Progress in Fishery Sciences (Dec 2023)
Transcriptomic Analysis and Stress Response of Chlamys farreri to Paralytic Shellfish Toxins
Abstract
Paralytic shellfish toxins (PSTs) are some of the most harmful algal neurotoxins in the world. They easily accumulate in bivalve shellfish and are transmitted through the food chain, causing symptoms such as nausea and vomiting, muscle paralysis, dyspnea and even asphyxiation in consumers, leading to food poisoning in humans. Therefore, a widely accepted limit standard of 800 μg STXeq/kg has been established as a safe limit for PSTs. PSTs are produced by some microalgae, among which Alexandrium tamarense is one of the predominant toxic algae found along the coast of China. It was found that the detection rate and over-standard rate of Chlamys farreri in bivalve shellfish sold in China are relatively high. PSTs are mainly stored in visceral masses and are characterized by their fast accumulation and slow metabolism.PSTs are neurotoxins that exert their toxic effects by blocking sodium channels and inhibiting nerve conduction. Studies have shown that PSTs can cause stress responses in bivalves, including production of a large amount of reactive oxygen species (ROS), antioxidant stress (including enzymatic and non-enzymatic defense), imbalance of intracellular redox homeostasis, and cell damage (i.e., lipid peroxidation). As one of the main products of lipid peroxidation, the content of malondialdehyde (MDA) can directly reflect tissue and cell membrane damage caused by PSTs. In addition, superoxide dismutase (SOD) and peroxidase (POD) are often used as indicators to evaluate the level of antioxidation, and glutathione peroxidase (GSH-Px) plays a key role in antioxidant defense. The changes in lipid peroxidation and antioxidant enzymes are commonly used in existing studies to reflect the injury and degree of stress in organisms. Some studies have also shown that PSTs can cause tissue damage and induce abnormal gene expression in C. farreri, but research on the changes of gene expression and regulatory mechanism of PST-induced tissue damage in C. farreri is still lacking. This information is important for establishing and perfecting food safety risk assessment technology.In this study, C. farreri was exposed to a strain of A. tamarense (AT5-3). We measured the toxin accumulation, oxidative stress kinase activity, and its transcriptional regulation in the visceral mass of C. farreri in control and experimental groups. Further, the ultrastructure of the visceral mass in the control group and the experimental group was observed to explore the initial stress response mechanism of C. farreri exposed to PSTs.The 2-year-old scallop C. farreri was selected as the experimental animal. AT5-3 was cultured in L1 medium at temperatures of (20±1) ℃, light intensity of 54 μ/Em2·s, and a light-dark ratio of 12 h: 12 h in the laboratory, and Chlorella vulgaris was cultured simultaneously. The experimental group was fed with algal solution in the exponential growth period and the cell density was 4×104–4.2×104 cells/mL. The control group was fed with the same amount of C. vulgaris. The experiment lasted 20 days, of which the first six (days 0–6) were the exposure stage and the remaining 14 days (days 7–20) were the metabolic stage. During the exposure stage, C. farreri were fed regularly with the AT5-3 strain in exponential growth period twice a day with a feeding dose of 8×106 cells/items/day. The same amount of C. vulgaris was fed in the metabolic stage.The results of toxin accumulation showed that paralytic shellfish toxins could accumulate rapidly in the visceral mass of C. farreri, but the metabolic rate was slow. The toxin content was highest on day 6 of the experiment, and the maximum accumulated content was approximately 15 times higher than the limit standard. The toxin residue reached its highest level (62.4%) on 20th day of the experiment.The results of enzyme activity tests showed that the stress due to MDA, GSH-Px, and POD in the visceral mass was significantly increase (P < 0.05), and the SOD activity was significantly inhibited (P < 0.05) after a brief increase. The results also showed that PSTs could induce lipid peroxidation in C. farreri, and POD, SOD, and GSH-Px were significantly stressed to eliminate the adverse effects of PSTs.The pathological changes in the visceral mass were observed under a transmission electron microscope and included vacuolation, chromatin aggregation, and nucleoplasmic pyknosis. Tissue damage worsened as the exposure time increased and although the toxin content in C. farreri decreased after the exposure period, the tissue damage was further aggravated. The results of transcriptome analysis showed that 933 differentially expressed genes (DEGs) were screened from the visceral mass of C. farreri after PST exposure. The results of KEGG and GO annotations showed that DEGs were mainly annotated in amino acid metabolism, energy metabolism, and other metabolic processes. Weighted gene co-expression network analysis showed that apoptosis and the glutathione metabolic detoxification pathway were significantly up-regulated, mapping ALOX5, AfGST-σ11, caspase-8 and Bax4 key transcription factors. In the experimental group, the expression of ALOX5 and AfGST-σ11 increased significantly when the accumulation rate was highest (P < 0.05). The expression of caspase-8 and Bax4 was highest when toxin accumulation was high, which was significantly higher than that in the control group (P < 0.05).In summary, PSTs can cause lipid peroxidation stress and cell damage to C. farreri. In addition to antioxidant stress, C. farreri can activate characteristic apoptosis and resist PSTs toxicity by glutathione detoxification metabolism. However, this effect is limited and the persistent damage caused by high residual PSTs cannot be eliminated. This study provides a basis for further study on the potential toxicity of PSTs to the scallop C. farreri and its immunomolecular mechanisms.
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