proPO System Research Articles

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.

Immune response and gene expression in hemocytes of Portunus trituberculatus inoculated with the parasitic dinoflagellate Hematodinium

The parasitic dinoflagellates in the genus of Hematodinium infect broad range of crustaceans around the world, causing fatal diseases in multiple species of wild and cultured crabs and subsequent economic loss. In order to explore this host–parasite interaction in the early development stage of infection, mRNA transcript levels of eight key immune-related genes, including LGBP, proPO, PPAF, serpin, α2m, and three PTcSPs were quantitatively assessed in Portunus trituberculatus artificially inoculated with the Hematodinium parasites. The fluctuation of proPO gene expression indicated that the host proPO system was disturbed overtly due to the intrusion of the parasites. And as manifested by the suppressed expression of LGBP and PPAF, an immunosuppressive mechanism was likely induced by the parasites against being entrapped or killed by the host proPO system. Furthermore, the significant variations of the transcript levels of serpin, α2m, and the three PTcSPs suggested that the parasites affected the proteinase cascade reactions associated with the immune response by destroying the balance between serine proteinases and the proteinase inhibitors. The hemocytes counts and PO activity varied accordingly over the time course of infection, showing that hemocytes were actively involved in the immune response against the parasitic invasion. This study primarily highlighted the anti-parasitic immune response of crab hosts, and presented the first report of the immune response of P. trituberculatus to the parasitic dinoflagellate Hematodinium.

PtPLC, a pacifastin-related inhibitor involved in antibacterial defense and prophenoloxidase cascade of the swimming crab Portunus trituberculatus

Pacifastin-related inhibitor is a new family of serine protease inhibitors that regulate the proteolytic cascade in multiple biological processes. Contrary to the knowledge on the structure and inhibitory mechanism of pacifastin-like members in locust, very little is known about their functions. Here, we report the inhibitory activities in relation to the structural characteristics of pacifastin light chain (PtPLC) gene identified from the swimming crab Portunus trituberculatus. The mature PtPLC and five PLD-related domains with critical residues were expressed in Escherichia coli, and assayed for their activities. The recombinant PtPLC (rPtPLC) displayed inhibitory activities against trypsin and chymotrypsin in a dose dependent manner, with a preference for trypsin. Except for rPtPLC-D4, the other four rPtPLC-related domains could inhibit at least one of serine proteases. The enzyme specificity of PtPLC domains generally corresponded to the nature of the P1 residue at the reactive site. rPtPLC was able to inhibit the growth of Gram-negative bacteria Vibrio alginolyticus and Pseudomonas aeruginosa, but not the Gram-positive bacterium and fungus tested. Further phenoloxidase (PO) assay showed the rPtPLC could depress the crab proPO system activation in vitro, and lead to 72.8% inhibition of PO activity at the concentration of 9.11 μM. It also suppressed proPO activation induced by rPtcSP and rPtSPH1. As the first functional study of the recombinant PLC protein in crustaceans, the present results together indicate that PtPLC functions in the crab immune response possibly via inhibiting bacterial growth and regulating the proPO system.

Modulation of immune responses of Rhynchophorus ferrugineus (Insecta: Coleoptera) induced by the entomopathogenic nematode Steinernema carpocapsae (Nematoda: Rhabditida)

Aim of this study was to investigate relationships between the red palm weevil (RPW) Rhynchophorus ferrugineus (Olivier) and the entomopathogenic nematode Steinernema carpocapsae (EPN); particularly, the work was focused on the immune response of the insect host in naive larvae and after infection with the EPN. Two main immunological processes have been addressed: the activity and modulation of host prophenoloxidase-phenoloxidase (proPO) system, involved in melanization of not-self and hemocytes recognition processes responsible for not-self encapsulation. Moreover, immune depressive and immune evasive strategies of the parasite have been investigated. Our results suggest that RPW possess an efficient immune system, however in the early phase of infection, S. carpocapsae induces a strong inhibition of the host proPO system. In addition, host cell-mediated mechanisms of encapsulation, are completely avoided by the parasite, the elusive strategies of S. carpocapsae seem to be related to the structure of its body-surface, since induced alterations of the parasite cuticle resulted in the loss of its mimetic properties. S. carpocapsae before the release of its symbiotic bacteria, depress and elude RPW immune defenses, with the aim to arrange a favorable environment for its bacteria responsible of the septicemic death of the insect target.

