Journal of Xenobiotics 2018; volume 8:7820

Behavioral effects of the stage, an idea accepted under the concept of neurotoxin b-N-methylamino- Developmental Origin of Health and Correspondence: Alessandra Carion, Disease (DOHaD).2 However, even if Laboratory of Evolutionary and Adaptive L-alanine on the mangrove experimental and epidemiological studies Physiology, Institute of Life, Earth and ( support this concept, the mechanisms Environment, University of Namur, 61 Rue de Bruxelles, 5000 Namur, Belgium. explaining long-term delayed effects of marmoratus) larvae E-mail: [email protected] early life NCs exposure remain largely unclear.3 Alessandra Carion, Julie Hétru, Key words: Mangrove rivulus; Nowadays, an increasing number of Developmental origin of health and disease; Angèle Markey, people suffer from neurological disorders Neurotoxin; Behavior; b-N-methylamino-L- Victoria Suarez-Ulloa, Silvestre Frédéric such as Alzheimer’s, Parkinson’s or alanine.alanine. Laboratory of Evolutionary and Amyotrophic lateral sclerosis diseases, Conflict of interest: the authors declare no Adaptive Physiology, Institute of Life, which could be partly related to environ- potential conflict of interest. Earth and Environment, University of mental influences during ELS. Such dis- Namur, Namur, Belgium eases are triggered by a complex interplay Funding: this study was supported by the between genes, ageing, and environmental FRS-FNRS (Fonds de la Recherche conditions plus a random component.4 In Scientifique) PhD fellowship to A. Carion and this context, the need for understanding the a research grant number N°T.0174.14. role played by exposure to NCs has been Abstract growing in importance.5 Of particular inter- Conference presentation: part of this paper was presented at the 2018 EcoBIM meeting, Mangrove rivulus, Kryptolebias mar- est is b-N-methylamino-L-alanine May 22-25, Bordeaux, France. moratus, is a fish capable of (BMAA), a non-protein amino acid NC pro- self-fertilization. This particularity allows duced by the extremely ubiquitous Received for publication: 6 September 2018. to naturally produce highly homozygous cyanobacteria, dinoflagellates and diatoms.6 Accepted for publication: 17 October 2018. and isogenic individuals. Despite the low Although the first link between BMAA and only genetic diversity, rivulus can live in neurodegenerative diseases was established This work is licensed under a Creative extremely variable environments and adjust more than 50 years ago, evidence of BMAA Commons Attribution NonCommercial 4.0 its phenotype accordingly. This species rep- effects on human brain is still inconclusive License (CC BY-NC 4.0). resents a unique opportunity to clearly dis- 7 and remains controversial. On the other ©Copyright A. Carion et al., 2018 tinguish the genetic and non-genetic factors use hand, considering that BMAA can be found Licensee PAGEPress, Italy implicated in adaptation and evolution, such in aquatic food webs at high Journal of Xenobiotics 2018; 8:7820 as epigenetic mechanisms. It is thus a great concentrations,8 further studies about doi:10.4081/xeno.2018.7820 model in aquatic ecotoxicology to investi- BMAA risk assessment on aquatic organ- gate the effects of xenobiotics on the isms are urgently required. epigenome, and their potential long-term With this aim in mind, the present work impacts. In the present study, we used the are one of the most often cited mechanisms has been developed using a strategic model potentially explaining neuronal disorders mangrove rivulus to investigate the effects organism, the mangrove rivulus fish. 3 b and DOHaD. Effects on the epigenome do of the neurotoxin -N-methylamino-L-ala- Together with its sister species, the man- not involve changes in the DNA sequence nine (BMAA) on larvae behaviors after 7 grove rivulus (Kryptolebias marmoratus) is but can modify gene expression patterns in days exposure to two sub-lethal concentra- the only known that naturally a heritable manner through mechanisms tions. Results show that BMAA can affect reproduce by self-fertilization.9 In nature, such as DNA methylation (DNAme), his- the maximal speed and prey capture (trials this Cyprinodontiform oviparous fish is tone modifications, and non-coding RNAs. and