Effects of dietary exposure to ciguatoxin P-CTX-1 on the reproductive performance in marine medaka (Oryzias melastigma)

Marine Pollution Bulletin 152 (2020) 110837

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Effects of dietary exposure to ciguatoxin P-CTX-1 on the reproductive performance in marine medaka (Oryzias melastigma)

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Meng Yana,b, , Maggie Y.L. Maka,b, Jinping Chenga,c, Jing Lia,d, Jia Rui Gua,d, ⁎ Priscilla T.Y. Leunga,b, , Paul K.S. Lama,b,d a

State Key Laboratory of Marine Pollution, City University of Hong Kong, Hong Kong, China Research Centre for the Oceans and Human Health, City University of Hong Kong Shenzhen Research Institute, Shenzhen, China c State Key Laboratory of Marine Pollution and Department of Ocean Science, The Hong Kong University of Science and Technology, Hong Kong, China d Department of Chemistry, City University of Hong Kong, Hong Kong, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Gambierdiscus Ciguatoxin Bio-encapsulation Reproduction Oryzias melastigma Maternal transfer

Ciguatoxins are natural compounds produced by benthic dinoflagellates Gambierdiscus and Fukuyoa spp., which cause fish intoxication by ciguatera fish poisoning. This study aimed to assess the dietary exposure effects of ciguatoxin P-CTX-1 on the reproductive performance in marine medaka (Oryzias melastigma). Fish which ingested > 1.16 pg·day−1 for 21 days exhibited abnormal behaviors including diarrhea, abnormal swimming, loss of appetite and decreased egg production. After 7-day exposure to P-CTX-1 at a dose of 1.93 pg·day−1, significant gender-specific differences in reproductive performance and decreased hatching rate of the offspring were observed. Chemical analysis of P-CTX-1 showed that the P-CTX-1 accumulation rates were 24.1 ± 1.4% in female fish and 9.9 ± 0.4% in male fish, and 0.05 pg·egg−1 was detected. The results illustrate that dietary exposure to P-CTX-1 affected the reproductive performance and survival of offspring, and caused bioaccumulation and maternal transfer of P-CTX-1 in marine medaka.

1. Introduction Ciguatoxins (CTXs) are polycyclic polyether compounds mainly biotransformed from precursor gambiertoxins which are produced by the toxic benthic dinoflagellates Gambierdiscus and Fukuyoa spp. (Murata et al., 1989, 1990; Lewis, 2006; Lewis et al., 2016). CTXs are generally lipophilic and can be biomagnified in top predatory fish through marine food chains (Holmes and Lewis, 1994; Yasumoto and Satake, 1996; Larsson et al., 2019). Bio-oxidation of CTXs leads to species-specific toxin profiles (Randall, 1958; Helfrich and Banner, 1963; Oshiro et al., 2009; Mak et al., 2013). Ciguatera fish poisoning (CFP) is the most common food-borne illness in humans caused by the consumption of marine fish contaminated by CTXs (Friedman et al., 2008; Nuñez et al., 2012; Boucaud-Maitre et al., 2018). Marine fish are the primarily affected taxa in subtidal ecosystems dominated by CTX-producing toxic dinoflagellates (Mak et al., 2013). More than 90 species of fish including many commercially important species belonging to families such as Serranidae (grouper), Carangidae (jacks), Acanthuridae (surgeon fish) and Muraenidae (moray eel), are linked to CFP (Kohli et al., 2015; U.S. Food and Drug Administration, 2019). Based on the geographic distribution and structural variants, CTXs

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are classified into three groups, i.e., Pacific ciguatoxins (P-CTXs), Caribbean ciguatoxins (C-CTXs) and Indian Ocean ciguatoxins (I-CTXs). Among the CTX congeners, P-CTX-1 is the most potent member (Dickey and Plakas, 2010; Hossen et al., 2015) and is the major toxin found in contaminated carnivorous fish. Several studies reported that P-CTX-1 can pose a health risk to humans at levels above 0.01 μg·kg−1 fish (Dickey, 2008; Dickey and Plakas, 2010). Being a neurotoxin, P-CTX-1 can elicit various effects on the electrophysiological properties of voltage-gated sodium channels (Nav) and subsequently enhances neuronal excitability and causes ciguatera poisoning (Mattei et al., 1999; Strachan et al., 1999; Pearn, 2001; Dechraoui et al., 2006; Inserra et al., 2017). Given the strong linkage between marine fish and CTXs, several studies have attempted to elucidate the possible toxic mechanisms that are associated with different responses in fish upon exposure to CTXs. For example, in surgeonfish (Acanthuridae) toxins accumulate in muscle after dietary exposure, causing no apparent harmful effect (Helfrich and Banner, 1963; Clausing et al., 2018). In contrast, mullet (Mugilidae) which ingested an equivalent quantity of CTXs to that in surgeonfish (Acanthuridae), exhibited strong signs of intoxication, with rapid distribution of toxins into the systemic circulation and little

Corresponding authors at: State Key Laboratory of Marine Pollution, City University of Hong Kong, Hong Kong, China. E-mail addresses: [email protected] (M. Yan), [email protected] (P.T.Y. Leung).

https://doi.org/10.1016/j.marpolbul.2019.110837 Received 30 September 2019; Received in revised form 14 December 2019; Accepted 18 December 2019 0025-326X/ © 2020 Elsevier Ltd. All rights reserved.

