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Exposure to aflatoxin B1 interferes with locomotion and neural development in zebrafish embryos and larvae
Accepted Manuscript Exposure to aflatoxin B1 interferes with locomotion and neural development in zebrafish embryos and larvae Ting-Shuan Wu, Ya-Chih Cheng, Pei-Jen Chen, Ying-Tzu Huang, Feng-Yih Yu, BiingHui Liu PII:
S0045-6535(18)32161-1
DOI:
https://doi.org/10.1016/j.chemosphere.2018.11.058
Reference:
CHEM 22542
To appear in:
ECSN
Received Date: 31 August 2018 Revised Date:
7 November 2018
Accepted Date: 8 November 2018
Please cite this article as: Wu, T.-S., Cheng, Y.-C., Chen, P.-J., Huang, Y.-T., Yu, F.-Y., Liu, B.-H., Exposure to aflatoxin B1 interferes with locomotion and neural development in zebrafish embryos and larvae, Chemosphere (2018), doi: https://doi.org/10.1016/j.chemosphere.2018.11.058. 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.
6 hpf zebrafish embryos
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Locomotion interference mL)
abnormal neuron morphology Scale bar = 50 m
gfap huc
ngfa
prtga atp1b1b
gene level alteration
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Exposure to aflatoxin B1 interferes with locomotion and
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neural development in zebrafish embryos and larvae
Biing-Hui Liua* a
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Taipei, Taiwan b
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Department of Agricultural Chemistry, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan
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Graduate Institute of Toxicology, College of Medicine, National Taiwan University,
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Ting-Shuan Wu a‡, Ya-Chih Chenga‡, Pei-Jen Chen b, Ying-Tzu Huanga, Feng-Yih Yucd*,
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c
Department of Biomedical Sciences, Chung Shan Medical University, Taiwan
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d
Department of Medical Research, Chung Shan Medical University Hospital,
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Taichung, Taiwan
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Running titles:
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‡ These authors contributed equally to this work
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*Corresponding authors: Biing-Hui Liu, Feng-Yih Yu
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Address: Graduate Institute of Toxicology
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College of Medicine, National Taiwan University
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No. 1,Sec 1, Jen-Ai Rd, Taipei, Taiwan. 10043
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Tel : 886-2-23123456 ext 88602
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Fax: 886-2-23410217
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E-mail: [email protected]
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[email protected] 1
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Abstract Aflatoxin B1 (AFB1) is the major mycotoxin that contaminates aquafeeds and
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regarded as a causative agent in illnesses and the mortality of aquacultural species.
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However, the effects of AFB1 on developing fish and associated toxic mechanism are
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still unknown. This study examines the behavioral changes, neuronal morphology and
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gene expression in zebrafish embryos and larvae upon exposure to aflatoxin solutions.
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Treatment of 6 hour post fertilization (hpf) embryos with AFB1 at 15-75 ng/mL
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significantly changed the swimming patterns of seven days post-fertilization (dpf)
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zebrafish larvae. Larvae in the 15 ng/mL group demonstrated a hypolocomotor
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activity in free swimming, but hyperlocomotion was observed in the larvae exposed to
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30-75 ng/mL AFB1. AFB1 at 75 ng/mL also significantly reduced the startle response
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of 7 dpf larvae after tapping stimulus. Exposure to AFB1 resulted in an aberrant
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morphology of
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(HuC:eGFP); this finding was supported by acetylated alpha-tubulin staining in
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wild-type fish. Additionally, AFB1 altered the levels of neurotoxic markers, including
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gfap and huC. The transcriptomic profile of AFB1-treated embryos revealed several
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differentially expressed genes that are related to neuroactivity and neurogenesis. PCR
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analysis verified that AFB1 significantly down-regulated the expression of ngfa and
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atp1b1b genes and increased that of prtga gene. The results herein indicate the
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toxicological impacts of AFB1 on the behaviors and neurodevelopment of fish in the
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early embryonic stage. Disruption of neural formation and synapse dysfunction may
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be responsible for the behavioral alteration.
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trigeminal ganglia and hindbrain neurons in transgenic embryos
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1. Introduction Aquaculture supplies nearly half of all seafood that is intended for human
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consumption and is a vital part of the global food industry. Over the past few years,
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the replacement of fishmeal with cheaper plant-based proteins in commercial feed
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formulations has gained widespread acceptance (Dirican et al., 2015). However,
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increasing reliance on the use of plant-based ingredients has increased the risk of
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introducing mycotoxins into the feed at the point of manufacture and storage (Barbosa
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et al., 2013). Mycotoxins are a rising threat to aquaculture and exposure to aflatoxin,
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which is one of them, is the primary risk to farm-raised fish. Aflatoxin-contaminated
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feed not only has direct negative effects on the health of the cultured fish, but also
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increases the risk of passing along the contaminants to consumers via the food chain
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(Anater et al., 2016).
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Aflatoxins are secondary metabolites that are primarily generated by Aspergillus
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flavus and A. parasiticus. Several surveys reveal the serious contamination of
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Aspergillus and aflatoxins in fish feed worldwide. In South America, Aspergillus is
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the most abundant species isolated from 60 to 70 % of fish feeds and aflatoxins are
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found in 55% of finished fish feeds (Barbosa et al., 2013). In Asia and Africa, the
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mean levels of AFB1 in aquafeeds were ranged from 50 to 100 ng/g (Anater et al.,
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2016; Fallah et al., 2014; Rodrigues et al., 2011). Additionally, co-occurrence of other
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mycotoxins with aflatoxins in aquafeeds is very common and may induce synergistic
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effects to increase the negative impact of aflatoxins at lower levels (Goncalves et al.,
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2016).
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Fish that are exposed to AFB1, the most prevalent and toxic compound in the
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aflatoxin group, usually demonstrate symptoms that are associated with weight loss,
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growth retardation, immunosuppression and even high mortality (Dhanasekaran et al., 3
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that are fed with feedstuffs contaminated with AFB1 exhibits unusual swimming
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patterns that are associated with loss of equilibrium (El-Sayed and Khalil, 2009). A
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study by Baldissera et al. indicates that an AFB1-containing diet results in the
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hyperlocomotion of silver catfish. This behavioral dysfunction is associated with
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impairment of the blood brain barrier and alteration of various neurotransmitters in
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the fish brain (Baldissera et al., 2018).
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The utility of zebrafish (Danio rerio) in studying neurotoxicity and aberrant
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behavior has grown remarkably recently; optical transparency of embryos and the
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establishment of transgenic lines make individual neurons in zebrafish be visualized
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throughout
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physiological and genetic information regarding zebrafish neurogenesis are available
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(Guo, 2009; Kalueff et al., 2014). Glial fibrillary acidic protein (Gfap) is critical to
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guide the neural stem cell migration in central nervous system and serves as a
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sensitive biomarker of neurotoxicity in zebrafish (Noctor et al., 2001; McGrath and Li,
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2008). HuC is a neuron-specific RNA binding protein that is regarded as an early
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neuronal differentiation marker in developing zebrafish (Kim et al., 1996: St John and
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Key, 2012). Nerve growth factor a (Ngfa) is related to the division and differentiation
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of sympathetic and embryonic sensory neurons (Dethleffsen et al., 2003). Protogenin
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homolog (Prtga) signaling is crucial to the suppression of premature neuronal
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differentiation during early neural development (Vesque et al., 2006; Wong et al.,
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2010).
development
(Schmidt,
2013).
Additionally,
more
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Information about the effect of aflatoxins on aquaculture are very limited (Anater
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et al., 2016). To examine the potential adverse effects of AFB1 on the early
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neurodevelopment of fish, zebrafish embryos and larvae were exposed to AFB1 at
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concentrations relevant to the levels in contaminated aquafeeds Attempts were also 4
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neurotoxicity.
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2. Materials and Methods
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2.1. Test species and husbandry.
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The Wild-type (WT) AB laboratory strain zebrafish (Danio rerio) and the
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transgenic line Tg(huC:eGFP)as8 were obtained from Taiwan Zebrafish Core Facility
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at Academia Sinica (TZCAS) (Taipei, Taiwan). Tg(huC:GFP)as8 is known to use the
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promoter of the zebrafish HuC gene to drive the expression of green fluorescent
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protein (GFP) in the telencephalic cluster, retinal ganglion cells, medial longitudinal
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fasciculus, dorsal longitudinal fasciculus, trigeminal ganglion and Rohon-Beard
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neurons (Huang et al., 2011). Zebrafish were kept in a water circulating system
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(Aquazoo Fish housing system. Taiwan) at 27~28 °C with a 14-h light/10 h-dark cycle.
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Water used for all the tanks passed through 120-micron filter pad, 5-micron filter
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cartridge, activated carbon filter, biological filter, and UV disinfection filter before
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being circulated into every tank. To maintain the water quality of circulating system,
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the values of pH, general hardness, carbonate hardness, nitrite and nitrate were
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monitored weekly. Daily feeding included artificial feed pellets (Taikong Corp.
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Taiwan) or live brine shrimp (Ocean Star International, Inc. U.S.A.).
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breeding were based on the animal research protocols approved by Institutional
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Animal Care and Use Committee (National Taiwan University, approval No.1334).
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2.2. Viability and morphology after AFB1 exposure
Adult care and
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AFB1 (CAS:1162-65-8) was purchased from the Sigma-Aldrich Company (St.
