Effects of microplastic combined with Cr(III) on apoptosis and energy pathway of coral endosymbiont

The combined effect of polyethylene (PE) microplastics and chromium (Cr(III)) on the scleractinian coral Acropora pruinosa (A. pruinosa) was investigated. The endpoints analysed in this study included the endosymbiont density, the chlorophyll a + c content, and the activity of enzymes involved in apoptosis (caspase-1, caspase-3), glycolysis (lactate dehydrogenase, LDH), the pentose phosphate pathway (glucose-6-phosphate dehydrogenase, G6PDH) and electron transfer coenzyme (nicotinamide adenine dinucleotide, NAD+/NADH). During the 7-day exposure to PE and Cr(III) stress, the endosymbiont density and chlorophyll content decreased gradually. The caspase-1 and caspase-3 activities increased in the high-concentration Cr(III) exposure group. Furthermore, the LDH and G6PDH activities decreased significantly, and the NAD+/NADH was decreased significantly. In summary, the results showed that PE and Cr(III) stress inhibited the endosymbiont energy metabolism enzymes and further led to endosymbiont apoptosis in coral. In addition, under exposure to the combination of stressors, when the concentration of Cr(III) remained at 1 × 10–2 mg/L, the toxic effects of heavy metals on the endosymbiont were temporarily relieved with elevated PE concentrations. In contrast, when coral polyps were exposed to 5 mg/L PE and increasing Cr(III) concentrations, their metabolic activities were seriously disturbed, which increased the burden of energy consumption. In the short term, the toxic effect of Cr(III) was more obvious than that of PE because Cr(III) exposure leads to endosymbiont apoptosis and irreversible damage. This is the first study to provide insights into the combined effect of microplastic and Cr(III) stress on the apoptosis and energy pathways of coral endosymbionts. This study suggested that microplastics combined with Cr(III) are an important factor affecting the apoptosis and energy metabolism of endosymbionts, accelerating the collapse of the balance between the coral host and symbiotic endosymbiont.


Introduction
Coral reefs are one of the most important ecosystems in the world due to their abundant biodiversity and high primary productivity (Mooney et al. 2009;Liao et al. 2021). In recent years, the dual effects of natural and human factors have led to increasing degradation of coral reef ecosystems, and many coral species are on the verge of extinction (Li et al. 2017;Hughes et al. 2013). In the past 5 years, these systems have shown an increasing degradation trend, and coral coverage dropped to 16.8% by 2015 (Smith et al. 2016;Suchley and Alvarez-Filip 2018). Currently, the effects of toxic and harmful substances on coral reef ecosystems have attracted extensive attention because coral reef ecosystems face the joint toxicological effects of microplastics (Thompson et al. 2004), heavy metals, persistent pollutants, antibiotics, and pathogens (Wardrop et al. 2016;Tanaka et al. 2013). Heavy metals have a serious interference effect on the growth of marine organisms, and trace concentrations of heavy metals can lead to poisoning of organisms (Gissi et al. 2019;Bielmyer et al. 2010;Nystrom et al. 2001).
Heavy metals are major hazardous substances as they are nondegradable and remain in the ecosystem for relatively long periods (Biscere et al. 2017;Basirun et al. 2019;Das et al. 2019). Although heavy metal pollution has received widespread concern, approximately 1.79 × 10 7 tons of multiple heavy metals enters the ocean every year (zhang et al. 2020). Heavy metals in seawater can be absorbed by corals, resulting in toxicity. For example, Cu, Zn, and Ni were shown to cause toxicological injury to coral growth and calcification (Rodriguez et al. 2016). In detail, the effects of heavy metals on the growth of corals are mainly manifested in the serious inhibition of coral metabolism. These effects not only lead to the DNA damage, but also cause genetic material mutations in organisms, resulting in slow growth and abnormal symptoms (Jones 2004;Zhou et al. 2017). Heavy metals can decrease the survival rate of biological larvae and affect the diversity of marine organisms, which is a major threat to the marine ecosystem (Hudspith et al. 2017;Horwitz et al. 2014). Thompson's and Reichelt's results showed that when the concentration of heavy metals was increased, the growth of symbiotic algae in corals was inhibited. Moreover, Cu(II), Zn(II), and Cd(II) affected the survival rate of gametophyte fertilization, which restrained the growth process of corals (Thompson 1971;Reichelt-Brushett and Harrison 1999). In addition, a previous report showed that an increase in heavy metals exerts a certain inhibitory effect on the development of coral larvae into corals, and leads to synergistic toxicity (Reichelt-Brushett and Harrison 1999). The endosymbiont was dissociated in the tissue of corals, which significantly reduced the growth rate and calcification rate of corals, thus causing serious damage to the coral reef reconstruction process (Sabdono 2009).
The degradation rate of plastics in the natural environment is very low Lebreton et al. 2017). In the environment, weathering processes immediately begin to reduce the stability of polymers, resulting in increased surface roughness, shape changes, fragmentation and alteration of the chemical composition of plastics (Lebreton et al. 2017;Wright et al. 2013;Cooper and Corcoran 2010). These changes may enhance the adsorption of harmful substances and promote the release of chemical additives to the surrounding environment (Carbery et al. 2018). The adsorption process of heavy metals by microplastic particles occurs through a complex mechanism.
The precipitation of inorganic minerals and organic matter on the surface of plastic particles causes the surface properties of the plastic particles to change, and active binding sites for various metal ions are formed (Rochman et al. 2014). Several studies have reported that heavy metals are adsorbed on the surface of microplastics by electrostatic interactions or complexation and that microplastics can act as carriers to transport heavy metals to remote locations, which increases the ecotoxicity of heavy metals (Holmes et al. 2014;Wen et al. 2018). Lu showed that polystyrene microplastics enhanced the toxicity of Cd to zebrafish to a certain extent and combined exposure to polystyrene microplastics and Cd resulted in oxidative damage and inflammation in zebrafish (Lu et al. 2018). Therefore, if microplastic pollution continues to increase, coral reef ecosystem exposure to microplastics and heavy metals may reach high levels in the future. Such exposure will potentially reduce coral resilience to environmental change and make ecosystems more vulnerable (Rodriguez et al. 2016;Lajeunesse et al. 2018).
In our previous study , the combined toxic effects of microplastics and heavy metals copper (Cu) on Tubastrea aurea coral were studied. However, the toxic effects of microplastics and heavy metals on reef-building corals have not been reported. A recent survey (Patterson et al. 2020) of microplastic and heavy metal pollution in coral reef area showed that heavy metals are more likely to be distributed on the surface of microplastic than in sediment. It is urgent to analyse the physiological changes in corals and their endosymbionts in response to microplastic and Cr(III) stress. In this study, Acropora pruinosa, an important species in the South China Sea, was used to study the response of coral to combined microplastic and Cr(III) stress in the laboratory. The physiological characteristics of the endosymbiont in A. pruinosa were detected, including chlorophyll content, endosymbiont number, apoptosisrelated protein enzymes, and energy metabolism pathway enzyme activity. The overall goals of this study were to determine the sensitivity and physiological response of coral and its endosymbiont to microplastic and Cr(III) exposure and to elucidate the toxicity mechanism in A. pruinosa, which is of great significance for the protection of marine biological resources.