Marine Pollution Bulletin 152 (2020) 110837

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2.3. Behavioral responses upon P-CTX-1 exposure

retention potential for CTXs (Ledreux et al., 2014). In some other studies, developmental toxicity induced by CTXs was observed in medaka fish (Oryzias latipes) embryos microinjected with CTX in the yolk sac, including hyperkinetic twitching, severe spinal deformities and hatching failure (Edmunds et al., 1999; Colman et al., 2004). A similar exposure approach with microinjection of CTXs was also utilized in marine medaka (O. melastigma) in some recent studies. It was found that exposure to CTXs could lead to behavioral changes, altered physiological performance and reduced survivability in medaka larvae (Mak et al., 2017). Another study showed toxic effects on embryos and larvae such as abnormalities in development, reduced heart rate, hatching failure, abnormal swimming, hyperkinetic twitching, and altered expression of genes related to stress and immune responses, abnormal cardiac and bone development, and apoptosis (Yan et al., 2017) Apart from the primary route through dietary uptake, maternal transfer could also be another possible course of CTX exposure in fish. A study reported that the concentration of CTXs in wild-caught barracuda was higher in eggs than in muscle tissue (Colman et al., 2004), suggesting that toxins contaminating a fish are transferable to their eggs. However, information on the ciguatoxicity on reproduction and parental effect in marine fish is largely unknown. This study aimed to elucidate the effects of P-CTX-1 on the reproductive ability of adults and the parental effects in the developing embryos of the common marine model fish, marine medaka. Concentrations of P-CTX-1 in treated adults and their larvae were also analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

A toxicity test was firstly performed to determine the various general symptoms of medaka fish that could be caused by dietary exposure to P-CTX-1 by using male adult individuals. Female adult medaka fish were not chosen for this test, because CTXs could be concentrated in eggs compared with liver and muscle oviparous fish (Colman et al., 2004) and female medaka fish are able to spawn daily, which could result in underestimating the toxicity effects in adult fish. In this pilot study, P-CTX-1 solutions at five concentrations (33.3, 66.6, 166.5, 333.0 and 666.0 ng·mL−1) were injected into fish eleuthero-embryos (equiv. 0.23, 0.46, 1.15, 2.31 and 4.62 pg·eleuthero-embryo−1), five healthy adult male medaka fish (4-month-old) were exposed to P-CTX-1 at each dose by feeding them with five eleuthero-embryos daily. The exposure period lasted 21 days. Sub-lethal effects on behavior including swimming, feeding and features of fish feces were recorded daily. Three symptoms that could reflect body condition were recorded during the exposure period: (1) diarrhea, (2) abnormal swimming and (3) low appetite. Diarrhea refers to fish feces that turned lighter in color and shorter in shape and the water appeared turbid (Mitchell et al., 2017). Abnormal swimming is loss of equilibrium, erratic swimming or loss of orientation of a fish (Davin et al., 1986), while low appetite refers to a fish individual that only ate < 3 eleuthero-embryos daily. After the exposure, the fish were weighed after anesthetization with drops of 0.1% MS-222 solution (Sigma-Aldrich, St. Louis, USA). 2.4. Acute toxicity tests to evaluate the effect on egg production

2. Material and methods

In order to further determine an appropriate dose of P-CTX-1 for subsequent dietary exposure, five male fish and five female fish were grouped in one tank with 1.5 L of artificial seawater and four replicates (tanks) for each exposure dose. The new set of exposure doses were adjusted based on the results of the abnormal behavioral effects, at 0.08, 0.19, 1.93 and 3.86 pg·fish−1·day−1. Fish were fed individually before the dark cycle started and put back into the original tanks gently every day. The exposure period lasted 7 days. About 1/3 of the water was changed with fresh artificial seawater twice per week. Eggs in each tank were collected in the morning, and then counted and observed daily. Fish samples were weighed after anesthetization with drops of 0.1% MS-222 solution (Sigma-Aldrich, St. Louis, USA).