Double-dose β-glucan treatment in WSSV-challenged shrimp reduces viral replication but causes mortality possibly due to excessive ROS production

In our research efforts to reduce the impact of white spot syndrome virus (WSSV) disease outbreaks in shrimp aquaculture, we studied the effect of β-glucan administration to activate the prophenoloxidase (proPO) enzymatic cascade prior to WSSV challenge. Injection of a single dose of β-glucan (5 μg/g) prior to WSSV challenge resulted in activation of the proPO system and reduced shrimp mortality (25–50%) when compared to controls (100%). By contrast, no significant reduction was observed using yellow head virus (YHV) in a similar protocol. We subsequently hypothesized that administration of a second dose of β-glucan after WSSV challenge might reduce shrimp mortality further. Surprisingly, the opposite occurred, and mortality of the WSSV-infected shrimp increased to 100% after the second β-glucan dose. Both immunofluorescence and RT-PCR assays revealed low WSSV levels in hemocytes of shrimp collected after the second dose of β-glucan administration, suggesting that the cause of increased mortality was unlikely to be increased WSSV replication. We found from measured phenoloxidase acitivity (PO) and H2O2 production that the higher mortality may have resulted from a combination of WSSV infection plus over-production of reactive oxygen species (ROS) stimulated by two doses of β-glucan. Thus, caution may be prudent in continuous or prolonged activation of the shrimp immune system by β-glucan administration lest it exacerbate shrimp mortality in the event of WSSV infection.

Hemocyte morphology and phagocytic activity in the common cuttlefish (Sepia officinalis)

Little is known about the immune system of cephalopods, in spite of their many highly derived characters within the molluscan clade, including a vertebrate-like high-pressure closed circulatory system. Further the economic importance of cephalopod fisheries, potential for aquaculture, and use as ecotoxicology models demand a thorough understanding of their immune system. In this study, we present a comprehensive characterization of hemocytes in the common cuttlefish Sepia officinalis. Cytological stainings, electron microscopy- and flow cytometry-observations highlight a single granulocyte population with various densities of eosinophilic granules and unstained vesicles. These hemocytes contain acid phosphatase-, lysozyme- and proPO system enzymes, and have high activity in bead phagocytosis assays. Interestingly, bead pre-incubation in plasma results in time-dependent aggregation perhaps resulting from hemocyanin-coating, and decrease in phagocytosis. This study provides the basis for understanding hemocyte-mediated immunity in the common cuttlefish, and essential background for future studies on cephalopod immunity.

Melanization reaction products of shrimp display antimicrobial properties against their major bacterial and fungal pathogens

Melanization is a rapid defense mechanism in invertebrates. The substrate specificity of phenoloxidases (POs) and the role of melanization reaction products were investigated in the black tiger shrimp, Penaeus monodon. Two PmPOs (PmproPO1 and PmproPO2) were found to display a substrate specificity towards monophenols and diphenols, and exhibit relatively weak activity against 5,6-dihydroxyindole (DHI). Systemic infection of the PmproPO1/2 co-silenced shrimp with the fungus, Fusarium solani, led to a significantly increased mortality, suggesting an important role of PmproPOs in shrimp’s defense against fungal infection. Using l-DOPA, dopamine or DHI as a substrate, the melanization reaction products exhibited in vitro antimicrobial activities towards Gram-negative bacteria (Vibrio harveyi and Vibrioparahaemolyticus) and Gram-positive bacteria (Bacillus subtilis), whereas the lower effect was detected against the fungus (F. solani). SEM analysis revealed the morphological changes and damage of cell membranes of V. harveyi and F. solani after treatment with shrimp melanization reaction products. Together, these findings demonstrate the crucial functions of the proPO system and the importance of melanization reaction products in the shrimp’s immune defense.

