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. Author manuscript; available in PMC: 2022 Jul 10.
Published in final edited form as: Sci Total Environ. 2021 Feb 26;777:146075. doi: 10.1016/j.scitotenv.2021.146075

Study of locomotion response and development in zebrafish (Danio rerio) embryos and larvae exposed to enniatin A, enniatin B, and beauvericin.

Ana Juan-García 1,3, Cristina Juan 1, Marie-Abèle Bind 2, Florian Engert 3
PMCID: PMC8154722  NIHMSID: NIHMS1677909  PMID: 33677298

Abstract

Mycotoxins are secondary metabolites produced by a variety of fungi that contaminate food and feed resources, and are capable of inducing a wide range of toxicity. Here, we studied the developmental and behavioral toxicity in zebrafish (Danio rerio) embryos and larvae exposed to three mycotoxins: beauvericin (BEA), Enniatin A (ENN A), and Ennitain B (ENN B). Zebrafish embryos were collected after fertilization, treated individually from 1 to 6 dpf with BEA at 8, 16, 32 and, 64 μM and for both enniatins at 3.12, 6.25, 12.5 and, 25 μM. Mixture of mycotoxins were assayed as follows: i) for BEA + ENN A and BEA + ENN B at [32 + 12.5] μM and [16 + 6.25] μM; ii) for ENN A + ENN B at [12.5 + 12.5] μM and [6.25 + 6.25] μM and, iii) for BEA + ENN A + ENN B at [32 + 12.5 + 12.5] μM and [16 + 6.25 + 6.25] μM. Response was collected after a white light-flash intermittent coming on for 5 seconds during 2 hours with a imaging platform. Outcomes measured were: time to death, response to light, and circadian rhythm. This last outcome was measured in a plate where embryos had evolved in natural intervals of light and dark until day 7 or in a plate maintained in darkness. Images of all stages and evolution were collected. Results indicated that mycotoxins induced toxicity at the concentrations tested. All exposed zebrafish induced developmental defects, specifically hatching time and motion activity. After exposure, fish showed enhanced baseline activity but they lost their responsiveness to light.

Keywords: zebrafish, mycotoxins, mixture, locomotion, toxicological studies, circadian rythm

Graphical Abstract

graphic file with name nihms-1677909-f0006.jpg

1. Introduction

The analysis of food, feed, and its products show the presence of secondary metabolites produced for filamentus fungi, known as mycotoxins and reported previously (Stanciu et al. 2017, Juan et al 2019; Oueslati et al 2020). Mycotoxins are causing several problems in aquaculture due to contaminated feed, which constitutes a primary risk to farm-raised fish. This implies a direct effect in cultured fish but also in consumers by passing along the contaminants via the food chain (Anater et al., 2016). Few mycotoxins have been classified by the International Agency for Research in Cancer (IARC), but effects in vivo and in vitro have been reported. In vitro effects include altering mitochondrial membrane potential, cell cycle distribution, cell death, and the production of reactive oxygen species (ROS) (Prosperini et al., 2013a, Juan-García 2019, Mallebrera 2015, Dornetshuber et al., 2007; Gammelsrud et al., 2012); while in vivo effects consist of diarrhea, vomiting, respiratory and cardial failure, genotoxicity, sepsis, estrogenicity, embryotoxicity, carcinogenicity, and death in acute toxicity assays (Yang et al., 2014; Sherif et al., 2009; EFSA 2010, 2011)

To reduce in vivo studies and to implement the 3R′s principle (reduction, refinement and replacement), model systems as zebrafish (Danio rerio) have been proposed (Juan-García et al., 2020). Zebrafish is a good candidate due to the characteristics associated to its initial development stages (embryos and larvae); among that, the Organization for Economic Co-operation and Development (OECD) established zebrafish as a good standard for ecotoxicological test and toxicology studies, such as developmental and neuro behavioral toxicity, cardiotoxicity, measurement of estrogen receptor activation, (Hill et al., 2005; Reimers et al., 2006; Strähle et al., 2012; OECD 236, 2013; Rodríguez-Fuentes et al., 2015; Juan-García et al., 2020). Zebrafish and fish in general were the earliest vertebrates in the planet able to adapt to changing environments. Brainstem, midbrain and diencephalon are well-developed areas with implications in sensor, motor and integrative central nervous circuits comparable with humans (Zdanova, 2011). Circadian rhythm in zebrafish, although not completely understood form the mechanistic point of view, it seems to oscillate closely to that of mammals. It has been suggested that the circadian information can be transmitted throughout animals by mechanism that are autonomous and non-cell based but with implications of molecular circadian oscillations which also modulate the distinct phases in brain regions (Mosser et al., 2019).

