Antibacterial activity and immunomodulatory role of a proline-rich antimicrobial peptide SpPR-AMP1 against Vibrio campbellii infection in shrimp Litopenaeus vannamei.

Antimicrobial peptides (AMPs) constitute one of the most promising sources of natural molecules used for the design of effective antimicrobial agents alternative to antibiotics. Previously, we have showed that a crab proline-rich AMP designated as SpPR-AMP1 is a potent AMP that exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. Here, we demonstrated the importance of SpPR-AMP1 peptide in treating a virulent acute hepatopancreatic necrosis disease (AHPND) Vibrio campbellii VH-639 isolate and eliciting the innate immune response to counter the AHPND infection in shrimp Litopenaeus vannamei. SpPR-AMP1 exhibited a strong antimicrobial activity against V. campbellii VH-639 at MIC value of 0.195-0.39 µM. Scanning electron microscopy (SEM) revealed the membrane disruption potential of SpPR-AMP1 against the V. campbellii VH-639 cells. The in vivo effect of SpPR-AMP1 in shrimp L.vannamei was investigated and the results showed that SpPR-AMP1 was capable of modulating the innate immune response by stimulating the expression levels of AMP transcripts in shrimp hemocytes. Moreover, treatments with SpPR-AMP1 could promote the resistance of shrimp against V. campbellii VH-639 infection as demonstrated by a significant increase in shrimp survival rate and decrease in both the bacterial load and the expression levels of bacterial PirA and PirB toxin gene transcripts in the infected shrimp. These results suggest the potential of SpPR-AMP1 peptide with the combined antimicrobial and immunoenhancing capabilities as promising antimicrobial agent to treat V. campbellii VH-639 causing AHPND infection in shrimp aquaculture.

Characterization and functional analysis of fibrinogen-related protein (FreP) in the black tiger shrimp, Penaeus monodon.

Ficolin is classified as an immune related protein containing collagen-like and fibrinogen-related domain (FreD). In invertebrates, the functions of fibrinogen-related proteins (FrePs) are of importance to innate immunity. In this study, a FreP in the black tiger shrimp Penaeus monodon was identified and characterized. The PmFreP cDNA is 1,007 bp long with a 921 bp-open reading frame that encodes for 306 amino acids. The deduced PmFreP sequence consists of a signal peptide, an unknown region and the FreD. Phylogenetic analysis showed that PmFreP was clustered with fibrinogen-like proteins in crustaceans which was separated from vertebrate ficolin-like proteins. The deduced fibrinogen-like domain contains four conserved cysteine residues (Cys96, Cys127, Cys249, and Cys262) that are responsible for the formation of disulfide bridges. Gene expression analysis shows that Pmfrep is mainly expressed in the intestine and the expression is significantly upregulated after Vibrio harveyi and white spot syndrome virus (WSSV) challenge. Recombinant PmFreP (rPmFreP) were successfully expressed and purified, and forms a trimeric structure as judged by native-PAGE. Bacterial binding assay showed that the rPmFreD can bind and agglutinate Gram-negative and Gram-positive bacteria in the presence of calcium (Ca2+) ions. Moreover, the rPmFreP facilitates the clearance of V. harveyi in vivo. Overall, our results suggested that the PmFreP may serve as pattern recognition receptors implicated in shrimp innate immunity.

Stimulator of interferon gene (STING) and interferon regulatory factor (IRF) are crucial for shrimp antiviral defense against WSSV infection.

The cytosolic DNA-sensing immune response is essential for recognizing and establishing an effective host immune response to pathogens. However, the importance of the cytosolic signalling molecules responsible for facilitating an appropriate immune response following infection with a DNA virus in shrimps remains unknown. Here, we report the discovery of the Penaeus monodon stimulator of interferon gene (PmSTING) and interferon regulatory factor (PmIRF) genes and their important roles in the host defense against viral infection. High expression levels of PmSTING transcripts were detected in the midgut, hepatopancreas, and hindgut, with lower levels in foregut, while PmIRF was highly expressed in the hindgut, foregut, and hepatopancreas of P. monodon. The mRNA expression level of both PmSTING and PmIRF was up-regulated in the foregut in response to white spot syndrome virus (WSSV; dsDNA virus) infection. RNA-interference-mediated gene silencing of PmSTING and PmIRF rendered shrimps to be more susceptible to WSSV infection; suppression of PmIRF decreased the mRNA transcript level of PmSTING; and silencing of the cytosolic sensor PmDDX41 suppressed both PmSTING and PmIRF gene transcript levels. Thus, PmSTING and PmIRF are likely to be important for the antiviral innate response against the dsDNA WSSV pathogen and may mediate the antiviral immune defenses via PmDDX41/PmSTING/PmIRF signaling cascade in P. monodon.

