Identification and functional analysis of a Hemolin like protein from Litopenaeus vannamei

• The first Hemolin gene identified in crustaceans.
• LvHemolin from Litopenaeus vannamei shows similarity with insect hemolins.
• LvHemolin exhibits agglutination activity against bacteria.
• LvHemolin plays a role in shrimp immunity against bacterial and viral infections.

Hemolin is a specific immune protein belonging to immunoglobulin superfamily and firstly identified in insects. Growing evidences suggest that Hemolin can be activated by bacterial and viral infections and may play an important role in antimicrobial immunity. In this paper, we firstly identified a Hemolin-like protein from Litopenaeus vannamei (LvHemolin). Sequence analysis showed that LvHemolin shares high similarity with insect Hemolins and is mainly composed of seven immunoglobulin (Ig) domains which form a ‘horseshoe’ tertiary structure. Tissue distribution analysis demonstrated that LvHemolin mainly expressed in stomach, gill, epithelium and pyloric cecum of L. vannamei. After challenge with pathogens or stimulants, expression of LvHemolin was significantly up-regulated in both gill and stomach. Agglutination analysis demonstrated that recombinant LvHemolin protein purified from Escherichia coli could accelerate the agglutination of Vibrio parahaemolyticus, E. coli, Staphylococcus aureus, and Bacillus subtilis in the presence of Ca2+. To verify the immune function of LvHemolin in vivo, shrimps were injected with gene-specific dsRNA, followed by challenge with white spot syndrome virus (WSSV) or V. parahaemolyticus. The results revealed that silence of LvHemolin could increase the cumulative mortalities of shrimps challenged by pathogens and increase the WSSV copies in shrimp tissues. These suggested that Hemolin could play an important role in shrimp innate immune defense against bacterial and viral infections.