In biological evolution, gene duplication (GD) generates new genes to facilitate new functions. C-type lectins (CTLs) in crayfish have been extended by GD to expand their family members. In this ...study, four CTL genes generated by GD were identified from Procambarus clarkii (PcLec1–4). Among these four genes, PcLec1 can also generate new isoforms with different numbers of tandem repeats through DNA slip mispairing. PcLec1–4 was widely expressed in multiple tissues. The expression levels of PcLec1–4 were upregulated in the intestine of P. clarkii upon white spot syndrome virus (WSSV) challenge at multiple time points. Further analysis indicated that GATA transcription factor regulated PcLec1–4 expression. RNA interference and recombinant PcLec1–4 protein injection experiments suggested that PcLec1–4 promoted the expression of calreticulin (PcCRT) and negatively regulated the expression of antimicrobial peptides, thereby promoting WSSV replication. This study contributes to the understanding of the function of CTLs produced by GD during WSSV invasion in crustaceans.
•Four CTL genes were identified from Procambarus clarkii generated by gene duplication.•The transcription factor GATA controls the expression of PcLec1-4.•PcLec1-4 promotes viral replication through regulation of Pccalreticulin and antimicrobial peptides, respectively.
Carcinin, a member of the crustin family, plays important roles in crustacean innate immunity. In this study, we identified two carcinin isoforms (MnCarc1 and MnCarc2) produced by alternative ...splicing from Macrobrachium nipponense. The full length of MnCarc1 and MnCarc2 cDNA are 1554 and 1495 bp with 687 and 609 bp open reading frame-encoding proteins that contain 228 and 202 amino acids, respectively. The genome of carcinin has nine exons and eight introns. MnCarc1 transcript contains all nine exons, whereas MnCarc2 only contains eight exons and lacks exon 4. MnCarc1 and MnCarc2 proteins contain a signal peptide, cysteine-rich regions, and a whey acidic protein domain. The phylogenetic tree shows that MnCarc1 and MnCarc2 are not grouped with other crustins and carcinins. MnCarc1 and MnCarc2 form a subgroup. MnCarc1 and MnCarc2 are widely distributed in various tissues. The expression of MnCarc1 and MnCarc2 were evidently upregulated at multiple time points in hemocytes and the intestine of M. nipponense after white spot syndrome virus, Vibrio parahaemolyticus, and Staphylococcus aureus challenges. Further studies showed that knockdown of MnDorsal or MnStat transcription factor could remarkably inhibit the upregulated expression of MnCarc1 and MnCarc2 caused by viral or bacterial challenges. In addition, recombinant MnCarc1 and MnCarc2 proteins could bind to various bacteria and polysaccharides and inhibit the growth of S. aureus and V. parahaemolyticus in vitro. This study indicated that carcinins from M. nipponense were involved in prawns innate immunity.
•Two Carcinin isoforms (MnCarc1 and MnCarc2) were identified from M. nipponense.•MnCarc1 and MnCarc2 could respond to viral and bacterial infection.•Two Carcinin isoforms were positively regulated by Dosal and Stat during microbial infection.•MnCarc1 and MnCarc2 proteins have binding activity against bacteria and polysaccharides.•MnCarc1 and MnCarc2 proteins could inhibit the growth of bacteria in vitro.
•A novel LGR gene (MnLGR2) was identified from M. nipponense.•The expression level of MnLGR2 was regulated by bacterial and viral infection.•MnLGR2 negatively regulates Relish mediated AMP genes ...expression.•MnLGR2 positively regulates the expression of proPO activating pathway-related genes.•MnLGR2 promotes the activation of PO.
Leucine-rich repeat-containing G-protein-coupled receptors (LGRs) form a subfamily of the large superfamily of G-protein-coupled receptors. LGRs can be divided into three groups. LGR2 from Drosophila melanogaster is involved in cuticle tanning (melanization and sclerotization). In this study, one LGR2 (MnLGR2) was identified from Macrobrachium nipponense. MnLGR2 has an open reading frame of 4515 bp encoding a protein with 1504 amino acids. MnLGR2 is comprised of a 7-transmembrane domain, 12 leucine-rich repeats, and 5 low-complexity regions. The highest expression level of MnLGR2 was observed in gills. The expression levels of MnLGR2 in gills and stomach could be regulated by bacterial challenge. Knockdown of MnLGR2 upregulated the expression of anti-microbial peptide (AMP) genes. Further study indicated that inhibition of AMP expression by MnLGR2 was through inhibition of relish-mediated AMP expression. In addition to the negative regulation of AMP expression, MnLGR2 participated in positive regulation of phenol oxidase (PO) activity and expression of proPO activating pathway-related genes (proPO-activating factor and proPO-activating enzymes). Therefore, MnLGR2 plays an important role in prawn innate immunity.
•A new dorsal gene (MnDorsal) was identified from Macrobrachium nipponense.•Bacteria, virus, and stimulant challenges could up-regulate the transcription of MnDorsal.•MnDorsal positively regulates ...the expression of AMPs during S. aureus infection.