One of the most studied mycotoxin for environmental effects has been zearalenone (ZEA), which has estrogenic activity associated in the range of natural steroid estrogens (Arukwe et al., 1999; Celius et al., 2000; Keles et al., 2002). However, in the last decade, the prevalence of emergent mycotoxins such as beauvericin (BEA), enniatins (ENNs), and fusaproliferin (FUS), has increased. Although studies are still scarce compared to traditional studies examining mycotoxins, the mycotoxin scientific community does not consider them anymore as emergent.

BEA and ENNs are cyclohexadepsipeptides belonging to emerging mycotoxins group and are considered potential anticarcinogenic drugs. BEA is synthesized by various toxigenic fungi from genus Fusarium, and the entomopathogenic Beauveria bassiana; while ENNs are produced by Fusarium fungi. The main variants of ENNs are ENN A, A1, B, and B1 together with minor amounts of other ENNs (e.g., J1, J3, A2, B4) (Feudjio et al., 2010). BEA induces mitochondrial modifications, cell cycle disruption, cell death, while ENNs are known to have ionophoric properties able to incorporate in cell membranes and for cation selective pores (Ivanova et al., 2006; Tonshin et al., 2010).

It has been demonstrated that some mycotoxins cause neurotoxicity. It has been reported that AFB1 causes alterations in locomotion and swimming patterns in zebrafish revealing an impairment of the blood brain barrier and neurotransmitters in the fish brain (Baldissera et al., 2018); similarly, this happened for patulin (PAT) in zebrafish at different concentrations, reporting increases of locomotion (Ciornea et al., 2019) and for ZEA, abnormal swimming behavior (circular) (Muthulakshmi et al., 2018). Information of toxic effects of ENN A, ENN B, and BEA exposures on aquaculture has not yet been reported. The aim of this study was to investigate the potency of ENN A, ENN B and BEA (individually and in binary and tertiary combinations) (Table 1) on zebrafish embryos and larvae (at the stage of 6 hpf to 7 dpf) to various concentrations relevant to the levels in contaminated food as reported in several studies (Juan et al., 2019, 2017 and 2012). The effect was studied by investigating possible effects of continuous exposure and measuring survival status, time to death, hatching status, motion activity (stimulus of movement/locomotion of embryonic zebrafish), and circadian response.

Table 1.-.

Concentrations of mycotoxins assayed in larvae zebrafish (Danio rerio) for individually and combined treatment.

Concentrations assayed
Mycotoxins
ENN A 3.15, 6.25, 12.5 and 25 μM
ENN B
BEA 8, 16, 32 and 64 μM
Mycotoxin’s mixture High Low
ENN A + BEA [12.5 + 32] μM [6.25 + 16] μM
ENN B + BEA [12.5 + 32] μM [6.25 + 16] μM
ENN A + ENN B [12.5 + 12.5] μM [6.25 + 6.25] μM
Triple (ENN A + ENN B + BEA) [12.5 + 12.5 + 32] μM [6.25 + 6.25 + 16] μM
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2. Materials and Methods

2.1. Reagents

All reagents and cell culture components were standard laboratory grade from Sigma-Aldrich (St. Louis Mo. USA). The standard BEA (MW: 783.95 g/mol), ENN A (MW: 681.90 g/mol), and ENN B (639.82 g/mol) were purchased from Sigma-Aldrich (Milwaukee, WI USA), as well as methanol (MeOH). The final concentration of MeOH in the medium for all assays was ≤ 1% (v/v) as per established.

2.2. Test species and husbandry

Wild-type (WT) AB strain zebrafish (Danio rerio) were maintained on a 14-hour light/10-hour dark cycle at 28°C and fed two times daily with live Artemia nauplii and/or powder food. To generate embryos, adults were placed in spawning tanks in the afternoon and then spawned following the onset of light the next day. Larvae from bucket crosses were collected and pooled in blue water [density of > 5 ml per larva (pH 7.2); sodium bicarbonate buffer, methylene blue (1 g/liter), and instant ocean salt (0.2 g/liter)]. Live embryos were selected ~24 hours post-fertilization (hpf) and transferred to the 96 well/plate with one embryo per well. All protocols and procedures involving zebrafish were approved by the Harvard University/Faculty of Arts and Sciences Standing Committee on the Use of Animals in Research and Teaching (Institutional Animal Care and Use Committee).