Materials and chemical reagents
Polyethylene (PE) is one of the most commonly used plastics globally and is commonly used in packaging. PE was selected for this study because it is a common plastic polymer detected in microplastics sampled from marine environments and beaches (Ding et al. 2019;Jensen et al. 2019). PE microplastics were purchased from Yineng Plastic Materials Co., Ltd. (Dongguan, China). The PE suspension was prepared in aerated seawater following ultraviolet disinfection. The composition of PE microplastics was confirmed by Raman spectroscopy (RS, SR-510 Pro, Ocean Optics Asia, 785-nm laser, Raman shifts 50-3500 cm −1 ), and the results are shown in Fig S1. Analytically, pure acetone (AR 99.9% purity) and analytical-grade chromium chloride hydrate (2:5) (99.0% purity) were purchased from Macklin Chemical Co., Ltd. (Shanghai, China). Cr(III) stock solutions of 0.1 mg/L and 5 × 10 -2 mg/L were prepared from filtered seawater. Assay kits for measuring the levels of nicotinamide adenine dinucleotide (NAD + /NADH), glucose-6-phosphate dehydrogenase (G6PDH), lactate dehydrogenase (LDH), caspase-3 and caspase-1 were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A BCA protein assay kit was provided by the Beyotime Institute of Biotechnology (Jiangsu, China). The seawater used in the experiment was artificial.

Coral collection and treatment
Colonies of stony corals were collected from a coral reef in Nan ′ ao Bay, Guangdong Province, China, after which they were transferred to a facility located at the Shenzhen Institute of Guangdong Ocean University and cultured in a flow-through aquarium (ca. 200 L) filled with seawater (the corals were domesticated for 1 year). The branches in the colonies were split into nubbins (3-5 cm long) and attached to ceramic matrix bases with two-component glue (Coral Glue, EcoTech Marine, Allentown, PA, USA), and 300 nubbins were generated in total. The light source in the laboratory was a Chihiros LED lighting system (21 W), and 12 h of light were applied every day from 06:00 to 18:00 with an irradiance of 70 ± 10 μmol quanta m −2 s −1 for 1 year. The sea-water was regularly renewed to ensure the sufficient supply of nutrients. All corals were not fed any exogenous food during the experiment. The culture temperature of the seawater was controlled at 23-24 ℃, and the salinity was approximately 35.0 ± 0.2 ppt.