Rel/nuclear factor (NF)-κB family of transcription factors paly vital roles in innate immunity response to bacterial and viral infection. Here, we cloned and identified a dorsal homologue (named as MnDorsal) from Macrobrachium nipponense. The full-length cDNA of MnDorsal is 2573 bp with a 1986 bp open reading frame that encodes 661 amino acids. Predicted MnDorsal protein contained a RHD (Rel homology domain), an IPT (Iglike, plexins, and transcriptions factors) domain, and two low complexity regions. Phylogenetic analysis showed that MnDorsal has a closer genetic distance with dorsal homologues from invertebrates. MnDorsal was widely expressed in a variety of tissues, including hemocytes, heart, hepatopancreas, gills, stomach, and intestine. Expression patterns analysis showed that the transcriptional level of MnDorsal in the gills was evidently up-regulated after Staphylococcus aureus, Vibrio parahaemolyticus, white spot syndrome virus, or polyinosinic-polycytidylic acid challenge, suggesting that MnDorsal participates in the immune defenses against pathogens and stimulant challenges. Additionally, the dsRNA-mediated RNA interference analysis showed that knockdown of MnDorsal can significantly inhibit the expression of anti-lipopolysaccharide factor (ALF) and crustin. Further studies revealed that the up-regulated expression of ALFs (MnALF2, MnALF3, and MnALF4) and crustins (MnCrustin3 and MnCrustin4) caused by S. aureus infection were obviously decreased after silencing MnDorsal. These findings suggest that MnDorsal positively regulate the expression of antibacterial peptides (AMPs) during S. aureus infection. Our study will promote to better understand the role of Toll-Dorsal-AMPs pathway in innate immunity response to gram-positive bacterial infection in crustacean.
Micro-payment systems have the potential to provide non-intrusive, high-volume and low-cost pay-as-you-use services for a wide variety of web-based applications. We propose an extension, P2P-NetPay, ...a micro-payment protocol characterized by off-line processing, suitable for peer-to-peer network services sharing. Our approach provides high performance and security using one-way hashing functions for e-coin encryption. In our P2P-NetPay protocol, each peer’s transaction does not involve any broker and double spending is detected during the redeeming transaction. We describe the motivation for P2P-NetPay and describe three transactions of the P2P-NetPay protocol in detail to illustrate the approach. We then discuss future research on this protocol.
The Wnt signal transduction pathway is involved in a wide variety of cellular processes, including cell proliferation, differentiation, apoptosis, and immunity against microbial infection. In the ...current study, we cloned and characterized two Wnt homologues (Mn-Wnt4 and Mn-Wnt16) in Macrobrachium nipponense. The full length cDNA of Mn-Wnt4 was 3144 bp with a 1074 bp open reading frame (ORF) that encoded a protein containing 358 amino acid residues. The full length cDNA of Mn-Wnt16 transcript was 2893 bp with a 1281 bp ORF that encoded a 427 amino acid protein. Mn-Wnt4 and Mn-Wnt16 proteins contained a highly conserved WNT1 domain. Tissue distribution analysis showed that Mn-Wnt4 and Mn-Wnt16 were highly expressed in the stomach. The transcriptional levels of Mn-Wnt4 and Mn-Wnt16 in the stomach were upregulated at most tested time points after bacterial (Staphylococcus aureus and Vibrio parahaemolyticus) and viral (White spot syndrome virus) infection. Moreover, the expression levels of some antimicrobial peptides (AMPs) (including anti-lipopolysaccharide factor ALF and crustin CRU) were upregulated after V. parahaemolyticus infection. We further used dsRNA-mediated RNA interference technology to explore the relationship between these two Wnt genes and the expression levels of AMPs during V. parahaemolyticus infection. Mn-Wnt4 knockdown could significantly inhibit the expression of ALF1 and CRU4 in the stomach of V. parahaemolyticus-injected prawns, whereas Mn-Wnt16 silencing could result in the inhibition of the expression level of CRU3 and CRU4 in the stomach of V. parahaemolyticus-infected prawns. These findings indicated that the Wnt gene family might participate in the body's innate immune response to Vibrio infection by regulating the synthesis of a variety of AMPs. Our study will help to understand the role of the Wnt signaling pathway in the immune response of crustaceans.
•Two Wnt genes (Mn-Wnt4 and Mn-Wnt16) were identified from Macrobrachium nipponense.•Vibrio infection promotes the transcription of Mn-Wnt4, Mn-Wnt16, and some AMPs.•Mn-Wnt4 and Mn-Wnt16 knockdown inhibit the expression levels of different AMPs in Vibrio-infected prawns.