failures), suggesting potential impacts characterized by an androdioecious mixed- Together, the emerging fields of neuroepi- on the organism’s fitness. mating reproductive system, in which her- genetics and environmental epigenetics maphrodites coexist with a low proportion Non-commercialof males (between 5 and 25%). While out- show that exposure to environmental NCs crossing with males is known to happen, it can affect the brain epigenome and conse- Introduction is much less frequent than self-fertilization quently lead to impacts on behavior and/or cognitive faculties.11 In this context, the Pollution involving neurotoxic com- of . Consistent self-fertiliza- rivulus constitutes an optimal model choice pounds (NCs) is one of the emerging issues tion is an extreme form of inbreeding and it for human health but also for wild species consequently naturally produces isogenic to investigate the role of epigenetic mecha- and ecosystems. In wild organisms, the lineages after a few generations.10 nisms in DOHaD and in transgenerational adverse outcome of exposure to NCs is a Additionally, despite its low genetic vari- inheritance via sexual reproduction, while change in behavior, which can affect an ability, the rivulus displays a high level of minimizing the confounding effects of 12,13 individual’s fitness and potentially lead to phenotypic plasticity and it is capable to genetic variation. population decline.1 Moreover, it is well efficiently acclimate to the highly variable The main objective of this study was to established that embryonic development environment of mangrove forests. These determine the immediate effects of BMAA and early life stages (ELS) are periods dur- features have recently put the mangrove exposure during ELS on locomotion and ing which the organism is particularly sen- rivulus in the spotlight of scientific prey capture behaviors in the rivulus. sitive to environmental stress, and conse- research, as it constitutes an innovative and Moreover, this preliminary work paves the quently to NCs exposure. In addition to valuable model for the identification of true ground towards a better understanding of immediate effects on ELS, developmental cause-effect relationships between the envi- the conspicuous consequences that NCs exposure at relatively low dose can lead to ronment, the phenotype and the epigenome. may have for populations of aquatic organ- adverse outcomes later in life, at the adult Impacts of NCs on the brain epigenome isms and their link with molecular mecha-

[Journal of Xenobiotics 2018; 8:7820] [page 7] Article nisms, such mechanisms including modifi- Ethovision® software. Afterwards, 10 nau- 1 and Figure 1A), while no effect has been cations of the expression level of some key plii of Artemia were provided in each well observed at the highest concentration. The genes involved in behavior and in nervous to measure larvae prey capture ability dur- prey capture assay revealed significant cells metabolism as well as potential varia- ing 5 min. Results are expressed as the per- effects of BMAA exposure on the percent- tions in epigenetic mechanisms. centage of success and failure, calculated as age of failure in prey capture and on the the proportion between successful capture total trials expressed by larvae to capture or failed capture trials over the total number prey (Figure 1B-C). The 20 µg/L and 15 of trials. mg/L groups respectively expressed a 14% Materials and Methods Agostino and Pearson tests were per- and 17% increases in total trials compared formed to confirm data normality. Rivulus larvae were exposed to 2 doses to group-controls. Moreover, these two Accordingly, one-way ANOVA or non- of BMAA directly after hatching for 7 days: exposure concentrations respectively parametric Kruskal-Wallis tests were 20 µg/L and 15 mg/L, plus a control unex- expressed a 13% and 15% increases in per- applied followed by Dunn’s multiple com- posed group (n = 24). Larvae were fed ad centage of prey capture failure compared to parisons test in order to evaluate an effect of libitum with Artemia salina every day. The controls. BMAA on larvae locomotion, thigmotaxis BMAA working solution was made from L- According to these results, BMAA or capability to capture prey. Percentages of BMAA