Stress experimental design
After the nubbins were transferred into acrylic laboratory aquaria (ca. 15 L), the growth status was recorded regularly, and the stress experiment was started when the nubbin condition was stable. Except for the presence of stressors, the experimental environment was consistent with the conditions of the adaptation period. Thirty acrylic aquaria were used to store coral nubbins (3-5 cm in length), including 6 aquaria as the control group and 24 aquaria as the experimental group. Coral growth was monitored in real time. A total of 240 nubbins were used for the PE and Cr(III) stress experiments (8 nubbins per tank). Since there is no unified standard for the actual concentration of microplastics in seawater, we selected a low concentration based on recent studies on the toxicity of microplastics to corals (Tan et al. 2020;Ding et al. 2019). Moreover, the concentration of Cr(III) was determined according to studies on related heavy metals (Ahsanullah and Williams 1991;Urrutia et al 2008;Rodriguez et al. 2016;Fonseca et al. 2019a, b). In detail, PE microplastics were added to filtered artificial seawater with concentrations of 2 mg/L, 10 mg/L and 20 mg/L. Similarly, chromium chloride (CrCl 3 ) was added to filtered artificial seawater with final concentrations of 2 × 10 -3 mg/L, 1 × 10 -2 mg/L and 2 × 10 -2 mg/L. The detailed operation is shown in Fig. S2. Then, 96 nubbins were transferred to seawater with elevated Cr(III) contents, hereinafter referred to as the Cr(III) group. Another 96 coral nubbins were transferred to seawater with elevated PE contents and defined as the MP group. The other 48 coral nubbins served as the control group and 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 PE microplastics and Cr(III) were added at the same time. After 1 day and 7 days of incubation, 3 nubbins were randomly sampled from the Cr(III), PE, Cr(III)-10&PE (2 mg/L, 10 mg/L and 2 mg/L), PE-5&Cr(III) ( 2 × 10 -3 mg/L, 1 × 10 -2 mg/L and 2 × 10 -2 mg/L) and control groups.
Before the beginning of the experiment, the mass of CrCl 3 and PE was accurately weighed to calculate the actual Cr (III) and PE concentrations in each treatment group. During the experiment, the seawater pH was measured daily by a pH meter calibrated with standard liquid, and the water salinity and temperature were measured by a salinometer and a thermometer, respectively. The related results are shown in Table S1.

Endosymbiont density measurement
The endosymbiont density was measured according to Higuchi's work with slight modification (Higuchi et al. 2015). In short, the collection fluid was obtained by stripping the coral tissue from the coral skeleton with a Waterpik water jet (WP670EC, USA, www. water pik. com). The collected-fluid was shaken violently to destroy coral mucus. To obtain a relatively pure endosymbiont, the extracts were repeatedly filtered with a sieve (40 μm) until no impurities were observed under the microscope. The filtered algal extract was centrifuged at 4000 rpm and 4℃ for 3 min, and then, the endosymbiont deposition at the bottom of the centrifuge tube was collected and fixed in 1 mL 10% formaldehyde for 2-4 h. The number of endosymbionts was calculated with a blood cell counting plate (QIUJING, China). The quantity of endosymbionts contained in the total volume of solution (A, cell/mL) was obtained by conversion.
The area of the aluminium foil was calculated according to the reported density and weight , and this value was assigned to the coral bone surface area (S, cm 2 ).
The endosymbiont density (D, cell/cm 2 ) was defined as the number of endosymbionts per unit coral nubbin surface area according to the following formula: D = A/S.

Chlorophyll measurement
The chlorophyll content was determined according to Jeffrey's method (Jeffrey and Humphrey 1975). On days 1 and 7, the coral nubbins with sizes of 4-5 polyps and diameters of 1 cm were quickly removed with forceps under water. The excess seawater was wiped off with absorbent paper. The coral nubbins were then transferred to a centrifuge tube containing 10 mL acetone and extracted for 24 h at 4 ℃ in the dark. Then, the acetone extract was centrifuged at 4000 rpm for 10 min. The chlorophyll concentration was determined by a Thermo Nanodrop 2000 visible light spectrophotometer. The calculation formula is as follows: where CHl-a/c is the chlorophyll content per unit coral surface area (μg/cm 2 ) and A is the light absorption value at different wavelengths. The chlorophyll content per unit coral surface area was thus obtained, and combined with the measured endosymbiont density per unit coral surface area (S) described above.