Mangrove reserves promote the protection of mangroves, but they also lead to the restriction of land development rights in the region, which is unfair to society. This paper aims to evaluate the ...impact of the spillover of coastal development. Taking the Shankou Mangrove Ecological Reserve in the Beibu Gulf of Guangxi as an example, this paper conducts a research design using the choice experiment. Conditional logit and a structural equation model are used to analyze data and to calculate the respondents' choice preferences and willingness to pay for the limited right to develop the coastal zone. Also, to reveal preference heterogeneity, individual characteristic factor analysis is added. The results show that: after the establishment of the protected area, regional differences are evident in the limited space of coastal development rights. Among them, the limitation degree of sea area use is the highest, followed by beach area; the degree of limitation on land area use is the lowest. The limited spatial differences of coastal development rights may lead to an imbalance in the development of coastal land resources among regions. Other problems include the forced idleness of coastal land resources and the threat to the livelihood of residents. Overall, this is not conducive to the coordinated development of an ecological economy in the reserve. This study can provide a decision-making basis for optimizing the coastal development rights management of coastal zone ecological protection areas. A reference for the coastal development rights management in other countries is also provided.
•Coastal farmers’ dependence on land use is different from that of mainland farmers.•The externality of ecological protection policies decreases from coastal to inland.•Differences exist in preferences for land, tidal flat and sea development rights.•Land resource development in coastal zones is unbalanced between sea and land.
Activating transcription factor 2 (ATF2), a member of the bZIP transcription factor family, is involved in multiple physiological and developmental processes, yet its role in the innate immunity ...remains unclear. In this study, two isoforms (named as MnATF2a and MnATF2b) of ATF2 gene were identified in Macrobrachium nipponense and were produced by exon skipping. The full length of MnATF2a is 2328 bp with an open reading frame of 2079 bp that encode 692 amino acids. MnATF2a has 237 bp nucleotides more than MnATF2b and the extra 237 bp is a complete exon. MnATF2a and MnATF2b proteins contain the same conserved and typical bZIP domain at the C-terminus. MnATF2a has 79 amino acids more than MnATF2b. MnATF2a and MnATF2b are widely distributed in a variety of immune tissues. After Vibrio parahaemolyticus and Staphylococcus aureus infection, the expression levels of MnATF2a and MnATF2b were significant up-regulated in the gills and stomach at 12 h. RNA interference analysis showed that knockdown of the total MnATF2 gene significantly inhibits the transcription of tumor necrosis factor (TNF) and promotes the expression of crustins (including Cru3, Cru4, and Cru7). Further study showed that knockdown of MnTNF evidently increase the expression of Cru3, Cru4, and Cru7. Our research indicates that ATF2 negatively regulate the expression of AMPs by regulating the transcription of TNF in M. nipponense. This study provides valuable information about the function of ATF2 family in the innate immunity in crustacean.
•Two isoforms of ATF2 gene were found in Macrobrachium nipponense.•The expression of MnATF2a and MnATF2b were increased after Vibrio parahaemolyticus and Staphylococcus aureus infection.•MnATF2 can positively regulate the expression of MnTNF.•MnATF2 and MnTNF can negatively regulate the expression of AMPs.
Transcription factor activator protein 1 (AP1) plays an irreplaceable role in the response to a variety of external stimulants, such as cellar stress, bacterial and viral infections, and inflammatory ...cytokines. In this study, we identified a novel AP1 gene from Macrobrachium nipponense and named it MnAP1, which has a full length of 1747 bp contains an 882 bp open reading frame, and encodes a protein with 293 amino acids. The MnAP1 protein contains Pfam and bZIP domains. MnAP1 is widely distributed in hemocytes, heart, hepatopancreas, gill, stomach, and intestinal tissues. The expression levels of MnAP1 in the gills and stomach were significantly upregulated after Vibrio parahaemolyticus and Staphylococcus aureus attacks. We studied the relationship between MnAP1 and the transcripts of antimicrobial peptides (AMPs) in gills through RNA interference. Interestingly, the regulatory effects of MnAP1 on the expression of different AMPs were different. We found that the expression levels of crustins, including Cru1, Cru3, and Cru4 in the gills were evidently decreased, whereas the synthesis of Cru5 and anti-lipopolysaccharide factors (ALF3 and ALF4) were obviously increased. We further explored the effect of MnAP1 on the expression of transcription factor relish from M. nipponense. The result showed that the knockdown of MnAP1 can remarkably upregulate the expression of MnRelish. Relish as a member of the nuclear factor κB family that regulates the expression of AMPs in the innate immunity of crustacean. Hence, we also detected the expression levels of Cru5, ALF3, and ALF4 in the gills of MnRelish-silenced prawns. The Data showed that the expression levels of these three AMPs were evidently reduced after MnRelish silencing. Our results indicated that MnAP1 plays a positive role in regulating the expression of AMPs, promotes the JNK/AP1 signaling pathway, and exerts a negative regulatory effect on the synthesis of AMPs by inhibiting the transcription of NF-κB factor in the innate immunity of M. nipponense.
•A novel activator protein 1 (AP1) was identified from Macrobrachium nipponense.•The expression of MnAP1 was regulated by bacterial challenge.•MnAP1 could positive and negative regulate the expression of different AMPs.
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.
•MncSP and MncSP-isoform were identified from M. nipponense by exon jumping.•The expressions of MncSP and MncSP-isoform were increased after bacterial invasion.•Silencing MncSP inhibited the up-regulation of MncSP-isoform caused by bacteria.•Silencing MncSP significantly reduced the synthesis of some AMPs.•Silencing of MncSP leads to a significant decrease in PO activity.