2.1. Chemical preparation The in-house P-CTX-1 standard (purity ≥ 95%) was isolated and purified from the viscera of P-CTX-1 contaminated moray eels (Gymnothorax flavimarginatus and G. undulatus) by Wu et al. (2011) and Mak et al. (2013) according to methods described previously (Lewis et al., 1991; Hamilton et al., 2002). P-CTX-1 was quantified by highperformance liquid chromatography-tandem mass spectrometry (HPLCMS/MS) analysis, as described previously (Wu et al., 2011). Serially diluted P-CTX-1 solutions were prepared in PBS with 5% Tween 60 (Sigma-Aldrich, Irvine, UK) (Yasumoto et al., 1984; Jacquet et al., 2004). The concentrations of P-CTX-1 solutions ranged from 11.1 to 666.0 ng·ml−1 as described in a previous study (Yan et al., 2017). PBS with 5% Tween 60 (5% Tween 60 PBS) was used as the vehicle solution.

2.5. Reproductive toxicity and gender differences induced by P-CTX-1 For the exposure, a dose of 1.93 pg·fish−1·day−1 (8.39 pg·g−1 of body wet weight·day−1) was applied. This concentration is based on the sub-lethal responses observed in tests from Sections 2.3 and 2.4. To reveal the gender-specific differences in reproductive toxicity induced by P-CTX-1, three experiments were set up: (1) with both male and female fish exposed, (2) with only female fish exposed and (3) with only male fish exposed (Fig. 1). In each treatment tank, three pairs of fish were placed and maintained as mentioned in Section 2.4. Four replicate tanks were used for each set of experiment. The exposure period lasted for 7 days and the fish were fed eleuthero-embryos with P-CTX-1 as described in Section 2.4. Eggs in each tank were collected and counted in the morning, and the hatching rate, hatching time and heart rate of the developing embryos and larvae were recorded every day. To estimate the hatching rate, every 10 eggs were randomly selected and incubated in a petri dish (9 cm in diameter) with sterile artificial seawater. The time for hatching (days) of each treatment was estimated using 30 surviving embryos randomly selected from each treatment at 8 days after fertilization. The record of heart rate was collected from the surviving individuals at the three time points (3 days post fertilization, 7 days post fertilization and 3 days post hatching) in each experiment. In order to detect the tissue burden of P-CTX-1 in different sexes of medaka and to examine the maternal transfer of P-CTX-1, another exposure experiment was performed. The limit of quantification for P-

2.2. Experimental fish and microinjection Adult marine medaka used for the exposure experiments (4-monthold, 0.23 ± 0.04 g wet weight, n = 28) were obtained from the fish colonies stock facility of the State Key Laboratory of Marine Pollution at City University of Hong Kong. Adult fish were maintained in artificial seawater (salinity 28‰), fed live brine shrimp larvae, Artemia sanfransisca, twice a day, at 24 °C, with a 14 h: 10 h light-dark cycle. Fish eggs were collected daily and eleuthero-embryos were hatched after 10–12 days post fertilization (dpf) at 24 °C in artificial seawater. Eleuthero-embryo is defined as the phase from hatching to yolk sac absorption (Clay et al., 2008). Eleuthero-embryos (within 12 h posthatch) were microinjected with P-CTX-1 using a nitrogen-driven pneumatic picopump micro-injector (World Precision Instruments LLC, Sarasota, FL, USA) as described in Yan et al. (2017). Fish eleutheroembryos freshly injected with P-CTX-1 were employed as bio-vehicles of P-CTX-1 for the dietary exposure in adult medaka fish. P-CTX-1 in eleuthero-embryos was quantified using UPLC-MS/MS. It was assumed that all P-CTX-1 in eleuthero-embryos were consumed by the adult medaka fish and not dissolved in seawater. The concept of bio-encapsulation was firstly proposed by Poncelet (1996). 2

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Fig. 1. An overview of the design of P-CTX-1 exposure to adult medaka. The gender symbols (♂♀) in red with plus signs indicate the fish were under the exposure to P-CTX-1.

CTX-1 was 12.5 pg · g−1 dry weight, four pairs of male and female medaka adult fish were maintained and exposed to a single dose of PCTX-1 at 1.93 pg · fish−1 · day−1 as mentioned previously for 28 days so as to accumulate a considerable amount of P-CTX-1 for HPLC-MS/MS detection. Freshly produced eggs were collected each morning and stored at −20 °C immediately, the fish parents were weighed and sampled after anesthetization with drops of 0.1% MS-222 solution (Sigma-Aldrich, St. Louis, USA). Fish samples were stored at −20 °C for chemical analysis.

washed with 65% aqueous methanol (6.5 mL), and the target analyte was eluted with acetonitrile (16 mL). The eluent was dried under a stream of high-purity nitrogen. The residue was then re-dissolved in 3 mL of acetonitrile and passed through a PSA cartridge (GL Sciences INC, 500 mg, 6 mL) that was preconditioned with 15 mL of methanol and 15 mL of acetonitrile. The cartridge was washed with 3 mL of acetonitrile and then P-CTX-1 was eluted with 12 mL of methanol. The eluent was dried under a stream of high-purity nitrogen and then redissolved in 100 μL of methanol.