Transcriptome analysis identifies immune-related genes and antimicrobial peptides in Siamese fighting fish (Betta splendens).

Siamese fighting fish (Betta splendens) is one of the most widely cultivated ornamental fish in global trade. However, transcriptomic data, which can reveal valuable genetic data for disease control and prevention, are extremely limited for this species. In this study, whole-body transcriptome sequencing of juvenile betta fish generated 4.457 GB of clean data and a total of 71,775 unigenes using the Illumina HiSeq4000 platform. These unigenes were functionally classified using 7 functional databases, yielding 45,316 NR (63.14%), 47,287 NT (65.88%), 39,105 Swiss-Prot (54.48%), 16,492 COG (22.98%), 37,694 KEGG (52.52%), 4,506 GO (6.28%), and 35,374 Interpro (49.28%) annotated unigenes. Furthermore, we also detected 13,834 SSRs distributed on 10,636 unigenes and 49,589 predicted CDSs. Based on KEGG analysis, five innate immune pathways (997 unigenes) were reported, including the NOD-like receptor signaling pathway, complement and coagulation cascades, toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway and cytosolic DNA-sensing pathway. Moreover, four antimicrobial peptide (AMP) families (hepcidin, piscidin, LEAP-2, and defensins) from the betta fish transcriptome were also identified. Additionally, cDNA and genomic DNA of two ß-defensins was successfully isolated from four betta fish species. RT-PCR analysis showed that BsBD1 transcripts were most abundant in the muscle and kidney and BsBD2 transcripts were most abundant in the gill. The genomic organization showed that the BD1 and BD2 genes consisted of three exons and two introns according to the GT-AG rule. Most importantly, this is the first report of the betta fish whole-body transcriptome obtained by high-throughput sequencing. Our transcriptomic data and the discovery of betta fish AMPs should promote a better understanding of molecular immunology for disease prevention for further ornamental fish aquaculture.

Shrimp hemocyte homeostasis-associated protein (PmHHAP) interacts with WSSV134 to control apoptosis in white spot syndrome virus infection.

Hemocyte homeostasis-associated protein (PmHHAP) was first identified as a viral-responsive gene, due to a high upregulation in transcription following white spot syndrome virus (WSSV) infection. Functional studies using RNA interference have suggested that PmHHAP is involved in hemocyte homeostasis by controlling apoptosis during WSSV infection. In this study, the role of PmHHAP in host-viral interactions was further investigated. Yeast two-hybrid assay and co-immunoprecipitation revealed that PmHHAP binds to an anti-apoptosis protein, WSSV134. The viral protein WSSV134 is a late protein of WSSV, expressed 24 h post infection (hpi). Gene silencing of WSSV134 in WSSV-infected shrimp resulted in a reduction of the expression level of the viral replication marker genes VP28, wsv477, and ie-1, which suggests that WSSV134 is likely involved in viral propagation. However, co-silencing of PmHHAP and WSSV134 counteracted the effects on WSSV infection, which implies the importance of the host-pathogen interaction between PmHHAP and WSSV134 in WSSV infection. In addition, caspase 3/7 activity was noticeably induced in the PmHHAP and WSSV134 co-silenced shrimp upon WSSV infection. Moreover, PmHHAP and WSSV134 inhibited caspase-induced activation of PmCasp in vitro in a non-competitive manner. Taken together, these results suggest that PmHHAP and WSSV134 play a role in the host-pathogen interaction and work concordantly to control apoptosis in WSSV infection.

Anti-melanization mechanism of the white spot syndrome viral protein, WSSV453, via interaction with shrimp proPO-activating enzyme, PmproPPAE2.

