Response of coral reef dinoflagellates to nanoplastics under experimental conditions

Plastic products contribute heavily to anthropogenic pollution of the oceans. Small plastic particles in the micro- and nanoscale ranges have been found in all marine ecosystems, but little is known about their effects upon marine organisms. In this study we examine changes in cell growth, aggregation, and gene expression of two symbiotic dinoflagellates of the family Symbiodiniaceae, Symbiodinium tridacnidorum (clade A3) and Cladocopium sp. (clade C), under exposure to 42-nm polystyrene beads. In laboratory experiments, cell number and aggregation were reduced after 10 days of nanoplastic exposure at 0.01, 0.1, and 10 mg/L concentrations, but no clear correlation with plastic concentration was observed. Genes involved in dynein motor function were upregulated compared to control conditions, while genes related to photosynthesis, mitosis, and intracellular degradation were downregulated. Overall, nanoplastic exposure led to more genes being downregulated than upregulated and the number of genes with altered expression was larger in Cladocopium sp. than in S. tridacnidorum, suggesting different sensitivity to nanoplastic between species. Our data show that nanoplastic inhibits growth and alters aggregation properties of microalgae, which may negatively affect the uptake of these indispensable symbionts by coral reef organisms.


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Coral reefs provide habitat for marine invertebrate and vertebrate species alike, sustaining the 28 highest biodiversity among marine ecosystems [1]. Formed primarily by scleractinian corals and 29 coralline algae, coral reefs are complex and vulnerable ecosystems. Structural complexity of coral 30 reefs, and by extension, the capability to sustain biodiversity often declines due to natural and human-31 related stressors [2,3].

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Cladocopium goreaui, to 1-µm polystyrene spheres, leading to diminished detoxification activity, 41 nutrient uptake, and photosynthesis, as well as increased oxidative stress, apoptosis levels, and ion 42 transport. Plastic particles seem to negatively impact symbiotic relationships between corals and their microalgae, thereby degrading the entire coral reef ecosystem, but this has not been systematically 44 investigated.

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Nanoplastics, particles smaller than 1 µm [5], can originate by fragmentation of larger plastic 46 objects through photochemical and mechanical degradation. There are also primary sources of 47 nanoplastics. Medical and cosmetic products, nanofibers from clothes and carpets, 3D printing, and 48 Styrofoam byproducts find their way into coral reef ecosystems via river drainages, sewage outfalls, 49 and runoff after heavy rainfall, as well as via atmospheric input and ocean currents. Nanoplastic has 50 recently been reported in ocean surface water samples [14].

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In this study we focused on the microalgal symbionts of mollusks that inhabit fringing coral reefs

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Laboratories Inc., catalog number FSDG001, polystyrene density 1.05 g/cm 3 , nanoPS) were added to 82 the treatment tanks (Tables S1). Treatment tanks as well as control tanks (no nanoPS) were 83 established in triplicate. Three tanks without algae were prepared as negative controls (at 10 mg/L, 0.01 mg/L, 0 mg/L nanoplastic). In each culture tank, the final cell density of the two strains was 85 adjusted to ~7 x 10 5 cells/mL. Tanks were harvested after 9-11 days, for logistical reasons, making 86 replicates a day apart (Supplementary Table 2

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In addition, Su et al. [13] reported a reduction in cell size in Cladocopium goreaui. Further

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investigations are needed to see if this is the case under nP exposure. Interesting to note is that the 141 biggest growth rate reduction observed was at 0.01 mg/L nanoPS42, far below the 5 mg/L used by Su 142 et al. [13]. The nutrient deficiency is also a reason discussed in (Long2017) which could explain the   Figure S2). All tanks showed aggregation, which was expected, as self-aggregation 158 of Symbiodiniaceae has been observed previously [13].

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Changes in aggregation and resulting sedimentation was observed under nanoPS exposure. It is 208 interesting to see that the biggest changes in sinking velocity correspond to increases in aggregation 209 and are observed in the lowest plastic treatment at 0.01 mg/L. On the other hand, the 10 mg/L 210 treatment did not have any significant effect on the sinking rates but did affect sedimentation indirectly 211 through changes in the aggregate size distribution (see Figure 2). These changes, both sinking 212 velocities and aggregate sizes distribution, are most likely due to hetero-aggregation between algae 213 and nanoPS. Under different treatments, the size distribution of aggregates was significantly different (see Figure 2). In combination, it is likely that the same effect that led to that difference in aggregation 215 is also responsible for the difference in sinking velocities. Changes in EPS production and stickiness

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Cladocopium seems more sensitive to nanoPS42 exposure, as overall more genes responded than in  . Interestingly, dynein light chain genes were also shown to be 239 upregulated in gill cells of zebra mussels exposed to polystyrene microplastic [34].

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Annotations by Blast2GO show the presence of microtubule-or photosynthesis-related genes among DEGs.

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Previous studies have shown that nanoplastic has adverse effects on different algae groups 267 [27,29,30,42,43], and a recent study shows that microplastic has similarly negative effects on an 268 endosymbiotic dinoflagellate Cladocopium goreaui [13]. No previous studies have been conducted 269 on nanoPS42 effects on Symbiodiniaceae. We found significant changes in aggregation and aggregate

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Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1; Figure S1: Cell abundance 280 in treatment tanks, control tanks, and outside controls; Figure S2: NanoPS exposure changes aggregation 281 behaviour, reduces cell numbers, and alters size class distributions; Table S1: Relationship between nanoPS42 282 concentration and particles per Tank; Table S2: Sampling days of each tank;