2.6. Extraction of P-CTX-1 in fish samples

2.7. Instrumental analysis

The extraction of P-CTX-1 referred to previously established methods (Yogi et al., 2011; Mak et al., 2013). Briefly, each fish sample i.e. female fish fed P-CTX-1, male fish fed P-CTX-1, and the eggs produced by adult fish fed P-CTX-1 was homogenized and mixed with 3.5 g of diatomaceous earth (Thermo Fisher Scientific, Waltham, MA, USA) as a dispersant and drying agent, and was transferred into a 22 mL stainless steel extraction cell (Dionex, Sunnyvale, CA, USA) with two Whatman glass fiber filters (Thermo Fisher Scientific, Waltham, MA, USA) placed at the bottom of the cell. Extraction of P-CTX-1 was carried out using an ASE 200 system (Dionex, Sunnyvale, CA, USA) at 75 °C and 1500 psi with 5 min of heating followed by two 5-minute static extractions. Methanol was used as the extraction solvent. The extraction cell was flushed with 60% methanol with a purge time of 100 s. Extracts were concentrated to dryness using rotary evaporation. Solid phase extraction (SPE) was employed for cleaning up the crude extract for instrumental analysis. The crude extract was dissolved in 11 mL of 50% aqueous methanol before loading into C18 cartridges (Bond Elute, Agilent, 500 mg, 6 mL) which were preconditioned with acetonitrile (15 mL, Merck GmbH, Darmstadt, Germany), methanol (15 mL, Merck GmbH, Darmstadt, Germany) and MilliQ water (10 mL) successively. The crude extract was then passed through the pre-conditioned cartridges at a rate of 1 drop s−1. The cartridges were subsequently

Instrumental analysis was performed as previously described (Mak et al., 2013). Briefly, the levels of P-CTX-1 in various tissues were detected by the Agilent 1290 HPLC system (Agilent, USA) equipped with a 5500 QTRAP (AB Sciex, USA). Purified extract (10 μL) was separated by a Phenomenex Kinetex C18 column (100 × 2.1 mm i.d., 1.7 μm, Phenomenex, Torrance, CA, USA) with a flow rate of 200 μL/min. Mobile phases consisted of (A) Milli-Q water and (B) 95% acetonitrile in MilliQ water, both of which contained 0.1% formic acid (98%–100%, Sigma-Aldrich, St. Louis, MO, USA) and 2 mM ammonium formate (≥ 99.995%, Sigma-Aldrich, St. Louis, MO, USA). The initial gradient condition was set at 50% B and then maintained for 1 min. It was ramped to 100% B within 1 min and kept for 8 min before returning to 50% B in 0.1 min, followed by equilibrating at 50% B for 5 min until the next injection. Multiple reaction monitoring (MRM) HPLC-MS/MS analysis for P-CTX-1 was run using [M + H-2H2O]+ as target parent ions and the fragment ions in Q1 and Q3, as follows: P-CTX-1: m/z 1128.4 > 1075.5, m/z 1128.4 > 1057.5 and m/z 1128.4 > 1039.5. 2.8. Data analysis Abnormality levels in response to the different treatments including number of eggs, percentage of hatching success and heart rate were 3

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production in experiment Set 1, with both female and male fish exposed to P-CTX-1 and experiment Set 2, with female fish exposed to P-CTX-1. However, in experiment Set 3, with male fish exposed to P-CTX-1, the number of eggs laid by three fish pairs increased significantly (271 ± 11 eggs · week−1) compared to those in control groups (172 ± 83 eggs·week−1 and 201 ± 61 eggs·week−1, F = 4.12, x2 = 0.05, n = 4, p < 0.05) (Fig. 3).

compared using one-way analysis of variance (ANOVA) followed by a post-hoc Student–Newman–Keulstest using SPSS Statistics 25 software (SPSS Inc., Chicago, IL, USA). Statistical significance was accepted at p < 0.05. 3. Results 3.1. Behavioral responses

3.4. Parental effects on the survival and early development of offspring Two adult male fish died on exposure day 3 and day 4 after ingestion of 46.20 pg P-CTX-1 (Table S2). The accumulated amount of PCTX-1 in adult fish was estimated with the numbers of bio-vehicles consumed. The toxic behavioral responses observed in adult male fish included diarrhea, abnormal swimming and loss of appetite in a dosedependent manner. The occurrence of these three main symptoms varied at different exposure doses and different times. Diarrhea was the earliest symptom in comparison with abnormal swimming and loss of appetite, with the time (days) to onset of symptom outbreak with maximal individuals within 7 days. The daily dose at or over 2.31 pg fish−1 caused severe loss of appetite (< 50%) from day 8 (Table S1). After calculation with the number of eleuthero-embryos with P-CTX-1 ingested by fish, the average accumulated amounts (estimated value) of P-CTX-1 in fish for each group were 205.80 ± 32.59 pg · fish−1, 137.41 ± 65.15 pg · fish−1, 43.42 ± 9.89 pg · fish−1, 30.99 ± 11.59 pg · fish−1 and 23.00 ± 0.21 pg · fish−1 (Table S2).