Inhibition of the host melanization reaction, activated by the prophenoloxidase activating (proPO) system, is one of the crucial evasion strategies of pathogens. Recently, the shrimp pathogen, white spot syndrome virus (WSSV), was found to inhibit melanization in the shrimp Penaeus monodon. The viral protein WSSV453 was previously shown to interact with PO-activating enzyme 2 (PmPPAE2) and reported to be involved in suppressing the shrimp melanization response after WSSV infection. Here, we characterized how WSSV453 inhibits melanization. WSSV453 is a non-structural viral protein, which was first detected in shrimp haemocytes at 6 hours post-infection (hpi) by WSSV and in shrimp plasma at 24 hpi. We produced recombinant proteins for three components of the P. monodon proPO system: PmproPPAE2, PmproPO1 and PmproPO2. Functional assays showed that active PmPPAE2 processed PmproPO1 and 2 to produce functional PO. Incubation of WSSV453 with PmproPPAE2 dose-dependently reduced PmPPAE2 activity toward PmPO1 or PmPO2. In contrast, WSSV453 had no effect on activated PmPPAE2. The addition of active PmPPAE2 to WSSV-infected shrimp plasma at day 2 post-infection also rescued PO activity. Taken together, these results indicate that the anti-melanization activity of WSSV is due to WSSV453, which interacts with PmproPPAE2 and interferes with its activation to active PmPPAE2.

A Natural Vibrio parahaemolyticus ΔpirA Vp pirB Vp+ Mutant Kills Shrimp but Produces neither Pir Vp Toxins nor Acute Hepatopancreatic Necrosis Disease Lesions.

Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor a pVA plasmid encoding toxins PirA Vp and PirB Vp These are released from VPAHPND isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that V. parahaemolyticus isolate XN87 lacked pirA Vp but carried pirB Vp Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirB Vp was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VPAHPND isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirA Vp gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirA Vp gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirA Vp gene sequence and in the downstream pirB Vp gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirAB Vp mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However, the virulence of XN87 revealed that some V. parahaemolyticus isolates carrying mutant pVA plasmids that produce no Pir Vp toxins can cause mortality in shrimp in ponds experiencing an outbreak of early mortality syndrome (EMS) but may not have been previously recognized to be AHPND related because they did not cause pathognomonic AHPND lesions.IMPORTANCE Shrimp acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor the pVA1 plasmid encoding toxins PirA Vp and PirB Vp The toxins are produced in the shrimp stomach but cause death by massive sloughing of hepatopancreatic tubule epithelial cells (pathognomonic AHPND lesions). V. parahaemolyticus isolate XN87 harbors a mutant pVA plasmid that produces no Pir toxins and does not cause AHPND lesions but still causes ∼50% shrimp mortality. Such isolates may cause a portion of the mortality in ponds experiencing an outbreak of EMS that is not ascribed to VPAHPND Thus, they pose to shrimp farmers an additional threat that would be missed by current testing for VPAHPND Moribund shrimp from ponds experiencing an outbreak of EMS that exhibit collapsed hepatopancreatic tubule epithelial cells can serve as indicators for the possible presence of such isolates, which can then be confirmed by additional PCR tests for the presence of a pVA plasmid.

A novel C-type lectin in the black tiger shrimp Penaeus monodon functions as a pattern recognition receptor by binding and causing bacterial agglutination.

C-type lectins are pattern recognition proteins that play important roles in innate immunity in invertebrates by mediating the recognition of pathogens. In this study, a novel C-type lectin gene, PmCLec, was cloned and characterized from the black tiger shrimp Penaeus monodon. The open reading frame of PmCLec is 657 bp in length. It encodes a predicted protein of 218 amino acids with a calculated molecular mass and an isoelectric point of 24086 Da and 4.67, respectively. Sequence analysis of PmCLec showed similarity to members of the C-type lectin gene superfamily. The deduced protein contains a single carbohydrate recognition domain (CRD) and four conserved cysteine residues (Cys58, Cys126, Cys141, Cys149) that are involved in the formation of disulfide bridges. PmCLec transcripts are expressed in various tiger shrimp tissues, with the highest expression in the lymphoid organ. RNAi-mediated silencing of PmCLec resulted in higher cumulative mortality of knockdown shrimp after Vibrio harveyi infection compared to the control groups. Recombinant PmCLec was successfully expressed in the E. coli system. In the presence of Ca2+, purified rPmCLec protein binds and agglutinates Gram-positive bacteria (Staphylococcus aureus, S. hemolyticus), but only slightly binds and agglutinates E. coli and could not bind to the Gram-negative bacteria Bacillus megaterium and Vibrio harveyi. These results suggest that PmCLec functions as a pattern recognition receptor that is implicated in shrimp innate immunity.

