Materials and chemical reagents
Polyethylene (PE) is one of the most commonly used plastics globally, and commonly seen in packaging. PE was selected because it is a common plastic polymer used in microplastics sampling in marine and beaches (Ding et al., 2019; Jensen et al., 2019). Polyethylene microplastic was purchased from Yineng Platic Materials CO., Ltd. (Dongguan, China). Polyethylene suspension was prepared by ultraviolet disinfection of aerated seawater. The composition of polyethylene was confirmed by Raman spectroscopy (RS, SR-510 Pro, Ocean optics Asia, 785 nm laser, Raman shift 50-3500 cm-1), and it showed in Fig S1. Analytically pure acetone (AR 99.9% purity), chromium chloride hydrate of analytical grade (2:5) (99.0% purity) were purchased from Macklin Chemical Co., Ltd. (Shanghai, China). In filtered sea water, 100 μg/L and 50 μg/L Cr(III) stock solutions were prepared. Assay kits for measurement of levels of NDA+/NADH, G6DPH, LDH, Caspase-3 and Caspase-1 were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). BCA protein assay kit was offered by Beyotime Institute of Biotechnology (Jiangsu, China). The seawater used in the whole experiments was artificial.
The corals collection and treatment
The colonies of stony corals were collected from a coral reef in Nan'ao Bay, Guangdong Province, China, transferred and cultured in an in flow-through aquaria (ca. 200 L) filled with seawater in a facility located at the Shen Zhen Institute of Guangdong Ocean University. The branches in the colonies were split as nubbins (long 3-5 cm), they were attached to the ceramic matrix bases with two-component glue, and 300 nubbins were generated in total. In laboratory, the light source was Chihiros LED lighting system (21 W), and 12 hours of light was given every day from 06:00 to 18:00, with a light 70 ± 10 μmol quanta m−2 s−1 cycle for one year, and the sea water was regularly renewed to ensure the nutrition supply. All corals were not fed with any exogenous food during the experiment. The temperature of seawater for culture was controlled at 23-24 ℃, and the salinity was about 35.0 ± 0.2 ppt.
Stress experimental design
After the nubbins were transferred into the acrylic laboratorial aquariums (15 L), the growth status was recorded regularly, and the stress experiment was started when the nubbins condition was stable. In the experiment, except for stressors, the experimental environment was consistent with the conditions of adaptation period. 30 acrylic aquariums were used to store coral nubbins (3-5 cm in length), including 6 as the control group and 24 as the experimental group, and the growth of coral in this environment was monitored in real time. 240 nubbins were used for PE and Cr(III) stress experiments (8 nubbins per tank). In detail, PE-MP were added into filtered seawater to prepare seawater with the concentrations of 2 mg/L, 10 mg/L and 20 mg/L, respectively. Similarly, chromium chloride (CrCl3) was added into filtered seawater to prepare Cr(III) seawater with final concentration of 2 μg/L, 10 μg/L and 20 μg/L, respectively. The detailed operation is shown in Fig.S2. Then, 96 nubbins were transferred to the seawater with elevated Cr(III), hereinafter referred to as Cr(III) group. Another 96 of the coral nubbins were transferred to the seawater with elevated PE, hereinafter referred to as microplastic group. The other 48 coral nubbins were served as control group, which were incubated only in filtered seawater. There were 3 biological replicates in each treatment group. In addition, The seawater in all tanks was replaced once every 24 h with freshly filtered seawater from the coral culture system to ensure a suitable aquaculture water environment, and new PE and Cr(III) were also added at the same time. After 24 h and 7 days of incubation, the 3 nubbins were randomly sampled from Cr(III), PE, Cr(III)-10&PE(2, 10, 20 mg/L), PE -5&Cr(III)( 2, 10, 20 μg/L) and control groups.
Before the beginning of the experiment, the mass of CrCl3 and PE were accurately weighed to prepare the actual Cr (III) and PE concentrations in each treatment group. During the experiment, the pH of seawater was measured daily by a pH meter calibrated with standard liquid, and the salinity and temperature of water were measured by a salinometer and a thermometer respectively. The related results are shown in Table S1.
