Preliminary Study on Phenomenon and Mechanism of Immune Priming in Eriocheir Sinensis
|School||Graduate School , Chinese Academy of Sciences ( Institute of Oceanography )|
|Keywords||Eriocheir sinensis immune priming Dscam Peroxinectin Masquerade|
Immune priming plays an important role in the process of fighting againstpathogen invasion, especially when invertebrates encountered secondly with theparticular bacteria. And it could provide new insights into the strategy of diseasecontrol in invertebrates. This phenomenon has been found in many invertebrates, butthe relative study on its mechanism is still limited. In the present study, the survivalrate, cellular and humoral indicators were detected using immunological methodsafter Chinese mitten crab Eriocheir sinensis were “vaccinated” or successivelychallenged with different bacteria. Two adhesion molecules, Dscam (Down syndromecell adhesion molecule) and Masquerade in crabs were cloned and the putativefunctions of these three adhesion molecules, including Peroxinectin, in innate defenseand immune priming were explored using recombinant protein and RT-PCR methods.When crabs were challenged with live Aeromonas hydrophila, the survival ratein A. hydrophila-immuned group was significantly higher than that in non-immunedgroup, Micrococcus luteus-immuned group and saline group. And enhancedphagocytic rate were observed in A. hydrophila-immuned group compared with M.luteus-immuned group and the group which only received first immunization withkilled A. hydrophila. The antibacterial activity in serum of A. hydrophila-immunedgroup is higher to the same bacteria than the different class of bacteria, indicating acertain degree of specificity. But the antibacterial activity in serum of M. luteus-immuned group had no sign of specificity. The increasement of PO expression in A.hydrophila-immuned group is higher than that in M. luteus immuned group. Theactivity of lysozyme did not change between the two injections.The full-length cDNA sequences of Dscam and Masquerade were5125bp and1525bp, respectively. The open reading frame (ORF) of Dscam encoded apolypeptide consisted of10Ig domains and6FN3domains and there were diversealternative splicing forms in Ig2,3and7regions. The ORF of Masquerade encoded a polypeptide consisted of a serine protease domain. The mRNA of Dscam,Peroxinectin and Masquerade could be detected in all the tested tissues and waspredominantly expressed in heart, hemocyte and hepatopancreas, hemocyte,respectively. The protein of Dscam and Peroxinectin were mainly detected in serumand membrane in hemocyte. The mRNA profile of hemocytes after different bacteriachallenge showed that the mRNA of Dscam and Peroxinectin was higher expressedafter Pichia pastoris GS115stimulation than other bacteria, while Masquerade mRNAwas sensitive to Listonella anguillarum. After secondly challenged with A. hydrophila,Dscam mRNA was significantly higher than that after first immunization. The mRNAlevels of Peroxinectin and Masquerade both increased after first immunization, but thelevel of Peroxinectin significantly decreased and the level of Masquerade had nodistinct change after A. hydrophila challenge. Recombinant protein of Dscam,Peroxinectin and Masquerade could all modulate encapsulation of crab hemocyte.Peroxinectin and Masquerade could also enhance phagocytic rate in vitro.Furthermore, Masquerade had capacity to inhibit the growth of E. coli.All the results suggested that a second encounter with the particular pathogenresulted in the enhanced immunity with certain degree of specificity, indicating theexistence of immune priming in crabs. Three adhesion molecules including Dscam,Peroxinectin and Masquerade were involved in immune defense to pathogens andmight take part in immune priming with varying ways in crabs. The results providenew information for the enrichment and development of immune priming andcontribute to the further knowledge of mechanism on immune priming ininvertebrates as well.