Suppression of shrimp melanization during white spot syndrome virus infection.

The melanization cascade, activated by the prophenoloxidase (proPO) system, plays a key role in the production of cytotoxic intermediates, as well as melanin products for microbial sequestration in invertebrates. Here, we show that the proPO system is an important component of the Penaeus monodon shrimp immune defense toward a major viral pathogen, white spot syndrome virus (WSSV). Gene silencing of PmproPO(s) resulted in increased cumulative shrimp mortality after WSSV infection, whereas incubation of WSSV with an in vitro melanization reaction prior to injection into shrimp significantly increased the shrimp survival rate. The hemolymph phenoloxidase (PO) activity of WSSV-infected shrimp was extremely reduced at days 2 and 3 post-injection compared with uninfected shrimp but was fully restored after the addition of exogenous trypsin, suggesting that WSSV probably inhibits the activity of some proteinases in the proPO cascade. Using yeast two-hybrid screening and co-immunoprecipitation assays, the viral protein WSSV453 was found to interact with the proPO-activating enzyme 2 (PmPPAE2) of P. monodon. Gene silencing of WSSV453 showed a significant increase of PO activity in WSSV-infected shrimp, whereas co-silencing of WSSV453 and PmPPAE2 did not, suggesting that silencing of WSSV453 partially restored the PO activity via PmPPAE2 in WSSV-infected shrimp. Moreover, the activation of PO activity in shrimp plasma by PmPPAE2 was significantly decreased by preincubation with recombinant WSSV453. These results suggest that the inhibition of the shrimp proPO system by WSSV partly occurs via the PmPPAE2-inhibiting activity of WSSV453.

Knockdown of a novel G-protein pathway suppressor 2 (GPS2) leads to shrimp mortality by exuvial entrapment during ecdysis.

A novel G-protein pathway suppressor 2 (GPS2) has been identified from hemocytes of the whiteleg shrimp Penaeus vannamei (Pv) and appears to play a role in ecdysis. The full-length of PvGPS2 cDNA consisted of a 1230-bp open reading frame, encoding 409 deduced amino acids with significant sequence homology to GPS2 sequences of crustaceans and insects. RT-PCR revealed that PvGPS2 was expressed in all P. vannamei tissues examined, but that expression was molt stage specific in eyestalk tissue. Relative expression was higher in the period before molting (i.e., intermolt and pre-molt stages) than in the post-molt stage. When double-stranded RNA (dsRNA)-mediated RNA interference was employed to inhibit PvGPS2 formation in shrimp, it led to significant mortality due to unsuccessful separation of new cuticle from old cuticle (exuvial entrapment) during ecdysis.

Adjuvant Effects of a CC Chemokine for Enhancing the Efficacy of an Inactivated Streptococcus agalactiae Vaccine in Nile Tilapia (Oreochromis niloticus).

In this study, the ability of a CC chemokine (On-CC1) adjuvant to enhance the efficacy of a formalin-killed Streptococcus agalactiae vaccine (WC) in inducing immune responses against S. agalactiae in Nile tilapia was investigated through immune-related gene expression analysis, enzyme-linked immunosorbent assay (ELISA), transcriptome sequencing, and challenge tests. Significantly higher S. agalactiae-specific IgM levels were detected in fish in the WC+CC group than in the WC alone or control groups at 8 days postvaccination (dpv). The WC vaccine group exhibited increased specific IgM levels at 15 dpv, comparable to those of the WC+CC group, with sustained higher levels observed in the latter group at 29 dpv and after challenge with S. agalactiae for 14 days. Immune-related gene expression analysis revealed upregulation of all target genes in the control group compared to those in the vaccinated groups, with notable differences between the WC and WC+CC groups at various time intervals. Additionally, transcriptome analysis revealed differential gene expression profiles between the vaccinated (24 and 96 hpv) and control groups, with notable upregulation of immune-related genes in the vaccinated fish. Differential gene expression (DGE) analysis revealed significant upregulation of immunoglobulin and other immune-related genes in the control group compared to those in the vaccinated groups (24 and 96 hpv), with distinct patterns observed between the WC and WC+CC vaccine groups. Finally, challenge with a virulent strain of S. agalactiae resulted in significantly higher survival rates for fish in the WC and WC+CC groups compared to fish in the control group, with a notable increase in survival observed in fish in the WC+CC group.