Biochemical evaluation of endosymbiont homogenates
The preparation method of used to obtain pure endosymbiont sediment was consistent with that used for endosymbiont density. To measure the enzyme activity of symbiotic algae, the endosymbiont sediment was homogenized in 5 mL of filtered artificial seawater by using an Automatic Sample Rapid Grinding Instrument (JingXin, Shanghai, China). The homogenate was centrifuged at 5500 rpm for 15 min. The supernatant was transferred to a new tube for analysis of biochemical parameters. The activities of caspase-1, caspase-3, LDH, G6PDH and NAD + /NADH were measured by commercial kits (Nanjing Jiancheng Biological Engineering Research Institute Co. LTD). Caspase is the general term for cysteinyl aspartate-specific protein.
Caspase, the first signal protein identified in mammalian cells, mediates the apoptosis of certain types of cells (Zhou et al. 2017;Hengartner 2000). Nicotinamide adenine dinucleotide (NAD + ), also known as coenzyme I, is an essential coenzyme in the redox process. NAD + is involved in many physiological activities, such as cell metabolism, energy synthesis and DNA repair (Hosseini et al. 2014; Marangoni et al. 2017). NADH (reduced coenzyme I) is the reduced state of NAD + (Ying 2008;Babot et al. 2014). As a carrier and electron donor of biological hydrogen, NADH transfers energy for ATP synthesis through the oxidative phosphorylation process in mitochondrial inner membrane. NADH plays an important role in cell growth, differentiation, and maintenance (Tanner 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 the BCA method (Zhou et al. 2018a, b).

Statistical analysis
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 analysis of variance (ANOVA) of nonparametric equivalents was applied for statistical analysis of the significance of differences by SPSS 20, followed by the Student-Newman-Keuls post-test. The statistical significance of Cr/PE-induced changes was evaluated using an independent two-sample Student's t test. For all tests, p < 0.05 was considered statistically significant. The commercial statistical software Origin 2019 was used to prepare the column diagrams. The symbol "*" indicates a significant difference between the treatment group and the control group on day 1 (p < 0.05). The symbol " + " indicates a significant difference between the treatment group and the control group on day 7 (p < 0.05). The letter "a" represents differences in Cr(III)-1 × 10 -2 vs. PE-2&Cr(III)-1 × 10 -2 , PE-10&Cr(III)-1 × 10 -2 and PE-20&Cr(III)-1 × 10 -2 respectively (p < 0.05), and "b" represents differences in PE-5 vs. Cr(III)-2 × 10 -3 &PE-5, Cr(III)-1 × 10 -2 &PE-5 and Cr(III)-2 × 10 -2 &PE-5 (p < 0.05). Figure 1A shows that the endosymbiont density in corals decreased under the stress of Cr(III) (1 × 10 -2 mg/L) and PE (2 m/L, 10 mg/L, 20 mg/L). On day 1, when 1 × 10 -2 mg/L Cr (III) was present, the number of endosymbionts decreased with increasing PE concentration. On day 7, the corals exposed to Cr(III) (1 × 10 -2 mg/L) alone showed a significantly lower endosymbiont density (2.68 × 10 6 cell/ cm 2 , P < 0.05) than all other exposure groups. Similarly, the endosymbiont density in corals was also decreased with increasing PE concentration, and it reached the lowest value (2.90 × 10 6 cell/cm 2 , 3.01 × 10 6 cell/cm 2 , 3.46 × 10 6 cell/cm 2 , P < 0.05) in the PE group (PE 2 mg/L, 10 mg/L, 20 mg/L) on day 7. Figure 1B presents the effect of PE exposure alone as well as the joint effect of PE and Cr(III) (2 × 10 -3 mg/L, 1 × 10 -2 mg/L, 2 × 10 -2 mg/L) on the endosymbiont density in corals after 7 days of exposure. In detail, endosymbiont density in coral decreased after exposure to PE (5 mg/L) combined with Cr(III) (2 × 10 -3 mg/L, 1 × 10 -2 mg/L, 2 × 10 -2 mg/L). In particular, the endosymbiont density in corals decreased gradually in the high-concentration group (2 × 10 -2 mg/L Cr(III)) and it reached the lowest level on day 7. However, in the presence of PE, the effect of increasing Cr(III) concentration on endosymbiont density decreased (Fig. 1B).
As shown in Fig. 2B, adverse effects on chlorophyll a + c content due to the combination of stressors were observed under exposure after seven days. The chlorophyll a + c content decreased with exposure time to Cr(III) stress in the PE and Cr(III) groups (2 μg/L, 10 μg/L, 20 μg/L). In particular, it decreased gradually and reached the lowest level (3.62 µg/cm 2 , 3.21 µg/cm 2 , 2.28 µg/cm 2 , P < 0.05) on day 7 in all concentration groups. On the day 1 and 7, in the presence of a fixed concentration of PE microplastics, the endosymbiont chlorophyll content decreased with increasing Cr(III) concentration.