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Louis, MO) and first dissolved in DMSO at a concentration of 6 mg/mL and storage
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at -20
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embryos/larvae, AFB1 in stock solution would be diluted with 0.01 M phosphate
as a stock solution. To examine the survival rate and morphology of
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ACCEPTED MANUSCRIPT buffered saline (PBS, pH7.0) to 0.3 mg/mL and further diluted with egg water (60 mg
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sea salts/L, Instant Ocean® sea salts) to the tested concentrations (15, 30, 75, 150
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ng/mL). Healthy and normally developing WT embryos at 6 hour post-fertilization
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(hpf) were collected and exposed to vehicle or various concentrations of AFB1 in egg
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water. The survival rate of embryos (at least 20 embryos per treatment group in each
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independent experiment) was determined at the designate time (from 1 to 7 dpf). The
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morphology of surviving larvae at 7 dpf was observed from the lateral view under a
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dissecting microscope (magnification ×25).
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2.3. The larval locomotion test
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For the assessment of behavioral effects, four AFB1 concentrations were selected
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based on the survival rate of embryos at 7 dpf (Fig 1A): lethal concentration causing
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around
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no-observable-effects-concentration (NOEC), and half the NOEC. These were 75, 50,
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30 and 15 ng/mL, respectively. WT zebrafish embryos were treated with vehicle or
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indicated concentrations of AFB1 from 6 hpf to 7 dpf. There were three replicates for
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each exposure concentration, and at least 18 larvae were included per dose in each
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replicate. Since the larvae obtained their fully free swimming ability in our system at
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7 dpf, the swimming behaviors of 7 dpf larvae after vehicle or AFB1 treatment were
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observed under stereomicroscope. The number of larvae with the patterns of unstable
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shaking, swimming upside down and swimming on a side was recorded and divided
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by the number of total examined larvae in each treatment. Data were presented as the
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percentage of abnormal swimming behaviors (%).
of
mortality
(LC10),
2/3
the
concentration
of
LC10,
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The locomotor activity of 7 dpf larvae were further evaluated with the animal
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movement tracking system and software (EthoVison XT, Noldus Information
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Technology, Netherlands) as described by Liao et al. (2018). Briefly, after 5 mins of 6
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turn angle (degree) and the ratio of moving to nonmoving duration time were
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recorded for each larvae in dark with IR illumination for 3 mins (tracking rate of 25
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frames/s). The velocity was defined as the distance traveled by the center column of
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larvae per unit time. The absolute turn angle was the change in movement direction
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either clockwise or counterclockwise. During the following 3 mins, the holding stage
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was tapped every 30 s with a consistent intensity by machine, and the movement of
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larvae was recorded to calculate the maximum velocity (mm/s) after tapping stimulus.
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2.4. mRNA detection
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The alteration of fish behavioral response was a highly sensitive indicator
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corresponding to low/sublethal doses of toxins (Little and Finger, 1990), but high
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doses of toxin (75 and 150 ng/mL AFB1) were required to identify its biochemical
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and molecular targets at the early developing stages by 48 hpf. Therefore, total RNA
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samples were isolated from vehicle or AFB1 (75 and 150 mg/mL) treated-embryos (25
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embryos per dose in each independent experiment) at 24 and 48 hpf with
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TRIzol-reagent (Invitrogen, Carlsbad, CA) and then transcribed into cDNA with
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reverse
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semi-quantitative PCR was conducted using designed primers, including gfap
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(NM_131373.2), huC (ELAV like neuron-specific RNA binding protein 3,
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NM_131449.1), and ef (elongation factor, NM_131263); the primer sequences and
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reaction conditions are shown in Table S1. The ef gene served as an internal control.
(Super
Script
III,
Invitrogen,
Carlsbad,
CA).
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Real-time PCR (qPCR) was applied to analyze gfap, huC, ngfa (NM_131064),
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atp1b1b (ATPase Na+/K+ transporting subunit beta 1b, NM_131671), prtga
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(NM_001045030) and ef. The diluted cDNA sample was mixed with 55 nM designed
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primers (Table S1) and SYBR Green I master mix (Roche, Mannheim, Germany), and
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then the reaction was performed in the cycler StepOnePlus™ (Applied Biosystems, 7
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at 50 °C for 2 min, polymerase activation at 95 °C for 10 min, 40 cycles at 95 °C for
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15 s and 60 °C for 1 min. Data of relative mRNA expression level were calculated by
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StepOne™Software with the comparative CT (∆∆CT) method.
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2.5. Microarray analysis
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For the microarray experiment, WT embryos at 6 hpf were exposed to vehicle or
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150 ng/mL AFB1 (n= 25 for each treatment) and then collected at 48 hpf for RNA
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extraction with TRIzol reagent in each experiment. Three independent experiments
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were conducted and extracted RNA samples were pooled together for Agilent
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Zebrafish V3 array (4 x44 K) analysis based on the protocol of Welgene Biotech Co.
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(Taipei, Taiwan). The gene expression data obtained were submitted to Gene
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Expression Omnibus and assigned a GEO accession number GSE121125.
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2.6. Whole mount immunostaining
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Acetylated α-tubulin is one of the earliest markers for neuronal differentiation
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(Yeh and Hsu, 2016). In addition, the trigeminal ganglia and hindbrain neurons can be
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clearly and easily observed at the very early developmental stage, without the
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interference of other neural networks. Thus, 24 hpf embryos were used for the
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observation of neuron morphology after whole mount immunostaining based on the
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protocol describe by Wu et al., (2012). WT embryos exposed to vehicle or AFB1 (150
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ng/mL) (40 embryos for each treatment from two independent replicates) were
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collected at 24 hpf and fixed in 100% methanol at -20°C overnight. After progressive
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rehydration with PBST (0.05% Tween 20 in 0.01M PBS), the embryos were treated
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with 2N HCl at room temperature for 1 h. Next, the embryos were blocked with 1%
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blocking reagent in PBST for 1 h and then incubated at 4°C overnight with
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monoclonal antibodies specific to acetylated α-tubulin (1:400 dilution in blocking
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reagent)(Sigma, USA). Following PBST wash, the embryos were incubated with goat
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temperature for 1 h to develop red fluorescent signals. The images were taken under a
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fluorescence microscope equipped with a Rhodamine filter (DMi8, LEICA with 200x
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magnification).
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2.7. Statistical analysis
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The statistical analyses were performed with one-way ANOVA analysis plus
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post-hoc Dunnett test in the software GraphPad Prism (version 4.0, GraphPad
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Software Inc., San Diego, CA). Data for locomotor activity, non-normally distributed,
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were analyzed with a nonparametric Kruskal-Wallis test. Statistically significant
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differences were considered at p < 0.05.
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3. Results
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3.1. Viability and morphology of zebrafish embryos after AFB1 treatment
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In order to identify the suitable concentrations and time points for the following
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experiments, AFB1 ranging from 15 to 150 ng/mL, concentrations reported in
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contaminated aquafeeds, was applied to observe the survival rate and morphology of
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larvae until 7 dpf. The survival rates were recorded at the indicated time points. As
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presented in Fig. 1A, AFB1 concentrations up to 30 ng/mL did not alter the zebrafish
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viability until 7 dpf. The survival rate of 6 and 7 dpf larvae was found to be 93.3 %
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after 75 ng/mL AFB1 treatment. Embryos that were exposed to 150 ng/mL AFB1 did
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not suffer any mortality at 4 dpf, but their viability was dramatically reduced to 20%
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of the control at 7 dpf. Furthermore, the morphology of larvae at 7 dpf was observed
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under a microscope. None of the surviving larvae in AFB1 treated groups exhibited
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any obviously phenotypic defect as determine by comparison with the control (Fig.
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1B).
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3.2. AFB1 disturbed locomotion of embryonic zebrafish Although administrating AFB1 did not alter the morphology of 7 dpf zebrafish
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larvae, some of larvae showed patters of unstable shaking, swimming upside down
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and swimming on a side in all AFB1-treated group (15, 30, 50, 75 ng/mL). The
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percentages of 7 dpf larvae with abnormal swimming behaviors were significantly
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increased in a dose-dependent manner (Fig. 2A). In the 50 and 75 ng/mL AFB1 groups,
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around 19.1± 5.4 % (p<0.05) and 27.9 ± 5.5 % (p<0.01) of larvae exhibited abnormal
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behaviors, respectively. The AFB1 at 150 ng/mL was excluded from the locomotion
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test due to a high mortality rate since 6 dpf.
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The locomotor activity during the free swimming stage of 7 dpf larvae was
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evaluated. A low dose of AFB1 (15 ng/mL) slightly suppressed the average velocity of
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7 dpf larvae comparing to the control group, but higher doses (30-75 ng/mL) evoked
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hyperactivity of the larvae (Fig. 2B). Both 30 and 50 ng/mL AFB1 significantly
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increased the velocity of free swimming larvae to approximately 133 % that of control.
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This biphasic dose response was also found in the ratios of moving time to
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nonmoving time after AFB1 was administered (Fig. S1). Additionally, larvae exposed
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to 15 ng/mL AFB1 showed a significant increase in absolute turn angle (110% of
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control, p<0.01); in contrast, higher than 30 ng/mL decreased the turn angle of larvae
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(Fig. 2C). On the other hand, as depicted in Fig. 2D, the maximum velocity after
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tapping stimulation was significantly suppressed in 75 ng/mL AFB1-treated larvae,
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their maximum velocity decreasing to 75.5% that of the control (p<0.01).
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3.3 AFB1 increased gfap gene expression
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The expression pattern of gfap gene was examined to investigate the effects of
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AFB1 on the early neural development. The gfap expression and most transcriptional
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factors involved in zebrafish neurogenesis have been developed by 42 hpf (Schmidt et
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al., 2013), so the expression pattern of gfap were examined at 24 and 48 hpf. 10
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of gfap mRNA were apparently elevated in the 48 hpf embryos that had been treated
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with 75 and 150 ng/mL AFB1. Further confirmation by qPCR demonstrated that gfap
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transcripts in 150ng/mL AFB1-treated embryos significantly increased to 1.3 and 1.7
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fold over control groups at 24 and 48 hpf (p<0.05), respectively (Fig. 3B). The
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morphology of 24 and 48 hpf embryos was not apparently altered after 75 and 150
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ng/mL AFB1 treatment (Fig. S2).