2.3. Mycotoxin exposure (individual and combined)

BEA (CAS:26048-05-5), ENN A (CAS:2503-13-1), and ENN B (CAS:917-13-5) were first dissolved in MeOH at a concentration of 6400 μM for BEA and 2500 μM for ENNs (ENN A and ENN B), and stored at 20°C as a stock solution. Healthy and normally developing WT-AB zebrafish embryos at 1day post-fertilization (dpf) were collected and exposed to vehicle or various concentrations of mycotoxins in egg water. To examine the survival rate and morphology of embryos/larvae, mycotoxins in stock solution were diluted with 0.01M phosphate buffered saline (PBS, pH 7.0), and further diluted with egg water (60 mg sea salts/L, Instant Ocean® sea salts) to the tested concentrations. In detail, assays were performed by exposure to mycotoxins as reported in Table 1: i) individually at 8, 16, 32 and 64 μM for BEA, and at 3.12, 6.25, 12.5 and 25 μM for ENNs (ENN A and ENN B); ii) in binary and tertiary combination was assessed in two scenarios of high and low concentrations at [12.5 + 32] μM and [6.25 + 16] μM, respectively for [ENN A + BEA] and [ENN B + BEA]; and [12.5 + 12.5] μM and [6.25 + 6.25] μM, respectivley for [ENN A + ENN B]. Lastly, tertiary combination [ENN A + ENN B + BEA] was assayed at [12.5 + 12.5 + 32] μM and [6.25 + 6.25 + 16] μM for high and low scenarios, respectively. Exposure time was different according to the parameter and the measurement in each experiment.

2.4. Larvae zebrafish development assay

The day after crossing zebrafish, fertilized embryos were visualized in the microscope, selected and placed in flat-bottom 96 well plate containing 200μL per well of larvae′s growth water. Growth development was followed by taking well images with a stereomicroscope (Olympus MVX10) from 6 hpf to 6 dpf. 96 well plates were maintained on a 14-hour light/10-hour dark cycle at 28°C in an incubator. Columns 1 and 12 were firstly treated with the mycotoxins at concentrations reported in Table 1 or MeOH. Every day, zebrafish were treated from columns 1 and 12 in inwards direction (from column 1st to 6th and from 12th to 7th) serving as a control of untreated the images taken the previous day. The assay was performed twice and replicates were each time a total of four wells.

2.5. Zebrafish embryos dead, alive, and hatched

Fertilized embryos were visually selected and placed in flat-bottom six-well plate containing 3mL per well of larvae′s growth water (i.e., 20 eggs per well). Embryos were exposed to different concentrations of mycotoxins of ENN A, ENN B, and BEA (individual and combined treatment, Table 1). The number of eggs dead, alive, and hatched (evolved) were visually counted under a stereomicroscope after 6, 24, 48, 72, and 96 hpf. The assay was performed twice with 20 eggs per well.

2.6. Larvae zebrafish phototaxis (light stimulus)

The system design to measure the light stimulus assay is described in Jordi et al., (2018). Briefly, 96 well plates with one larvae zebrafish per well treated were tracked for 2 h to a light stimulus (IR light source) from above. The instrument had a zebrafish imaging platform that permitted intermittent flash lights that came on for 5 seconds during 2h. A camera (IDS UI-3370CP-NIR) was positioned above the plate to record the signal. Assays were performed by collecting the response after light-flashes and saving a total of 40–80 trials per run. For data analysis, a program designed in Prof. Florian Engert′s lab (MCB Department, Harvard University) and described in Jordi et al. (2018) was used. This program focuses on measuring a time period of larvae zebrafish movement in a coordinate area. For each well, it was reported an amplitude signal (arbitrary units, AU) which was analyzed and compared with control wells (no-treated). Outcomes of interest measured were: response to light, variation circadian rhythm, and time to death. All assays were performed twice by using 96 well plates and replicates were each time a total of four wells.

