Three tetraspanins, Tspan1, Tspan2A and Tspan3 from Litopenaeus vannamei involved into Vibrio parahaemolyticus infection

Tetraspanins have essential roles in immune defense in eukaryotes. However, the research of tetraspanins in the immune system of crustaceans is relatively limited. Our previous transcriptome analysis found that the expression of three members of the tetraspanin family - Tspan1, Tspan2A and Tspan3 - was significantly changed in Litopenaeus vannamei after Vibrio parahaemolyticus infection. In this study, these three genes were cloned from L. vannamei, and the results showed that LvTspan1, LvTspan2A and LvTspan3 contained open reading frames (ORFs) of 1188 bp, 717 bp and 720 bp, encoding 395, 238 and 239 amino acids, respectively, all of which possessed a typical tetraspanin domain. Expression analysis showed that these tetraspanins were highly expressed in immune- related tissues including intestine, hemocytes and hepatopancreas and responded to V. parahaemolyticus infection. To investigate their innate immune functions, they were further subjected to dsRNA interference experiments. Results showed that the knockdown of LvTspan1 and LvTspan3 significantly reduced shrimp survival rates, whereas LvTspan2A knockdown showed no significant difference compared to the control (dsRNA-GFP). Furthermore, LvTspan1 and LvTspan3 knockdown increased bacterial loads in hemolymph and hepatopancreas, and exacerbated damage of intestine and hepatopancreas from dsRNA-LvTspan1 silencing shrimps under V. parahaemolyticus challenge. Additionally, the knockdown of LvTspan1 downregulated the expression of the IMD and JAK/STAT pathways (Stat and Domeless), leading to the reduced expression of associated antimicrobial peptides (Pens, Crus and Lyzs) and proPO-activating system genes (proPO1 and proPO2). These results indicated that LvTspan1 and LvTspan3 participated in the response of L. vannamei to V. parahaemolyticus challenge by regulating shrimp innate immunity.