Effects of Cr(III) stress on caspase-3 activity
Effects on caspase-3 activity were observed after 7 days of exposure; the activity of caspase-3 (1.88 U/g prot, 2.29 U/g prot, P < 0.05) increased with the increasing stress duration (Fig. 3A). On both day 1 and 7, the higher the concentration of microplastics in the combined exposure group was, the lower the effect on endosymbiont caspase-3 activity. Moreover, corals exposed to 10 µg/L Cr(III) combined with PE presented lower caspase-3 activity than those exposed to Cr(III) alone (P < 0.05).
As shown in Fig. 3B, the caspase-3 activity in corals exposed to PE (5 mg/L) increased with the stress time.
In addition, under exposure to 5 mg/L PE combined with Cr(III) (2 × 10 -3 mg/L, 1 × 10 -2 mg/L, 2 × 10 -2 mg/L), caspase-3 activity increased with increasing Cr(III) concentration. On day 7, there was a significant difference in the activity of caspase-3 (2.02 U/g prot, 2.51 U/g prot, 2.95 U/g prot, P < 0.05) between the Cr(III) exposure group and the control group, and the activity of caspase-3 in groups with all concentrations of Cr(III) was higher than that in the PE group. Figure 4A shows that caspase-1 activity significantly changed (3.95 U/g prot, P < 0.05) after 7 days of exposure to Cr(III) stress. Increased caspase-1 activity was observed in corals exposed to different combinations of PE concentrations (2 mg/L, 10 mg/L, 20 mg/L) and Cr(III) (1 × 10 -2 mg/L). On day 7, the caspase-1 activity in the low-concentration PE (2 mg/L) exposure group reached the highest level (3.66 U/g prot, P < 0.05). Compared with that in the Cr(III) exposure group, the caspase-1 activity in the combined exposure group decreased gradually with increasing of microplastic concentration.

Activity of glycolysis enzymes
As shown in Fig. 5A, the LDH content in the control group did not change significantly with the extension of time (P > 0.05). On day 7, the LDH activity decreased significantly under Cr(III) (1 × 10 -2 mg/L) exposure alone, and the LDH activity reached the lowest level (0.013 U/mg prot, P < 0.05). Corals exposed to Cr(III) (1 × 10 -2 mg/L) combined with PE (2 mg/L, 10 mg/L, 20 mg/L) showed lower LDH activity. Compared with the Cr(III) exposure group, the activity of LDH in the combined exposure group increased with increasing of microplastic concentration. LDH activity significantly decreased (0.023 U/mg prot, 0.021 U/mg prot, 0.0249 U/mg prot, P < 0.05) in all PE groups.

NAD + /NADH ratio
As shown in Fig. 7A, the NAD + /NADH ratio was reduced in the coral symbiotic algae under Cr(III) stress alone and under combined Cr(III) and PE stress. The NAD + /NADH ratio was significantly reduced (P < 0.05) after exposure to Cr(III) (1 × 10 -2 mg/L). Additionally, after exposure to Cr(III) combined with PE (2 mg/L, 10 mg/L, 20 mg/L), the NAD + /NADH ratio was lower. Interestingly, the NAD + / NADH ratio under the combined stress of Cr(III) and PE was higher than that under Cr(III) stress alone.