Significant hatching failure and delay of hatching were observed in the developing embryos from the treated pairs of Experiment Set 1 or either one of the parents of the pair of Experiment Set 2 and Experiment Set 3. The survival rates of eggs laid by medaka fed P-CTX-1 dropped sharply to 5 ± 2% in Experiment Set 1 with both treated male and female fish of the pair. The survival rate was, 15 ± 10% in Experiment Set 2, with the treated female fish in the pairs. The survival rate was 8 ± 4% in Experiment Set 3, with the treated male fish in the pairs (Fig. 4-A). The hatching time was delayed significantly; for example, in Experiment Set 1, the hatching period was 18 ± 5 days (Fig. 4-B). The heart rates of the developing embryos laid by fish exposed to PCTX-1 were decreased (SNK test, p < 0.05), i.e., in the Experiment Set 1, the heart rates were 63 ± 3 beats·min−1 at 3 dpf (days post fertilization) and 87 ± 6 beats·min−1 at 7 dpf; in Experiment Set 2, the heart rates were 60 ± 4 beats·min−1 at 3 dpf and 87 ± 2 beats·min−1 at 7 dpf; in Experiment Set 3, the values were 60 ± 4 beats·min−1 at 3 dpf and 87 ± 6 beats·min−1 at 7 dpf, which is less than those observed in the vehicle solution group (5% Tween 60 PBS, 71 ± 5 beats·min−1 at 3 dpf and 100 ± 2 beats·min−1 at 7 dpf). There was no difference in heart rate in all groups at a later developmental stage of larval fish at 3 dph (days post hatching) (Fig. 5).

3.2. Reproductive performance: egg production capability upon exposure to P-CTX-1 Exposure to a high dose of P-CTX-1 (3.86 pg · fish−1 · day−1) corresponded to a significant (p < 0.05) decline in egg production from each pair of fish with a mean value at 5 ± 4 (mean ± S.D., n = 4) eggs · week−1. In fish with ingested P-CTX-1 at the dose of 1.93 pg · fish−1 · day−1, the mean numbers of eggs laid per week was 108 ± 45 (mean ± S.D., n = 4), showing no significant difference from the values for lower doses and the control (Fig. 2). Based on the aforementioned results, a maximum concentration that could cause no apparent effect on egg production, that is 1.93 pg · fish−1 · day−1, was selected as the exposure dose for the subsequent investigation of the maternal transfer of P-CTX-1 in marine medaka fish.

3.5. Bioaccumulation of P-CTX-1 in fish samples Analysis of P-CTX-1 levels accumulated in fish samples revealed that both female and male fish had accumulated detectable quantities of PCTX-1. During the 28d exposure, both male and female fish were fed with eleuthero-embryos that pre-injected with P-CTX-1 1.93 pg·eleuthero-embryo−1). The females consumed the eleuthero-embryos everyday during the course of the exposure period, but the males ceased eating after Day 22 due to the negative effect on their appetite. The estimated totally ingested amount of P-CTX-I was 53.93 pg per female and 42.37 pg per male. P-CX-1 in female fish were 11.43 to 15.25 pg·fish−1 (dry weight, 44.37 ± 7.57 mg, n = 4), and it was 3.72 to 4.92 pg·fish−1 in male fish (dry weight, 32.62 ± 3.96 mg, n = 4). In female fish, 24.1 ± 1.4% of the total ingested P-CTX-1 was accumulated, and the value in male fish was lower at 9.9 ± 0.4% after ingestion of 42.37 pg P-CTX-1 (Fig. 6). P-CTX-1 in eggs laid by parents contaminated with P-CTX-1 was also detected at 0.05 pg·egg−1. The recovery of P-CTX-1 in the analytical process was 74.9%; concentrations reported were not corrected for recovery (more details are shown in the Supplementary data).

3.3. Reproductive performance: Gender-specific difference in reproductive performance Dietary exposure to P-CTX-1 exhibited no effects on the egg

4. Discussion 4.1. The feasibility of dietary exposure to P-CTX-1 in fish using bio-capsules In this study, we established a method to produce bio-capsules of PCTX-1 using fish eleuthero-embryos. The method of bio-encapsulation has been used to deliver biologically or pharmacologically active substances to living animals such as fish. Several examples exist such as, planktonic crustacean (brine shrimp, daphnia, copepod and rotifers) eggs of the polychaete worm Nereis virens, enriched with active materials such as fatty acids, GFP-expressing yeast, beneficial bacteria or antibiotics which are utilized as functional fish feed in the aquaculture industry (Katharios et al., 2005; Barroso et al., 2015; Kolman et al.,

Fig. 2. Number of eggs laid by medaka exposed to four doses of P-CTX-1. Data are presented as mean (+S.D.), and bars with different letters indicate significantly different means (p < 0.05; SNK test, n = 4). 4