LYSOZYME AND DEFENSE PEPTIDES AS SUPPRESSORS OF PHENOLOXIDASE ACTIVITY IN Galleria mellonella

The prophenoloxidase (proPO) cascade supplies quinones and other reactive compounds for melanin formation, protein cross-linking, hemolymph coagulation, and killing of microbial invaders as well as parasites. The high cytotoxicity of the generated compounds requires a strict control of the activation of the proPO system and phenoloxidase (PO) activity to minimize damage to host tissues and cells. The PO activity in hemolymph of Escherichia coli challenged Galleria mellonella larvae increased, with a temporal drop 1 h after the challenge, reaching the highest level 24 h after the challenge. In the present study, a potential role of G. mellonella defense peptides and lysozyme in controlling the proPO system was investigated. The effects of purified defense peptides (anionic peptides 1 and 2, cecropin D-like peptide, Galleria defensin, proline-rich peptides 1 and 2) and lysozyme were analyzed. Four compounds, namely lysozyme, Galleria defensin, proline-rich peptide 1, and anionic peptide 2, decreased the hemolymph PO activity considerably, whereas the others did not affect the enzyme activity level. Our results indicate that these hemolymph factors could play multiple and distinct roles in the insect immune response.

PtSerpin from the swimming crab Portunus trituberculatus, a putative regulator of prophenoloxidase activation with antibacterial activity

Serpin or serine protease inhibitor is the largest family of protease inhibitors involved in many innate immune pathways, particularly the prophenoloxidase (proPO) activating system in arthropod. Here, we report the molecular and functional characterization of PtSerpin identified from the swimming crab Portunus trituberculatus. The genomic sequence encoding mature peptide of PtSerpin gene contained two exons of 84 and 1098 bp separated by one intron of 111 bp. The recombinant PtSerpin (rPtSerpin) with a predicted size of 44 kDa was expressed in Escherichia coli system, purified and assayed for its activities. The rPtSerpin exhibited inhibitory activity against trypsin in a dose-dependent manner, but did not affect chymotrypsin, which could define a role for PtSerpin as a trypsin inhibitor. The rPtSerpin could inhibit the growth of Gram-negative bacterium Vibrio alginolyticus, but not the tested Gram-positive bacterium and fungus. Further phenoloxidase (PO) assay showed PO activity was dramatically increased in hemocyte lysate supernatant of P. trituberculatus upon bacterial challenge. The rPtSerpin could depress the crab proPO system activation in vitro, and it could lead to 100% inhibition of PO activity under the concentration of 8.62 μM. Moreover, the rPtSerpin was able to inhibit the PO activity induced by rPtcSP and rPtSPH1. These results together indicate that PtSerpin is a potential trypsin inhibitor and may participate in crab innate immunity by the inhibition of bacterial growth and the regulation of proPO system.

Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity.

Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.

Gene expression responses of white shrimp (Litopenaeus vannamei) infected with necrotizing hepatopancreatitis bacterium

Abstract Necrotizing hepatopancreatitis bacterium (NHPB) affects the production of shrimp farms along Latin America. NHPB is one of the main threats to shrimp culture; therefore the defense mechanisms and molecular mediators that act against this bacterium ought to be well understood. The aim of this study was to study the changes in the immune response and the expression of genes coding for proteins of the immune system in the white shrimp, Litopenaeus vannamei, infected with NHPB. Shrimp infected with NHPB and healthy shrimp were maintained under laboratory conditions during 24 d. Haemolymph and hepatopancreas were collected from shrimp every six days. Total hemocyte count (THC), activities of α-2-Macroglobulin (A-2-M), phenoloxidase and total phenoloxidase from haemolymph were monitored. The expression of clottable protein (CP) and lipopolysaccharide & β-glucan binding protein (LGBP) were determined from hepatopancreas, whereas the expression of A-2-M, prophenoloxidase (proPO) and transglutaminase (TGase) was determined from hemocytes. THC showed no significant differences among infected and healthy shrimp at any of the four times. The specific activity of A-2-M significantly decreased on days 18th and 24th post-infection. In the case of the specific activity of total phenoloxidase and phenoloxidase a significant decrease was detected only in day 18 post-infection. CP gene expression decreased at 6th day post-infection, while at 18 days showed an increase of about 2.5-fold. LGBP expression increased in the first six days and at the 24th day post-infection had a drastic decline. The expression of A-2-M showed no significant changes in any of the four times. proPO expression increased more than 4-fold at day 12th post-infection but decreased at 18th day post-infection. TGase expression increased 3.5-fold at day 12th post-infection. The results found in this study suggest an active participation of the proPO system and the clotting reaction against NHP infection, mostly on days 12th and 18th post-infection.

