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.

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.

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.

Molecular isolation and characterization of a spätzle gene from Macrobrachium rosenbergii.

Spätzle protein is an extracellular ligand of Toll receptor in Toll signaling pathway involved in the embryonic dorsoventral patterning and in the innate immunity. In this study, a spätzle gene of freshwater prawn, Macrobrachium rosenbergii (MrSpz) was isolated and characterized. The open reading frame of MrSpz consisted of 747 nucleotides encoding 248 amino acid residues containing a signal peptide and C-terminal spätzle activated domain. MrSpz shared high similarity to spätzle of Fenneropenaeus chinensis (FcSpz) at 92% identity and Marsupenaeus japonicus (MjSpz) at 83% identity. Phylogenetic analysis was performed and the results revealed that MrSpz was a member of the clade containing LvSpz3 of Litopenaeus vannamei, FcSpz and Penaeus monodon spätzle protein. The expression distribution at transcriptional level in various tissues of normal prawn revealed that the MrSpz was detected in gills, heart and hepatopancreas while no expression was observed in hemocyte, muscle and stomach. In the Aeromonas caviae challenged prawn, the expression level of MrSpz in hemocyte was increased gradually at 6, 12 and 24 h post-injection. Furthermore, in MrSpz knocked down prawn injected with Aeromonas caviae, the mortality rate were higher than that of non-related dsRNA group and control group. These results suggest that MrSpz protein may play a key role in the innate immunity of M. rosenbergii, especially in response to Gram-negative bacteria A. caviae invasion.

Two host gut-derived lactic acid bacteria activate the proPO system and increase resistance to an AHPND-causing strain of Vibrio parahaemolyticus in the shrimp Litopenaeus vannamei.

Lactic acid bacteria (LAB) are group of beneficial bacteria that have been proposed as relevant probiotics with immunomodulatory functions. In this study, we initially isolated and identified host-derived LAB from the gut of the Pacific white shrimp Litopenaeus vannamei. Analysis of the bacterial 16S rRNA gene sequence revealed two candidate LAB, the Lactobacillus plantarum strain SGLAB01 and the Lactococcus lactis strain SGLAB02, which exhibited 99% identity to the L. plantarum strain LB1-2 and the L. lactis strain R-53658, which were isolated from bee gut, respectively. The two LAB displayed antimicrobial activities against gram-positive and gram-negative bacteria, including the virulent acute hepatopancreatic necrosis disease (AHPND)-causing strain of Vibrio parahaemolyticus (VPAHPND). Viable colony count and SEM analysis showed that the two candidate LAB, administered via oral route as feed supplement, could reside and adhere in the shrimp gut. Double-stranded RNA-mediated gene silencing of LvproPO1 and LvproPO2 revealed a significant role of two LvproPOs in the proPO system as well as in the immune response against VPAHPND infection in L. vannamei shrimp. The effect of LAB supplementation on modulation of the shrimp proPO system was investigated in vivo, and the results showed that administration of the two candidate LAB significantly increased hemolymph PO activity, the relative mRNA expression of LvproPO1 and LvproPO2, and resistance to VPAHPND infection. These findings suggest that administration of L. plantarum and L. lactis could modulate the immune system and increase shrimp resistance to VPAHPND infection presumably via upregulation of the two LvproPO transcripts.

A novel white spot syndrome virus protein WSSV164 controls prophenoloxidases, PmproPOs in shrimp melanization cascade.

Melanization, mediated by the prophenoloxidase (proPO)-activating system, is an important innate immune response in invertebrates. The implication of the proPO system in antiviral response and the suppression of host proPO activation by the viral protein have previously been demonstrated in shrimp. However, the molecular mechanism of viral-host interactions in the proPO cascade remains largely unexplored. Here, we characterized the viral protein, namely, WSSV164, which was initially identified from the forward suppression subtractive hybridization (SSH) cDNA library of the PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon that was challenged with white spot syndrome virus (WSSV). Using the yeast two-hybrid system, WSSV164 was found to interact with the PmproPO2 protein. The subsequent validation assay by co-immunoprecipitation revealed that WSSV164 directly bound to both PmproPO1 and PmproPO2. The gene silencing experiment was carried out to explore the role of WSSV164 in the control of the proPO pathway in shrimp, and the results showed that suppression of WSSV164 can restore PO activity in WSSV-infected shrimp hemolymph. The recombinant proteins of PmproPO1 and PmproPO2 were produced in Sf-9 cells and were shown to be successfully activated by exogenous trypsin and endogenous serine proteinases from shrimp hemocyte lysate supernatant (HLS), yielding PO activity in vitro. Moreover, the activated PO activity in shrimp HLS was dose-dependently reduced by the recombinant WSSV164 protein, suggesting that WSSV164 may interfere with the activation of the proPO system in shrimp. Taken together, these results suggest an alternative infection route of WSSV through the encoded viral protein WSSV164 that binds to the PmproPO1 and PmproPO2 proteins, interfering with the activation of the melanization cascade in shrimp.