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Fig. 3. Egg production by three pairs of adult fish exposed to P-CTX-1 (1.93 pg·fish-1·day-1, 7 days). Number of eggs laid by adult medaka pairs in (A) experiment Set 1 treatment (both female and male fish were exposed to P-CTX-1), (B) in experiment Set 2 treatment (only female fish were exposed to P-CTX1) and (C) experiment Set 3 (only male fish were exposed to P-CTX1). Vehicle: adult medaka fed eleuthero-embryos injected with 5% Tween 60 PBS; Blank control: adult medaka fed eleuthero-embryos without treatment. Data are presented as mean (+ S.D.), and bars with different letters indicate significantly different means (p < .05; SNK test, n = 4). Red indicates exposure to P-CTX-1. The gender symbols (♂♀) in red with plus signs indicate the fish were under the exposure to P-CTX-1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

CTXs varied among fish species (Helfrich and Banner, 1963; Clausing et al., 2018; this study). An early laboratory study revealed that toxins causing ciguatera are transferred through feeding without apparent harm to the carrier, the herbivorous fish Acanthurus triostegus (Acanthuridae) (Helfrich and Banner, 1963). Similar results were found in the herbivorous and planktivorous fish species, Naso brevirostris (Acanthuridae). The juvenile fish were given gel food containing toxic cells of Gambierdiscus polynesiensis for 16 weeks (89 cells g−1 fish daily, 0.4 μg CTX3C equiv. kg−1 fish). Although the ciguatoxin CTX3C accumulated in the fish muscle, no behavioral signs of intoxication were observed during the exposure (Clausing et al., 2018). In contrast, ichthyotoxic effects have been reported in blue-headed wrasse (Thalassoma bifasciatum) and largemouth bass (Micropterus salmoides), showing signs of skin color variations, inactivity, loss of equilibrium and jerky feeding movements after dietary exposure to Gambierdiscus toxicus (Davin et al., 1986, 1988). Marine medaka, Oryzias melastigma (Adrianichthyidae) are naturally found occupying shallow lagoons and swamps among roots and mangroves (Menon, 1999), with less opportunity to be exposed to CTXs compared with other fish such as reef-associated species. Previously, marine medaka have exhibited developmental toxicity to P-CTX-1 at the embryonic and larval stages (Mak et al., 2017; Yan et al., 2017). In this study, adult medaka fish exhibited toxic responses including

2018; Loh et al., 2018; Embregts et al., 2019). Moreover, as an ideal feed, the brine shrimp Artemia has been used to study the toxicity of pollutants such as halogenated acetic acids (HAAs) and polybrominated diphenyl ether (PBDE-47) in Japanese medaka fish (O. latipes) or marine medaka fish (O. melastigma) (Muirhead et al., 2006; Schultz et al., 2007; Ye et al., 2012). Due to the limited availability of existing highly purified P-CTX-1, the enrichment method by feeding brine shrimps as described in previous studies was not suitable to be applied in this study. Facilitated by microinjection, a very small volume of P-CTX-1 solution (pg level) could be injected into the early stages of medaka fish, e.g., eleutheroembryos of medaka fish (Mak et al., 2017; Yan et al., 2017). Adult medaka fish prey on smaller animals such as juvenile fish, larvae and eleuthero-embryos (Kinoshita et al., 2012; our study, data not shown), in particular newly hatched eleuthero-embryos with a jerky swimming movement which can be caught the most easily by adult fish with a normal appetite (our study, data not shown). Thus, the use of microinjected eleuthero-embryos as bio-capsules for dietary exposure is an efficient method when small-dose application is needed. 4.2. Sensitivity of fish with ingested P-CTX-1 and changes in behaviors Feeding experiments showed that the sensitivity of fish exposed to 5

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Fig. 4. (A) Hatching success and (B) day of hatching of eggs laid by P-CTX-1 treated medaka pairs from Experiments Set 1, Set 2 and Set 3. Data are presented as mean (+ S.D.), and bars with different letters indicate significantly different means (p < .05; SNK test). Replications were n = 4 for each treatment in regard to hatching success and n = 30 for day of hatching. The gender symbols (♂♀) in red with plus signs indicate the fish were under the exposure to P-CTX-1. Fig. 5. Heart rate of developing embryos and larvae from parent fish exposed to P-CTX-1. Data are presented as mean (+ S.D.), and bars with different letters indicate significantly different means (p < .05; SNK test, n = 4). dpf: days post fertilization, dph: days post hatching. The gender symbols (♂♀) in red with plus signs indicate the fish were under the exposure to P-CTX-1.