Exploration of protein–protein interaction effects on α-2-macroglobulin in an inhibition of serine protease through gene expression and molecular simulations studies

In Prophenoloxidase (ProPO) cascade, two targets namely serine protease and α-2-macroglobulin are key regulators involved in the defense system of crustaceans. In biological systems, routine role of cell systems requires the understanding in protein–protein interactions through experimental and theoretical concepts, which might yield useful insights into the cellular responses. Response of cells to regulating the immune system is governed by the interactions-involved biomolecular simulations. Unfortunately, studies on the inhibitors (SP and α-2M) that negatively regulate the proPO system or melanization in penaeid shrimp are not yet available. In order to understand how these interactions change the proPO mechanism in Indian white shrimp Fenneropenaeus indicus was determined. In F. indicus, innate immune system is in a sensitive balance of intricate interactions; elucidating these interactions by the integration of in silico and in vitro has great potential. We have determined the expression of both the SP and α-2M enzymes in regulatory mechanism, which are analyzed through qRT-PCR, protein–protein docking, and simulation studies. From this work, we propose a novel approach for studying an organism at the systems level by integrating genome-wide computational analysis and the gene expression data.

The roles of serine protease, intracellular and extracellular phenoloxidase in activation of prophenoloxidase system, and characterization of phenoloxidase from shrimp haemocytes induced by lipopolysaccharide or dopamine

We investigated the effects of lipopolysaccharide (LPS) and dopamine (DA) on the activation of the prophenoloxidase (proPO) system of Litopenaeus vannamei. LPS and DA were shown with a negative dose-dependent effect on hyalne cells (HC), semi-granular cells (SGC), large granular cells (LGC), and total haemocyte count (THC). When haemocytes were treated with LPS or DA, serine proteinase activity and intracellular phenoloxidase (PO) activity were significantly reduced, but extracellular PO activity increased significantly. These findings indicated that the reduction in haemocyte counts was mainly because of the degranulation and activation of the proPO system from semi-granule and large granule cells. The PKC inhibitor, chelerythrine, and the TPK inhibitor, genistein, had an inhibitory effect on extracellular PO activity, while serine proteinase and intracellular PO activity increased. This suggests that the LPS and DA induce the activation of proPO in haemocytes via PKC and TPK-related signaling pathways, but serine proteinase may be activated only by PKC, as the genistein effects were not statistically significant. Electrophoresis analysis revealed that POs induced by LPS or DA have the same molecular mass and high diphenolase activity. Two PO bands at 526 kDa and 272 kDa were observed in PAGE, while in the haemocyte lysate supernatant (HLS), only a 272-kDa band was observed. This band was resolved after SDS-PAGE under non-reducing and reducing conditions into two groups of POs, 166 kDa and 126 kDa, and 78.1 kDa and 73.6 kDa, respectively, suggesting that PO in L. vannamei is an oligomer, which may have different compositions intra- and extracellularly.

Effects of phosphatidyl serine on immune response in the shrimp Litopenaeus vannamei

Abstract Phosphatidyl serine plays an important role in animal innate immunity. Given its important functions, numerous investigations have been carried out on its immunological function in many animals. However, studies of phosphatidyl serine in the white shrimp Litopenaeus vannamei, an economically important animal, are rare. In this paper, we demonstrated influences of injecting phosphatidyl serine (PS) on immune response including some parameters from pro-phenol oxidase activating system (pro-PO system) and hemocyanin-derived phenol oxidase activity (Hd-PO) along with antibacterial and bacteriolytic activities in the white shrimp Litopenaeus vannamei with different PS concentrations (5, 10 and 20 μg mL−1). The results showed that PS could affect immune response of L. vannamei significantly (P<0.05), including total hemocyte counts (THC), PO activity from hemocyte, phenol oxidase (PO) activity from plasma, hemocyanin concentration, Hd-PO activity as well as antibacterial and bacteriolytic activities in the plasma. Among the lines, 20 μg mL−1 PS had the strongest effect on the above parameters, whereas 5 μg mL−1 had the least effect. The experimental results indicated that PS was able to activate exocytosis of pro-PO and formation of Hd-PO in white shrimp after injection, further regulating the immune process reflected by variation of antibacterial and bacteriolytic activities in a certain way.