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3.4. AFB1 interfered with neural development of embryonic zebrafish
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Transgenic zebrafish Tg (HuC: eGFP) showing green fluorescence in neurons
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were exposed to AFB1 to observe the phenotypic change of trigeminal ganglia and
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hindbrain neurons in 24 hpf embryos. Under a fluorescent microscope, cells in the
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trigeminal ganglia neuron were found to be arranged in a denser and less clear pattern
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in 150 ng/mL AFB1-treated embryos than in the vehicle control (Fig. 4 A (a, a’, c, c’)).
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Additionally, 76.8% of the 150 ng/mL AFB1-treated Tg embryos (43 out of 56
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embryos) was deteriorated in the hindbrain neurons (Fig. 4A (d,f)). On the other hand,
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WT embryonic zebrafish was treated with AFB1 to examine the endogenous
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expression of huc mRNA. As shown in Fig. 4B, results from qPCR analysis indicated
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that AFB1 at 150 ng/mL significantly reduced the levels of huc transcripts in whole
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embryos at 24 and 48 hpf to 65.9% (p<0.05) and 73.7% (p<0.01) of those in control
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groups, respectively. The inhibitory pattern was also consistent with data obtained
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from semi-quantitative PCR.
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Further immunostaining the neurons in 24 hpf embryos was conducted with
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antibodies against acetylated α-tubulin. Results revealed that exposure to 150 ng/mL
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AFB1 visibly disturbed the development of trigeminal ganglia neurons (Fig. 5A (b, b’))
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in 74% of examined embryos (26 out of 35). Similar phenomena were observed with
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the neurons in hindbrain areas (Fig. 5 B (b, b’)). Most of 24 hpf embryos in the 11
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control group displayed vivid images for the neural structures of trigeminal ganglia
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and hindbrain regions (Figs. 5A (a,a’) and Fig. 5B (a,a’)).
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3.5. AFB1 altered gene expression involved in neural activity and neurogenesis in
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embryos To elucidate the mechanism of AFB1-induced neurotoxicity, the transcriptome
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profiling of 48 hpf WT embryos that had been exposed to 150 ng/mL AFB1 was
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obtained. AFB1 at 150 ng/mL did not change the viability or morphology of embryos
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at 48 hpf (Fig. 1A and Fig. S2). Data from microarray analysis indicated that nine
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genes that are known to be involved in neural activity and neurogenesis were
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differentially expressed (>1.5 fold) between the embryos treated with AFB1 and
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solvent, as shown in Table 1. To validate the data from microarray, qPCR was applied
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to measure mRNA levels of ngfa, atp1b1b and prtga genes in 24 and 48 hpf embryos.
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AFB1 at 75 and 150 ng/mL significantly reduced the strength of ngfa signals to 48.9
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and 37.2% of those in the control group at 24 hpf (p<0.01), respectively (Fig. 6A).
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Similarly, both the levels of atp1b1b at 24 and 48 hpf were significantly
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down-regulated after exposure to 150 ng/mL AFB1 (Fig. 6B). On the other hand, the
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levels of prtga transcripts were significantly elevated to 2.2±0.4 fold those of the
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control (p<0.05) in the 48 hpf group exposed to 150 ng/mL AFB1 (Fig. 6C).
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4. Discussion
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The presence of mycotoxins within feeds significantly constrains the
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aquacultural productivity (Bryden, 2012). It is estimated that annual losses due to the
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ingestion of mycotoxin-contaminated animal feed in the United States and Canada are
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of the order of 5 billion. In Asia and Europe, around 60 % of commercial aquafeeds
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are contaminated with aflatoxins; the average toxin concentration of positive samples
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is around 50 ng/g, with the maximum concentration reaching 221 ng/g (Fallah et al., 12
ACCEPTED MANUSCRIPT 2014; Goncalves et al., 2016). Similarly, the fish feeds collected from east Africa were
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contaminated with aflatoxins at a mean level of 100 ng/g (Anater et al., 2016;
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Olorunfemi et al., 2013). Since there is no document to discuss the environmental
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relevant level of aflatoxin in surface water, in this study we applied the AFB1
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concentrations which are relevant to aquafeed contamination to demonstrate the
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potential adverse effects of AFB1 on the early life stage of fish through food chain.
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The number of larvae that exhibited abnormal unbalanced swimming patterns
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significantly increased upon the treatment of AFB1 in a dose-dependent manner (Fig.
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2A); this result is consistent with the finding concerning sea bass by EL-Sayed and
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Khalil (2009). Adult fish Dicentrarchus labrax were fed with AFB1 (18 ng/g body
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weight) for a period of 42 days and showed abnormal behavioral changes, such as
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swimming imbalance (El-Sayed and Khalil, 2009). Kalueff et al. (2013) have
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cataloged all major zebrafish behaviors using both larval and adult models. According
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to Kalueff’s work, our observations of unbalanced swimming, swimming upside
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down and swimming on a side in AFB1-exposed larvae can be closely associated with
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neuroactive/neurotoxic syndromes.
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AFB1-induced neurotoxicity remain uncertain.
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the molecular mechanisms of
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Both average velocity and the ratio of moving time to nonmoving time were
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decreased at 15 ng/mL group, but significantly increased upon the exposure of larvae
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to 30 to 50 ng/mL AFB1 (Fig. 2B and Fig. S1). An U-shaped dose response may
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account for low-dose inhibition and high-dose stimulation of locomotion activity
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during AFB1 treatment, but the reason for this biphasic dose response is not clear. The
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hyperlocomotion caused by higher levels of AFB1 herein is consistent with a recent
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finding that adult catfish after 14 days feeding of AFB1-contaminated diet (1177 ng/g
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feed) demonstrated hyperlocomotion (Baldissera et al., 2018). On the other hand,
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exposure to AFB1 reduced the maximum velocity of larvae following tapping stimulus,
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suggesting interference with the startle response of the larvae (Fig. 2D). The startle
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response is critical for the survival of neonatal fish, helping them escape from
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predators and other danger in the environment (Wolter and Arlinghaus, 2003). An AFB1-contaminated diet is known to inhibit Na+/K+-ATPase activity in the
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synaptosomes in the brains of catfish, which may contribute to their hyperlocomotion
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(Baldissera et al., 2018). Our findings also demonstrated that AFB1 suppresses the
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expression of atp1b1b gene, encoding Na+/K+-ATPase subunit in neurons. The
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Na+/K+-ATPase plays an important role in maintaining ionic homeostasis in fish and
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is a heterodimeric protein composed of one α and one β subunit (Doganli et al., 2013).
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Eight Na+/K+-ATPase α subunits and five β subunits are identified in zebrafish, and
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these various isoenzymes are likely to exhibit distinct functions within specific cell
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and tissue types. The transcripts of atp1b1b, an ortholog of the vertebrate β1 subunit,
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are majorly detected in the brain and eye tissues of adult zebrafish (Rajarao et al.,
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2001). It implies that down regulation of atp1b1b expression by AFB1 may involve in
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the neurological and behavioral change in zebrafish. AFB1 treatment did not
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obviously affect the mRNA signals of other α or β isoforms based on the microarray
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data.
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Among the nine differentially expressed genes that presented in Table 1, nlgn2b,
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syngr2a, syt5a, and grid 1b genes are located within presynapse/synapse/postsynapse
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areas. The taar 64, adra1bb, and grid1b genes are grouped in the category of
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“neuroactive ligand-receptor interaction”, based on the KEGG pathway. Baldissera et
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al. demonstrate that AFB1-induced behavioral dysfunction is associated with the
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alteration of neurotransmitters in fish brains (2018). AFB1 treatment also interferes
349
with the formation of biogenic catecholamine neurotransmitters and the regulation of
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hypothalamic neuropeptides in adult rodents and chickens (Ahmed and Singh, 1984;
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Jayasekara et al., 1989; Trebak et al., 2015). This information suggests that the
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AFB1 may interfere with synapse functions and the interaction among
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neurotransmitters in various species, including zebrafish. The onset of primary neurogenesis in zebrafish begins at 10.5 hpf and is
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completed within 24 hpf (Dam et al., 2011). The spatial and temporal patterns of
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neural induction and axonal tract formation are usually assessed before 48 hpf (Dou
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and Zhang, 2011). Therefore, in the present study the neuron morphology in
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trigeminal ganglia and hindbrain areas was identified at 24 hpf under microscope to
359
avoid the interference of latter neural outgrowth. In addition, the mRNA levels of all
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the neurogenesis-related factors, including gfap, huc, ngfa, atp1b1b, and prtga, were
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detected at both 24 and 48 hpf. The trigeminal ganglia in zebrafish with a cluster of
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30 neurons mediates the responses to mechanical stimuli and chemical irritants and
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are responsible for later abnormal behavior (Saint-Amant and Drapeau, 1998).
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Interrupting the formation of trigeminal ganglia neurons could be one of the factors
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that contributed to the abnormal swimming behavior triggered by AFB1.
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The AFB1 concentrations we used (15-150 ng/mL) did not cause any apparent
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morphological changes in embryos and larvae from 6 hpf to 7 dpf, as presented in Fig.
