Abstract
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 Aeromonas hydrophila, among which anti-lipopolysaccharide factor 1 (EsALF-1) and 3 (EsALF-3) were identified as the most significant (p < 0.05). After being challenged with A. hydrophila, EsALF-1 and EsALF-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 EsTLR1-EsMyd88-EsPelle-EsALF pathway also increased significantly (p < 0.05). The EsALF-3 and EsMyd88 were even significantly higher expressed in response to the second A. hydrophila challenge, but their expressions all decreased (p < 0.05) when EsTLR1 was knocked down by RNAi. After the naïve crabs received an injection with the recombinant protein of EsALF-1 (rEsALF-1) or EsALF-3 (rEsALF-3), their survival rate increased significantly (p < 0.05) upon A. hydrophila stimulation. In contrast, the survival rate of the primed crabs reduced significantly (p < 0.05) after they received an injection with the antibody of EsALF-1 or EsALF-3. The enhanced expressions of EsALF-1 and EsALF-3 after A. hydrophilap riming stimulation could sustain for four weeks. All the results suggested that the EsTLR1-mediated productions of EsALF-1 and EsALF-3 in haemolymph played an indispensable role in the month-long humoral immune protection induced by A. hydrophila, which provides solid evidence of immune priming in crabs and a valuable reference for further understanding immune memory in invertebrates.
Highlights
The passive immune protection of sera from pre-exposed individuals was uncovered and applied in humans a hundred years ago, leading to subsequent study of immune memory and the development of vaccines
The survival rates of crabs in the priming haemolymph transferred group (PHT), blank haemolymph transferred group (BHT), and naïve crab group (Naïve) were monitored after the crabs were challenged with live bacteria A. hydrophila
A total of 13 individuals died in the Naïve group, which was more than that in the BHT group (10 individuals) and the PHT group (6 individuals)
Summary
The passive immune protection of sera from pre-exposed individuals was uncovered and applied in humans a hundred years ago, leading to subsequent study of immune memory and the development of vaccines. Elevated specific antibodies and related complement components in serum after pre-infection or vaccination provide long-lasting enhanced protection from re-infection [1] This convalescent plasma can even confer enhanced resistance in naïve individuals against pathogens when it is transferred into recipients, which is used as an emergent clinical therapy in humans for fatal infectious diseases [2, 3]. Enhanced immune protection after pre-infection is increasingly reported in mollusks, insects, and crustaceans, indicating the existence of immune memory (named immune priming) or immune enhancement in invertebrates [4,5,6] In these animals, the immune defense capacity and survival rate increase significantly when they encounter the same pathogen for the second time. A number of effector molecules and immune related signal pathways have been suggested to be involved in these humoral and cellular protective responses
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