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with better mate-guarding behavior; otherwise, they resist laying eggs, showing the ability to determine the timing of their spawning (Okuyama et al., 2014; Yokoi et al., 2016). In this study, three female and three male fish were maintained in one tank after consumption of P-CTX-1 individually. Male-male competition is believed to affect females' preferences regarding potential mates, and our results may suggest that the male medaka fish exposed to P-CTX-1 could have performed better in regard to mate-guarding behavior. However, the survival rate of the offspring was extremely low, suggesting underlying toxic effects during the reproduction process. On the other hand, in the group with female medaka treated with P-CTX-1, the exposure to PCTX-1 did not increase their egg production. It is interesting that the male medaka contaminated with P-CTX-1 promoted egg production. It is believed that the female fish may have been exposed to higher P-CTX1 toxicity because a higher level of P-CTX-1 was found to have accumulated in these females. Such disruption of reproduction in marine fish by algal toxins deserves further investigation. 4.4. Maternal transfer of P-CTX-1 and parental effects Fig. 6. P-CTX-1 ingested and accumulated in fish samples. Data are presented as mean (+ S.D.) (n = 4).

This study showed that ciguatoxin (P-CTX-1) accumulates in adult medaka fish and is transferrable to the eggs laid by fish after dietary exposure. This is the first laboratory study to reveal the possibility of the maternal transfer of CTXs in fish. Maternal transfer of other organic compounds to offspring in oviparous fish has been reported as well, such as organochlorine, tetrabromodiphenyl ether (PBDE) and polychlorinated biphenyls (PCBs) (Miller, 1993; Serrano et al., 2008; Arnoldsson et al., 2012; González-Doncel et al., 2017). It is reported that lipophilic contaminants could be transferred from the liver to oocytes during the vitellogenesis process in fish (Serrano et al., 2008). A high concentration (4.5-fold that in muscle tissue) of lipophilic CTXs has been detected in eggs collected from great barracuda (Sphyraena barracuda) in the Caribbean (Colman et al., 2014). It has also been found that ciguatoxin poisoning is associated with fish egg ingestion (Hung et al., 2005) indicating the potential of maternal transfer of the toxins. However, the health of eggs contaminated with CTXs in the natural environment remains unknown. In the laboratory, we found that the eggs from P-CTX-1 contaminated fish parents suffered severe hatching failure. Similar results were reported after direct exposure via microinjection of P-CTX-1 into fertilized medaka fish oocytes with a 96 h-LD50 at 1.50 pg egg−1 (Yan et al., 2017). The approach of exposure by using microinjection into fish eggs was used in several studies to simulate the maternal transfer CTXs (Edmunds et al., 1999; Colman et al., 2004; Yan et al., 2017). Although the mortality rate of the developing eggs was extremely high (> 90%), the amount of maternal transferred P-CTX-1 (0.05 pg·egg−1) was much lower than the 96 h-LD50 (1.50 pg·egg−1) in our previous study. Additionally, the developing embryos contaminated with maternally transferred P-CTX-1 showed decreased heart rates. In our previous study, P-CTX-1 exposure by microinjection could also induce decrease of heart rates and the overexpression of heart development-related gene Cox-2 in the developing embryos of marine medaka (Yan et al., 2017). A recent report showed that the heart rate of larval marine medaka could be significantly reduced under P-CTX-1 exposure using microinjection approach (Mak et al., 2017). However, our results revealed a normal heart rate of the exposed larvae as in the control group. This may be due to the acclimation to the maternal transferred P-CTX-1 by the larval fish. Further studies on the transgenerational toxicity and recovery in fish are needed to provide additional data on the effects of maternal transferred P-CTX-1 and potential detoxification strategies in female fish. Our results have provided supportive evidence for a previous perspective that maternal transfer of CTXs can happen in fish and it may represent an unrecognized threat to the reproductive success of reef fish (Edmunds et al., 1999). Bioaccumulation of CTXs was absent in a feeding study which used mullet Mugil cephalus (fish symptomatic to CTXs in the laboratory), and