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1 B and Fig. S2. Those 150 ng/mL AFB1-exposed fish which eventually died at 6 or 7
369
dpf exhibited an extremely abnormal swimming pattern, but still remained their
370
normal morphology right before their death. The main factor which contributes to the
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death of AFB1-treated larvae without any morphological change remains unclear. In
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addition to neurotoxicity, we also found that the liver size of 75 and 150
373
ng/mL-treated embryos was slightly shrunk compared to that of control. Other major
374
organs, including heart and kidney, are morphologically normal in larvae after 150
375
ng/mL AFB1 exposure (data not shown). AFB1 at a higher dose may play a role in
376
disrupting central nervous system development or neurotransmitter interaction,
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leading to the high mortality of larvae at 6 and 7 dpf.
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5. Conclusion Aflatoxin B1 is the major mycotoxin which contaminates plant-based aquafeeds.
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AFB1-containing feed not only impairs the health of the cultured fish, but also has
381
potential threats against the public health through the food chain. This study exposed
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zebrafish embryos to AFB1 concentrations relevant to aquafeed contamination and
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found that AFB1 at high dose promoted the locomotor activity of larvae, but reduced
384
their startle response after environmental stimulation. AFB1 also disrupted the neural
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morphology of embryonic zebrafish, including trigeminal ganglia and hindbrain
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neurons. The transcriptome profiling of AFB1-treated embryos revealed that several
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differentially expressed genes are located within synaptic areas and functionally
388
related to neurogenesis or neuroactive ligand-receptor interaction. Neural mal
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-formation and synapse dysfunction may contribute to the behavioral alteration
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induced by AFB1. Data presented herein can provide more information for estimating
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the health risk associated with the fish and the public.
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Acknowledgment
This work was financially supported by the National Science Council of the Republic of China, Taiwan, under Contract No. 104-2320-B-002-037-MY3.
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Conflict of Interest
Authors declare that there are no conflicts of interest.
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Baldissera, M.D., Souza, C.F., Zeppenfeld, C.C., Descovi, S.N., Moreira, K.L.S., da Rocha, M.I.U., Baldisserotto, B., 2018. Aflatoxin B1-contaminated diet disrupts the blood–brain barrier and affects fish behavior: Involvement of neurotransmitters in brain synaptosomes. Environ. Toxicol. Pharmacol. 60,
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45-51. Barbosa, B.T.S., Pereyra, C.M., Soleiro, C.A., Dias, E.O., Oliveira, A.A., Keller, K.A., Silva, P .P.O., Cavaglieri, L.R., Rosa, C.A.R., 2013. Mycobiota and mycotoxins present in finished fish feeds from farms in the Rio de Janeiro State, Brazil. Int. Aquat. Res. 5, 2-9.
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Saint-Amant, L., Drapeau, P., 1998. Time course of the development of motor behaviors in the zebrafish embryo. J. Neurobiol. 37, 622-632. St John, J.A., Key, B., 2012. HuC-eGFP mosaic labelling of neurons in zebrafish enables in vivo live cell imaging of growth cones. J. Mol. Histol. 43, 615-623. Trebak, F., Alaoui, A., Alexandre, D., El Ouezzani, S., Anouar, Y., Chartrel, N.,
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Magoul, R., 2015. Impact of aflatoxin B1 on hypothalamic neuropeptides regulating feeding behavior. Neurotoxicology. 49, 165-173. Vesque, C., Anselme, I., Couvé, E., Charnay, P., Schneider Maunoury, S., 2006. Cloning of vertebrate Protogenin (Prtg) and comparative expression analysis
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during axis elongation. Dev. dyn. 235, 2836-2844. Wong, Y.H., Lu, A.C., Wang, Y.C., Cheng, H.C., Chang, C., Chen, P.H., Fann, M.J., 2010. Protogenin defines a transition stage during embryonic neurogenesis and prevents precocious neuronal differentiation. J. Neurosci. 30, 4428-4439.
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Wolter, C., Arlinghaus, R., 2003. Navigation impacts on freshwater fish assemblages: the ecological relevance of swimming performance. Rev. Fish Biol. Fisher. 13,
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63-89. Wu, T. S., Yang, J. J., Yu, F. Y., Liu, B. H., 2012. Evaluation of nephrotoxic effects of
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mycotoxins, citrinin and patulin, on zebrafish (Danio rerio) embryos. Food Chem. Toxicol. 50, 4398-4404. Yeh, C.W., Hsu, L.S., 2016. Zebrafish diras1 Promoted Neurite Outgrowth in Neuro-2a Cells and Maintained Trigeminal Ganglion Neurons In Vivo via Rac1-Dependent Pathway. Mol. Neurobiol. 53, 6594-6607.
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Legends of figures
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Figure 1. The viability and morphology of zebrafish embryos after AFB1 exposure.
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The 6 hpf WT embryos were treated with vehicle (0 ng/mL) or various concentrations
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of AFB1 (15-150 ng/mL). (A) The survival rate of embryos was determined at the
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designate time. Values are presented as the mean ± SD from four independent
520
experiments with at least 20 fish embryos per treatment group in each experiment. (B)
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Morphology of 7 dpf larvae after exposure to AFB1. Images were taken from the
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lateral view under a dissecting microscope (magnification ×25). Scale bar, 500 µM.
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Figure 2. The locomotor activity of zebrafish larvae after AFB1 treatment. The WT
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embryos were treated with vehicle or various concentrations of AFB1 (15-75 ng/mL)
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from 6 hpf to 7 dfp. (A) The percentage of fish with an abnormal swimming pattern
526
were recorded at 7 dpf and presented as means ± SD from three independent
527
experiments with at least 18 larvae per exposure concentration in each experiment.
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The average velocity (B), absolute turn angle (C) and maximum velocity after
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stimulation (D) at 7 dpf are shown with box plots, in which boxes represent the 25th
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and 75th percentiles and whiskers represent the 10th and 90th percentiles. Solid lines
531
and dashed lines within the boxes indicate the median and mean values, respectively.
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Data are collected from at least 52 larvae (control, n=70; 15 ng/mL, n=52; 30 ng/mL,
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n=52; 50 ng/mL, n=53; 75 ng/mL, n=53). Black dots are data points not within the
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range of 10th to 90th percentile. *, p<0.05 ; **p<0.01 vs the control.
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Figure 3. AFB1 activated gfap gene expression. WT embryos at 6 hpf were exposed to
536
vehicle, 75 and 150 ng/mL of AFB1, and then the levels of gfap transcript were
537
analyzed with semi-quantitative PCR and qPCR at 24 and 48 hpf. (A) The
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representative data from semi-quantitative PCR. (B) The expression of gfap was
539
determined by qPCR. Each set of data represents mean ± SEM from four independent
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experiments. The data of vehicle control is arbitrarily regarded as 1.0. *p < 0.05 vs the
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Figure 4. AFB1 caused neurogenesis defects in Tg(HuC: eGFP) embryos. Transgenic
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embryos showing GFP in neurons were exposed to vehicle, 75 and 150 ng/mL AFB1
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at 6 hpf. (A) The images of 24 hpf embryos were taken under a fluorescent
545
microscope in a lateral view. The TG areas are demonstrated with square shapes (a, b,
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c); (a’, b’, c’) are enlarged figures of (a, b, c). The morphology of Hb neurons is
547
displayed in (d, e, f). (B) The levels of huc mRNA in 24 and 48 hpf embryos were
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detected by qPCR (left) and semi-quantitative PCR (right). The qPCR data are mean ±
549
SEM from four independent experiments. The data of vehicle control is arbitrarily
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regarded as 1.0. * p < 0.05; ** p < 0.01 vs control. TG, trigeminal ganglion; Hb,
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hindbrain; Scale bar, 50µm.
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Figure 5. AFB1 disrupted neural formation in WT embryos. WT embryos were treated
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with vehicle (0 ng/mL) or 150 ng/mL AFB1 from 6 to 24 hpf. Whole-mount
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immunostaining was conducted with anti-acetylated α-tubulin antibodies. (A) The
555
representative figures of TG areas. The (a’, b’) derived from (a, b) are enlarged
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figures of TG. (B) The representative figures of Hb areas from the dorsal view. The
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(a’, b’) derived from (a, b) are enlarged figures of Hb area. TG, trigeminal ganglion;
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Hb, hindbrain; Scale bar, 100 µm.
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Figure 6. AFB1 suppressed the expression of neurodevelopment-related genes. WT
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embryos at 6 hpf were exposed to vehicle, 75 and 150 ng/mL AFB1 and then mRNAs
561
were collected at 24 and 48 hpf for qPCR analysis of ngfa (A), atp1b1b (B), and prtga
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(C). The data were mean ± SEM from at least three independent experiments. The
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data of vehicle control is arbitrarily regarded as 1.0. *, p < 0.05; **, p < 0.01 vs the
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control .
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Table 1. Characteristics of differential expression genes related to neural functiona symbol
gene name
FCb
cellular component by GOc term
NM_131064
ngfa
nerve growth factor a
3.5 ↓
membrane-bounded vehicle
NM_001166329
nlgn2b
neuroligin 2b
3.1 ↓
NM_001083102
taar 64
Trace amine associated 2.7 ↓ receptor 64
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Accession code
Postsynaptic membrane integral to membrane
NM_001080585
syngr2a
synaptogyrin 2a
1.8 ↓
NM_131671
atp1b1b
Na+/K+ ATPase, beta 1b
1.6 ↓
NM_001103137
syt5a
Synaptotagmin Va
1.5 ↓
Presynapse/calcium ion binding
NM_001045030
prtga
protogenin homolog a
2.0 ↑
integral to membrane
NM_001007358
adra1bb adrenoreceptor alpha 1Bb
ENSDART
grid1b
SC
2.1↑
ionotropic 2.8 ↑
integral to membrane postsynaptic membrane
neuroactivity-related genes from microarray analysis of WT embryos treated with vehicle or 150 ng/mL AFB1 from 6 to 48 hpf.