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.

Shrimp humoral responses against pathogens: antimicrobial peptides and melanization.

Diseases have caused tremendous economic losses and become the major problem threatening the sustainable development of shrimp aquaculture. The knowledge of host defense mechanisms against invading pathogens is essential for the implementation of efficient strategies to prevent disease outbreaks. Like other invertebrates, shrimp rely on the innate immune system to defend themselves against a range of microbes by recognizing and destroying them through cellular and humoral immune responses. Detection of microbial pathogens triggers the signal transduction pathways including the NF-κB signaling, Toll and Imd pathways, resulting in the activation of genes involved in host defense responses. In this review, we update the discovery of components of the Toll and Imd pathways in shrimp and their participation in the regulation of shrimp antimicrobial peptide (AMP) synthesis. We also focus on a recent progress on the two most powerful and the best-studied shrimp humoral responses: AMPs and melanization. Shrimp AMPs are mainly cationic peptides with sequence diversity which endues them the broad range of activities against microorganisms. Melanization, regulated by the prophenoloxidase activating cascade, also plays a crucial role in killing and sequestration of invading pathogens. The progress and emerging research on mechanisms and functional characterization of components of these two indispensable humoral responses in shrimp immunity are summarized and discussed. Interestingly, the pattern recognition protein (PRP) crosstalk is evidenced between the proPO activating cascade and the AMP synthesis pathways in shrimp, which enables the innate immune system to build up efficient immune responses.

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.

Characterization and antimicrobial evaluation of SpPR-AMP1, a proline-rich antimicrobial peptide from the mud crab Scylla paramamosain.

Antimicrobial peptide (AMP) is an important molecule in the innate immune system. Here, we report the cloning and functional studies of proline-rich AMPs (PR-AMPs) from the three species of mud crab: Scylla paramamosain, S. serrata, and the swimming crab Portunus pelagicus. The deduced peptides revealed that they contain the putative signal peptides and encode for mature peptides, which contain sequence architecture similar to a 6.5-kDa proline-rich AMP of the shore crab, Carcinus maenas which showed similarity with the bactenecin7. Tissue distribution analysis indicated that the SpPR-AMP1 was expressed in a wide range of adult tissues, with the highest expression levels in the crab hemocyte. Challenge experiments showed that the levels of SpPR-AMP1 mRNA expression were up-regulated in the hemocyte after peptidoglycan stimulation. To evaluate the biological properties of mature SpPR-AMP1, peptides were chemically synthesized and recombinantly expressed. SpPR-AMP1 showed strong antibacterial activity against both Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Vibrio harveyi. The results indicate that the SpPR-AMP1 plays a role in crab immunity.

The expression and purification of WSSV134 from white spot syndrome virus and its inhibitory effect on caspase activity from Penaeus monodon.

WSSV134 or VP36A protein of white spot syndrome virus was previously reported to be able to reduce apoptosis in Sf-9 cells transfected with caspase of Penaeus monodon (PmCasp). The protein was therefore believed to have a role in supporting the survival of WSSV inside the host cells during infection. However, the anti-apoptosis activity of WSSV134 involved in the inhibition of PmCasp is still unclear. In this study, we produced a recombinant WSSV134 (rWSSV134) and tested for its ability to inhibit PmCasp in vitro. The results from a caspase inhibition assay revealed that rWSSV134 could inhibit PmCasp in a dose-dependent manner. Since WSSV134 was predicted to contain three potential caspase binding sites, corresponding to the D54, D104 and D259, we then employed site-directed mutagenesis to investigate the involvement of these sites in PmCasp inhibition. D54A and D259A mutants could still inhibit PmCasp while D104A mutant lacks this activity. Our results confirmed that the WSSV134 is an inhibitor for PmCasp and that residue D104 is important for PmCasp inhibition.

