Inhibitory effects of neurotoxin β-N-methylamino-L-alanine on fertilization and early development of the sea urchin Lytechinus pictus

Highlights

• Embryos and sperm of sea urchin Lytechinus pictus exposed to BMAA were assessed.

• Fertilization and development of embryos were inhibited by BMAA above 300 μg L⁻¹.

• The EC₅₀ for larval mortality at 96 h post fertilization was 329 μg L⁻¹.

• Fertilization of eggs was decreased by sperm exposed to BMAA for 10 min.

• The ABC transport activity of sea urchin blastula was not affected by BMAA.

Abstract

Neurotoxin β-N-methylamino-L-alanine (BMAA) has been widely detected in diverse aquatic organisms and hypothesized as an environmental risk to neurodegenerative diseases in humans. However, the knowledge of its toxicity to marine organisms requires attention. In the present study, embryos and sperm of the sea urchin, Lytechinus pictus, were used to assess the toxicity of BMAA. Effects of BMAA on fertilization and development of sea urchin embryos were measured, and its impacts on efflux transport of sea urchin blastula were also assayed. Results demonstrated that the fertilization and development of embryos were significantly inhibited by high concentrations of BMAA above 300 μg L⁻¹. The EC₅₀ values indicated by active swimming larvae and total larvae numbers at 96 HPF (hours post fertilization) were 165 μg L⁻¹ (1.4 μmol L^-1) and 329 μg L⁻¹ (2.8 μmol L^-1), respectively. Additionally, sperm exposed to BMAA for 10 min significantly reduced the fertilization ratio of sea urchin eggs. However, the ABC transport activity on the cytomembrane of sea urchin blastula was not inhibited by the presence of BMAA at 50 μg L⁻¹, even up to 500 μg L⁻¹. Abnormal division and developmental malformations occurred at different developmental stages for sea urchin embryos exposed to BMAA at 500 μg L⁻¹. The inhibitory effects of BMAA on sea urchin embryos were reported at the first time in this study, for which the toxicological mechanisms will be explored in future studies.

Introduction

Neuroscientists have paid attention to one derivative of alanine, β-N-methylamino-L-alanine (BMAA), due to its toxicity to motor neurons (Rao et al., 2006; Buenz and Howe, 2007; Lobner et al., 2007). BMAA was inferred as an environmental trigger for neurodegenerative disease such as amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of the Chamorro people in Guam (Spencer et al., 1987; Cox et al., 2003). It was a further evidence for this hypothesis that researchers detected BMAA in the brain tissues of two Canadians died from progressive neurodegenerative disease (Murch et al., 2004). So far the neurotoxin was reported in diverse cyanobacterial samples in freshwater or seawater environments (Cox et al., 2003; Johnson et al., 2008; Brand et al., 2010; Violi et al., 2019) and marine eukaryotic diatoms (Jiang et al., 2014; Réveillon et al., 2016a), which hints a risk of BMAA exposure to wild organisms in aquatic ecosystems. Additionally, BMAA has also been found in marine invertebrates, such as mussels, from different sea area (Beach et al., 2015; Réveillon et al., 2016b; Li et al., 2018). Experiments carried out on the arthropod Daphnia magna, and the mussels Mytilus galloprovincialis and Dreissena polymorpha, also demonstrated the accumulation of BMAA along food chains under laboratory conditions (Lürling et al., 2011; Baptista et al., 2015; Esterhuizen-Londt et al., 2015; Lepoutre et al., 2018). Negative effects of BMAA were tested on organisms in diverse trophic levels, including the macrophyte (Ceratophyllum demersum), the zooplankter (D. magna), and the brine shrimp (Artemia salina) (Lürling et al., 2011; Esterhuizen-Londt et al., 2011, 2015; Brooke-Jones et al., 2018; Purdie et al., 2009a). Therefore, the risk of BMAA exposure to marine animals should be considered and assessed to understand the ecological risk of BMAA to wild organisms.

Developmental toxicity of BMAA to aquatic animals should be performed to the potential release of BMAA from its producers in water environments. For example, the neuromuscular abnormality occurred in zebrafish when the exposure concentration of BMAA was above 50 μg L⁻¹, while the heart rate of zebrafish reduced when the exposure of BMAA increased above 500 μg L⁻¹ (Purdie et al., 2009b). According to the animal model of developmental toxicity test, sea urchins have been accepted internationally as a standardized marine pollutant assay for more than 30 years due to some advantages such as simplicity, speed, sensitivity, unambiguity of results, uniformity and accuracy (Kobayashi, 1984). A standard ecotoxicology test procedure developed on the sea urchin embryos was adopted by Environmental Protection Agency, USA in 1995 (US EPA 600R95136). Sea urchin bioassays have been used to study diverse toxic chemicals, such as phosphate pollutants and mercuric chloride (Böttger and McClintock, 2001; Marc et al., 2002). The early development of sea urchins has also been used to study the effects of neurotoxins due to similarities between sea urchin and vertebrate development (Buznikov et al., 2001, 2007; Quiao et al., 2003). For example, the sea urchin embryos were used to test the effects of organophosphate pesticides on gene expression (Aluigi et al., 2008) and the effects of pesticide monocrotophos on serotonin metabolism (Xu et al., 2012).

In addition to being a model for toxic exposure, the sea urchin embryo is also a model for cellular defense by ATP-binding cassette (ABC) transporters. More than half of the chemical defense genes that regulate ABC-transporters are expressed during embryonic or larval life stages (Goldstone et al., 2006). The apical transporters ABCB1a, ABCB4a, and ABCG2a overexpressed in the embryos of sea urchin (Stronglycentrotus purpuratus) can account for as much as 87 % of the observed efflux activity, providing a robust assay for their substrate selectivity (Gökirmak et al., 2012). The ABC transporters can be inhibited by ubiquitous pollutants found in fish and other foods due to specific binding of drug efflux transporter P-glycoprotein by pollutants, which argue for further consideration of transporter inhibition in the assessment of the risk of exposure to these chemicals (Nicklisch et al., 2016).

This study utilized the advantages of sea urchin (Lytechinus pictus) embryos to assess the effect of BMAA exposure to fertilization, development, and ABC transporter activity. Gametes and embryos were exposed to a series of BMAA concentrations and compared by microscopic observation. Possible effects of BMAA on ABC transporter activity were also assessed using a fluorescent substrate efflux assay.