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FC: fold change GO: gene ontology
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b c
integral to membrane
receptor delta Type 1b
00000152431 a
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glutamate
Presynatic vesicles
Figure 1
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(A)
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40 20 0
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4
5
6
7 (dpf)
Figure 2
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(B)
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mL)
(C)
(D)
2D Graph 2
**
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0 Plot 1
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100
2
15
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30
X Data
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50
5
75 (ng/mL)
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(B)
2
24 hpf
48 hpf
*
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gfap mRNA level (fold of control)
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(A)
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75 150
75 150
AFB1 (ng/mL)
50 mm
Figure 4
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50 mm
(A)
50 mm
AFB1
TG neuron
TG neuron (enlarged)
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Hb neuron d
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24 hpf
*
**
*
48 hpf
*
0.5
0.0 75 150
75 150
AFB1 (ng/mL)
f
43/56
50 mm
31/43
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(A) ngfa mRNA level (fold of control)
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1 .0
0 .5
*
**
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prtga mRNA level (fold of control)
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ACCEPTED MANUSCRIPT Aflatoxin B1 (AFB1) is the major mycotoxin in plant-based aquafeeds
AFB1 interfered with locomotor activity of zebrafish larvae
AFB1 disrupted neural development of embryonic zebrafish
AFB1 altered the marker genes involved in neuroactivity and neurogenesis
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S0045-6535(18)32161-1
DOI:
https://doi.org/10.1016/j.chemosphere.2018.11.058
Reference:
CHEM 22542
To appear in:
ECSN
Received Date: 31 August 2018 Revised Date:
7 November 2018
Accepted Date: 8 November 2018
Please cite this article as: Wu, T.-S., Cheng, Y.-C., Chen, P.-J., Huang, Y.-T., Yu, F.-Y., Liu, B.-H., Exposure to aflatoxin B1 interferes with locomotion and neural development in zebrafish embryos and larvae, Chemosphere (2018), doi: https://doi.org/10.1016/j.chemosphere.2018.11.058. 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.
6 hpf zebrafish embryos
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Locomotion interference mL)
abnormal neuron morphology Scale bar = 50 m
gfap huc
ngfa
prtga atp1b1b
gene level alteration
ACCEPTED MANUSCRIPT 1
Exposure to aflatoxin B1 interferes with locomotion and
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neural development in zebrafish embryos and larvae
Biing-Hui Liua* a
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Taipei, Taiwan b
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Department of Agricultural Chemistry, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan
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Graduate Institute of Toxicology, College of Medicine, National Taiwan University,
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Ting-Shuan Wu a‡, Ya-Chih Chenga‡, Pei-Jen Chen b, Ying-Tzu Huanga, Feng-Yih Yucd*,
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c
Department of Biomedical Sciences, Chung Shan Medical University, Taiwan
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d
Department of Medical Research, Chung Shan Medical University Hospital,
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Taichung, Taiwan
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Running titles:
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‡ These authors contributed equally to this work
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*Corresponding authors: Biing-Hui Liu, Feng-Yih Yu
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Address: Graduate Institute of Toxicology
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College of Medicine, National Taiwan University
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No. 1,Sec 1, Jen-Ai Rd, Taipei, Taiwan. 10043
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Tel : 886-2-23123456 ext 88602
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Fax: 886-2-23410217
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E-mail: [email protected]
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[email protected] 1
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Abstract Aflatoxin B1 (AFB1) is the major mycotoxin that contaminates aquafeeds and
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regarded as a causative agent in illnesses and the mortality of aquacultural species.
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However, the effects of AFB1 on developing fish and associated toxic mechanism are
26
still unknown. This study examines the behavioral changes, neuronal morphology and
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gene expression in zebrafish embryos and larvae upon exposure to aflatoxin solutions.
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Treatment of 6 hour post fertilization (hpf) embryos with AFB1 at 15-75 ng/mL
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significantly changed the swimming patterns of seven days post-fertilization (dpf)
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zebrafish larvae. Larvae in the 15 ng/mL group demonstrated a hypolocomotor
31
activity in free swimming, but hyperlocomotion was observed in the larvae exposed to
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30-75 ng/mL AFB1. AFB1 at 75 ng/mL also significantly reduced the startle response
33
of 7 dpf larvae after tapping stimulus. Exposure to AFB1 resulted in an aberrant
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morphology of
35
(HuC:eGFP); this finding was supported by acetylated alpha-tubulin staining in
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wild-type fish. Additionally, AFB1 altered the levels of neurotoxic markers, including
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gfap and huC. The transcriptomic profile of AFB1-treated embryos revealed several
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differentially expressed genes that are related to neuroactivity and neurogenesis. PCR
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analysis verified that AFB1 significantly down-regulated the expression of ngfa and
40
atp1b1b genes and increased that of prtga gene. The results herein indicate the
41
toxicological impacts of AFB1 on the behaviors and neurodevelopment of fish in the
42
early embryonic stage. Disruption of neural formation and synapse dysfunction may
43
be responsible for the behavioral alteration.
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1. Introduction Aquaculture supplies nearly half of all seafood that is intended for human
46
consumption and is a vital part of the global food industry. Over the past few years,
47
the replacement of fishmeal with cheaper plant-based proteins in commercial feed
48
formulations has gained widespread acceptance (Dirican et al., 2015). However,
49
increasing reliance on the use of plant-based ingredients has increased the risk of
50
introducing mycotoxins into the feed at the point of manufacture and storage (Barbosa
51
et al., 2013). Mycotoxins are a rising threat to aquaculture and exposure to aflatoxin,
52
which is one of them, is the primary risk to farm-raised fish. Aflatoxin-contaminated
53
feed not only has direct negative effects on the health of the cultured fish, but also
54
increases the risk of passing along the contaminants to consumers via the food chain
55
(Anater et al., 2016).
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Aflatoxins are secondary metabolites that are primarily generated by Aspergillus
57
flavus and A. parasiticus. Several surveys reveal the serious contamination of
58
Aspergillus and aflatoxins in fish feed worldwide. In South America, Aspergillus is
59
the most abundant species isolated from 60 to 70 % of fish feeds and aflatoxins are
60
found in 55% of finished fish feeds (Barbosa et al., 2013). In Asia and Africa, the
61
mean levels of AFB1 in aquafeeds were ranged from 50 to 100 ng/g (Anater et al.,
62
2016; Fallah et al., 2014; Rodrigues et al., 2011). Additionally, co-occurrence of other
63
mycotoxins with aflatoxins in aquafeeds is very common and may induce synergistic
64
effects to increase the negative impact of aflatoxins at lower levels (Goncalves et al.,
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2016).
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Fish that are exposed to AFB1, the most prevalent and toxic compound in the
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aflatoxin group, usually demonstrate symptoms that are associated with weight loss,
68
growth retardation, immunosuppression and even high mortality (Dhanasekaran et al., 3
ACCEPTED MANUSCRIPT 2011). AFB1 also has negative effects on the nervous system. Marine-water sea bass
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that are fed with feedstuffs contaminated with AFB1 exhibits unusual swimming
71
patterns that are associated with loss of equilibrium (El-Sayed and Khalil, 2009). A
72
study by Baldissera et al. indicates that an AFB1-containing diet results in the
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hyperlocomotion of silver catfish. This behavioral dysfunction is associated with
74
impairment of the blood brain barrier and alteration of various neurotransmitters in
75
the fish brain (Baldissera et al., 2018).
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The utility of zebrafish (Danio rerio) in studying neurotoxicity and aberrant
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behavior has grown remarkably recently; optical transparency of embryos and the
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establishment of transgenic lines make individual neurons in zebrafish be visualized
79
throughout
80
physiological and genetic information regarding zebrafish neurogenesis are available
81
(Guo, 2009; Kalueff et al., 2014). Glial fibrillary acidic protein (Gfap) is critical to
82
guide the neural stem cell migration in central nervous system and serves as a
83
sensitive biomarker of neurotoxicity in zebrafish (Noctor et al., 2001; McGrath and Li,
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2008). HuC is a neuron-specific RNA binding protein that is regarded as an early
85
neuronal differentiation marker in developing zebrafish (Kim et al., 1996: St John and
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Key, 2012). Nerve growth factor a (Ngfa) is related to the division and differentiation
87
of sympathetic and embryonic sensory neurons (Dethleffsen et al., 2003). Protogenin
88
homolog (Prtga) signaling is crucial to the suppression of premature neuronal
89
differentiation during early neural development (Vesque et al., 2006; Wong et al.,
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2010).
development
(Schmidt,
2013).
Additionally,
more
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Information about the effect of aflatoxins on aquaculture are very limited (Anater
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et al., 2016). To examine the potential adverse effects of AFB1 on the early
93
neurodevelopment of fish, zebrafish embryos and larvae were exposed to AFB1 at
94
concentrations relevant to the levels in contaminated aquafeeds Attempts were also 4
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96
neurotoxicity.
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2. Materials and Methods
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2.1. Test species and husbandry.
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The Wild-type (WT) AB laboratory strain zebrafish (Danio rerio) and the
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transgenic line Tg(huC:eGFP)as8 were obtained from Taiwan Zebrafish Core Facility
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at Academia Sinica (TZCAS) (Taipei, Taiwan). Tg(huC:GFP)as8 is known to use the
102
promoter of the zebrafish HuC gene to drive the expression of green fluorescent
103
protein (GFP) in the telencephalic cluster, retinal ganglion cells, medial longitudinal
104
fasciculus, dorsal longitudinal fasciculus, trigeminal ganglion and Rohon-Beard
105
neurons (Huang et al., 2011). Zebrafish were kept in a water circulating system
106
(Aquazoo Fish housing system. Taiwan) at 27~28 °C with a 14-h light/10 h-dark cycle.
