Toll/Toll-like receptor (TLR) is an important pattern recognition receptor that plays an important role in the immunity of animals. Six Toll genes were identified in Macrobrachium rosenbergii, ...namely, MrToll, MrToll1, MrToll2, MrToll3, MrToll4, and MrToll5. SMART analysis showed that all six Tolls have a transmembrane domain, a TIR domain, and different number of LRR domains. The phylogenetic tree showed that six Tolls were located in six different branches. Among these six Tolls, only MrToll4 contains the QHR motif, which is similar to insect Toll9. MrToll4 belongs to V-type/scc Toll with only one LRRCT domain. MrToll1 and MrToll5 are classical P-type/mcc Toll with two LRRCT domains and an LRRNT. MrTolls were distributed in the hemocytes, heart, hepatopancreas, gills, stomach, and intestine. During the infection of Enterobacter cloacae, the expression level of MrToll and MrToll1-4 was upregulated in the intestine of M. rosenbergii. RNA interference experiments showed that the expression of most antimicrobial peptide (AMP) genes was negatively regulated by MrTolls during E. cloacae infection. On the contrary, crustin (Cru) 3 and Cru4 were inhibited after the knockdown of MrToll, and Cru1 and Cru4 were significantly downregulated with the knockdown of MrToll4 during E. cloacae challenge. These results suggest that MrTolls may be involved in the regulation of AMP expression in the intestine during E. cloacae infection.
The forkhead box transcription factor O family protein (FOXO) acts as a transcription factor that regulates biological processes regarding DNA repair, immunity, cell cycle regulation, and other ...biological processes. In this study, EcFOXO was identified from the ridgetail white prawn, Exopalaemon carinicauda. EcFOXO protein contains multiple low-complexity regions and a forkhead (FH) domain. Phylogenetic tree showed that EcFOXO is clustered with crustacean FOXOs. The amino acid sequences of its FH domain are highly similar to the FH domain of FOXOs from other crustaceans. The expression of EcFOXO is altered after white spot syndrome virus (WSSV) stimulation in hepatopancreas and gills. The relationship between EcFOXO and EcRelish was explored by RNA interference (RNAi). Results showed that EcFOXO and EcRelish could positively regulate each other's expression. The expression levels of various antimicrobial peptides (AMPs) significantly reduced after interfering with EcFOXO or EcRelish. These results suggest a positive regulatory loop between EcFOXO and EcRelish, which participates in the innate immunity of ridgetail white prawn by regulating the expression of AMPs during WSSV infection. This study enriches the knowledge about the regulatory mechanism of FOXO in the innate immunity of crustaceans.
In insects, Taiman (Tai) participates in the juvenile hormone, 20-hydroxyecdysone, insulin, and Hippo signaling pathways. However, the role of Tai in crustacean innate immunity is less known. In this ...study, four Tai isoforms (MnTai-A, MnTai-B, MnTai-C, and MnTai-D) produced by alternative splicing were identified from Macrobrachium nipponense. The obtained genome sequences indicated that MnTai DNA has more than 20 exons and 19 introns. The second to last (-exon2) and the third to last (-exon3) exons can be alternatively spliced. The loss of -exon2 or -exon3 produces MnTai-B or MnTai-C, respectively. Both exons are absent in MnTai-D. The full-length cDNA of MnTai-A (including all exons) was 6894 bp with an open reading frame of 4998 bp that encoded a protein of 1665 amino acids. MnTaiA contains the conservative structure of the Tai family and clustered with nuclear receptor coactivator from shrimp. All these four isoforms were widely distributed in a variety of tissues with the highest expression level in the hepatopancreas except MnTaiC. The transcriptional levels of total Tai genes (designated as MnTaiT) in the hepatopancreas and gills were regulated by bacterial or viral challenge. Knockdown of MnTaiT increased the expression of anti-microbial peptides (AMPs) during Vibrio parahaemolyticus infection. Further study indicated that the negative regulation of AMP gene expression by prawn Tai was mediated through its positive regulation of cactus. Our research provides valuable information that prawn Tai isoforms are involved in innate immunity.
•Four Tai isoforms produced by alternative splicing were identified from Macrobrachium nipponense.•The transcriptional level of MnTaiT were regulated by bacterial or viral infection.•Knockdown of MnTaiT inhibited the transcription of MnCactus and promoted the expression of AMPs during Vibrio infection.•MnCactus silencing promoted the synthesis of AMPs during Vibrio infection.•MnTaiT negatively regulates the expression of AMPs by promoting the transcription of MnCactus during Vibrio infection.