Duplex PCR assay and in situ hybridization for detection of Francisella spp. and Francisella noatunensis subsp. orientalis in red tilapia.

Conventional isolation and identification based on phenotypic characteristics is challenging with the highly fastidious, intracellular bacterium Francisella noatunensis subsp. orientalis (Fno). Here, we developed a duplex PCR method for simultaneous detection of the Francisella genus and Fno in one PCR reaction and an in situ hybridization method for paraffin section based diagnosis of Fno. The PCR results showed genus- and species-specific bands (1140 and 203 bp) from Fno but only one genus-specific band (1140 bp) from F. noatunensis subsp. noatunensis. Sensitivity of the duplex PCR assay revealed a detection limit of 20 to 200 fg genomic DNA (~10 to 100 genome equivalents) depending on DNA template extraction methods. The newly developed duplex PCR assay could be used to detect Fno from clinically sick fish exhibiting signs of visceral granulomas and would also be able to detect Fno infection in naturally diseased fish without symptoms of francisellosis, indicating potential application for diagnosis of field samples. The in situ hybridization assay using Fno species-specific probe revealed positive signals in multiple organs including the spleen, liver, kidney, gills and intestine of infected fish.

A shrimp pacifastin light chain-like inhibitor: molecular identification and role in the control of the prophenoloxidase system.

Pacifastin is a recently classified family of serine proteinase inhibitors that play essential roles in various biological processes, including in the regulation of the melanization cascade. Here, a novel pacifastin-related gene, termed PmPacifastin-like, was identified from a reverse suppression subtractive hybridization (SSH) cDNA library created from hemocytes of the prophenoloxidase PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon. The full-length sequences of PmPacifastin-like and its homologue LvPacifastin-like from the Pacific white shrimp Litopenaeus vannamei were determined. Sequence analysis revealed that both sequences contained thirteen conserved pacifastin light chain domains (PLDs), followed by two putative kunitz domains. Expression analysis demonstrated that the PmPacifastin-like transcript was expressed in all tested shrimp tissues and larval developmental stages, and its expression responded to Vibrio harveyi challenge. To gain insight into the functional roles of PmPacifastin-like protein, the in vivo RNA interference experiment was employed; the results showed that PmPacifastin-like depletion strongly increased PO activity. Interestingly, suppression of PmPacifastin-like also down-regulated the expression of the proPO-activating enzyme PmPPAE2 transcript; the PmPacifastin-like transcript was down-regulated after the PmproPO1/2 transcripts were silenced. Taken together, these results suggest that PmPacifastin-like is important in the shrimp proPO system and may play an essential role in shrimp immune defense against bacterial infection. These results also expand the knowledge of how pacifastin-related protein participates in the negative regulation of the proPO system in shrimp.

Knockdown of Litopenaeus vannamei HtrA2, an up-regulated gene in response to WSSV infection, leading to delayed shrimp mortality.

HtrA2 is an apoptosis-activating gene that enhances the apoptotic process by preventing the formation of the IAP-caspase complex, thereby freeing caspase to trigger the apoptosis pathway. In this study, we presented the full-length cDNA sequence of HtrA2 from Litopenaeus vannamei (LvHtrA2). The full-length LvHtrA2 was 1335 bp, encoding 444 amino acids. This deduced amino acid sequence contained five conserved domains: a mitochondrial targeting signal (MTS), a transmembrane (TM) domain, an IAP-binding motif (IBM), a trimerization motif, a serine protease domain, and a PDZ domain normally found in the HtrA2 proteins of other organisms. A phylogenetic analysis revealed that LvHtrA2 clustered with the HtrA2 from other invertebrates and was closely related to Penaeus monodon HtrA2 (PmHtrA2). RT-PCR with RNA extracts from L. vannamei revealed that LvHtrA2 expression was found in several tissues, including the lymphoid organs, the haemocytes, the hepatopancreas, the gill, and the stomach, with different expression levels. When determining the role of LvHtrA2 in WSSV infection, it was found that LvHtrA2 transcription was early up-regulated in the WSSV-infected shrimp at 8h post-infection (p.i.) and expression still remained high at 48 h p.i.. It also demonstrated that dsRNA specific to LvHtrA2 reduced the cumulative mortality in the WSSV-infected shrimp compared with the control group. Additionally, depletion of the LvHtrA2 transcripts reduced expression levels for caspase-3 (Cap-3) gene in shrimp. This result could suggest that LvHtrA2 may involved in apoptosis mediated mortality rather than providing immune protection during WSSV infection.

