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Eriocheir sinensis CD63 activate mitochondria-mediated apoptosis to resist Spiroplasma eriocheiris infection
Wenbo Li1, Guanzheng Yang1, Yangzhi Fan1
1Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Marine Science and Engineering, Nanjing Normal University, 2 Xuelin Road, Nanjing, 210023, China.
Abstract:
CD63, a member of the tetraspanins, is involved in cell movement, adhesion, immune response. Nevertheless, the role of CD63 in combating pathogen infections in invertebrates remains largely unclear. Tremor disease, whose pathogen is Spiroplasma eriocheiris, is one of the most prevalent illnesses affecting Eriocheir sinensis. EsCD63 is 1474 bp, with a 756 bp open reading frame that encodes for 252 amino acids. The qPCR data demonstrated that gills showed significant levels of transcription for EsCD63, followed by hemocytes, hepatopancreas, intestines and nerves, while showing low levels of transcription in the heart and muscles. After infection with S. eriocheiris, an obvious drop in the transcription level of EsCD63 was observed. Both the amount of S. eriocheiris copies in hemocytes and the mortality of E. sinensis significantly increased after the injection of chemically synthesized EsCD63 siRNA and stimulation with S. eriocheiris. After EsCD63 interference, the phagocytosis of hemocytes to S. eriocheiris, the apoptosis of hemocytes, and reactive oxygen species level of hemocytes were all decreased significantly, by laser scanning confocal microscopy and flow cytometry analysis. Meanwhile, the mitochondrial membrane potential of hemocytes was increased after EsCD63 interference. These findings indicated that EsCD63 was crucial for E. sinensis immunity and defense mechanisms against infection of S. eriocheiris.
Insights
The study reveals that EsCD63 is vital for the immune defense of Eriocheir sinensis against Spiroplasma eriocheiris infection. Silencing EsCD63 in crabs significantly impairs their ability to fight this pathogen.
Area of Science:
- Marine Biology
- Immunology
- Molecular Biology
Background:
- Tetraspanins, like CD63, play roles in cellular processes, but their function in invertebrate immunity against pathogens is not well understood.
- Tremor disease, caused by Spiroplasma eriocheiris, is a significant threat to the Chinese mitten crab, Eriocheir sinensis.
Purpose of the Study:
- To investigate the role of EsCD63 in the immune response of Eriocheir sinensis against Spiroplasma eriocheiris infection.
- To characterize the expression pattern and functional significance of EsCD63 in crab immunity.
Main Methods:
- Quantitative PCR (qPCR) was used to determine EsCD63 transcription levels in various tissues.
- RNA interference (siRNA) was employed to silence EsCD63 expression.
- Laser scanning confocal microscopy and flow cytometry were utilized to assess hemocyte functions, including phagocytosis, apoptosis, reactive oxygen species (ROS) levels, and mitochondrial membrane potential.
- Bacterial load and crab mortality were monitored after infection and EsCD63 interference.
Main Results:
- EsCD63 was highly transcribed in gills and hemocytes of E. sinensis, with levels decreasing significantly after S. eriocheiris infection.
- EsCD63 gene silencing led to increased S. eriocheiris loads in hemocytes and higher crab mortality.
- Interference with EsCD63 reduced hemocyte phagocytosis, apoptosis, and ROS production, while increasing mitochondrial membrane potential.
Conclusions:
- EsCD63 is a crucial component of the innate immune system in Eriocheir sinensis, playing a significant role in defense against Spiroplasma eriocheiris.
- EsCD63 is essential for maintaining hemocyte function, including phagocytosis and ROS generation, which are critical for combating bacterial infections.