2.6.1. Response to light

Response movement of larvae zebrafish to light flashes were recorded during 2h with the equipment described in the previous paragraph. One 96-well plate with larvae zebrafish exposed to different mycotoxins (individually and combined, see Table 1) were run each day with a total of 40 trials per run from 1dpf to 6dpf. A total of 240 trials per mycotoxin was recorded. The outcome measurement consists of the increases of the amplitude signal from the baseline signal after the flash light comes on. Measurement was reported as response to light-flash of zebrafish larvae.

2.6.2. Circadian rhythm

Zebrafish eggs placed in 96 well plate (one per well) had evolved in natural intervals of light and dark or in a plate maintained in darkness until day 7. Afterwards, larval zebrafish were exposed to different concentration of two mycotoxins individually (BEA and ENN B) (Table 1), and plates were run with the equipment detailed above for 3 days in a row (72h), arriving to 9 and 11 dpf for dark-light and dark plates, respectively. A total of 80 trials per day were recorded (i.e., 240 trials in total). Changes in the stimulus of light-flashes were recorded and compared with controls.

2.6.3. Time to death

For carrying out the time to death assay, we previously ensure that zebrafish larvae were alive; which was assessed with the equipment detailed above (section 2.6). Once zebrafish larvae were exposed to different concentration to mycotoxins (individually and combined, see Table 1) from 1dpf to 6dpf, the absence of movement to light-flash stimulus was recorded as time to death. Changes in the amplitude signal recorded from the detection of movement (of alive larvae) to absence of movement (plane line signal after light-flash), was used to establish the time to death, as well as the lapse time until this stopped to happen.

2.7.-. Statistical analysis.

Data were expressed as mean ± SEM (standard error of the mean) of four independent experiments. The statistical analysis of the results was included in the equipment designed for the measurement.

3. Results

3.1. Morphology and development-growth of zebrafish exposed to mycotoxins.

In order to observe the survival rate and morphology of larvae exposed to mycotoxins ENN A, ENN B, and BEA, images were taken, evaluated, and reported in Supplementary 1. We observed no obvious anatomical phenotype upon toxin exposures (for times and doses assayed) until death set in; which suggest that no change in development and growth of larvae zebrafish occurred after mycotoxin exposures.

3.2. Larvae zebrafish dead, alive, and hatched after mycotoxin exposure

A total of 120 larvae zebrafish were exposed to different concentrations of mycotoxins individually and combined (see section 2.5 and Table 1), and the number of dead larvae, alive, and hatched ones was calculated after visualization in a steromicroscope (Olympus MVX10). Figure 1A reports the effect of individual ENN A treatment, where dead larvae started to be observed at the lowest concentration of 3.12 μM and 48 hpf (70%). The higher the concentration, the higher the percentage of dead larvae and the sooner the detection of dead (Figure 1A). At 25 μM, the percentage of larvae dead was 90% at 24 hpf. After 48hpf, 100% larvae were detected as dead. Opposite to this, the percentage of alive larvae started to decrease after 24hpf; however, any larvae evolved (Figure 1A).

Figure 1.

Figure 1.

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Percentage of larvae zebrafish (Danio rerio) that were dead, alive and evolved after mycotoxins exposure (from 6hpf to 96 hpf) at different concentrations: ENN A (A) and ENN B (B) at 3.12, 6.25, 12.5 and 25 μM; while BEA (C) at 8, 16, 32 and 64 μM. Data are expressed as mean values ± SEM of 240 larvae zebrafish (Replicates of two 6 well-plates with 20 eggs per well).

For ENN B, the strongest effect was seen at 25 μM after 24hpf for the number of dead (100%) and alive (0%) larvae zebrafish (Figure 1B). However, the number of hatched larvae oscillated between 50% and 90% for 12.5 μM and 3.12 μM, respectively; while at 25 μM, none of the larvae evolved (Figure 1B).

Lastly, for BEA (Figure 1C), the highest and the lowest concentration had an opposite behavior for the percentage of dead larvae as well as for alive. Death: at 64 μM and 24hpf 100% of larvae were dead, while at 8 μM and 72hpf none of the larvae was dead (Figure 1C). Alive: at 64 μM and 24hpf none of larvae were alive, while at 8 μM and 72hpf, 100% of the larvae were dead (Figure 1C). Larvae exposed to BEA evolved only at 8 μM 48 hpf (60%), although it decreased at 96hpf (20%) (Figure 1C). An estimation of the lethal concentration of the 50% of the zebrafish larvae (LC50) was calculated for ENN A, ENN B and BEA (Table 1S).