Pattern recognition protein binds to lipopolysaccharide and ß-1,3-glucan and activates shrimp prophenoloxidase system.

The prophenoloxidase (proPO) system is activated upon recognition of pathogens by pattern recognition proteins (PRPs), including a lipopolysaccharide- and ß-1,3-glucan-binding protein (LGBP). However, shrimp LGBPs that are involved in the proPO system have yet to be clarified. Here, we focus on characterizing the role of a Penaeus monodon LGBP (PmLGBP) in the proPO system. We found that PmLGBP transcripts are expressed primarily in the hemocytes and are increased at 24 h after pathogenic bacterium Vibrio harveyi challenge. The binding studies carried out using ELISA indicated that recombinant (r)PmLGBP binds to ß-1,3-glucan and LPS with a dissociation constant of 6.86 × 10(-7) M and 3.55 × 10(-7) M, respectively. Furthermore, we found that rPmLGBP could enhance the phenoloxidase (PO) activity of hemocyte suspensions in the presence of LPS or ß-1,3-glucan. Using dsRNA interference-mediated gene silencing assay, we further demonstrated that knockdown of PmLGBP in shrimp in vivo significantly decreased the PmLGBP transcript level but had no effect on the expression of the other immune genes tested, including shrimp antimicrobial peptides (AMPs). However, suppression of proPO expression down-regulated PmLGBP, proPO-activating enzyme (PmPPAE2), and AMPs (penaeidin and crustin). Such PmLGBP down-regulated shrimp showed significantly decreased total PO activity. We conclude that PmLGBP functions as a pattern recognition protein for LPS and ß-1,3-glucan in the shrimp proPO activating system.

Prophenoloxidase system and its role in shrimp immune responses against major pathogens.

The global shrimp industry still faces various serious disease-related problems that are mainly caused by pathogenic bacteria and viruses. Understanding the host defense mechanisms is likely to be beneficial in designing and implementing effective strategies to solve the current and future pathogen-related problems. Melanization, which is performed by phenoloxidase (PO) and controlled by the prophenoloxidase (proPO) activation cascade, plays an important role in the invertebrate immune system in allowing a rapid response to pathogen infection. The activation of the proPO system, by the specific recognition of microorganisms by pattern-recognition proteins (PRPs), triggers a serine proteinase cascade, eventually leading to the cleavage of the inactive proPO to the active PO that functions to produce the melanin and toxic reactive intermediates against invading pathogens. This review highlights the recent discoveries of the critical roles of the proPO system in the shrimp immune responses against major pathogens, and emphasizes the functional characterizations of four major groups of genes and proteins in the proPO cascade in penaeid shrimp, that is the PRPs, serine proteinases, proPO and inhibitors.

RNA-seq transcriptome analysis and identification of the theromacin antimicrobial peptide of the copepod Apocyclops royi.

Copepods, including Apocyclops royi, are small aquatic crustaceans and one of the important foods for fish and shellfish larvae. However, studies of the host-pathogen interactions and understanding of infectious disease in copepods are still very limited, yet they are likely to be a significant factor in the sustainable development of copepod aquaculture. In the present study, we performed de novo RNA sequence analysis of A. royi-TH (a Thai isolate of A. royi), which yielded 4.80 Gb bases of clean data and a total of 29,786 unigenes. Annotation was then performed by comparison against seven functional databases, yielding 17,617 (NR: 59.15%), 2,969 (NT: 9.97%), 15,023 (SwissProt: 50.44%), 14,543 (KOG: 48.82%), 15,077 (KEGG: 50.62%), 6,763(GO: 22.71%), and 15,841 (InterPro: 53.18%) unigenes. In comparison to the components of the shrimp Toll pathway, LGBP, Spätzle, Toll receptors, MyD88, Pelle, TRAF6, Dorsal, and Cactus homologs were successfully identified in A. royi-TH. Additionally, a novel antimicrobial peptide (Theromacin-like) was characterized in A. royi (ArTM-like). The ArTM-like ORF was 279 bp and predicted to encode for 92 amino acid residues, with a mature peptide of 75 amino acids and a molecular mass of 8.56 kDa. The genomic organization of the ArTM-like gene consisted of three exons and two introns. Expression analysis indicated that ArTM-like mRNA was abundantly expressed in copepodid and adult stages as an immune responsive gene after infection with the pathogenic Vibrio parahaemolyticus-(AHPND)-causing strain. Altogether, the knowledge obtained in this study will provide a basis for future functional studies of the molecular mechanisms in copepod immunity that may eventually be applied for disease prevention in copepod aquaculture.