A novel pattern recognition protein of the Chinese oak silkmoth, Antheraea pernyi, is involved in the pro-PO activating system

In this paper, we firstly reported a C-type lectin cDNA clone of 1029 bps from the larvae of A. Pernyi (Ap-CTL) using PCR and RACE techniques. The full-length cDNA contains an open reading frame encoding 308 amino acid residues which has two different carbohydrate-recognition domains (CRDs) arranged in tandem. To investigate the biological activities in the innate immunity, recombinant Ap-CTL was expressed in E. coli with a 6-histidine at the amino-terminus (Ap-rCTL). Besides acted as a broad-spectrum recognition protein binding to a wide range of PAMPs and microorganisms, Ap-rCTL also had the ability to recognize and trigger the agglutination of bacteria and fungi. In the proPO activation assay, Ap-rCTL specifically restored the PO activity of hemolymph blocked by anti-Ap-rCTL antibody in the presence of different PAMPs or microorganisms. In summary, Ap-rCTL plays an important role in insect innate immunity as an pattern recognition protein. [BMB Reports 2013; 46(7): 358-363]

A serine proteinase PmClipSP2 contributes to prophenoloxidase system and plays a protective role in shrimp defense by scavenging lipopolysaccharide

Serine proteinases (SPs) participate in various biological processes and play vital role in immunity. In this study, we investigated the function of PmClipSP2 from shrimp Penaeus monodon in defense against bacterial infection. PmClipSP2 was identified as a clip-domain SP and its mRNA increased in response to infection with Vibrio harveyi. PmClipSP2-knockdown shrimp displayed a significantly reduced phenoloxidase (PO) activity and increased susceptibility to V. harveyi infection. Injection of LPS and/or β-1,3-glucan induced a dose-dependent mortality and a significant decrease in the number of total hemocytes, with clear morphological changes in the hemocyte surface, of the PmClipSP2-knockdown shrimp. Recombinant PmClipSP2 was shown to bind to LPS and β-1,3-glucan and to activate PO activity. These results reveal that PmClipSP2 acts as a pattern-recognition protein, binding to microbial polysaccharides and likely activating the proPO system, whilst it may play an essential role in the hemocyte homeostasis by scavenging LPS and neutralizing its toxicity.

Purification and characterization of a 1,3-β-D-glucan recognition protein from Antheraea pernyi larve that is regulated after a specific immune challenge

Pattern recognition receptors are known to participate in the activation of Prophenoloxidase system. In this study, a 1,3-β-D-glucan recognition protein was detected for the first time in Antheraea pernyi larvae (Ap-βGRP). Ap-βGRP was purified to 99.9% homogeneity from the hemolymph using traditional chromatographic methods. Ap-βGRP specifically bind 1,3-β-D-glucan and yeast, but not E. coli or M. luteus. The 1,3-β-D-glucan dependent phenoloxidase (PO) activity of the hemolymph inhibited by anti-Ap-βGRP antibody could be recovered by addition of purified Ap-βGRP. These results demonstrate that Ap-βGRP acts as a biosensor of 1,3-β-Dglucan to trigger the Prophenoloxidase system. A trace mount of 1,3-β-D-glucan or Ap-βGRP alone was unable to trigger the proPO system, but they both did. Ap-βGRP was specifically degraded following the activation of proPO with 1,3-β-Dglucan. These results indicate the variation in the amount of Ap-βGRP after specific immune challenge in A. pernyi hemolymph is an important regulation mechanism to immune response. [BMB Reports 2013; 46(5): 264-269]

PmSERPIN3 from black tiger shrimp Penaeus monodon is capable of controlling the proPO system

