Molecular Characterization And Expression Analysis Of Complement Component 3 In Dojo Loach(Misgurnus Anguillicaudatus)

The dojo loach(Misgurnus anguillicaudatus)is one of the most commercially cultured fish species in Eastern Asian countries,with rapid development of loach culture industry,the infectious disease caused by pathogens are becoming more and more severe,resulting in great economic losses.However,little is known about the immunological basis of loach.The complement system is an important link between innate and adaptive immunity in vertebrates,playing a pivotal role in the defense against pathogens infection.The complement component 3(C3)is a central component of complement system.All three pathways converge at formation of C3 convertases and share the terminal pathways of membrane attack complex(MAC)formation.In this study,we used molecular cloning technique to clone C3 of Misgurnus anguillicaudatus(Ma-C3),constructed the phylogenetic trees of Ma-C3 and analyzed m RNA expression levels of C3 in different tissues.Further,we constructed a model of soaking infecting by Aeromonas hydrophila.We assessed the effects of bacterial challenge on the morphological changes in gill and skin,used q PCR to analysis the m RNA expression of Ma-C3 and complement relevant genes in response to A.hydrophila infection.1.In the present study,three isoforms of C3 were discovered in Misgurnus anguillicaudatus,named “C3-1”,“C3-2” and “C3-3”,respectively.The full-length of C3-1 c DNA sequence was firstly identified and analyzed from dojo loach(Misgurnus anguillicaudatus).The Ma-C3-1 c DNA sequence comprised of 4509 bp encoding 1454 amino acids with a putative signal peptide of 20 amino acid residues.The deduced amino acid sequence showed that Ma-C3-1 has conserved residues and domain,which are known to be crucial for C3 function.Interestingly,an amino acid substitution of the highly conserved GCGEQ was discovered in Ma-C3-1.Phylogenetic analysis showed that Ma-C3-1 was closely related to Cyprinidae.The m RNA expression levels of three isoforms of C3 were detected in kidney,eye,spleen,gonad,heart,fin ray,gut,muscle,brain,gill,skin,blood and liver.The expression of Ma-C3-1 and Ma-C3-3 were mainly detected in liver,followed by spleen,gonad.However,the high expression of Ma-C3-2 was found in kidney,followed by blood and gonad.2.In the present study,challenge with A.hydrophila(1 x 10~7 CFU/ml)was performed.the lesions were observed from gill and skin in the loach obviously worsened after infected with A.hydrophila.The length-width ratio of the secondary lamellae(SL)of infected loach gill was detected smaller than control fish.Similar results also observed in skin tissue,the width of skin epidermis become wider than control fish after infected with A.hydrophila.3.In the present study,challenge with A.hydrophila(1 x 10~7 CFU/ml)was performed.Three isoforms of C3 expression levels were found up-regulated to various extents in four tested tissues at different times.In loach gill,the expression level of MaC3-1m RNA was found obviously up-regulated at 36 h post infection,whereas its expression in the skin was found significantly up-regulated at 24 h.Similarly,the expression of Ma-C3-3 in gill showed significant up-regulated at 36 h.However,the expression of Ma-C3-3 in skin showed significant up-regulated at 24 h.Ma-C3-2 transcripts in the gill were substantially increased at 12 h after infected with A.hydrophila,and reached a peak at 48 h.The expression of Ma-C3-2 in skin was found up-regulated at 48 h after infected with A.hydrophila.Three isoforms of C3 expression in the liver and spleen were found up-regulated after bacterial challenge.To gain insights into the kinetics of the complement system immune responses that takes place following infection,ten complement relevant genes including BF,C1 qb,C2b,C3 a R,C3 b,C4,C5,C8 b and MASP-1 were measured by q PCR.The studies show that following infection with A.hydrophila,most complement relevant genes were significantly up-regulated in gill,liver and skin.In conclusion,three isoforms of C3 were discovered in Misgurnus anguillicaudatus,we identified and characterized full-length c DNAs of C3-1 from M.anguillicaudatus for the first time.The deduced amino acid sequence of Ma-C3-1 showed similarity with other teleost fish.Three isoforms of C3 were expressed in various tissues,but with different tissue-specific expression profiles.Morphological analysis in loach gill and skin tissues showed significant changed after infection with A.hydrophila.Challenge experiments resulted in significant up-regulated expression of three isoforms of C3 and other complement-relevant genes in gill,liver and skin.Taken together,these results suggest that complement C3 might play an important role not only in live,but also in the mucosal tissues as gill and skin of teleost fish after bacterial infection.The results can also provide a foundation for subsequent functional studies of complement C3.