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Water used for all the tanks passed through 120-micron filter pad, 5-micron filter
108
cartridge, activated carbon filter, biological filter, and UV disinfection filter before
109
being circulated into every tank. To maintain the water quality of circulating system,
110
the values of pH, general hardness, carbonate hardness, nitrite and nitrate were
111
monitored weekly. Daily feeding included artificial feed pellets (Taikong Corp.
112
Taiwan) or live brine shrimp (Ocean Star International, Inc. U.S.A.).
113
breeding were based on the animal research protocols approved by Institutional
114
Animal Care and Use Committee (National Taiwan University, approval No.1334).
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2.2. Viability and morphology after AFB1 exposure
Adult care and
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AFB1 (CAS:1162-65-8) was purchased from the Sigma-Aldrich Company (St.
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Louis, MO) and first dissolved in DMSO at a concentration of 6 mg/mL and storage
118
at -20
119
embryos/larvae, AFB1 in stock solution would be diluted with 0.01 M phosphate
as a stock solution. To examine the survival rate and morphology of
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ACCEPTED MANUSCRIPT buffered saline (PBS, pH7.0) to 0.3 mg/mL and further diluted with egg water (60 mg
121
sea salts/L, Instant Ocean® sea salts) to the tested concentrations (15, 30, 75, 150
122
ng/mL). Healthy and normally developing WT embryos at 6 hour post-fertilization
123
(hpf) were collected and exposed to vehicle or various concentrations of AFB1 in egg
124
water. The survival rate of embryos (at least 20 embryos per treatment group in each
125
independent experiment) was determined at the designate time (from 1 to 7 dpf). The
126
morphology of surviving larvae at 7 dpf was observed from the lateral view under a
127
dissecting microscope (magnification ×25).
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2.3. The larval locomotion test
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For the assessment of behavioral effects, four AFB1 concentrations were selected
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based on the survival rate of embryos at 7 dpf (Fig 1A): lethal concentration causing
132
around
133
no-observable-effects-concentration (NOEC), and half the NOEC. These were 75, 50,
134
30 and 15 ng/mL, respectively. WT zebrafish embryos were treated with vehicle or
135
indicated concentrations of AFB1 from 6 hpf to 7 dpf. There were three replicates for
136
each exposure concentration, and at least 18 larvae were included per dose in each
137
replicate. Since the larvae obtained their fully free swimming ability in our system at
138
7 dpf, the swimming behaviors of 7 dpf larvae after vehicle or AFB1 treatment were
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observed under stereomicroscope. The number of larvae with the patterns of unstable
140
shaking, swimming upside down and swimming on a side was recorded and divided
141
by the number of total examined larvae in each treatment. Data were presented as the
142
percentage of abnormal swimming behaviors (%).
of
mortality
(LC10),
2/3
the
concentration
of
LC10,
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The locomotor activity of 7 dpf larvae were further evaluated with the animal
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movement tracking system and software (EthoVison XT, Noldus Information
145
Technology, Netherlands) as described by Liao et al. (2018). Briefly, after 5 mins of 6
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147
turn angle (degree) and the ratio of moving to nonmoving duration time were
148
recorded for each larvae in dark with IR illumination for 3 mins (tracking rate of 25
149
frames/s). The velocity was defined as the distance traveled by the center column of
150
larvae per unit time. The absolute turn angle was the change in movement direction
151
either clockwise or counterclockwise. During the following 3 mins, the holding stage
152
was tapped every 30 s with a consistent intensity by machine, and the movement of
153
larvae was recorded to calculate the maximum velocity (mm/s) after tapping stimulus.
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2.4. mRNA detection
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The alteration of fish behavioral response was a highly sensitive indicator
156
corresponding to low/sublethal doses of toxins (Little and Finger, 1990), but high
157
doses of toxin (75 and 150 ng/mL AFB1) were required to identify its biochemical
158
and molecular targets at the early developing stages by 48 hpf. Therefore, total RNA
159
samples were isolated from vehicle or AFB1 (75 and 150 mg/mL) treated-embryos (25
160
embryos per dose in each independent experiment) at 24 and 48 hpf with
161
TRIzol-reagent (Invitrogen, Carlsbad, CA) and then transcribed into cDNA with
162
reverse
163
semi-quantitative PCR was conducted using designed primers, including gfap
164
(NM_131373.2), huC (ELAV like neuron-specific RNA binding protein 3,
165
NM_131449.1), and ef (elongation factor, NM_131263); the primer sequences and
166
reaction conditions are shown in Table S1. The ef gene served as an internal control.
(Super
Script
III,
Invitrogen,
Carlsbad,
CA).
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Real-time PCR (qPCR) was applied to analyze gfap, huC, ngfa (NM_131064),
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atp1b1b (ATPase Na+/K+ transporting subunit beta 1b, NM_131671), prtga
169
(NM_001045030) and ef. The diluted cDNA sample was mixed with 55 nM designed
170
primers (Table S1) and SYBR Green I master mix (Roche, Mannheim, Germany), and
171
then the reaction was performed in the cycler StepOnePlus™ (Applied Biosystems, 7
ACCEPTED MANUSCRIPT Foster City, CA) with the following condition: activation of uracil-DNA glycosylase
173
at 50 °C for 2 min, polymerase activation at 95 °C for 10 min, 40 cycles at 95 °C for
174
15 s and 60 °C for 1 min. Data of relative mRNA expression level were calculated by
175
StepOne™Software with the comparative CT (∆∆CT) method.
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2.5. Microarray analysis
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For the microarray experiment, WT embryos at 6 hpf were exposed to vehicle or
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150 ng/mL AFB1 (n= 25 for each treatment) and then collected at 48 hpf for RNA
179
extraction with TRIzol reagent in each experiment. Three independent experiments
180
were conducted and extracted RNA samples were pooled together for Agilent
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Zebrafish V3 array (4 x44 K) analysis based on the protocol of Welgene Biotech Co.
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(Taipei, Taiwan). The gene expression data obtained were submitted to Gene
183
Expression Omnibus and assigned a GEO accession number GSE121125.
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2.6. Whole mount immunostaining
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Acetylated α-tubulin is one of the earliest markers for neuronal differentiation
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(Yeh and Hsu, 2016). In addition, the trigeminal ganglia and hindbrain neurons can be
187
clearly and easily observed at the very early developmental stage, without the
188
interference of other neural networks. Thus, 24 hpf embryos were used for the
189
observation of neuron morphology after whole mount immunostaining based on the
190
protocol describe by Wu et al., (2012). WT embryos exposed to vehicle or AFB1 (150
191
ng/mL) (40 embryos for each treatment from two independent replicates) were
192
collected at 24 hpf and fixed in 100% methanol at -20°C overnight. After progressive
193
rehydration with PBST (0.05% Tween 20 in 0.01M PBS), the embryos were treated
194
with 2N HCl at room temperature for 1 h. Next, the embryos were blocked with 1%
195
blocking reagent in PBST for 1 h and then incubated at 4°C overnight with
196
monoclonal antibodies specific to acetylated α-tubulin (1:400 dilution in blocking
197
reagent)(Sigma, USA). Following PBST wash, the embryos were incubated with goat
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199
temperature for 1 h to develop red fluorescent signals. The images were taken under a
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fluorescence microscope equipped with a Rhodamine filter (DMi8, LEICA with 200x
201
magnification).
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2.7. Statistical analysis
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The statistical analyses were performed with one-way ANOVA analysis plus
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post-hoc Dunnett test in the software GraphPad Prism (version 4.0, GraphPad
205
Software Inc., San Diego, CA). Data for locomotor activity, non-normally distributed,
206
were analyzed with a nonparametric Kruskal-Wallis test. Statistically significant
207
differences were considered at p < 0.05.
208
3. Results
209
3.1. Viability and morphology of zebrafish embryos after AFB1 treatment
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In order to identify the suitable concentrations and time points for the following
211
experiments, AFB1 ranging from 15 to 150 ng/mL, concentrations reported in
212
contaminated aquafeeds, was applied to observe the survival rate and morphology of
213
larvae until 7 dpf. The survival rates were recorded at the indicated time points. As
214
presented in Fig. 1A, AFB1 concentrations up to 30 ng/mL did not alter the zebrafish
215
viability until 7 dpf. The survival rate of 6 and 7 dpf larvae was found to be 93.3 %
216
after 75 ng/mL AFB1 treatment. Embryos that were exposed to 150 ng/mL AFB1 did
217
not suffer any mortality at 4 dpf, but their viability was dramatically reduced to 20%
218
of the control at 7 dpf. Furthermore, the morphology of larvae at 7 dpf was observed
219
under a microscope. None of the surviving larvae in AFB1 treated groups exhibited
220
any obviously phenotypic defect as determine by comparison with the control (Fig.
221
1B).
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3.2. AFB1 disturbed locomotion of embryonic zebrafish Although administrating AFB1 did not alter the morphology of 7 dpf zebrafish
225
larvae, some of larvae showed patters of unstable shaking, swimming upside down
226
and swimming on a side in all AFB1-treated group (15, 30, 50, 75 ng/mL). The
227
percentages of 7 dpf larvae with abnormal swimming behaviors were significantly
228
increased in a dose-dependent manner (Fig. 2A). In the 50 and 75 ng/mL AFB1 groups,
229
around 19.1± 5.4 % (p<0.05) and 27.9 ± 5.5 % (p<0.01) of larvae exhibited abnormal
230
behaviors, respectively. The AFB1 at 150 ng/mL was excluded from the locomotion
231
test due to a high mortality rate since 6 dpf.