Serpin or serine proteinase inhibitor is a family of protease inhibitors that are involved in controlling the proteolytic cascade in various biological processes. In shrimp, several serpins have been identified but only a few have been characterized. Herein, the PmSERPIN3 gene identified from Penaeus monodon EST database was studied. By using the 5′- and 3′-Rapid Amplification of cDNA Ends (RACE) techniques, the full-length of PmSERPIN3 cDNA was obtained. The cDNA contained an open reading frame of 1233bp encoding for 410 amino acid residue protein. Genome sequence analysis revealed that the PmSERPIN3 was an intronless gene. RT-PCR analysis revealed that it was constitutively expressed in all developmental stages, all shrimp tissues tested, and upon pathogen infections. The recombinant mature PmSERPIN3 protein (rPmSERPIN3) produced in Escherichia coli exhibited inhibitory activity against subtilisin. The rPmSERPIN3 also inhibited the shrimp prophenoloxidase system activation in vitro. Injecting the rPmSERPIN3 along with Vibrio harveyi into the shrimp decreased the clearance rate of bacteria in the hemolymph. Potentially, the PmSERPIN3 functions as a regulator of the proPO activating system.

Transcript and protein profiling analysis of the Destruxin A-induced response in larvae of Plutella xylostella.

BackgroundDestruxins (dtxs) are the mycotoxin produced by certain entomopathogenic fungi, such as Metarhizium anisopliae, Aschersonia sp, Alternaria brassicae and Ophiosphaerella herpotrichae. It can affect a wide variety of biological processes in insects, including innate immune, Ca2+ channel in cells, and apoptosis in a dose-dependent manner. Dtxs have been used as biological control agent for a long time, however, their molecular mechanism of action is still unknown.Principal FindingsIn this study, both digital gene expression (DGE) and two-dimensional electrophoresis (2-DE) approaches were adopted to examine the effects of dtx A on Plutella xyllostella (L.) larvae. By using DGE and 2-DE analyses, 1584 genes and 42 protein points were identified as being up- or down regulated at least 2-fold in response to dtx A. Firstly, injection of dtx A to larvae accelerated the increase of peptidoglycan recognition protein (PGRP), which could activate the Toll signal pathway inducing production of antibacterial substances such as cecropin and gloverin. Dtx A also stimulated prophenoloxidase (proPO) system which plays an important role in innate immunity and leads to melanization of external organisms. Secondly, dtx A suppressed the expression of genes related to the Toll pathway, and induced expression of serine proteinase inhibitors (serpins), especially the serpin 2 that blocked process of the proPO system. Finally, other physiological process like xenobiotics detoxification, apoptosis, calcium signaling pathway and insect hormone biosynthesis, were also mediated in response to dtx A toxicity.ConclusionsTranscript and protein profiling analyses will provide an insight into the potential molecular mechanism of action in P. xylostella larvae in response to dtx A.

Feeding truncated heat shock protein 70s protect Artemia franciscana against virulent Vibrio campbellii challenge

The 70 kDa heat shock proteins (Hsp70s) are highly conserved in evolution, leading to striking similarities in structure and composition between eukaryotic Hsp70s and their homologs in prokaryotes. The eukaryotic Hsp70 like the DnaK (Escherichia coli equivalent Hsp70) protein, consist of three functionally distinct domains: an N-terminal 44-kDa ATPase portion, an 18-kDa peptide-binding domain and a C-terminal 10-kDa fragment. Previously, the amino acid sequence of eukaryotic (the brine shrimp Artemia franciscana) Hsp70 and DnaK proteins were shown to share a high degree of homology, particularly in the peptide-binding domain (59.6%, the putative innate immunity-activating portion) compared to the N-terminal ATPase (48.8%) and the C-terminal lid domains (19.4%). Next to this remarkable conservation, these proteins have been shown to generate protective immunity in Artemia against pathogenic Vibrio campbellii. This study, aimed to unravel the Vibrio-protective domain of Hsp70s in vivo, demonstrated that gnotobiotically cultured Artemia fed with recombinant C-terminal fragment (containing the conserved peptide binding domain) of Artemia Hsp70 or DnaK protein were well protected against subsequent Vibrio challenge. In addition, the prophenoloxidase (proPO) system, at both mRNA and protein activity levels, was also markedly induced by these truncated proteins, suggesting epitope(s) responsible for priming the proPO system and presumably other immune-related genes, consequently boosting Artemia survival upon challenge with V. campbellii, might be located within this conserved region of the peptide binding domain.