Antimicrobial peptide (AMP) is an important component of crustaceans’ innate immune system. In this study, a short neuropeptide F (sNPF) gene (Pc-sNPF) and a Forkhead box O (FOXO) gene (PcFOXO) from ...Procambarus clarkii were identified. Analysis findings showed that the expression level of AMP genes differed between male and female P. clarkii. Furthermore, Pc-sNPF and PcFOXO were related to the sex dimorphism of AMP. Knockdown of Pc-sNPF in the eyestalk significantly upregulated the expression of PcFOXO and two anti-lipopolysaccharide factors (PcALF4 and PcALFL) in the intestine of P. clarkii. The expression of PcFOXO in the intestine of female P. clarkii was higher than in that of males. Results from RNA interference revealed that PcFOXO positively regulated the expression of PcALF4 and PcALFL in the intestine of male and female P. clarkii. In summary, our study showed that differences in Pc-sNPF expression in eyestalk of male and female P. clarkii leading to sex dimorphism of AMP expression in the intestine are mediated by the sNPF-FOXO-AMP signal pathway called the eyestalk–intestine axis.
•Short neuropeptide F (sNPF) gene (PcsNPF) and forkhead box O (FOXO) gene (PcFOXO) from Procambarus clarkii.•PcsNPF in P. clarkii eyestalk inhibits the expression of PcFOXO, PcALF4 and PcALFL in the intestine.•Antimicrobial peptides are regulated by PcFOXO in P. clarkii.•Sexual dimorphism in the expression of AMPs is mediated by sNPF-FOXO-AMPs signal pathway called the eyestalk-intestine axis.
Innate immunity is the primary defense of crustaceans against pathogens. Crustins, as antimicrobial peptides, are important to crustacean innate immunity. In this study, two kinds of Gly-rich crustin ...genes were cloned from Macrobrachium nipponense and were referred to as Mn-Gly-Cru1 and Mn-Gly-Cru2. These crustins belong to type II crustins with typical type II crustin structures. The full-length cDNA of Mn-Gly-Cru1 is 677 bp and contains a 576 bp open reading frame (ORF) encoding 191 amino acids. The full-length cDNA of Mn-Gly-Cru2 is 727 bp, with 573 bp ORF encoding 190 amino acids. The constructed phylogenetic tree indicated that Mn-Gly-Cru1 and Mn-Gly-Cru2 belong to the type IIa subfamily. RT-PCR analysis showed that Mn-Gly-Cru1 and Mn-Gly-Cru2 are widely distributed in various tissues. qRT-PCR results indicated that Mn-Gly-Cru1 is mainly expressed in the gills, whereas Mn-Gly-Cru2 is expressed at the highest level in hemocytes. The transcripts of Mn-Gly-Cru1 and Mn-Gly-Cru2 respond to bacterial or white spot syndrome virus (WSSV) stimuli. After injection of 48 h dsMnRelish, the expression of MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 were all inhibited. After WSSV, Vibrio parahaemolyticus, or Staphylococcus aureus challenge, MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 were all upregulated. However, the expression levels of MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 at 6 h bacteria or 36 h WSSV challenge were downregulated in Relish-silenced prawns when compared with the control (bacteria or WSSV challenge only, bacteria or WSSV challenge plus dsGFP injection). Results suggest that Mn-Gly-Cru1 and Mn-Gly-Cru2 play essential roles in M. nipponense innate immunity against bacteria or WSSV, and the expression levels of both genes are regulated by Relish transcriptional factor.
•Two type II crustins with glycine rich region were identified in prawns.•Mn-Gly-Cru1 and Mn-Gly-Cru2 could respond to viral or bacterial challenge.•Mn-Gly-Cru1 and Mn-Gly-Cru2 were regulated by Relish during pathogen infection.
•A novel Duox gene (designated as EsDuox) was identified from Eriocheir sinensis.•Nitrite stress activates the expression of EsDuox that induces the production of ROS.•The synthetic ROS activates ...Dorsal that further regulates the expression of AMPs.•Inhibited replication of WSSV in crabs under 48 h nitrite stress was found.