Hydrochloride powder (Sigma- exposure during development impacts lar- success and failure were arcsine square root Aldrich®) mixed with 25 ppt (± 1) water. vae locomotion and capabilities to behave transformed. Statistical analyses were per- About 2/3 of water was renewed every day. appropriately to efficiently hunt their prey. formed using GraphPad Prism 7 Software. During exposure period, mortality was BMAA appears to mainly disrupt brain Significance level was set at P<0.05. assessed and larvae were individually meas- functions. Larvae maximum velocity was ured at 1, 3, 5 and 7 days post-hatching impacted by BMAA exposure at lowest (dph) using a Nikon Digital Camera USB3 concentration (20 µg/L) while no effect was ½.5 15 IM/SEC mounted on a Nikon evident at higher concentration (15 mg/L). SMZ1270 stereomicroscope and the NIS- Results and Discussion This suggests that an environmentally rele- Elements® program. Larvae locomotion No effect of BMAA exposure was vant only concentration of BMAA applied dur- and thigmotaxis (test adapted from Norton, observed on larvae growth, body shape and ing fish development can elicit changes in 2012) were video recorded after 7 days of mortality. Locomotion test showed a signif- maximum velocity possibly due to neuro- exposure (10 min of acclimation followed icant effect of 20 µg/L BMAA exposure on muscular effects and/or impairments in fish by 5 min of test) in 6-wells microplate. larvae maximum velocity with a use 61% perception of its environment.14,15 However, Larvae behaviors were analyzed using increase compared to group controls (Table these effects were not observed at a higher concentration suggesting a possible activa- tion of repair mechanisms limiting brain damages.16 Interestingly, the total number of prey capture for larvae exposed to BMAA did not differ from control group while larvae showed a higher rate of failure ABC since they tried more to catch a prey. This indicates that larvae exposed to BMAA need more trials to achieve the same suc- cess in prey capture with probably more energy expenditure. These observations support the assumptions of movement impairments induced by BMAA exposure and are consistent with literature in behalf Figure 1. Immediate effects of 7 days b-N-methylamino-L-alanine (BMAA) exposure on newly hatched rivulus larvae behaviors. A) A significant effect at 20 µg/L BMAA was of assumptions that BMAA can disrupt observed on larvae maximum velocityNon-commercial (P-value = 0.035); B) Larvae exposed to 20 µg/L synaptic signaling.17 Effects observed on and 15 mg/L BMAA expressed significant higher rate of failure during prey capture assay larvae serve as a baseline to assess possible (P-value = 0.016 and 0.004, respectively); C) significant higher trials to capture prey (P- consequences later in life. Movement value = 0.04 and 0.027, respectively) compared to controls. Results are expressed by mean ± SD. Different letters (a-b) mean significant differences (i.e., P-value <0.05) between impairments can lead to lower food intake conditions. and consequently impact fish growth, repro- duction and therefore organism fitness.

Table 1. Summary table including the number of biological replicates per treatment and results of locomotion and prey capture tests. Mean ± SD. Treatments Number of Locomotion test Prey capture test replicates Distance Thigmotaxis Maximum Total Prey capture Prey capture Maximum moved (cm) velocity (cm/s) trials success (%) failure (%) velocity (cm/s)

Ctl 24 39.33±36.14 0.83±0.48 2.06±1.46 10.08±2.12 94±17 7±10 3.09±5.11 20 µg/L 23 55.92±39.13 0.92±0.34 3.32±1.90 11.52±2.27 89±26 20±19 5.47±9.41 15 mg/L 24 49.45±25.30 0.91±0.22 2.72±1.36 11 .83±2.37 97±22 22±22 4.31 ± 6.34

