of delayed gonadal development caused by altered sex

372

steroids and Vtg. The exposure of F0 zebrafish to 20.0 µg/L AZO resulted in lethality, hatching delay,

373

morphological abnormality, and increased malformation rate in F1 embryos. These results were in good

374

agreement with our previous findings that impaired reproduction was induced in adult zebrafish after a

375

21-day exposure to a higher dose of 200 µg/L AZO.14 Mortality, hatching delay, morphological

376

abnormity, elevated malformation rate, and disrupted gene expression in the HPG axis were detected in

377

F1 embryos from exposed parents of 200 µg/L AZO, with or without continuous exposure to this

378

fungicide; adverse effects were also detected after F1 embryos from exposed parents of 20 µg/L AZO

379

were continuously exposed to the same dose as their parents.13 Taken together, AZO appears to affect

380

the reproduction of zebrafish with both short-term exposures at higher concentrations and long-term

381

exposures at lower, environmentally relevant concentrations. 19



382

In conclusion, this study showed that AZO adversely influenced a number of endpoints related to

383

sexual development and reproduction in zebrafish. Transcriptional responses may be the underlying

384

molecular mechanisms in terms of disrupted sexual development and impaired reproduction in F0

385

generation exposed to 20.0 µg/L AZO, and changes in mRNA levels were detected in females and males

386

exposed to 2.0 µg/L at 120 dpf. Parental exposure to 20.0 µg/L AZO for 120 days affected survival,

387

hatching and morphology in F1 embryos that were not exposed to AZO. These data provide new

388

understanding as to the effects of AZO on sexual development and reproduction in zebrafish after long-

389

term exposure to environmentally relevant concentrations.

390

ASSOCIATED CONTENT

391

Supporting Information (SI)

392

Descriptions of feeding procedure, sex steroid hormones and Vtg measurement, and analytical

393

chemistry are presented in Page S2−S9. Experimental design (Figure S1), sample size (Table S1),

394

primer sequences (Table S2), the stability of β-actin (Figure S2), melt curves of tested genes (Figure

395

S3−S6), concentrations of AZO in exposure solution (Table S3), growth and GSI (Table S4 and S5), Sex

396

steroid hormone and Vtg (Table S6 and S7), gene expression data (Figure S7−S10 and Table S8),

397

reproductive capacity in F0 generation (Table S9), developmental success in F1 embryos (Table S10),

398

and representative pictures in F1 offspring (Figure S11) are also presented in Supporting information.

399

AUTHOR INFORMATION

400

Corresponding author

401

*E-mail: [email protected]; Tel: +86 (0)10 62733924

402

Notes

403

The authors have no conflict of interest to declare. 20


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Page 21 of 27


404

ACKNOWLEDGMENTS

405

This work was financially supported by the National Key R&D Program of China (Grant No.

406

2017YFD0200504).

407

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Table of Contents Art of this manuscript


Long-Term Exposure to Environmental Concentrations of Azoxystrobin

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