Role of Penaeus monodon hemocyte homeostasis associated protein (PmHHAP) in regulation of caspase-mediated apoptosis.

The viral responsive protein, PmHHAP, plays an important role in the control of hemocyte homeostasis in shrimps during viral infection. In this study, we further investigate the role of PmHHAP in the regulation of hemocyte apoptosis. RNA interference (RNAi) mediated gene silencing was used to suppress the PmHHAP expression and the change in hemocyte apoptosis was determined in the knockdown shrimp. Within circulating hemocytes, PmHHAP knockdown increased the number of annexin V-positive apoptotic cells and the combined caspase-3/-7 activity and induced the characteristic apoptotic DNA ladder. Furthermore, PmHHAP down-regulation was accompanied by significantly altered expression of apoptosis-related proteins including the effector caspases, PmCaspase and PmCasp. Yeast two-hybrid and co-immunoprecipitation assays showed that PmHHAP binds to the p20 domain of PmCasp. Moreover, the recombinant PmHHAP protein was able to reduce the caspase activity in the actinomycin D-treated hemocyte cells and rPmCasp-treated hemocyte cells. Taken together, our data indicate that PmHHAP regulates hemocyte homeostasis by inhibits apoptotic cell death through caspase activation.

Shrimp serine proteinase homologues PmMasSPH-1 and -2 play a role in the activation of the prophenoloxidase system.

Melanization mediated by the prophenoloxidase (proPO) activating system is a rapid immune response used by invertebrates against intruding pathogens. Several masquerade-like and serine proteinase homologues (SPHs) have been demonstrated to play an essential role in proPO activation in insects and crustaceans. In a previous study, we characterized the masquerade-like SPH, PmMasSPH1, in the black tiger shrimp Penaeus monodon as a multifunctional immune protein based on its recognition and antimicrobial activity against the Gram-negative bacteria Vibrio harveyi. In the present study, we identify a novel SPH, known as PmMasSPH2, composed of an N-terminal clip domain and a C-terminal SP-like domain that share high similarity to those of other insect and crustacean SPHs. We demonstrate that gene silencing of PmMasSPH1 and PmMasSPH2 significantly reduces PO activity, resulting in a high number of V. harveyi in the hemolymph. Interestingly, knockdown of PmMasSPH1 suppressed not only its gene transcript but also other immune-related genes in the proPO system (e.g., PmPPAE2) and antimicrobial peptides (e.g., PenmonPEN3, PenmonPEN5, crustinPm1 and Crus-likePm). The PmMasSPH1 and PmMasSPH2 also show binding activity to peptidoglycan (PGN) of Gram-positive bacteria. Using a yeast two-hybrid analysis and co-immunoprecipitation, we demonstrate that PmMasSPH1 specifically interacted with the final proteinase of the proPO cascade, PmPPAE2. Furthermore, the presence of both PmMasSPH1 and PmPPAE2 enhances PGN-induced PO activity in vitro. Taken together, these results suggest the importance of PmMasSPHs in the activation of the shrimp proPO system.

Laminin receptor protein is implicated in hemocyte homeostasis for the whiteleg shrimp Penaeus (Litopenaeus) vannamei.

Here we show that knockdown of laminin receptor (Lamr) with PvLamr dsRNA in the whiteleg shrimp Penaeus (Litopenaeus) vannamei (Pv) caused a dramatic reduction specifically in hyaline hemocytes prior to death. Since apoptosis was not detected in hemocytes or hematopoietic cells, other possible causes of hemocyte loss were investigated. Reports that suppression of crustacean hematopoietic factor (CHF)-like protein or hemocyte homeostasis-associated protein (HHAP) also reduced shrimp hemocyte counts led us to carry out yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) assays to test for interactions between Lamr and Pv homologues to these proteins (PvCHF-like and PvHHAP). The assays revealed that Lamr bound to both these homologues, but that the homologues did not bind to each other. Subsequent RT-PCR assays confirmed that PvLamr dsRNA injection significantly reduced expression levels for both PvCHF-like and PvHHAP genes. Further work is needed to determine how interaction among these three proteins can regulate shrimp hemocyte homeostasis.

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.