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Epigenetics can explain long-term latent or model species to test long-term effects of Algae 2010;9:620-35. transgenerational effects. These hypotheses pollutants. 9. Costa WJEM, Lima SMQ, Bartolette R. will be tested in future experiments. in Kryptolebias killifish and the evolution of self-fertilizing her- maphroditism. Biol J Linn Soc 2010;99: References 344-9. 10. Tatarenkov A, Earley RL, Taylor DS, Conclusions 1. Hellou J. Behavioural ecotoxicology, an Avise JC. Microevolutionary Overall, 7 days exposure of sub-lethal “early warning” signal to assess envi- Distribution of isogenicity in a self-fer- concentrations of BMAA on newly hatched ronmental quality. Environ Sci Pollut tilizing fish (Kryptolebias marmoratus) larvae of mangrove rivulus revealed signif- Res 2011;18:1-11. in the Florida Keys. Integr Comp Biol icant effects on fish behavior. BMAA 2. Heindel JJ, Skalla LA, Joubert BR, 2012;52:1-10. showed non-monotonic effects, as 20 µg/L Dilworth CH, Gray KA. Review of 11. Blanco B, Barco A. Impact of environ- exposure increased maximum larvae speed, developmental origins of health and mental conditions and chemicals on the while 15 mg/L exposure had no effect. This disease publications in environmental neuronal epigenome. Curr Opin Chem emphasizes the need to test low concentra- epidemiology. Reprod Toxicol 2017;68: Biol 2018;45:157-65. tions when assessing the environmental 34-48. 12. Fellous A, Voisin A, Labed-veydert T, risk. Moreover, both concentrations under 3. Barouki R, Melén E, Herceg Z, Beckers Locrel M, Earley RL, Silvestre F. DNA study increased the number of trials neces- J, Chen J, Karagas M, et al. Epigenetics methylation in adults and during deve- sary for the rivulus to catch the same as a mechanism linking developmental lopment of the self- fertilizing man- amount of prey, suggesting impairment in exposures to long-term toxicity. grove rivulus, Kryptolebias marmora- energy expenditure and possible impacts on Environ Int Pergamon 2018;114:77-86. tus. Ecol Evol 2018;1-18. ’s fitness. Further molecular analyses 4. Riancho J, Bosque-Varela P, Perez- 13. Voisin A-S, Fellous A, Earley RL, such as gene-specific DNAme and gene Pereda S, Povedano M, de Munaín AL, Silvestre F. Delayed impacts of deve- expression in brain will provide new Santurtun A. The increasing importance lopmental exposure to 17-alpha-ethiny- insights into the modes of action of BMAA. of environmental conditions in amyo- only lestradiol in the self-fertilizing fish Further experiments on the delayed effects trophic lateral sclerosis. Int J Kryptolebias marmoratus. Aquat in adults will afford a better understanding Biometeorol 2018;62:1361-74. Toxicol 2016;180:247-57. of long-term consequences of BMAA pres- 5. Chin-Chan M, Navarro-Yepes J, 14. Norton WHJ. Measuring larval zebra- ence in the environment and the putative Quintanilla-Vega B. Environmentaluse pol- fish behavior: locomotion, thigmotaxis, roles of epigenetic mechanisms in neuro- lutants as risk factors for neurodegene- and startle. Zebrafish Protoc toxicity. These preliminary findings stress rative disorders: Alzheimer and Neurobehav Res Neuromethods 2012; the importance of ecotoxicological studies Parkinson diseases. Front Cell Neurosci about BMAA and other NCs on wild organ- 2015;9:1-22. 66:3-20. isms, such as the mangrove rivulus, in addi- 6. Faassen EJ. Presence of the neurotoxin 15. Purdie EL, Metcalf JS, Kashmiri S, tion to studies on human neurodegenerative BMAA in aquatic ecosystems: What do Codd GA. Toxicity of the cyanobacte- diseases, as its presence in the environment we really know? Toxins (Basel) rial neurotoxin β-N-methylamino-L- could have consequences on populations’ 2014;6:1109-38. alanine to three aquatic animal species. survival even at environmental concentra- 7. Nunn PB. 50 years of research on α- Amyotroph Lateral Scler 2009;10:67- tions, jeopardizing whole communities amino-β-methylaminopropionic acid 70. interconnected in trophic networks. (β-methylaminoalanine). 16. Zupanc GKH. Towards brain repair: Phytochemistry 2017;144:271-81. Insights from fish. Semin Cell Research highlights 8. Brand LE, Pablo J, Compton A, Dev Biol 2009;20:683-90. BMAA negatively impacts rivulus’ Hammerschlag N, Mash DC. 17. Frøyset AK, Khan EA, Fladmark KE. capacity of prey hunting. Cyanobacterial blooms and the occur- Quantitative proteomics analysis of Needs to test low concentrations of tox- rence of the neurotoxin, beta-N-methy- zebrafish exposed to sub-lethal dosages icants to assess environmental risk.Non-commerciallamino-l-alanine (BMAA), in South of β-methyl-amino-L-alanine (BMAA). The mangrove rivulus is a new valuable Florida aquatic food webs. Harmful Sci Rep 2016;6:29631.

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