Endosymbiont density measurement
The density of endosymbiont was measured according to Higuchi's work with slight modification (Higuchi et al., 2015). In short, the tissue homogenate was prepared with Waterpik water jet, and then it was stripped from the coral skeleton into about 10 mL filtered seawater (0.22 μm). The flushing liquid containing endosymbiont was poured into a cleaning measurement barrel, and finally it was recorded the total volume. The flushing liquid was centrifuged at 4000 rpm 4℃ for 3min, and the supernatant was collected. Re-suspended endosymbiont precipitate with filtered seawater, it repeated this operation until no impurities were detected under microscope. A total of 3 copies of homogenate (mixture of coral soft tissue and endosymbiont, 9 mL/share) were centrifuged. And then the endosymbiont deposition at the bottom of the centrifuge tube was collected and it was fixed in 1 mL 10% formaldehyde for 2-4 h. The preserved endosymbiont solution was mixed evenly, and the number of endosymbiont (n =10-12) was calculated with a neubauer hemocytometer (QIUJING, China). The quantity of endosymbiont contained in the total volume solution was obtained by conversion (A: cell/mL). The aluminum foil was wrapped on the surface of coral bone, and the weight of aluminum foil was weighed. The area of aluminum foil was calculated according to the density and weight of the known aluminum foil (Johannes et al., 1970). That is, the surface area of coral bones (S, cm2). According to the following formula: density (D, cells/cm2): D = A/S, the density of endosymbiont was defined as the number of endosymbiont per unit coral nubbins surface area.
The chlorophyll content was determined according to Jeffrey's method (Jeffrey et al., 1975). At 1 and 7 d, the coral nubbins with size of 4-5 polyps and diameter of 1 cm were quickly removed with forceps under water. The excess seawater was wiped off with absorbent paper, and then coral nubbins was transferred to a centrifuge tube containing 10 mL acetone, following it was extracted for 24 h at 4 ℃ under dark condition. Then the acetone extract was centrifuged at 4000 rpm for 10 min. The chlorophyll concentration was determined by thermo nanodrop 2000 visible light spectrophotometer. The calculation formula is as follows:
Where Chl-a/c is the chlorophyll content per unit area of coral surface (μg/cm2), and A is the light absorption value under different wavelengths. The chlorophyll content per unit area of coral surface can be obtained, which combined with the above-mentioned method.
Biochemical evaluation of coral tissue homogenates
To get the homogenates, the endosymbiont deposition were homogenized in 5 mL of filtered seawater by using an Automatic Sample Rapid Grinding Instrument (JingXin, Shanghai, China), and it was centrifuged at 5500 rpm for 15 min. The precipitation was washed thrice with millipore filtered seawater. The supernatant was transferred to a new tube for analyzing the contents of biochemical parameters. The activities of Caspase-1, Caspase-3, LDH, G6DPH and NAD+/NDAH were measured by commercial kits. Caspase is the general term of cysteinyl aspartate specific protein, as the first signal protein identified in mammalian cells, it mediates the apoptosis of certain types of cells (Zhou et al., 2017; Hengartner et al., 2000). Nicotinamide adenine dinucleotide (NAD+), abbreviated as coenzyme I, is an essential coenzyme in the redox process. The NAD+ involves in many physiological activities such as cell metabolism, energy synthesis, DNA repair and so on (Hosseini et al., 2014; Marangoni et al., 2017). NADH (reduced coenzyme I) is the reductive state of nicotinamide adenine dinucleotide (Ying et al., 2008; Babot et al., 2014). As a carrier and electron donor of biological hydrogen, NADH transfers energy to ATP synthesis through oxidative phosphorylation process in mitochondrial inner membrane. NADH plays an important role in cell growth, cell differentiation and maintenance (anner et al., 2000; Sauve et al., 2006). After the total enzyme activities were obtained, the concentration of total protein in the supernatant was quantified using BCA method (Zhou et al., 2018).
All experiments were repeated at least three times to ensure the accuracy and reproducibility of the results. All data are presented as the mean ± standard error of the mean. One-way ANOVA test of non-parametric equivalent was applied for statistical analysis on the significance of difference by SPSS 20, And they were followed by the Student-Newman-Keuls post-test. For all test, p<0.05 was considered statistically significant. The commercial statistical Origin 2019 was used to finish the column diagrams. The letter a expressed as the significant difference among the control, Cr(III) and microplastic treatment groups respectively (p<0.05). Letter of b represented the differences of Cr(III)-10 vs PE-2, 10, 20 respectively (p<0.05), and c represented the differences of PE-5 vs Cr(III)-2, 10, 20, respectively (p<0.05).