Okadaic acid (OA) is the main marine toxin responsible for the DSP that causes gastrointestinal symptoms in humans following consumption of contaminated bivalves. OA, a potent and non-selective inhibitor of serine/threonine phosphatases, has been shown to be cytotoxic in a variety of cell lines [20]. Previous reports revealed that constant contact with OA induced chromosome loss, apoptosis, DNA damage, and inhibited phosphatases in contaminated bivalves. In this study, immune-system responses and expression of immune-system-related genes were determined in the hemolymph of bay scallops following exposure to different concentrations of OA.

Several immune parameters have been successfully employed to evaluate the immune status of bivalves subjected to various stresses [21]. The total hemocyte count (THC) is one of the most widely used parameters to assess bivalve health status as the number of circulating hemocyte changes under stressful conditions [9]. In this study, THC decreased significantly in the medium to high concentrations of OA-treatment groups after 12 hpe. A similar situation was observed in the clam, Ruditapes philippinarum, where the THC decreased upon exposure to the dinoflagellate Prorocentrum minimum, which produces algal diarrheic shellfish toxins, such as OA and dinophysistoxins [9,22]. Exposure to higher concentrations of OA for longer periods resulted in higher apoptosis. This suggests that OA induced hemocyte death in a dose and time-dependent manner. Similarly, previous reports have revealed that OA induced apoptosis even in human monocytic U-937 cells [20]. Therefore, this result suggests that OA exposure could disturb and restrain the immune response of bay scallops. ROS production is another important mechanism of bivalve cellular immunity. Although a small amount of ROS is necessary to enhance the internal defense against pathogens, serious damage to lipids, proteins, and DNA occurs when the generation of ROS is excessive [13]. A significant increase in ROS in OA-treated scallops was observed during all time intervals, and ROS generation was time and dose-dependent. This is in agreement with the previous report of OA-induced ROS generation in human monocytic U-937 cells [20]. Therefore, our results indicated that exposure to OA generated ROS in scallops.

Malondialdehyde (MDA) is closely related to the membrane lipid peroxidation status; therefore MDA content assay is used to indirectly evaluate the extent of oxidative damage [23]. In this study, exposure to OA resulted in significant increases in MDA content in all time intervals, which continued to increase as the exposure time increased. The current results are agreement with previous study, which reported that MDA level in the hemolymph of the temperate scallop Pecten maximus significantly increases with exposure to phenanthrene [24].This phenomenon indicated that the hemolymph suffered from serious oxidative stress and oxidative damage to macromolecules. In addition, the NO level was significantly increased following exposure to OA, and sharply increased from 12 hpe in the group exposed to highest concentration of OA. In bivalves, NO is an essential molecule that is related to normal physiological functions [25], such as the regulation of neural transmission, vascular tone, and immune defense [19]. Although NO is not toxic in itself, during phagocytosis, in combination with superoxide anions synthesis, it generates the highly toxic peroxynitrite anion (ONOO-) [25]. Recently, it has been shown that production of the physiological messenger NO increased after treatment with the polyunsaturated aldehyde decadienal produced by diatoms in embryos of the sea urchin Paracentrotus lividus [26]. Moreover, a significant increase in NO production was reported in sea urchin embryos and in the different developmental stages of the offspring derived from females of the sea urchin exposed to heavy metal (e.g., cadmium and manganese ) contamination [27,28]. C.M. de Barros et al. [29] also reported that zymosan A and lipopolysaccharide (LPS) enhanced NO production in hemocytes of the ascidian Phallusia nigra. Our results of the present investigation are in accordance with the report of Migliaccio et al. [30] that adult sea urchin showed high nitric oxide (NO) levels, along with a low fertilization rate after toxic blooms of Ostreospsis cf. ovata. These previous studies indicated that Zymosan A, LPS, or other soluble or particulate non-self materials could stimulate NO production in mollusk. Therefore, the results of our study suggests that NO mediates the stress response of bay scallop against the toxic effects of OA. Lactate dehydrogenase (LDH) is a soluble cytosolic enzyme present in most living cells. This enzyme catalyzes the reversible oxidation of l-lactate to pyruvate, with nicotinamide adenine dinucleotide (NAD+) as a hydrogen acceptor in the final step of the metabolic chain of anaerobic glycolysis [17,31]. Therefore, LDH is widely used as a biomarker in toxicology and clinical chemistry to diagnose cell, tissue, and organ damage. In this study, bay scallops treated with different concentrations of OA produced notable increases in LDH activity in the hemolymph. This increase in LDH activity reflected OA damage to tissues or apoptosis. Ravindran et al. [20] also reported a significant increase in LDH activity in U-937 cells from OA exposure after 4 h, reaching a maximum at 16 h. Traoré et al. [32] reported that 15 ng/mL of OA increased the release of LDH in Vero cells.