Molecular cloning, genomic organization and antibacterial activity of a second isoform of antilipopolysaccharide factor (ALF) from the mud crab, Scylla paramamosain.

Antimicrobial peptides (AMPs) serve a major role in host defense systems against microbial invasion. In this study, a novel isoform (ALFSp2) of antilipopolysaccharide factors (ALFs) was cloned from the mud crab, Scylla paramamosain. The open reading frame of the ALFSp2 cDNA is 348 bp and encodes for a predicted 115 amino acid residues (12.92 kDa), and a mature protein of 94 amino acids and a molecular mass of 10.79 kDa. The amino acid sequence of ALFSp2 has an overall similarity of 74%, 66% and 52% to those of Eriocheir sinensis ALF, Penaeus monodon ALFPm3 and S. paramamosain ALFSp1, respectively. The genomic organization of the ALFSp2 gene consists of three exons and two introns, whilst the upstream region contains multiple putative transcription factor binding sites. In healthy crabs, ALFSp2 transcript levels were high in the hemocytes and gill tissues, intermediate levels in the intestine and muscles and at a low level in the hepatopancreas, as determined by RT-PCR. To characterize the in vitro antimicrobial activities of ALFSp2, the 24 amino acid LPS-binding domain encoding peptide was synthesized and revealed an antimicrobial activity against Gram-positive (Aerococcus viridans and Micrococcus luteus) and Gram-negative (Vibrio harveyi and Vibrio anguillarum) bacteria. Altogether these results suggest a potential involvement for ALFSp2 in the defense mechanism of the mud crab, S. paramamosain.

Two novel antimicrobial peptides, arasin-likeSp and GRPSp, from the mud crab Scylla paramamosain, exhibit the activity against some crustacean pathogenic bacteria.

Antimicrobial peptides (AMPs) are some of the important host molecules required to resist pathogen infection. Two novel AMPs (arasin-likeSp and GRPSp) were identified from the hemocytes of the mud crab, Scylla paramamosain, by analysis of a hemocyte expressed sequence tag library. The deduced open reading frames of the arasin-likeSp and GRPSp cDNAs are 198 and 168 bp, and encode for predicted peptides of 65 and 55 amino acid residues, respectively. The calculated molecular mass of the mature peptides was 4373 and 2995 Da with an estimated isoelectric point (pI) of 11.03 and 9.66, respectively. The mature peptide of arasin-likeSp is predicted to contain an N-terminal region rich in glycine and arginine and a C-terminal region containing four cysteine residues. Its amino acid sequence has an overall sequence identity of 53% to arasin-2 from the spider crab, Hyas araneus. The mature protein of GRPSp contains two cysteine residues at the C-terminus and two glycine-rich repeats (GGYG and GYGG). In healthy crabs, both arasin-likeSp and GRPSp transcript levels were found to be high in the hemocytes and were further increased at 3 h after challenge with the bacterium, Aerococcus viridans. A synthetic arasin-likeSp peptide revealed the antimicrobial activity against both Gram-positive and Gram-negative bacteria including some crustacean pathogens (A. viridans, Vibrio harveyi and V. anguillarum), whilst the synthetic GRPSp peptide exhibited antibacterial activity against some Gram-positive (A. viridans and Micrococcus luteus), but not Gram-negative, bacteria. These results indicate that arasin-likeSp and GRPSp are potentially novel AMPs involved in the immune responses of mud crab, S. paramamosain.

Penaeus monodon Interferon Regulatory Factor (PmIRF) Activates IFNs and Antimicrobial Peptide Expression via a STING-Dependent DNA Sensing Pathway.

