The Elevated Expressions of Anti-lipopolysaccharide Factors After Priming Stimulation Confer Lastingly Humoral Protection in Crab .

Evidence of immune memory in invertebrates (immune priming) has accumulated in various organisms, and both cellular and humoral immune reactions are speculated to be involved in immune priming. However, there is a lack of understanding of the molecular mechanisms involved. In the present study, the protective effect of primed haemolymph was further validated by the increased survival rate of naïve crabs receiving a transfusion of primed haemolymph. By proteomic analysis, there were 474 proteins identified from the primed haemolymph, and most of them were functionally annotated in transport and metabolism classes. A total of 70 proteins were found to be differentially expressed in haemolymph at 12 hours and 7 days after priming stimulation with , among which anti-lipopolysaccharide factor 1 (ALF-1) and 3 (ALF-3) were identified as the most significant ( < 0.05). After being challenged with , ALF-1 and ALF-3 were highly expressed at both mRNA (in haemocytes) and protein (in haemolymph) levels compared with blank crabs, and the mRNA expressions of components in the TLR1-Myd88-Pelle-ALF pathway also increased significantly ( < 0.05). The ALF-3 and Myd88 were even significantly higher expressed in response to the second . challenge, but their expressions all decreased ( < 0.05) when TLR1 was knocked down by RNAi. After the naïve crabs received an injection with the recombinant protein of ALF-1 (rALF-1) or ALF-3 (rALF-3), their survival rate increased significantly ( < 0.05) upon . stimulation. In contrast, the survival rate of the primed crabs reduced significantly ( < 0.05) after they received an injection with the antibody of ALF-1 or ALF-3. The enhanced expressions of sALF-1 and ALF-3 after . p riming stimulation could sustain for four weeks. All the results suggested that the TLR1-mediated productions of ALF-1 and ALF-3 in haemolymph played an indispensable role in the month-long humoral immune protection induced by . , which provides solid evidence of immune priming in crabs and a valuable reference for further understanding immune memory in invertebrates.