Anti-lipopolysaccharide factors regulated by Stat, Dorsal, and Relish are involved in anti-WSSV innate immune defense in Macrobrachium nipponense.

Anti-lipopolysaccharide factors (ALF) is an important antimicrobial peptide and critical effector molecule with a broad spectrum of antimicrobial activities in crustaceans. In addition to the previously reported five ALFs (MnALF1-5), another three ALFs [MnALF1, which is different from MnALF1 (ALF02818) that has been reported; MnALF6; and MnALF7] and an isoform of MnALF4 (MnALF4-isoform2) were newly identified from Macrobrachium nipponense in this study. MnALF6 has 134 amino acids and one single nucleotide polymorphism (SNP) in MnALF6 resulted in the change of 107th amino acid from E to D. Intron 1 retention produced longer transcript of MnALF6. The full length of MnALF7 has 691 bp with a 363 bp ORF encoding 120 amino acid protein. Three SNPs in MnALF2 resulted in the conversion of amino acids at positions 70, 73, and 91 from T70I73P91 to K70L73S91. The deletion of 13 bp in MnALF4 resulted in early termination of ORF, resulting in MnALF4-isoform2 with only 98 amino acids. The gDNAs of MnALF1, MnALF2, MnALF5, and MnALF6 contain three exons and two introns, while those of MnALF3 and MnALF7 contain three exons, one known intron, and one unknown intron. The MnALF1-7 in M. nipponense were widely distributed in multiple tissues. After white spot syndrome virus (WSSV) stimulation, the expression levels of MnALF1-7 changed. Knockdown of MnALF1-7 could evidently increase the expression of the envelope protein VP28 and the copy number of WSSV during viral infection. Further studies found that silencing of three transcription factors (Stat, Dorsal, and Relish) in M. nipponense significantly inhibit the synthesis of MnALF1-7 during the process of WSSV challenge. This study adds to the knowledge about the roles of ALFs in the innate immune responses to WSSV infection in M. nipponense.

Identification of four Spätzle genes (MnSpz1, MnSpz2, MnSpz2-isoform, and MnSpz3) and their roles in the innate immunity of Macrobrachium nipponense.

Spätzle, an extracellular ligand of the Toll receptor, is involved in the innate immunity of crustaceans. In this study, four Spätzle genes were cloned from Macrobrachium nipponense and designed as MnSpz1, MnSpz2, MnSpz2-isoform, and MnSpz3. The coding region of the four Spätzle genes all contained one intron and two exons, and they were predicted to be produced by gene duplication based on sequence similarities and phylogenetic tree. The predicted MnSpz1, MnSpz2, and MnSpz3 proteins all contained a signal peptide and a Spätzle domain. No signal peptide but a Spätzle domain existed in MnSpz2-isoform because of frameshift mutation caused by 50 bp nucleotide deletion compared with MnSpz2. Quantitative real-time polymerase chain reaction (RT-qPCR) analysis showed that MnSpz1, MnSpz2, and MnSpz3 were expressed in all the detected tissues of M. nipponense, and MnSpz2 was found to be the major isoform in the heart, gills, stomach, and intestine. After stimulation by Vibrio parahaemolyticus, Staphylococcus aureus, or White spot syndrome virus (WSSV), the expression levels of MnSpz1, MnSpz2, and MnSpz3 changed. Given the high similarities among MnSpz1-3, RNA interference (RNAi) using dsRNA of MnSpz1 inhibited the expression of the three Spätzle genes (MnSpz1, MnSpz2 and MnSpz3). Silencing of MnSpz1-3 down-regulated the expression levels of nine antimicrobial peptide (AMP) genes in M. nipponense. After Knockdown of MnSpzs, the number of V. parahaemolyticus, S. aureus and WSSV copies in M. nipponense increased significantly in vivo. Our results suggest that Spätzles are involved in the innate immunity of M. nipponense. The expansion of MnSpz genes through gene duplication is beneficial to enhance the innate immune defense ability of M. nipponense.

Characterization and functional analysis of a clip domain serine protease (MncSP) and its alternative transcript (MncSP-isoform) from Macrobrachium nipponense.

Clip domain serine protease (cSPs) play an important role in the innate immune defense of crustaceans. In this study, a clip domain serine protease (MncSP) and its alternative transcript (MncSP-isoform) were identified from Macrobrachium nipponense. The full-length cDNA sequences of MncSP and MncSP-isoform were 2447 and 2351 bp with open reading frames comprising 1497 and 1401 bp nucleotides and encoding 498 and 466 amino acids, respectively. The genome of MncSP had 10 exons and 9 introns. MncSP contained all 10 exons, whereas MncSP-isoform lacked the second exon. MncSP and MncSP-isoform contained a signal peptide, a clip domain, and a Tryp_SPc domain. Phylogenetic tree analysis showed that MncSP and MncSP-isoform clustered with cSPs from Palaemonidae. MncSP and MncSP-isoform were widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine. The expression profiles of MncSP and MncSP-isoform in the hemocytes of M. nipponense changed after simulation by Vibrio parahaemolyticus or Staphylococcus aureus. The RNAi of MncSP could inhibit the expression of antimicrobial peptides (AMPs), including crustins and anti-lipopolysaccharide factors. Phenoloxidase activity was also down-regulated in MncSP-silenced prawns. This study indicated that MncSP participated in the synthesis of AMPs and the activation of prophenoloxidase.

Characterization and functional analysis of tandem threonine containing C-type lectin (Thr-Lec) from the ridgetail white prawn Exopalaemon carinicauda.

As an important pattern-recognition receptor (PRR), C-type lectins (CTLs) play significant roles in recognizing microbes and battle against pathogenic microorganism in innate immunity. In this study, two tandem threonine containing CTLs (designated as EcThr-LecA and EcThr-LecB) were identified from Exopalaemon carinicauda. The full-length cDNA of EcThr-LecA and EcThr-LecB consisted of 1521 and 1518 bp with 1251 and 1242 bp open reading frame encoding a protein with 412 and 413 amino acids, respectively. The genome structure of EcThr-LecA included 10 exons and 9 introns, and the sequences of intron6 and intron7 were variable. The nucleotide sequence of intron2 in EcThr-LecB was specific and different with that of EcThr-LecA. EcThr-LecA and EcThr-LecB proteins were predicted to have a signal peptide, two conserved carbohydrate recognition domain (CRD), and tandem threonine region. The expression levels of EcThr-LecA and EcThr-LecB in the intestine were significantly up-regulated after Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenge. RNA interference (RNAi) was used to explore the effects of EcThr-LecB silencing on the mRNA expression of anti-lipopolysaccharide factor (ALF), crustin (CRU), and lysozyme (LYSO). Knock down of EcThr-LecB could evidently down-regulate the expression of eight different antibacterial peptides (AMPs), including EcALF2, EcCRU1, EcCRU3, EcCRU4, EcLYSO1, EcLYSO2, EcLYSO3, and EcLYSO4, whereas make no effect on the transcription of EcALF1, EcALF3, EcCRU2, and EcLYSO5. The recombinant two CRD domains and tandem threonine region (RLecB) of EcThr-LecB could bind diverse bacteria, lipopolysaccharide, and peptidoglycans in vitro. In addition, RLecB could accelerate the clearance of V. parahaemolyticus in vivo. The present data indicated that new-found tandem threonine containing CTLs in E. carinicauda may act as PRR to participate in the innate immune defense against pathogens by the recognition of non-self, regulation of AMPs, and clearance of invaders.