Effects of bifenthrin on the formation of extracellular traps in hemocytes of Haliotis discus hannai

Bifenthrin (BF) is widely used in agricultural production due to its excellent environmental stability and insecticidal activity, resulting in its widespread residue in the environment. In recent years, BF has been frequently detected in coastal areas of China, which would harm aquatic organisms. Extracellular traps play an important role in innate immunity in molluscs. However, knowledge of the formation of ETs is still limited in the marine mollusks induced by environmental contaminants so far. This study aimed to investigate the effects of bifenthrin on the formation of extracellular traps in hemocytes of Haliotis discus hannai. Here, H. discus hannai, with an average shell length of approximately 60 mm, was collected from a local farm and maintained in aerated seawater (temperature 20-22 °C; salinity 28-30) for a week before processing. Then hemocytes were obtained and stimulated by BF (0, 0.01, 0.10 and 1.00 mg/L) for an hour. The results showed that the cell viability of hemocytes decreased to 90.40%, 80.22% and 72.28%, respectively, in a dose-dependent manner. Meanwhile, observed formation of ETs outside hemocytes induced by BF, quantitative analysis revealed the amount of ETs formation significantly increases in a dose-dependent manne, compared to the control group. A more abundant structure of ETs could be induced by 1 mg/L BF. Furthermore, the mRNA expressions of phosphatidylinositol 3 kinase (PI3K) and protein kinase B (Akt) were significantly upregulated in a similar trend in response to the BF-induced reactive oxygen species (ROS) burst and hypoxia inducible factor 1α (HIF-1α) increased significantly (except 0.10 mg/L). Notably, we detected an increase of ROS production during ETs. Quantitative analysis revealed that ROS production increased significantly with the increased BF concentration, as well as in a dose-dependent manner. Furthermore, the formation of ETs was blocked with the inhibition of ROS production by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (DPI). Meanwhile, the mRNA expression of pyruvate kinase (PK) and hexokinase (HK) was significantly elevated, indicating that during the formation of extracellular traps, part of the energy was supplied by the glycolytic pathway. In summary, stimulation by bifenthrin causes toxic effects on H. discus hannai hemocytes, which in turn exert immune functions through the formation of ETs. The ROS-mediated mediates the formation of ETs, and the PI3K/AKT signaling pathway. HIF-1α, is involved in the formation of ETs, meanwhile energetically supplied by glycolysis. This study preliminarily explored the response of hemocytes of H. discus hannai under BF stress to ETs, to provied a reference for further research on the immune response of hemocytes in shellfish and expand the understanding of environmental pesticide residues on cellular immune toxicity.