A C-type lectin (CTL2) mediated both humoral and cellular immunity against bacterial infection in Tribolium castaneum

C-type lectins (CTLs) play essential roles in humoral and cellular immune responses of invertebrates. Previous studies have demonstrated the involvement of CTLs in the humoral immunity of Tribolium castaneum, a worldwide pest in stored products. However, the function of CTLs in cellular immunity remains unclear. Here, we identified a CTL gene located on chromosome X and designated it as CTL2 (TcCTL2) from T. castaneum. It encodes a protein of 305 amino acids with a secretion signal peptide and a carbohydrate-recognition domain. TcCTL2 was mainly expressed in the early pupae and primarily distributed in the hemocytes in the late larvae. It was significantly upregulated after larvae were infected with Escherichia coli or Staphylococcus aureus, while knockdown of TcCTL2 exacerbates larval mortality and bacterial colonization after infection. The purified recombinant TcCTL2 (rTcCTL2) can bind to pathogen-associated molecular patterns and microbes and promote hemocyte-mediated encapsulation, melanization and phagocytosis in vitro. rTcCTL2 also induced bacterial agglutination in a Ca²⁺-dependent manner. Knockdown of TcCTL2 drastically suppressed encapsulation, melanization, and phagocytosis. Furthermore, silencing of TcCTL2 followed by bacterial infection significantly decreased the expression of transcription factors in Toll and IMD pathways, antimicrobial peptides, and prophenoloxidases and phenoloxidase activity. These results unveiled that TcCTL2 mediates both humoral and cellular immunity to promote bacterial clearance and protect T. castaneum from infectious microbes, which will deepen the understanding of the interaction between CTLs and innate immunity in T. castaneum and permit the optimization of pest control strategies by a combination of RNAi technology and bacterial infection.