Effects of MPs and Cr(III) on the symbiotic coral endosymbiont relationship
The endosymbionts in corals provide most of the nutrients for the growth and reproduction of the hosts through photosynthesis (Okubo et al. 2018), and the chlorophyll content can affect the growth of endosymbionts. Heavy metals can accumulate in the tissues and endosymbionts of branching corals (Bielmyer et al. 2010;Mitchelmore et al. 2007). Reichelt-Brushett found that under heavy metal pollution, endosymbionts may accumulate certain heavy metals, and heavy metals seriously inhibited photosynthesis and led to Goniastrea aspera decline and death (Reichelt-Brushett and Harrison 1999). In the short term, the Fig. 6 Glucose-6-phosphate dehydrogenase (G6PDH) activity in the coral A. pruinosa exposed to microplastics and Cr(III) for day 1 and day 7. A Single exposure to heavy metal Cr(III) (1 × 10 -2 mg/L) and Cr(III) combined with different concentrations of PE (2 mg/L, 10 mg/L, 20 mg/L). B Single exposure to PE (5 mg/L) and PE combined with different concentrations of heavy metal Cr(III) (2 × 10 -3 mg/L, 1 × 10 -2 mg/L, 2 × 10 -2 mg/L). Data are expressed as mean ± standard error (n = 3). The symbol "*" indicates a significant difference between the treatment group and the control group on day 1 (p < 0.05). The symbol " + " indicates a significant difference between the treatment group and the control group on day 7 (p < 0.05). The letter "a" represents differences in Cr(III)-1 × 10 -2 vs. PE-2&Cr(III)-1 × 10 -2 , PE-10&Cr(III)-1 × 10 -2 and PE-20&Cr(III)-1 × 10 -2 (p < 0.05), and "b" represents differences in PE-5 vs. Cr(III)-2 × 10 -3 &PE-5, Cr(III)-1 × 10 -2 &PE-5 and Cr(III)-2 × 10 -2 &PE-5 (p < 0.05) endosymbiont density in corals decreased under the stress of PE and Cr(III), and the balance between the endosymbiont and the host was disturbed to a certain extent (Mendrik et al. 2020). Therefore, the damage to the coral symbiotic system in this study may correspond to direct toxicity caused by Cr(III) entering coral tissues and contacting the endosymbiont.
The chlorophyll content of the coral endosymbionts in the control group was the maximum, and it decreased with increasing PE and Cr(III) concentrations. The chlorophyll content decreased over time and reached the lowest level (3.62 µg/cm 2 , 3.21 µg/cm 2 , 2.28 µg/cm 2 , P < 0.05) on day 7 in all treatment groups. We assumed that the cause for the collapse under PE and Cr(III) stress may be similar to the response to copper stress (Juliana et al. 2021), which was attributed to changes in the internal environment of coral, such as an increase in reactive oxygen species (ROS) levels (Marangoni et al. 2017;Fonseca et al 2019a, b), and the activation of apoptosis (Zhou et al. 2017). The joint stress of microplastics and Cr(III) can inhibit the photosynthesis of coral endosymbionts. Moreover, the endosymbiont chlorophyll content under Cr(III) stress alone was significantly lower than that under PE stress alone, suggesting that the coral endosymbiont was more sensitive to Cr(III) stress. The inhibitory effect of heavy metals on photosynthesis may be ascribed to the toxic effect of Cr(III), which seriously interferes with electron transfer between photosystem (PS) I and PS II (Aro et al. 2005;Bhattacharya et al. 2010), resulting in a decrease in energy conversion and a decline in photosynthesis. It has been reported that microplastics damage the symbiotic system and reduce the chlorophyll content of endosymbionts Jiang et al. 2020;Rocha et al. 2020). Corals can block and remove microplastics from their surfaces by producing mucus (Martin et al. 2019). Due to the special properties of microplastics, they have a strong adsorption capacity for heavy metals in seawater (Brennecke et al. 2016;Mohsen et al. 2019). Therefore, within a certain pollutant concentration range, an increase in microplastic concentration can alleviate the combined toxicity of microplastics and Cr(III) to endosymbionts.

Effects of PE and Cr(III) on endosymbiont apoptosis
Caspase-1 and caspase-3 are commonly used as important indicators of apoptosis of coral host cells and symbiotic algae (Moya et al. 2016). As shown in Figs. 4A and 3A, deleterious effects due to the combination of PE and Cr(III) stressors were observed on caspase-1 and caspase-3 activity after short exposure. The combination of stressors led to higher expression of apoptotic proteases related to coral endosymbionts (Tang et al. 2020;Su et al. 2019). Caspase-3 is the key executive enzyme as well as the final effector of apoptosis. Therefore, caspase-3 activity was investigated to understand the apoptosis status of corals after short-term Cr(III) and PE stress. Caspase-3 activity was increased significantly, which indicated that Cr(III) and PE induced endosymbiont apoptosis. Heavy metals can lead to apoptosis (Morcillo et al. 2016;Di Paola et al. 2021), but there is little related literature on coral; the mechanisms underlying the response in coral may be that heavy metal stress induces apoptosis through the TNF signalling pathway and caspase-3. This proposed mechanism was confirmed by our results. On day 7, there was a significant difference in caspase-3 activity (2.02 U/g prot, 2.51 U/g prot, 2.95 U/g prot) between the Cr(III) Fig. 7 The ratio of NAD + /NADH in the coral A. pruinosa exposed to microplastic and Cr(III) for day 1 and day 7. A Single exposure to heavy metal Cr(III) (1 × 10 -2 mg/L) and Cr(III) combined with different concentrations of PE (2 mg/L, 10 mg/L, 20 mg/L). B Single exposure to PE (5 mg/L) and PE combined with different concentrations of heavy metal Cr(III) (2 × 10 -3 mg/L, 1 × 10 -2 mg/L, 2 × 10 -2 mg/L). Data are expressed as mean ± standard error (n = 3). The symbol "*" indicates a significant difference between the treatment group and the control group on day 1 (p < 0.05). The symbol " + " indicates a significant difference between the treatment group and the control group on day 7 (p < 0.05). The letter "a" represents differences in Cr(III)-1 × 10 -2 vs. PE-2&Cr(III)-1 × 10 -2 , PE-10&Cr(III)-1 × 10 -2 and PE-20&Cr(III)-1 × 10 -2 (p < 0.05), and "b" represents differences in PE-5 vs. Cr(III)-2 × 10 -3 &PE-5, Cr(III)-1 × 10 -2 &PE-5 and Cr(III)-2 × 10 -2 &PE-5 (p < 0.05) exposure group and the control group. Moreover, apoptosis eventually leads to the collapse of the symbiotic balance between the host and endosymbiont and induces the expulsion of the endosymbiont, resulting in coral bleaching (Tang et al. 2020;Su et al. 2019). In addition, programmed cell death (PCD) mediated by caspase-1 can effectively improve the body's ability to resist endogenous and exogenous stimuli and protects the host (Mariathasan et al. 2004).
In the presence of Cr(III), the endosymbiont apoptosis degree decreased with increasing microplastic concentration. This result is consistent with the changes in endosymbiont number and chlorophyll content, and the adsorption of Cr(III) by microplastics needs to be considered first. The adsorption capacity of microplastics for Cr(III) is limited (Fan et al. 2022), and it can be seen from the results of this study that a microplastics of 2 mg/L cannot effectively adsorb Cr(III) to reduce toxicity. With increasing microplastic concentration, more Cr(III) was adsorbed, and the toxic effect of the combined exposure group gradually decreased. As shown in Figs. 4B and 3B, when the microplastic concentration was 5 mg/L, the endosymbiont apoptosis level increased with increasing Cr(III) concentration and reached the peak on the seventh day of high-concentration combined exposure. Su et al. showed that the apoptotic effect of microplastics on coral symbiotic zooxanthellae was alleviated on the fourth day of exposure (Su et al. 2019). Therefore, Cr(III) plays a major role in the apoptosis of endosymbiont cells. In conclusion, our results provide preliminary evidence that PE combined with Cr(III) may induce apoptosis of coral endosymbionts, but the underlying mechanism still needs to be explored in the future.