diarrhea, abnormal swimming, loss of appetite and decline in egg production after dietary exposure to P-CTX-1 with a dose-dependent manner, and P-CTX-1 was detected in the fish (11.43 to 15.25 pg·fish−1 in female fish, and 3.72 to 4.92 pg·fish−1 in male fish). A similar response of losing of equilibrium and erratic swimming was also observed in blue-head Thalassoma bifasciatum (wrasse: Labridae) after exposure to Gambierdiscus toxicus cells, although the exposure dosage was not reported (Davin et al., 1986). This study for the first time reported the diarrhea, loss of appetite and decline in egg production as possible symptoms of toxic responses after dietary exposure to CTXs in fish. It is believed that the herbivorous surgeonfish A. triostegus and N. brevirostris with the chance to be natural carriers of CTXs (Helfrich and Banner, 1963; Gaboriau et al., 2014; Mak et al., 2013; Clausing et al., 2018) were found to be tolerant to CTXs in the laboratory, while other fish species that were not associated with ecological niches of toxic algae seemed to be more susceptible to the toxic effects which may even be lethal to them upon exposure to CTXs (Colman et al., 2004; Mak et al., 2017; Yan et al., 2017). However, the mechanism of the accumulation of CTXs and their distribution in the marine medaka fish remains unknown. The harmful impacts of CTXs on susceptible fish species which are non-endemic to current regions impacted by CTXs would be indicators of the presence of CTX-producing species in new areas. The risk of ciguatoxin-producing dinoflagellates on reef systems would be expected to increase because of the geographic expansion of Gambierdiscus spp. (Kohli et al., 2014; Vergés et al., 2014). 4.3. Gender-specific differences in reproductive performance As mentioned previously, lipophilic CTXs were more concentrated in eggs compared with other tissues in finfish (Colman et al., 2004), and gender-specific differences should be considered when assessing the reproductive toxicity of lipophilic materials in oviparous fish such as medaka fish. In this study, an increase in egg production was observed in fish pairs with male fish exposed to P-CTX-1. In general, the fecundity in fish is tightly linked to the process of differentiation of oocytes in the ovaries (Nishimura and Tanaka, 2014), which is affected by stocking density (Peck and Holste, 2006), light cycle (Koger et al., 1999), water temperature (Koger et al., 1999; Myers, 2001), age (Nasiadka and Clark, 2012), diet (Markovich et al., 2007), and the interaction with male fish (Eaton and Farley, 1974). In zebrafish, egg development may be triggered by the courtship behavior of male fish (Eaton and Farley, 1974), and a more recent study indicated that medaka females spawn when they accept the courtship from a male fish 7

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our study is the first to report on the detectable bioaccumulation of CTXs in marine medaka. However, the pathway of bioaccumulation of CTXs in marine medaka (fish symptomatic to CTXs in the laboratory) may differ from that in the natural carriers of CTXs such as surgeonfish (fish asymptomatic to CTXs in the laboratory) (Helfrich and Banner, 1963; Ledreux et al., 2014; Clausing et al., 2018). This study provides a novel perspective on the dynamics of CTXs in marine ecosystems, indicating that small oviparous fish like ricefish (fish symptomatic to CTXs) could contribute to the dynamics of CTXs or other phycotoxins in at least two ways, (a) transfer in food webs and (b) transfer between generations.

41306173 to MYL Mak and 41576113 to PTY Leung). The authors thank Dr. TC Wai, Dr. James CW Lam, Dr. Jack CH Ip and Mr. Roy JK Lam for their technical support during this study, and Mr. J Yan for the help on the illustration artworks. The authors also thank the anonymous reviewers for their useful comments and suggestions on the manuscript.

5. Conclusion

References

This study is a successful example demonstrating the use of eleuthero-embryos as a kind of bio-encapsulation to conduce dietary exposure to toxins with limited availability, e.g., CTXs in small fish models such as marine medaka fish. Our findings show that dietary exposure of P-CTX-1 to medaka can result in various behavioral responses including diarrhea, abnormal swimming, loss of appetite and impaired egg production. In the reproductive performance experiment, higher numbers of eggs were produced by females in the treatment pairs where only the males were exposed to P-CTX-1. The underlying reasons for this interesting observation deserve further investigation. For those eggs produced by adult fish treated with P-CTX-1 suffered from severe hatching failure. When compared with the two treated sexes, it is interesting to find that the amount of P-CTX-1 accumulated in the female fish was higher than in the male fish. Additionally, our results indicate that PCTX-1 is maternally transferrable to fish eggs. Altogether, this study provides a novel approach to use bio-encapsulation for the dietary exposure of CTXs to fish, advances our understanding of the reproductive toxicities in fish induced by P-CTX-1, and provides new information on the dynamics of P-CTX-1 in marine medaka.

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Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.marpolbul.2019.110837.

Author contributions Meng Yan, Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Validation, Visualization, Writing original draft; Maggie Y.L. Mak, Conceptualization, Funding acquisition, Investigation, Methodology; Jinping Cheng, Conceptualization, Methodology, Writing - review & editing; Jing Li and Jia Rui Gu, Investigation, Writing - original draft; Priscilla T.Y. Leung, Conceptualization, Formal analysis, Funding acquisition, Methodology, Resources, Supervision, Visualization, Writing - original draft, Writing review & editing; Paul K.S. Lam, Conceptualization, Funding acquisition, Resources, Supervision, Writing - review & editing. Declaration According to the Animals (Control of Experiments) Ordinance, Chapter 340 (Department of Health, Hong Kong SAR), permission was obtained to perform experiments involving fish in this project (Ref No. (16-36) in DH/HA&P/8/2/5 Pt. 5). Declaration of competing interest 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. Acknowledgments This project was conducted at the State Key Laboratory of Marine Pollution and supported by the following funding sources: Collaborative Research Fund (C1012-15G to PKS Lam); National Natural Science Foundation of China (41406184 to Meng YAN, 8

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