Fish and Shellfish Immunology 84 (2019) 451–457 Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Full length article Oxidative stress, apoptosis activation and symbiosis disruption in giant clam T Tridacna crocea under high temperature ∗ Zhi Zhoua, , Zhaoqun Liub, Lingui Wanga, Jian Luoa, Hailang Lic a State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, 570228, China b Liaoning Key Laboratory of Marine Animal Immunology & Disease Control, Dalian Ocean University, Dalian, 116023, China c Haikou No.4 Middle School, Haikou, 570203, China ARTICLE INFO ABSTRACT Keywords: Giant clams are one of the most important animals in coral reef ecosystem, and its growth and reproduction are Heat stress being threatened by heat stress due to global warming. In the present study, the symbiont density, the crucial Oxidative stress enzyme activities and the transcriptome were investigated in the outer mantle of giant clam Tridacna crocea after Apoptosis the acute exposure of high temperature. The density of symbiotic zooxanthellae decreased significantly during Tridacna 12–24 h, with the minimum level (7.75 × 105 cell cm−2, p < 0.05) at 12 h after heat stress. The activities of Symbiosis superoxide dismutase in the heat stress group was significantly lower than that in the control group at 24hafter heat stress, while no significant change in the activities of catalase was observed during the entire stress process. The activation level of caspase3 began to increase significantly at 12 h (1.22-fold, p < 0.05), and reached the highest level at 24 h (1.38-fold, p < 0.05) after heat stress. Six paired-end libraries were sequenced in two groups, including the heat stress and control group at 12 h after heat stress. Through the assembling of 187,116,632 paired-end reads with lengths of 2 × 150 bp, a total of 26,676 genes were obtained which derived from giant clam. Bioinformatics analysis revealed 47 significantly upregulated and 88 significantly down- regulated genes at 12 h after the treatment. There were 12 overrepresented GO terms for significantly upregu- lated genes, mostly related to unfolded protein binding and ATP binding, whereas no GO term was over- represented for significantly downregulated genes. These results collectively suggest high temperature could induce excessive oxidative stress through the repressed antioxidant ability, the apoptosis activated by the un- folded protein response, and further the collapse of the symbiosis between host and symbiont, which has been threatening the growth and reproduction of the giant clam T. crocea. 1. Introduction whereas populations of wild giant clams have been declining all over the world [5–9]. The shells of giant clams can provide substrate for Giant clams live mostly in coral reefs in the tropical Indo-Pacific epibionts and contribute to the complexity of the coral reef structure, through the symbiosis with the dinoflagellate Symbiodinium, colloqui- while their mantle cavities harbour substantial commensal and ecto- ally known as zooxanthellae [1]. The elaborately symbiosis endows parasitic organisms [10,11]. Furthermore, giant clams can expel Sym- giant clams with the successful adaptation to well-lit and oligotrophic biodinium, faeces and gametes to feed other organisms in the coral reef waters in coral reef ecosystem. Unlike the intracellular symbiosis in ecosystem [9]. Because the shifts of the mean annual sea surface tem- stony corals, symbiotic zooxanthellae reside extracellularly inside a perature due to global warming have caused the outbreak of coral tubular system permeating the outer mantle of giant clams [2]. The bleaching that increase both in frequency and severity, coral reef eco- symbionts provide giant clams with nutrients through photosynthesis, system has also been degrading over the past three decades [12]. The which can constitute up to 100% of the host's energy requirements [3]. destruction of environmental condition in coral reef ecosystem has In exchange, giant clams provide the symbionts with essential nutrients threaten the growth and reproduction of giant clams [13], and there- such as inorganic carbon, nitrogen, and phosphorus in support of their fore their conservation, reproduction and adaptation have been paid metabolism and growth [4]. more attentions for the restoration and protection of coral reef eco- Giant clams play various ecological roles in coral reef ecosystem, system [14]. ∗ Corresponding author. E-mail address: [email protected] (Z. Zhou). https://doi.org/10.1016/j.fsi.2018.10.033 Received 3 June 2018; Received in revised form 11 September 2018; Accepted 10 October 2018 Available online 11 October 2018 1050-4648/ © 2018 Elsevier Ltd. All rights reserved. Z. Zhou et al. Fish and Shellfish Immunology 84 (2019) 451–457 When giant clams suffer from heat stress, their symbiosis with homogenates with symbionts were centrifuged at 1500×g, 4 °C for zooxanthellae can collapse through the disordered metabolism, further 10 min, and the supernatants were used to determine enzyme activities resulting