Effects of PE and Cr(III) on the energy metabolism of coral endosymbionts
Glycolysis is considered to be the main energy production pathway of invertebrates with low metabolism, which can produce ATP and NADH under anaerobic conditions (Carvalho and Fernandes 2008). Pyruvate can be converted to lactic acid by LDH. This reaction replenishes the supply of NAD + , a cofactor required for the catabolism of glucose molecules (Murphy and Richmond 2016). After exposure to PE and Cr(III), LDH activity was inhibited in photobiont. In addition, LDH activity decreased in the corals exposed to different concentrations of Cr(III) and PE. LDH is a key enzyme in the glycolysis pathway. In aquatic animals showing low metabolism, such as corals, the inhibition of LDH activity can seriously compromise animal energy metabolism and homeostasis (da Silva Fonseca et al. 2019a, b). The results suggested that the corals exposed to 20 μg/L Cr(III) and 5 mg/L PE had lower enzyme activity than corals exposed to 10 μg/L Cr(III) alone. In these cases, the combination of stressors led to inhibitory effects on enzymatic activity (Carvalho and Fernandes 2008;Juliana et al. 2021). Interestingly, under the stress of 1 × 10 -2 mg/L Cr(III) and 2, 10 and 20 mg/L PE, the LDH activity of corals was higher than that of corals exposed to the same concentration of Cr(III) alone. Microplastics alleviated the effect of chromium on the energy metabolism of symbiotic algae. Previous study has shown that polyethylene terephthalate (PET), Polyamide 66 (PA66) and (PE) microplastic can reduce LDH activity in Acropora sp. As a result, microplastics can affect the energy metabolism of host coral and endosymbiont and disturb the symbiotic relationship. In general, the toxicity of Cr(III) to corals was significantly higher than that of PE during the short exposure period. A high concentration of PE can slightly alleviate the LDH damage to corals induced by Cr(III). When coral was exposed to the same Cr (III) concentration and different microplastic concentrations, the higher microplastic concentration was, the weaker effect on the coral energy metabolism enzyme LDH was, which may be due to the adsorption of heavy metals by microplastics (Mao et al. 2020;Zou et al. 2020;Wu et al. 2018). Previous studies have shown that both MPs and heavy metals have toxic effects on organisms, and their combination may lead to three effects, namely synergistic, antagonistic or potentiating effect (Bhagat et al. 2021). As for the antagonistic effect of microplastics and heavy metal toxicity, some studies have indicated that MPs can be used as the carrier of heavy metals to reduce the concentration of heavy metals in the exposure medium through adsorbing, thereby reducing the environmental biological toxicity of heavy metals (Bhagat et al. 2021). In the combined exposure experiment, the adsorption capacity of microplastics to heavy metals was positively correlated with the concentration of microplastics Liu et al. 2021). Unfortunately, the combined toxicity of microplastics, heavy metals or other marine pollutants to corals and commensal algae has been poorly studied. It is urgently necessary to explore the concentration thresholds for antagonism of microplastics and heavy metals on coral.
At the same concentration of PE (5 mg/L), an attenuation effect of Cr(III) on G6PDH activity was observed. Although Cr(III) and other metals are essential nutrients for the metabolism of organisms, especially invertebrates (Lee and Lee 2005;Rainbow 2002), excessive heavy metals can seriously interfere with the energy metabolism of host symbionts (Tang et al. 2020;Fonseca et al. 2019a, b). In fact, a short exposure time may have yielded the same results. Fonseca found that Cu(II) was assimilated by corals via adsorption or ingestion, which seriously interfered with glycolysis and pentose phosphate metabolism (Fonseca et al. 2019a, b). Exposure to PE combined with Cr(III) could inhibit the activity of G6PDH, and the inhibition of G6PDH activity may reduce NADH synthesis for antioxidant enzymes (Carvalho and Fernandes 2008;Juliana et al. 2021), leading to oxidative stress. Similarly, under the stress of 5 mg/L PE, an attenuating effect on G6PDH activity was observed, and exposure to combined stressors inhibited the activity of this enzyme. The exchange of nutrient and energy between coral and zooxanthellae is the essential condition for corals to survive. Nils et al. showed that the change of nutrient exchange function in coral directly or indirectly led to the breakdown of the coral-Symbiodiniaceae symbiosis (Radecker et al. 2021).