Regarding percentages of dead, alive, and hatched larvae zebrafish in mycotoxins mixtures (binary and tertiary), high and low concentrations exposures reported a very similar profile (Supplementary 2). Percentage of dead larvae went to 100% after 24hpf, none alive larvae zebrafish was recorded after 24hpf, and none larvae zebrafish evolved from 6hpf.

3.3. Larvae zebrafish activity (motion) exposed individually to ENN A, ENN B, and BEA

Response to flash-light was recorded for larvae zebrafish exposed to BEA, ENN A and ENN B at different concentrations from 1 dpf to 6 dpf for individual (Figure 2) and combined (Figure 3) treatment.

Figure 2.-.

Figure 2.-

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Motion activity of larvae zebrafish (Danio rerio) exposed to several concentrations of ENN A (A), ENN B (B) and BEA (C). Data are expressed as mean values ± SEM of 192 larvae zebrafish (Replicates of two 96 well-plates with one egg per well). (*) p ≤ 0.05, (**) p ≤ 0.01 and (***) p ≤ 0.001 represents significant difference as compared to control (no treatment).

Figure 3.-.

Figure 3.-

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Motion activity of larvae zebrafish (Danio rerio) from 1 dpf to 6dpf exposed to mycotoxin mixtures: ENN A + BEA (A), ENN B + BEA (B), ENN A + ENN B (C) and ENN A + ENN B + BEA (D). Data are expressed as mean values ± SEM of 192 larvae zebrafish (Replicates of two 96 well-plates with one egg per well). (*) p ≤ 0.05, (**) p ≤ 0.01 and (***) p ≤ 0.001 represents significant difference as compared to control (no treatment).

Among all three mycotoxins, ENN A showed the lowest values of motion followed by BEA and ENN B (Figure 2). For ENN A, the most marked records of motion compared with larvae of younger age, were observed at 5 dpf and 6 dpf, although for 5dpf, values reported were below the controls (Figure 2A). Specifically, for 5dpf, we observed a decrease in a concentration dependent manner; while for 6dpf, we observed an increase from the lowest concentrations assayed, until 12.5 μM which recorded the highest motion respect to the control, to decrease at the highest concentration assayed (25 μM) (Figure 2A).

ENN B jointly with BEA reported the highest motion of larvae zebrafish from 1 to 6 dpf. Larvae zebrafish of 2 dpf exposed at 3.12 μM of ENN B reached the highest motion with respect to the control to follow a decrease in a concentration dependent manner (Figure 2B). At 3 dpf similar tendency was observed although at 25 μM, an increase with respect to the control was observed. Older larvae zebrafish (4, 5 and 6 dpf) showed a similar behavior by increasing the motion with respect to the control at 3.12 μM and kept (in those levels of motion) for 5 dpf; while for 4 and 6 dpf, it was only maintained until 12.5 μM to end up decreasing at 25 μM (Figure 2B).

Figure 2C reports the amplitude of the response collected for larvae zebrafish exposed to BEA. At all concentrations, we observed increased motion activity compared to the control. An increase from 1dpf to 2 dpf was noticed at all concentrations; while at 3 dpf, a decrease was observed at concentrations above 16 μM For larvae zebrafish of 4 dpf, motion at 32 μM reached the highest increase to abruptly decrease at 64 μM (Figure 2C). At 5 dpf, motion increased at the lowest concentration assayed (8 μM) to describe a smooth decrease of motion with higher concentrations; however, an inversely increase was observed for 6 dpf until 32 μM to decrease at 64 μM (Figure 2C).

3.4. Larvae zebrafish activity (motion) exposed to combinations of ENN A, ENN B, and BEA

Larvae zebrafish for combination of mycotoxin is reported in Figure 3. Among all assays performed, only larvae fish exposed at the highest concentration ([12.5 + 32] μM) of ENN A + BEA and ENN B + BEA in 6 dpf and 5 dpf respectively, reported motion activity increases compared to the controls. Among that, for ENN A + BEA motion activity increased compared to younger larvae (Figure 3A). For ENN B + BEA, a decrease in motion compared to controls was observed for both high and low concentrations of exposure, although increases of motion were higher as older the larvae zebrafish (Figure 3B). Mixture of both ENNs revealed that the older the larvae zebrafish the higher the motion and being more marked for higher than lower concentrations (Figure 3C). Lastly, for tertiary or the three-factorial combination, some motion activity was noticed at 4 dpf, 5 dpf, and 6 dpf. The highest activity was observed at the highest concentration in 5dpf larvae zebrafish, all other scenarios reported decreases with respect to the control (Figure 3D).