in their bleaching. It has been observed that the exposure to of giant clams. The harvested symbionts were resuspended in the fil- increased water temperature could cause significant respiration in- tered seawater after the washing three times, and counted using a crease with a high photosynthetic rate in giant clam Tridacna squamosa Neubauer hemocytometer (QIUJING, China). The surface area of the [15], and induce the change of fatty acid diversity and the expression outer mantle was determined using the scaled photo method [22], and upregulation of genes involved in lipids in giant clam Tridacna maxima the density of symbiotic zooxanthellae was defined as the ratio of their [16]. Heat stress in combination with an irradiance increase has been number to the surface area of the outer mantle (cell cm−2). reported to cause the loss of symbiont, the retention of small symbiont and the chlorophyll content decrease of remaining symbiont in giant 2.4. Activity assay of antioxidant enzyme clam Tridacna gigas, which further resulted in the collapse of the clam- zooxanthella symbiosis, and finally the bleaching of giant clam[17]. The activities of superoxide dismutase (SOD, JIANCHENG, A001) Furthermore, it has also been considered that heat stress could lead to and catalase (CAT, JIANCHENG, A007) in the supernatants were two sequential cell death processes for the symbionts in giant clam measured using commercial kits, according to the manufacturer's pro- Tridacna maxima [16], and total mortality of trochophores in giant clam tocols. Total SOD activity was determined by the hydroxylamine Tridacna squamosal [18–20]. These studies have described the responses method [23], where 1 SOD activity unit was defined as the enzyme of giant clam to heat stress in the morphology and metabolism level, amount causing 50% inhibition in 1 mL reaction solution. Total CAT however, the detailed physiological mechanism underlying the heat activity was detected using spectrophotometry to measure the yel- stress response of giant clam and their bleaching remains unclear. lowish complex compound generated after the reaction between hy- Giant clam Tridacna crocea is a species of bivalve in the family drogen peroxide and ammonium molybdate. Here, 1 CAT enzyme ac- Cardiidae. It is native to the Indo-Pacific region, and its typical habitat tivity unit referred to the amount of enzyme needed to degrade 1 mmol is embedded in massive corals. The giant clam has been reported to hydrogen peroxide per second. The concentration of total protein in the suffer from the serious bleaching and even death, and thismass supernatant was quantified by BCA method [24]. SOD and CAT activ- bleaching accompanied with high sea surface temperatures [17]. To ities in the supernatants were the ratio of the total enzyme activity unit understand systemically the potential effects of high temperature on the to the total protein, and the results were expressed as U mg−1 protein. physiology and symbiosis of giant clam, the symbiotic zooxanthella density, the crucial enzyme activities and the metatranscriptome were 2.5. Activity assay of caspase3 investigated in the giant clam T. crocea after the acute exposure to high temperature. The present study would provide insights to further un- The caspase3 activities in the supernatant was measured by derstand the physiological mechanisms underlying heat stress response Caspase-3 Colorimetric Assay Kit (KeyGEN BioTECH) according to the of giant clam and its potential environmental adaptability. instruction. Briefly, the supernatants of all samples were diluted firstly to the same protein concentration. Then, 50 μL supernatants were 2. Materials and methods added in the reaction mixture containing 50 μL reaction buffer and 5 μL substrate. After an incubation in the dark at 37 °C for 4 h, the color 2.1. Giant clam change was detected spectrophotometrically at the wavelength of 405 nm. The activity of caspase3 was defined as the absorbance of the The giant clams T. crocea (Shell length: 8–10 cm) were collected and reaction solution at 405 nm (ABS405), and the activation level of cas- cultured in flow-through aquaria (ca. 300 L) filled with natural sea- pase3 in the mantle of giant clams was defined as the ratio 405of ABS in water (Temperature: 26 °C; Salinity: 34) in a facility located at Hainan samples to that of the blank group. University. Cultures were illuminated with metal halide bulbs in a 12 h/ 12 h light-dark cycle for two weeks to acclimatize in laboratory con- 2.6. Deep sequencing of outer mantle metatranscriptomes ditions. Total RNA was isolated from outer mantle samples of giant clams 2.2. Acute heat stress exposure following the TRIzol reagent (Invitrogen) protocol. The extracted total RNA was quantified by Nanodrop 2000 (Thermo Scientific) at260/ A total of 25 giant clams were employed in the heat stress experi- 280 nm (ratio > 2.0) and its integrity was checked with Agilent 2100 ment. Ten giant clams were incubated at 32 °C, which were referred Bioanalyzer (Agilent Technologies).