Nitrite stress and white spot syndrome virus (WSSV) infection are major problems threatening the sustainable and healthy development of Eriocheir sinensis. Some studies have found that nitrite stress can lead to the production of reactive oxygen species (ROS), whereas synthetic ROS plays a vital role in the signaling pathway. However, whether nitrite stress influences the infection of crabs by WSSV remains unclear. NADPH oxidases, including NOX1-5 and Duox1-2, are important for ROS production. In the present study, a novel Duox gene (designated as EsDuox) was identified from E. sinensis. The studies found that nitrite stress could increase the expression of EsDuox during WSSV infection and decrease the transcription of the WSSV envelope protein VP28. Moreover, nitrite stress could increase the production of ROS, and the synthesis of ROS relied on EsDuox. These results indicated a potential “nitrite stress–Duox activation–ROS production” pathway that plays a negative role in WSSV infection in E. sinensis. Further studies found that nitrite stress and EsDuox could promote the expression of EsDorsal transcriptional factor and antimicrobial peptides (AMPs) during WSSV infection. Moreover, the synthesis of AMPs was positively regulated by EsDorsal in the process of WSSV infection under nitrite stress. Furthermore, EsDorsal played an inhibitory role in the replication of WSSV under nitrite stress. Our study reveals a new pathway for “nitrite stress–Duox activation–ROS production–Dorsal activation–AMP synthesis” that is involved in the defense against WSSV infection in E. sinensis during short-term nitrite stress.
Nuclear factor κB (NF-κB) plays a key role in the innate immunity of invertebrates. Relish belongs to the NF-κB family. In insects, alternative splicing induces the sequence diversity of the Relish ...gene. However, information on the roles of various relish isoforms in crustacean innate immune response is limited. Here, two alternatively spliced Relish isoforms (designated as SPcRelish and LPcRelish) were identified from freshwater crayfish (Procambarus clarkii), and functional analysis was performed. The Relish gene has 25 exons and 24 introns. The long isoform LPcRelish is fully spliced, whereas the short isoform SPcRelish is alternatively spliced and contains exon 1–9 and a retention of intron 9. LPcRelish contains the Rel homology domain (RHD), the ig-like, plexins, transcription factors (IPT), and ankyrin-repeat (ANK) inhibitory domain. However, SPcRelish contains only the RHD and IPT domain, and does not have an ANK domain. The transcripts of SPcRelish and LPcRelish can be regulated by Vibrio parahaemolyticus. The intestinal immunological barrier and bacterial balance in the intestine play crucial roles in host health. In this study, we analyzed the connection between Relish isoforms and the transcripts of antimicrobial peptides (AMPs) in intestine. The transcripts of all the tested AMPs, except ALF-41125, were upregulated by V. parahaemolyticus. The knock down of the SPcRelish gene resulted in a significant decrease in the expression levels of ALF-7032, ALF-13162, and Crustin-42012 during V. parahaemolyticus invasion. The expression levels of four AMP genes (ALF-41125, ALF-42430, Crustin-41354, and Crustin-42993) were obviously increased in V. parahaemolyticus-challenged SPcRelish-silenced crayfish. ALF-7032, ALF-9228, ALF-13162, ALF-42430, Crustin-41354, Crustin-42012, and Crustin-42993 were evidently downregulated in V. parahaemolyticus-infected LPcRelish-silenced crayfish. Overall, generating the two Relish isoforms by alternative splicing may be an important mechanism of the host immune system to promote molecular diversity, which results in the functional diversity of the relish transcription factor.
•Two relish isoforms produced by alternative splicing were identified from Procambarus clarkii.•LPcRelish is fully spliced, while SPcRelish is alternative spliced with intron retention.•The expression of LPcRelish, SPcRelish, and seven AMPs were regulated by Vibrio parahaemolyticus challenge.•LPcRelish and SPcRelish show functional diversity in activating the AMP genes duringVibrio infection.
Trionyx sinensis Hemorrhagic Syndrome Virus (TSHSV), the first aquatic arterivirus identified in China, causes severe mortality to T. sinensis. In this study, we sought to determine the functions of ...T. sinensis mRNAs and non-coding RNAs (ncRNAs) that were differentially expressed (DE) over different periods of TSHSV infection of T. sinensis lung. We used RT-qPCR to validate the sequencing results of select RNAs, confirming their reliable and referable nature. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to predict multiple biological functions and signaling pathways in various comparison groups (1-day versus mock, 3-day versus 1-day, and 5-day versus 3-day). Multiple types of differentially expressed RNA, including mRNA, lncRNA, circRNA, and miRNA, were associated with cardiac dysfunction, coagulation abnormalities, and arachidonic acid metabolism at day 1. Pre-inflammatory cytokines and inflammatory factors such as PLA2G4A, cPLA2, γ-GGT1, TNFRSF14, TCP11L2, PTER CYP2J2 and LTC4S, were noticeably regulated at the same time. On day 3, multiple GO terms and KEGG pathways were implicated, including those related to virus defense, apoptosis, pyroptosis, and inflammatory response. Notably, key genes such as RSAD2, TRIM39, STAT4, CASP1, CASP14, MYD88, CXCL3, CARD11, ZBP1, and ROBO4 exhibited significant regulation. The lncRNAs and circRNAs that targeted the genes involved in viral recognition (TLR5), apoptosis (CARD11), pyroptosis (ZBP1), inflammatory processes (NEK7, RASGRP4, and SELE) and angiogenesis (ROBO4) exhibited significant regulation. Significantly regulated miRNAs were primarily linked to genes involved in apoptosis (Let-7f-3p, miR-1260a, miR-455-3p), and inflammation (miR-146a, miR-125a, miR-17a, miR-301b, and miR-30a-3p). The findings could advance our understanding of the host immunological response to TSHSV and offer new ideas for developing effective strategies to prevent infection of T. sinensis.
