Regulation of AMPs expression by six Tolls in stomach during WSSV infection in Macrobrachium nipponense

Tolls or Toll-like receptors (TLRs) are involved in innate immunity in invertebrates and vertebrates. In this study, six Toll genes (named MnToll1-6) were found in the oriental river prawn Macrobrachium nipponense, and MnToll1-4 were newly identified. All six Tolls have a transmembrane domain and a TIR domain, containing different numbers of leucine-rich repeats (LRR), LRR C-terminal, and LRR N-terminal domains. They can be classified into single cysteine cluster Toll, multiple cysteine clusters Toll, and MnToll2 (Toll without a cysteine cluster), and are located in the six branches (groups A-F) of the evolutionary tree. Intronless Tolls (MnToll1 and MnToll3-4) belong to groups A-C. MnToll5 and MnToll6 are clustered into groups D and E, and contain four introns. MnToll2 has more than four introns and belongs to group F. The six MnTolls were widely distributed in diverse tissues, and MnToll1-4 had the highest expression levels in the stomach. Upon white spot syndrome virus (WSSV) stimulation, the expression levels of MnToll1-6 mRNA were upregulated in the stomach of M. nipponense. The expression levels of four anti-lipopolysaccharide factors were downregulated in the stomach of MnToll1-6 knockdown M. nipponense upon WSSV infection. Moreover, knockdown of MnToll1-6 increased the number of WSSV copies and led to a significant reduction in the survival rate of M. nipponense. This study showed that diverse Tolls exist in M. nipponense and participate in innate immune defense by regulating antimicrobial peptide expression during WSSV infection.