Pollutants can produce free radical O₂ and H₂O₂ through biotransformation, both of which may cause oxidative stress if not metabolized quickly [33]. Glutathione (GSH), which catalyzes H₂O₂ to molecular water, plays a crucial role in protecting organisms from oxidative stress. Although a significant increase in GSH level was observed in the OA treatment groups at 12 hpe, which reflects an up-regulation of antioxidant defenses and, thereafter, it gradually decreased in all treatment groups, may be due to overwhelming of the antioxidant capacity that can lead to mass oxidation of GSH resulting in excretion of the oxidised molecule (GSSG) from the cell leading to a reduced intracellular concentration of GSH level [34]. Ravindran et al. [20] also reported a significant decrease in GSH in OA-treated U-937 cells. Moreover, Zhang et al. [35] revealed that GSH levels in one-month-old mice reduced at 24 h after intraperitoneal injection of OA. In addition, a similar reduction in total glutathione has been reported in scallop exposed to the acute oil after 48 h [34]. Superoxide dismutase (SOD) is another important antioxidant enzyme that catalyzes conversion of superoxide to oxygen and H₂O₂, and has been considered as a suitable indicator for ecological risk assessments [32]. Based on the metal ion cofactor in the active site, SODs are classified into iron SOD (Fe SOD), manganese SOD (Mn SOD), nickel SOD, and copper-zinc SOD (Cu, Zn SOD); the Cu/ZnSODs have two forms: cytosol dimmers and extracellular tetramers [36]. In the hemolymph of bay scallops, the expression of the Cu/ZnSOD gene was significantly down-regulated in all of the OA-treated groups at 6–48 hpe. This indicated that OA inhibited the expression of the Cu/ZnSOD gene, and inhibited the antioxidant abilities of scallops exposed to OA. Similarly, Zhang et al. [35] revealed that SOD activity in one-month-old mice was significant lower than that in the control group at 24 h after intraperitoneal injection of OA, similar to our results. Results of the present study suggest stimulated ROS production exceeded the neutralizing capabilities of the antioxidant system and reduced the GSH level.

In addition, heat shock proteins (HSPs) play a significant role in preventing irreversible protein denaturation, promoting either repair or destruction of damaged proteins. The expression of the HSP90 gene notably increased after exposure to OA in a dose and time-dependent manner, suggesting that HSPs (HSP90 gene) protected the bay scallop against higher concentrations of OA as the exposure time increased. These results are consistent with those reported by Manfrin et al. [37] who showed that the expression of the HSP90 gene in the mussel Mytilus galloprovincialis was significantly induced by OA exposure. Wang et al. [21] also reported that expression of the HSP90 gene was significantly induced after 24 h exposure to environmental ammonia-N. Although OA exposure stimulated the antioxidant systems of scallops, significant increases in MDA, ROS level, and LDH activity in the hemolymph of scallops were observed. This reflected the limited abilities of antioxidant systems in scallops to fully remove these harmful superoxide radicals, resulting in oxidative damage to macromolecules.

Metallothionein (MT) is a group of molecules involved in responses to oxidative stress, especially from toxic metals [25]. MT induction has also been found to respond to tissue injury, infection, and inflammation; therefore, MT is probably important in the immune system of scallops [38,39]. In our study, expression of the MT gene was significantly suppressed in the 50 and 100 nM OA-treated groups up to 6 hpe, and then markedly increased until 48 hpe; however, the result was opposite in the highest concentration of OA treated group. A previous study also revealed [23] that expression of the MT gene was up regulated by 80 mg/L of a chemical contaminant palmitoleic acid (PA) in a short-space of time at 12 hpe, and was suppressed up to 48 hpe. This phenomenon may be attributed to the high concentration of OA modulating MT gene expression in a short exposure period and inducing MT mRNA transcription to briefly counter the tissue injury or oxidative stress, but finally strongly inhibiting expression. However, the lower concentrations took more time to modulate MT gene expression, and it could be that the MT mRNA transcription was inhibited with the increasing exposure time. In general, these results reflect the weakened ability of scallops treated with OA to respond to oxidative stress, tissue injury, infection, and inflammation.

C-type lectins act as a first line of defense against pathogens; they recognize and bind to terminal sugars on glycoproteins and glycolipids, and play significant roles in non-self recognition and the clearance of foreign particles, either as cell surface receptors for microbial carbohydrates or as soluble proteins existing in scallop tissue fluids [37]. We found that scallops exposed to the lowest concentration of OA attenuated the expected expression of CLT-6 mRNA at 3–6 hpe, with the higher concentrations of OA producing significantly higher CLT-6 expression at each time interval. These results are consistent with the previous investigation in which PA exposure modulated the expected expression of the CLT-6 gene, suggesting that the antibacterial or antivirus ability of scallops was negatively impacted by OA exposure [23]. Fibrinogen-related protein (FREP) is another pattern recognition receptor in innate immune system responses, especially in the clearance of non-self materials, such as microorganisms and late apoptotic cells through the lectin pathway [40]. In this study, expression of the FREP gene was inhibited in all of the treatment groups at 3–48 hpe, except 100 and 500 nM at 6 hpe. These results indicated that OA exposure inhibited CLT-6 and FREP gene expression, and then restrained the ability of scallops to recognize and clear non-self particles, such as microorganisms and late apoptotic cells through the lectin pathway.

In conclusion, this study shows that the bay scallop, A. irradians was affected immunologically by OA exposure. Altogether, these results showed significant changes in several of the immune-system-related parameters that we monitored (THC, ROS, MDA, LDH, NO, and GSH) indicating that the toxin OA can slowly diminish the immune system of bay scallop, especially when they are exposed to high concentrations of toxins or a long exposure period. Furthermore, significant changes in expression of immune response related genes (CLT-6, FREP, HSP90, Cu/ZnSOD, and MT) suggests cellular stresses. This study revealed OA-induced immunological and physiological effects on a species of bivalve—the bay scallop—and helps to establish the species of bivalves that are immunologically more sensitive to algal toxins. This could serve to better control potential infections, inflammation, or other oxidative damage that may further affect cultivated scallops already immuno-depleted by the algal toxin OA.