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The locomotor activity during the free swimming stage of 7 dpf larvae was
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evaluated. A low dose of AFB1 (15 ng/mL) slightly suppressed the average velocity of
234
7 dpf larvae comparing to the control group, but higher doses (30-75 ng/mL) evoked
235
hyperactivity of the larvae (Fig. 2B). Both 30 and 50 ng/mL AFB1 significantly
236
increased the velocity of free swimming larvae to approximately 133 % that of control.
237
This biphasic dose response was also found in the ratios of moving time to
238
nonmoving time after AFB1 was administered (Fig. S1). Additionally, larvae exposed
239
to 15 ng/mL AFB1 showed a significant increase in absolute turn angle (110% of
240
control, p<0.01); in contrast, higher than 30 ng/mL decreased the turn angle of larvae
241
(Fig. 2C). On the other hand, as depicted in Fig. 2D, the maximum velocity after
242
tapping stimulation was significantly suppressed in 75 ng/mL AFB1-treated larvae,
243
their maximum velocity decreasing to 75.5% that of the control (p<0.01).
244
3.3 AFB1 increased gfap gene expression
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The expression pattern of gfap gene was examined to investigate the effects of
246
AFB1 on the early neural development. The gfap expression and most transcriptional
247
factors involved in zebrafish neurogenesis have been developed by 42 hpf (Schmidt et
248
al., 2013), so the expression pattern of gfap were examined at 24 and 48 hpf. 10
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250
of gfap mRNA were apparently elevated in the 48 hpf embryos that had been treated
251
with 75 and 150 ng/mL AFB1. Further confirmation by qPCR demonstrated that gfap
252
transcripts in 150ng/mL AFB1-treated embryos significantly increased to 1.3 and 1.7
253
fold over control groups at 24 and 48 hpf (p<0.05), respectively (Fig. 3B). The
254
morphology of 24 and 48 hpf embryos was not apparently altered after 75 and 150
255
ng/mL AFB1 treatment (Fig. S2).
256
3.4. AFB1 interfered with neural development of embryonic zebrafish
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Transgenic zebrafish Tg (HuC: eGFP) showing green fluorescence in neurons
258
were exposed to AFB1 to observe the phenotypic change of trigeminal ganglia and
259
hindbrain neurons in 24 hpf embryos. Under a fluorescent microscope, cells in the
260
trigeminal ganglia neuron were found to be arranged in a denser and less clear pattern
261
in 150 ng/mL AFB1-treated embryos than in the vehicle control (Fig. 4 A (a, a’, c, c’)).
262
Additionally, 76.8% of the 150 ng/mL AFB1-treated Tg embryos (43 out of 56
263
embryos) was deteriorated in the hindbrain neurons (Fig. 4A (d,f)). On the other hand,
264
WT embryonic zebrafish was treated with AFB1 to examine the endogenous
265
expression of huc mRNA. As shown in Fig. 4B, results from qPCR analysis indicated
266
that AFB1 at 150 ng/mL significantly reduced the levels of huc transcripts in whole
267
embryos at 24 and 48 hpf to 65.9% (p<0.05) and 73.7% (p<0.01) of those in control
268
groups, respectively. The inhibitory pattern was also consistent with data obtained
269
from semi-quantitative PCR.
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Further immunostaining the neurons in 24 hpf embryos was conducted with
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antibodies against acetylated α-tubulin. Results revealed that exposure to 150 ng/mL
272
AFB1 visibly disturbed the development of trigeminal ganglia neurons (Fig. 5A (b, b’))
273
in 74% of examined embryos (26 out of 35). Similar phenomena were observed with
274
the neurons in hindbrain areas (Fig. 5 B (b, b’)). Most of 24 hpf embryos in the 11
ACCEPTED MANUSCRIPT 275
control group displayed vivid images for the neural structures of trigeminal ganglia
276
and hindbrain regions (Figs. 5A (a,a’) and Fig. 5B (a,a’)).
277
3.5. AFB1 altered gene expression involved in neural activity and neurogenesis in
278
embryos To elucidate the mechanism of AFB1-induced neurotoxicity, the transcriptome
280
profiling of 48 hpf WT embryos that had been exposed to 150 ng/mL AFB1 was
281
obtained. AFB1 at 150 ng/mL did not change the viability or morphology of embryos
282
at 48 hpf (Fig. 1A and Fig. S2). Data from microarray analysis indicated that nine
283
genes that are known to be involved in neural activity and neurogenesis were
284
differentially expressed (>1.5 fold) between the embryos treated with AFB1 and
285
solvent, as shown in Table 1. To validate the data from microarray, qPCR was applied
286
to measure mRNA levels of ngfa, atp1b1b and prtga genes in 24 and 48 hpf embryos.
287
AFB1 at 75 and 150 ng/mL significantly reduced the strength of ngfa signals to 48.9
288
and 37.2% of those in the control group at 24 hpf (p<0.01), respectively (Fig. 6A).
289
Similarly, both the levels of atp1b1b at 24 and 48 hpf were significantly
290
down-regulated after exposure to 150 ng/mL AFB1 (Fig. 6B). On the other hand, the
291
levels of prtga transcripts were significantly elevated to 2.2±0.4 fold those of the
292
control (p<0.05) in the 48 hpf group exposed to 150 ng/mL AFB1 (Fig. 6C).
293
4. Discussion
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The presence of mycotoxins within feeds significantly constrains the
295
aquacultural productivity (Bryden, 2012). It is estimated that annual losses due to the
296
ingestion of mycotoxin-contaminated animal feed in the United States and Canada are
297
of the order of 5 billion. In Asia and Europe, around 60 % of commercial aquafeeds
298
are contaminated with aflatoxins; the average toxin concentration of positive samples
299
is around 50 ng/g, with the maximum concentration reaching 221 ng/g (Fallah et al., 12
ACCEPTED MANUSCRIPT 2014; Goncalves et al., 2016). Similarly, the fish feeds collected from east Africa were
301
contaminated with aflatoxins at a mean level of 100 ng/g (Anater et al., 2016;
302
Olorunfemi et al., 2013). Since there is no document to discuss the environmental
303
relevant level of aflatoxin in surface water, in this study we applied the AFB1
304
concentrations which are relevant to aquafeed contamination to demonstrate the
305
potential adverse effects of AFB1 on the early life stage of fish through food chain.
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The number of larvae that exhibited abnormal unbalanced swimming patterns
307
significantly increased upon the treatment of AFB1 in a dose-dependent manner (Fig.
308
2A); this result is consistent with the finding concerning sea bass by EL-Sayed and
309
Khalil (2009). Adult fish Dicentrarchus labrax were fed with AFB1 (18 ng/g body
310
weight) for a period of 42 days and showed abnormal behavioral changes, such as
311
swimming imbalance (El-Sayed and Khalil, 2009). Kalueff et al. (2013) have
312
cataloged all major zebrafish behaviors using both larval and adult models. According
313
to Kalueff’s work, our observations of unbalanced swimming, swimming upside
314
down and swimming on a side in AFB1-exposed larvae can be closely associated with
315
neuroactive/neurotoxic syndromes.
316
AFB1-induced neurotoxicity remain uncertain.
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the molecular mechanisms of
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Both average velocity and the ratio of moving time to nonmoving time were
318
decreased at 15 ng/mL group, but significantly increased upon the exposure of larvae
319
to 30 to 50 ng/mL AFB1 (Fig. 2B and Fig. S1). An U-shaped dose response may
320
account for low-dose inhibition and high-dose stimulation of locomotion activity
321
during AFB1 treatment, but the reason for this biphasic dose response is not clear. The
322
hyperlocomotion caused by higher levels of AFB1 herein is consistent with a recent
323
finding that adult catfish after 14 days feeding of AFB1-contaminated diet (1177 ng/g
324
feed) demonstrated hyperlocomotion (Baldissera et al., 2018). On the other hand,
325
exposure to AFB1 reduced the maximum velocity of larvae following tapping stimulus,
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suggesting interference with the startle response of the larvae (Fig. 2D). The startle
327
response is critical for the survival of neonatal fish, helping them escape from
328
predators and other danger in the environment (Wolter and Arlinghaus, 2003). An AFB1-contaminated diet is known to inhibit Na+/K+-ATPase activity in the
330
synaptosomes in the brains of catfish, which may contribute to their hyperlocomotion
331
(Baldissera et al., 2018). Our findings also demonstrated that AFB1 suppresses the
332
expression of atp1b1b gene, encoding Na+/K+-ATPase subunit in neurons. The
333
Na+/K+-ATPase plays an important role in maintaining ionic homeostasis in fish and
334
is a heterodimeric protein composed of one α and one β subunit (Doganli et al., 2013).
335
Eight Na+/K+-ATPase α subunits and five β subunits are identified in zebrafish, and
336
these various isoenzymes are likely to exhibit distinct functions within specific cell
337
and tissue types. The transcripts of atp1b1b, an ortholog of the vertebrate β1 subunit,
338
are majorly detected in the brain and eye tissues of adult zebrafish (Rajarao et al.,
339
2001). It implies that down regulation of atp1b1b expression by AFB1 may involve in
340
the neurological and behavioral change in zebrafish. AFB1 treatment did not
341
obviously affect the mRNA signals of other α or β isoforms based on the microarray
342
data.