Interferon regulatory factors (IRFs) are transcription factors found in both vertebrates and invertebrates that were recently identified and found to play an important role in antiviral immunity in black tiger shrimp Penaeus monodon. In this study, we investigated the mechanism by which P. monodon IRF (PmIRF) regulates the immune-related genes downstream of the cytosolic DNA sensing pathway. Depletion of PmIRF by double-stranded RNA-mediated gene silencing significantly reduced the mRNA expression levels of the IFN-like factors PmVago1, PmVago4, and PmVago5 and antilipopolysaccharide factor 6 (ALFPm6) in shrimp. In human embryonic kidney (HEK293T) cells transfected with PmIRF or co-transfected with DEAD-box polypeptide (PmDDX41) and simulator of IFN genes (PmSTING) expression plasmids, the promoter activity of IFN-ß, nuclear factor (NF-κB), and ALFPm6 was synergistically enhanced following stimulation with the nucleic acid mimics deoxyadenylic-deoxythymidylic acid sodium salt [poly(dA:dT)] and high molecular weight (HMW) polyinosinic-polycytidylic acid [poly(I:C)]. Both nucleic acid mimics also significantly induced PmSTING, PmIRF, and ALFPm6 gene expression. Co-immunoprecipitation experiments showed that PmIRF interacted with PmSTING in cells stimulated with poly(dA:dT). PmSTING, PmIRF, and PmDDX41 were localized in the cytoplasm of unstimulated HEK293T cells and PmIRF and PmDDX41 were translocated to the nucleus upon stimulation with the nucleic acid mimics while PmSTING remained in the cytoplasm. These results indicate that PmIRF transduces the pathogen signal via the PmDDX41-PmSTING DNA sensing pathway to induce downstream production of interferon-like molecules and antimicrobial peptides.

Establishment of hematopoietic tissue primary cell cultures from the giant freshwater prawn Macrobrachium rosenbergii.

The giant freshwater prawn Macrobrachium rosenbergii is one of the most important aquaculture species in Southeast Asia. In this study, in vitro culture of its hematopoietic tissue cells was achieved and characterized for use as a tool to study its pathogens that cause major farm losses. By transmission electron microscopy, the ultrastructure of the primary culture cells was similar to that of cells lining intact hematopoietic tissue lobes. Proliferating cell nuclear antigen (PCNA) (a marker for hematopoietic stem cell proliferation) was detected in some of the cultured cells by polymerase chain reaction (PCR) testing and flow cytometry. Using a specific staining method to detect phenoloxidase activity and using PCR to detect expression markers for semigranular and granular hemocytes (e.g., prophenoloxidase activating enzyme and prophenoloxidase) revealed that some of the primary cells were able to differentiate into mature hemocytes within 24 h. These results showed that some cells in the cultures were hematopoietic stem cells that could be used to study other interesting research topics (e.g. host pathogen interactions and development of an immortal hematopoietic stem cell line).

Immune Regulation, but Not Antibacterial Activity, Is a Crucial Function of Hepcidins in Resistance against Pathogenic Bacteria in Nile Tilapia (Oreochromis niloticus Linn.).

In this study, the functions of a recombinant propeptide (rProOn-Hep1) and the synthetic FITC-labelled mature peptides sMatOn-Hep1 and sMatOn-Hep2 were analyzed. Moreover, sMatOn-Hep1 and sMatOn-Hep2 were mildly detected in the lymphocytes of peripheral blood mononuclear cells (PBMCs) and strongly detected in head kidney macrophages. The in vitro binding and antibacterial activities of these peptides were slightly effective against several pathogenic bacteria. Immune regulation by sMatOn-Hep1 was also analyzed, and only sMatOn-Hep1 significantly enhanced the phagocytic index in vitro (p < 0.05). Interestingly, intraperitoneal injection of sMatOn-Hep1 (10 or 100 µg) significantly elevated the phagocytic activity, phagocytic index, and lysozyme activity and clearly decreased the iron ion levels in the livers of the treated fish (p < 0.05). Additionally, sMatOn-Hep1 enhanced the expression levels of CC and CXC chemokines, transferrin and both On-Hep genes in the liver, spleen and head kidney, for 1-96 h after injection, but did not properly protect the experimental fish from S. agalactiae infection after 7 days of treatment. However, the injection of S. agalactiae and On-Heps indicated that 100 µg of sMatOn-Hep1 was very effective, while 100 µg of rProOn-Hep1 and sMatOn-Hep2 demonstrated moderate protection. Therefore, On-Hep is a crucial iron-regulating molecule and a key immune regulator of disease resistance in Nile tilapia.