3.5. Larvae zebrafish circadian rhythm

Alterations in circadian rhythm of larvae zebrafish by ENN B and BEA are collected in Figure 4. Assays for ENN A were not performed as the motion activity detected in Figure 2A were very low after 5dpf as compared with ENN B and BEA, denoting a high potential toxicity (see discussion section). Larvae zebrafish kept in dark conditions reported lower motion activity than those that followed a normal light dark conditions cycle (Figure 4).

Figure 4.-.

Figure 4.-

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Motion activity in circadian rhythm (light and dark cycles) of larvae zebrafish (Danio rerio) exposed to several concentrations of ENN B (A) and BEA (B). Data are expressed as mean values ± SEM of 192 larvae zebrafish (Replicates of two 96 well-plates with one egg per well). (*) p ≤ 0.05, (**) p ≤ 0.01 and (***) p ≤ 0.001 represents significant difference as compared to control (no treatment).

In dark conditions, ENN B for larvae zebrafish of 9dpf and 10 dpf increased the motion activity for concentrations assayed with respect to the control; opposite to this happened for 11 dpf where a decrease was observed (Figure 4A). For BEA, increases with respect to the control were observed for 9 and 10 dpf for concentrations assayed; while for larvae zebrafish of 11 dpf a decrease with respect to the control was observed (Figure 4B).

In normal light-dark conditions, results of larvae zebrafish exposed to ENN B in 7, 8, and 9dpf revealed a decrease in motion activity with respect to the control at concentrations assayed; although at 6.25 μM, increases were higher than at 3.12 μM (lowest concentration assayed) except for 7 dpf (Figure 4A). Similar behave was observed for BEA, although in here lower concentrations revealed higher motility action values than lower concentrations (Figure 4B).

3.6. Time to death of larvae zebrafish exposed to ENN A, ENN B and BEA.

The absence of movement and the images taken allowed to establish the time-to-death for each mycotoxin assayed in larvae zebrafish (Figure 1, Figure 5 and Supplementary 2).

Figure 5.-.

Figure 5.-

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Time-to-death of 100% larvae zebrafish (Danio rerio) exposed to different concentrations of individual treatment (A) (ENN A, ENN B and BEA) and combined (B) ENN A + ENN B, ENN A + BEA, ENN B + BEA and ENN A + ENN B + BEA.

By measuring the absence of movement, it was noticed that 100% of dead larvae zebrafish occurred i) for ENN A from 25 μM to 6.25 μM after 48hpf and for 3.12 μM after 72hpf (Figure 5A); ii) for ENN B after 48hpf for 25 μM, after 72 hpf for 12.5 μM and 6.25 μM and only 20% of dead larvae zebrafish was detected after 96 hpf for ENN B 3.12 μM, and iii) for BEA after 24 hpf for 64 μM, after 72 hpf for 32 μM and 16 μM, 80% of dead larvae zebrafish were detected after 96 hpf for BEA 8 μM (Figure 5A).

Mixture treatment of high and low concentrations, revealed that 48 hpf was the critical point for all mixtures, except for triple and binary low concentration ENN A + ENN B that 72 hpf was the age at which 100% of larvae exposed were dead (Figure 5B).

4. Discussion

Mycotoxins constitute a global problem in productivity, as well as economical and health consequences with great problems associated (WHO, 2010). Mycotoxins as AFB1, CIT, DON, OTA (α-OTA), PAT, T-2 toxin, and ZEA (and its metabolites, α-ZOL, β-ZOL) have been studied individually and combined in zebrafish (Danio rerio) (Juan-García et al., 2020); however, only for some of them, locomotion activity in early stages has been assayed. AFB1 in aquafeed has been estimated to be present in more than 60% of samples production, as well as in fish (Fallah et al., 2014; Anater et al., 2016). Its potential adverse effect for the environment and surface water have been recently reported, indicating alterations in behavior and neurodevelopment in early embryonic stages (Wu et al., 2019).