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Among the nine differentially expressed genes that presented in Table 1, nlgn2b,
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syngr2a, syt5a, and grid 1b genes are located within presynapse/synapse/postsynapse
345
areas. The taar 64, adra1bb, and grid1b genes are grouped in the category of
346
“neuroactive ligand-receptor interaction”, based on the KEGG pathway. Baldissera et
347
al. demonstrate that AFB1-induced behavioral dysfunction is associated with the
348
alteration of neurotransmitters in fish brains (2018). AFB1 treatment also interferes
349
with the formation of biogenic catecholamine neurotransmitters and the regulation of
350
hypothalamic neuropeptides in adult rodents and chickens (Ahmed and Singh, 1984;
351
Jayasekara et al., 1989; Trebak et al., 2015). This information suggests that the
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AFB1 may interfere with synapse functions and the interaction among
353
neurotransmitters in various species, including zebrafish. The onset of primary neurogenesis in zebrafish begins at 10.5 hpf and is
355
completed within 24 hpf (Dam et al., 2011). The spatial and temporal patterns of
356
neural induction and axonal tract formation are usually assessed before 48 hpf (Dou
357
and Zhang, 2011). Therefore, in the present study the neuron morphology in
358
trigeminal ganglia and hindbrain areas was identified at 24 hpf under microscope to
359
avoid the interference of latter neural outgrowth. In addition, the mRNA levels of all
360
the neurogenesis-related factors, including gfap, huc, ngfa, atp1b1b, and prtga, were
361
detected at both 24 and 48 hpf. The trigeminal ganglia in zebrafish with a cluster of
362
30 neurons mediates the responses to mechanical stimuli and chemical irritants and
363
are responsible for later abnormal behavior (Saint-Amant and Drapeau, 1998).
364
Interrupting the formation of trigeminal ganglia neurons could be one of the factors
365
that contributed to the abnormal swimming behavior triggered by AFB1.
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The AFB1 concentrations we used (15-150 ng/mL) did not cause any apparent
367
morphological changes in embryos and larvae from 6 hpf to 7 dpf, as presented in Fig.
368
1 B and Fig. S2. Those 150 ng/mL AFB1-exposed fish which eventually died at 6 or 7
369
dpf exhibited an extremely abnormal swimming pattern, but still remained their
370
normal morphology right before their death. The main factor which contributes to the
371
death of AFB1-treated larvae without any morphological change remains unclear. In
372
addition to neurotoxicity, we also found that the liver size of 75 and 150
373
ng/mL-treated embryos was slightly shrunk compared to that of control. Other major
374
organs, including heart and kidney, are morphologically normal in larvae after 150
375
ng/mL AFB1 exposure (data not shown). AFB1 at a higher dose may play a role in
376
disrupting central nervous system development or neurotransmitter interaction,
377
leading to the high mortality of larvae at 6 and 7 dpf.
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5. Conclusion Aflatoxin B1 is the major mycotoxin which contaminates plant-based aquafeeds.
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AFB1-containing feed not only impairs the health of the cultured fish, but also has
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potential threats against the public health through the food chain. This study exposed
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zebrafish embryos to AFB1 concentrations relevant to aquafeed contamination and
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found that AFB1 at high dose promoted the locomotor activity of larvae, but reduced
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their startle response after environmental stimulation. AFB1 also disrupted the neural
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morphology of embryonic zebrafish, including trigeminal ganglia and hindbrain
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neurons. The transcriptome profiling of AFB1-treated embryos revealed that several
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differentially expressed genes are located within synaptic areas and functionally
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related to neurogenesis or neuroactive ligand-receptor interaction. Neural mal
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-formation and synapse dysfunction may contribute to the behavioral alteration
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induced by AFB1. Data presented herein can provide more information for estimating
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the health risk associated with the fish and the public.
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Acknowledgment
This work was financially supported by the National Science Council of the Republic of China, Taiwan, under Contract No. 104-2320-B-002-037-MY3.
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Conflict of Interest
Authors declare that there are no conflicts of interest.
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Legends of figures
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Figure 1. The viability and morphology of zebrafish embryos after AFB1 exposure.
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The 6 hpf WT embryos were treated with vehicle (0 ng/mL) or various concentrations
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of AFB1 (15-150 ng/mL). (A) The survival rate of embryos was determined at the
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designate time. Values are presented as the mean ± SD from four independent
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experiments with at least 20 fish embryos per treatment group in each experiment. (B)
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Morphology of 7 dpf larvae after exposure to AFB1. Images were taken from the
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lateral view under a dissecting microscope (magnification ×25). Scale bar, 500 µM.
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Figure 2. The locomotor activity of zebrafish larvae after AFB1 treatment. The WT
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embryos were treated with vehicle or various concentrations of AFB1 (15-75 ng/mL)
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from 6 hpf to 7 dfp. (A) The percentage of fish with an abnormal swimming pattern
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were recorded at 7 dpf and presented as means ± SD from three independent
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experiments with at least 18 larvae per exposure concentration in each experiment.
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The average velocity (B), absolute turn angle (C) and maximum velocity after
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stimulation (D) at 7 dpf are shown with box plots, in which boxes represent the 25th
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and 75th percentiles and whiskers represent the 10th and 90th percentiles. Solid lines
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and dashed lines within the boxes indicate the median and mean values, respectively.
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Data are collected from at least 52 larvae (control, n=70; 15 ng/mL, n=52; 30 ng/mL,
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n=52; 50 ng/mL, n=53; 75 ng/mL, n=53). Black dots are data points not within the
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range of 10th to 90th percentile. *, p<0.05 ; **p<0.01 vs the control.
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Figure 3. AFB1 activated gfap gene expression. WT embryos at 6 hpf were exposed to
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vehicle, 75 and 150 ng/mL of AFB1, and then the levels of gfap transcript were
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analyzed with semi-quantitative PCR and qPCR at 24 and 48 hpf. (A) The
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representative data from semi-quantitative PCR. (B) The expression of gfap was
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determined by qPCR. Each set of data represents mean ± SEM from four independent
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experiments. The data of vehicle control is arbitrarily regarded as 1.0. *p < 0.05 vs the
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Figure 4. AFB1 caused neurogenesis defects in Tg(HuC: eGFP) embryos. Transgenic
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embryos showing GFP in neurons were exposed to vehicle, 75 and 150 ng/mL AFB1
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at 6 hpf. (A) The images of 24 hpf embryos were taken under a fluorescent
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microscope in a lateral view. The TG areas are demonstrated with square shapes (a, b,
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c); (a’, b’, c’) are enlarged figures of (a, b, c). The morphology of Hb neurons is
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displayed in (d, e, f). (B) The levels of huc mRNA in 24 and 48 hpf embryos were
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detected by qPCR (left) and semi-quantitative PCR (right). The qPCR data are mean ±
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SEM from four independent experiments. The data of vehicle control is arbitrarily
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regarded as 1.0. * p < 0.05; ** p < 0.01 vs control. TG, trigeminal ganglion; Hb,
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hindbrain; Scale bar, 50µm.
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Figure 5. AFB1 disrupted neural formation in WT embryos. WT embryos were treated
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with vehicle (0 ng/mL) or 150 ng/mL AFB1 from 6 to 24 hpf. Whole-mount
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immunostaining was conducted with anti-acetylated α-tubulin antibodies. (A) The
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representative figures of TG areas. The (a’, b’) derived from (a, b) are enlarged
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figures of TG. (B) The representative figures of Hb areas from the dorsal view. The
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(a’, b’) derived from (a, b) are enlarged figures of Hb area. TG, trigeminal ganglion;
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Hb, hindbrain; Scale bar, 100 µm.
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Figure 6. AFB1 suppressed the expression of neurodevelopment-related genes. WT
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embryos at 6 hpf were exposed to vehicle, 75 and 150 ng/mL AFB1 and then mRNAs
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were collected at 24 and 48 hpf for qPCR analysis of ngfa (A), atp1b1b (B), and prtga
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(C). The data were mean ± SEM from at least three independent experiments. The
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data of vehicle control is arbitrarily regarded as 1.0. *, p < 0.05; **, p < 0.01 vs the
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control .
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Table 1. Characteristics of differential expression genes related to neural functiona symbol
gene name
FCb
cellular component by GOc term
NM_131064
ngfa
nerve growth factor a
3.5 ↓
membrane-bounded vehicle
NM_001166329
nlgn2b
neuroligin 2b
3.1 ↓
NM_001083102
taar 64
Trace amine associated 2.7 ↓ receptor 64
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Accession code
Postsynaptic membrane integral to membrane
NM_001080585
syngr2a
synaptogyrin 2a
1.8 ↓
NM_131671
atp1b1b
Na+/K+ ATPase, beta 1b
1.6 ↓
NM_001103137
syt5a
Synaptotagmin Va
1.5 ↓
Presynapse/calcium ion binding
NM_001045030
prtga
protogenin homolog a
2.0 ↑
integral to membrane
NM_001007358
adra1bb adrenoreceptor alpha 1Bb
ENSDART
grid1b
SC
2.1↑
ionotropic 2.8 ↑
integral to membrane postsynaptic membrane
neuroactivity-related genes from microarray analysis of WT embryos treated with vehicle or 150 ng/mL AFB1 from 6 to 48 hpf.
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b c
integral to membrane
receptor delta Type 1b
00000152431 a
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glutamate
Presynatic vesicles
Figure 1
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(A)
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40 20 0
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4
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7 (dpf)
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mL)
(C)
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**
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75 (ng/mL)
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48 hpf
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50 mm
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50 mm
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TG neuron (enlarged)
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AFB1 (ng/mL)
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(A) ngfa mRNA level (fold of control)
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prtga mRNA level (fold of control)
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ACCEPTED MANUSCRIPT Aflatoxin B1 (AFB1) is the major mycotoxin in plant-based aquafeeds
AFB1 interfered with locomotor activity of zebrafish larvae
AFB1 disrupted neural development of embryonic zebrafish
AFB1 altered the marker genes involved in neuroactivity and neurogenesis
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