•A novel Trim23 gene (MnTrim23) was identified from M. nipponense, which belongs to C-IX family.•MnTrim23 promotes the replication of WSSV and the expression of VP28.•MnTrim23 negatively regulates ...Relish mediated AMPs expression.•AMPs inhibits the VP28 expression and WSSV replication.
The family of TRIM proteins with E3 ubiquitin ligase activity plays important roles in virus infection in vertebrates and invertebrates. In this study, a novel Trim gene shows high similarity to Trim23 (designated as MnTrim23) was identified from Macrobrachium nipponense. The MnTrim23 protein contains three conserved domains (one RING finger domain, two B-box, and one Coiled-coil region) at its N-terminal and one ARF domain at its C-terminal. The ARF domain characterizes the members of the Trim23 family. MnTrim23 belongs to C-IX family. Phylogenetic analysis shows that MnTrim23 has a closer genetic distance with other Trim23 proteins from invertebrates than that from vertebrates. MnTrim23 has higher expression level in the intestine and hepatopancreas than in the other immune tissues. The expression levels of MnTrim23 in the gills, stomach, and intestines are significantly up-regulated after white spot syndrome virus (WSSV) infection. Moreover, knockdown of MnTrim23 inhibits WSSV replication and VP28 expression, suggesting that MnTrim23 plays a positive role in WSSV infection. Further studies revealed that MnTrim23 negatively regulates the Relish transcription factor-mediated expression of antimicrobial peptides (AMPs). Synthetic AMPs inhibit VP28 expression and WSSV replication. These findings indicate that Trim23 promotes WSSV replication by inhibiting the expression of AMPs that are positively regulated by the host NF-κB signal pathway.
C-type lectin (CTL) is an important pattern recognition receptor that play vital functions in the innate immunity. Many soluble CTLs in crustacean participate in the inhibition or promotion of white ...spot syndrome virus (WSSV) infection. However, whether transmembrane CTLs participate in WSSV infection in crustacean remains unknown. In the present study, four spliced isoforms of a transmembrane CTL (designated as PcTlec) from Procambarus clarkii were identified for the first time. The genome structure of PcTlec contains eight exons, six known introns, and one unknown intron. PcTlec-isoform1 is produced by intron retention, whereas PcTlec-isoform3 and PcTlec-isoform4 are produced by exon skipping. All of them contain the transmembrane domain and characteristic carbohydrate recognition domain (CRD). Four PcTlec isoforms were mainly expressed in the hepatopancreas, stomach, and intestine. After WSSV challenge, the expression levels of PcTlec-isoform1-4 in the intestine were upregulated. The knockdown of the region shared by four PcTlec isoforms evidently decreased the expression of WSSV envelope protein VP28 and the copies of viral particles. A recombinant protein (rPcTlec-CRD) containing the CRD that was shared by four PcTlec isoforms was acquired by procaryotic expression system. The injection of purified rPcTlec-CRD protein evidently increased the VP28 expression and WSSV copies during viral infection. Moreover, rPcTlec-CRD could directly bind to WSSV and interact with VP28 protein. These findings indicate that new-found transmembrane CTL isoforms in P. clarkii may act as viral receptors that facilitate WSSV infection. This study contributes to the recognition and understanding of the functions of transmembrane CTLs in crustacean in the infection of host by WSSV.
•Four spliced isoforms of a transmembrane CTL were identified in P. clarkii.•The expressions of four PcTlec isoforms were increased after WSSV challenge.•Knockdown of PcTlec remarkably decreased the VP28 expression and virus copies.•Injection of rPcTlec-CRD evidently increased the VP28 expression and virus copies.•rPcTlec-CRD could directly bind to WSSV and interact with VP28.