Mycotoxins alteration effects in live/dead of embryos/larvae zebrafish or on its growth development (hatch/evolve) for ENN A, ENN B, BEA, and its mixtures, showed effects with different implications happening at very low concentrations and short-term (24 hpf and 48hpf) (Figures 1 and 5 and Supplementary 2). Concentrations assayed comprise a wide range for ENN A and ENN B: upper value at 25 μM and lower value at 3.12 μM, corresponding to 16 times the values that can be found in food/feed and the minimum concentration found in food/feed, respectively (Prosperini et al., 2013a, 2013b). To the best of our knowledge, we cannot compare our results with other publications, as there are no previous studies in zebrafish with these mycotoxins. However, results suggest that in individual treatment, the strongest effect was reported for ENN A, followed by ENN B and BEA (Figure 1 and 5); to notice that BEA at the highest concentration assayed (64 μM) was lethal to all larvae zebrafish. In mixture exposures and scenario of high concentration, all larvae zebrafish were dead at 48h; while this happened in assays at low concentration for ENN A + BEA and ENN B + BEA. At low concentration for ENN A + ENN B and for ENN A + ENN B + BEA, all larvae zebrafish were dead after 72hpf (Figure 5).

Literature reveals that there are many mycotoxins (and mixture of them) exerting their toxic effect by producing oxidative stress in several cell lines and at different concentrations (Prosperini et al., 2013a, 2013b; Juan-García et al., 2018, 2020; Kang et al., 2019, Dai et al., 2019; Agahi et al., 2020b, Taroncher et al., 2020). Oxidative stress plays a crucial role in inhibiting acetylcholinesterase (AChE) (Schallreuter et al., 2004). Alterations of AChE in aquatic spp and organisms have been used as biomarker in neurotoxicity studies as it implies affectation in neurotransmission in cholinergic synapses and neuromuscular junction which it is translated in affectation in motion activity (movement/locomotion) (Schallreuter et al., 2004). Recently, Muthulakshmi et al. (2018) have postulated that oxidative stress detected in zebrafish embryos exposed to ZEA (750 and 950 mg/L) is the responsible of neurotoxicity effects associated to a significant inhibition of AChE. Mycotoxins studied in here have strong oxidative stress effect as previously reported in Caco-2 cells (at 1.5 and 3 μM) and in CHO-K1 cells for BEA (at 1 and 5 μM) (Prosperini et al., 2013a, 2013b; Mallebrera et al., 2015), and less strong in Jurkat-T cells exposed to ENN B and BEA (1.5, 3 and 5 μM) (Manyes et al., 2018). So that, there is no doubt that oxidative stress plays an important role on their mechanism of action, and subsequently in motion activity. In this regard and considering the alterations that this can cause in AChE, motion activity alterations in zebrafish were observed in here. There are studies of ROS detection in zebrafish exposed to OTA and PAT although implications in motion activity were not measured (Tschirren et al., 2018; Ciornea et al., 2019). Here, it is the first time where this is performed for BEA, ENN A, ENN B, and its mixtures. From results associated with these mycotoxins, and due to the different patterns of response to light according the concentration of the mycotoxin (Figures 2 and 3), it can be extracted that zebrafish have a response in motion associated to a level of oxidative stress generated by mycotoxins. This linked effect has been described when exposing zebrafish to other substances, and increases in ROS generation alter cholinergic system involved in activity modulation (Agostini et al., 2018; Giovanni et al., 2015).

Activity motion and response to light are also influenced by the exposure time to mycotoxins and the age (hpf/dpf) of zebrafish. At 48hpf, zebrafish develop the first cholinergic neurons in the zebrafish spinal cord (motoneurons); at 72hpf, additional cholinergic neurons are developed in the eyes and in optic tectum around 5dpf (Arenzana et al., 2015). The activity motion reported in Figures 2 and 3 covers from first development stages of zebrafish (embryos), so from cholinergic neurons (1dpf) to complete formation (6dpf), either in individual and in mixture treatment. In brain electrical activity modulation, it has been demonstrated that cholinergic system plays an important role, and alterations on it can be associated to damages in activity (Giovanni et al., 2015). In summary, neurotransmissions as well as its development can be altered by exposure to different contaminants, and a result of such is linked to a number of diseases, including movement disorders (Sarter et al., 2006; Werner et al., 2010).