Effects of PE and Cr(III) on the NAD + /NADH ratio of the endosymbiont
The NAD + /NADH ratio was significantly reduced in A. pruinosa under Cr(III) stress alone as well as under the combined stress of Cr(III) and PE. The joint stress of Cr(III) and PE inhibited the NAD + /NADH ratio in the coral endosymbionts. In addition, the stress of PE combined with Cr(III) reduced the NAD + /NADH ratio in the endosymbiont and may further inhibit the expression of SIRT1 protein (Fonseca et al. 2019a, b). We suspect that on the one hand, NAD + dysfunction blocked signal transduction and induced an antagonistic effect on coral tissue enzymes. On the other hand, it activated the senescence related pathway of coral cells, which induced endosymbiont apoptosis and led to the dysfunction of ATP conversion. NADH is a crucial factor in the SIRT1 signaling pathway and is produced in the citric acid cycle during glycolysis and cell respiration, which is involved in the metabolism of substances and energy in cells (Fonseca et al. 2019a, b;Babot et al. 2014). This accelerated the collapse of the coral-endosymbiont symbiosis system due to damaging effects from the above two aspects. Mitochondria are the energy source of ATP production in the cell and have various functions for cell energy metabolism. Two pathways, glycolysis and NAD biosynthesis, link cytoplasmic and mitochondrial NAD pools through complex processes. The NADH and pyruvate produced by glycolysis are transported from the cytoplasm into the mitochondrial matrix to provide reducing equivalents for the tricarboxylic acid (TCA) cycle and electron transport chain (ETC). Maintaining an optimal NAD/NADH ratio is essential for mitochondrial function (Stein and Imai 2012). Therefore, the decrease in the NAD + /NADH ratio may be the core mechanism of endosymbiont coral collapse. Notably, Cr(III) can promote MP-induced apoptosis of endosymbionts in corals by inhibiting the NAD + -SIRT1 pathway. This process increases the production of lysosomes in host cells to accelerate the formation of histiocytic lesions. The NAD + /NADH ratio of A. pruinosa was lowered as a result of exposure to PE and Cr(III), which may have been caused by ROS production and/ or damage to the glycolysis pathway. Based on this result, it is apparent that exposure to PE and Cr(III) will cause the breakdown of symbiosis between A. pruinosa and its algal endosymbionts. However, the mechanism of action still needs to be investigated.

Conclusion
This study is the first to investigate the isolated and joint effects of PE and Cr(III) exposure on the energy metabolism enzymes and apoptosis of endosymbionts in the coral A. pruinosa. In summary, the results showed that the stress sources of PE and Cr(III) resulted in the transient inhibition of chlorophyll and the endosymbionts of A. pruinosa. Caspase-1 and caspase-3 activity were significantly increased under these conditions, which led to endosymbiont apoptosis. In addition, LDH and G6PDH activities and the NAD + /NADH ratio decreased significantly after 7 days of exposure to PE and Cr(III), indicating that the combination of PE and Cr(III) had a significant adverse effect on the energy metabolism of endosymbionts in the coral A. pruinosa. Therefore, it can be inferred that an increase in marine microplastics combined with Cr(III) stress will lead to of disordered energy metabolism in corals and accelerate endosymbiont apoptosis in corals.