Zebrafish have by 6dpf an active swimming, response to stimuli, hunting for prey and displaying distinct periods of rest (Zhdanova 2011). Motion activity alterations by mycotoxins in circadian rhythm reported in Figure 4 (7 dpf to 11dpf) denote a stronger response in light-dark cycles than in continuous dark cycles (control values in Figure 4), coinciding with a study of Zhdanova (2011). It is not known if zebrafish have distinct sleep stages; however, sleep in zebrafish appears to have rhythms of increase depth of sleep or short arousals. In addition, melatonin production, photoreception, and autonomic oscillations are the major neurohumoral output of the circadian system in zebrafish (Danilova 2004) and it is not known the role of the central nervous system of zebrafish in circadian rhythmicity (Cahill et al., 1998).

Exposure to different concentrations of mycotoxins keeps the profile of lower amplitude in dark profile than in dark-light cycle; however, oscillations are noticeable for each concentration of mycotoxin, which cannot be associated to different stages of sleep as in humans, because it is unknown if those occur. Nonetheless, increase depth of sleep or short arousals have been detected as periodic event in sleep studies of Zhdanova (2011) as well as peak of melatonin production at night. Several studies have revealed the protective effect of melatonin against the toxicity of different mycotoxins (review of Iranshahy et al., 2020) in vivo and in vitro, especially against oxidative stress; however, in this study and for the dose assayed, it was impossible to get the entire toxic effect produced by these mycotoxins revealing that more studies are needed.

5. Conclusions

In summary, WT AB zebrafish exposed to ENN A, ENN B and BEA did not produce any obvious anatomical phenotype (for times and doses assayed) and no changes in development and growth of larvae zebrafish were affected. Measurement of larvae zebrafish dead, alive and hatched increased with the concentration of mycotoxins, as well as the percentage of dead larvae, thus detecting sooner dead larvae. In mixtures of mycotoxins, the percentage of dead larvae went to 100% after 24hpf, while no larva was alive neither evolved after 24hpf and 6hpf, respectively. For motion activity, among all three mycotoxins ENN A showed the lowest values of motion followed by BEA and ENN B. For circadian rhythm, larvae zebrafish kept in dark conditions reported lower motion activity than those that followed a normal light-dark conditions cycle for individual and mixture exposures. And finally, for time-to-death, it was noticed that 100% of dead larvae zebrafish occurred at different times in individual treatment; while in mixtures, 48 hpf was the critical point for all mixtures except for ENN A + ENN B, and triple combination which it was at 72 hpf.

Effects reported in this study manifest the danger of mycotoxins affecting environment and ecosystems revealing larvae/embryos zebrafish as a excellent alternative method for the study of toxic effects and the implementation of 3R principle.

Supplementary Material

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Highlights.

  • Mycotoxins affect environment and ecosystems as larvae/embryos zebrafish Danio rerio.

  • Zebrafish dead, alive and hatched increased with the concentration of mycotoxins.

  • ENN A showed the lowest values of motion followed by BEA and ENN B.

  • In all assays lower motion activity was reported for dark cycles than for light-dark cycles.

  • Time-to-death was critical at 48 hpf in binary mixtures and at 72 hpf in triple mixture.

Acknowledgements

This research was supported by the Spanish Ministry of Science and Innovation PID2019-108070RB-100ALI. Research reported in this publication was also supported by the John Harvard Distinguished Science Fellow Program within the FAS Division of Science of Harvard University, and by the Office of the Director, National Institutes of Health under Award Number DP5OD021412 (Marie-Abèle Bind). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. AJG acknowledges the Engert′s Lab at MCB Department and Harvard University for the welcome; and University of Valencia and RCC-Harvard University for the award received.

All persons who have made substantial contributions to the work reported in the manuscript (e.g., technical help, writing and editing assistance, general support), but who do not meet the criteria for authorship, are named in the Acknowledgements and have given us their written permission to be named. If we have not included an Acknowledgements, then that indicates that we have not received substantial contributions from non-authors.

Footnotes

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Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Supplementary Materials

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NIHMS1677909-supplement-1.pptx (160.5KB, pptx)
2
NIHMS1677909-supplement-2.pptx (59.3KB, pptx)
3
NIHMS1677909-supplement-3.docx (12.8KB, docx)

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