proPO System Research Articles

Variation in immune defence among populations of Gammarus pulex (Crustacea: Amphipoda)

Despite intensive studies in ecological immunology, few have investigated variation in immune defence among natural populations; in particular, there is a lack of knowledge of the sources of spatial variability in immune defence in the wild. Here we documented variation among twelve populations of the freshwater crustacean Gammarus pulex in the activity of the prophenoloxidase (ProPO) system, which is an important component of invertebrate immunity. We then tested for trade-offs between investment in immune defence and fitness-related traits such as survival and fecundity, as well as for environmental causes of variability (water temperature and conductivity, parasite prevalence). Levels of immune defence differed among populations, with environment partly explaining this population effect, as immune activities were negatively related to water conductivity and acanthocephalan parasite prevalence. There was a strong variation among populations for the maintenance of the ProPO system, while variation in its use was relatively weak. Such a pattern could be partly explained by the relative costs associated with the maintenance and/or the use of the ProPO system. Investment in the ProPO system was negatively correlated to survival, whereas it was positively related to female fecundity and resource storage. However, variation in immunity did not predict resistance to bacterial infection among populations, suggesting that measuring the activity of the ProPO system might not be sufficient to estimate immunocompetence at the population level. These results suggest that investment in immune function is a variable trait, which might be locally optimized as a result of both life history trade-offs and environmental conditions, highlighting the need to combine them in a common framework.

Effects of emersion and re-immersion on physiological and immunological variables in creel-caught and trawled Norway lobsterNephrops norvegicus

Immune defence in creel-caught and trawled Nephrops norvegicus was investigated to assess a possible relationship between phenoloxidase (PO) activation and the total haemocyte count (THC). Capture, capture method and emersion evoked physiological and immunological responses that may have implications for the ability of N. norvegicus to survive the effects of such stressors. Haemolymph THC was always negatively related to PO activity in the trawled samples, suggesting a decreased level of the plasma serine proteinase inhibitors which reportedly regulate the ProPO system (Le Moullac et al. 1998; Fish shellfish Immunol 8:621-629). In contrast, creel-caught samples showed increased levels of both PO and THC (cf. control N. norvegicus), after a 12 h emersion period. Trawling and emersion evoked progressive and significant increases (p < 0.05) in the mean levels of haemolymph L-lactate, glucose and total ammonia. The evidence of overt activity and measured haemolymph parameters suggest that creel fishing yields N. norvegicus that are more likely to survive post-harvest treatments than those that are trawled.

Molecular Control of Phenoloxidase-induced Melanin Synthesis in an Insect

The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Spätzle-processing enzyme is a key enzyme in the production of cleaved Spätzle from pro-Spätzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Spätzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses.

Comparison of antiviral efficiency of immune responses in shrimp

The antiviral effectiveness of three major immune responses including phagocytosis, apoptosis and proPO system in the shrimp Marsupenaeus japonicus was characterized. It was found that the suppression of apoptosis and phagocytosis in vivo by their corresponding inhibitors resulted in the increase of white spot syndrome virus (WSSV) copies and shrimp mortality, whereas the inhibition of phenol oxidase generated the least influence on WSSV infection and shrimp mortality. These results suggest that apoptosis and phagocytosis were the essential immune responses to protect shrimp from virus infection, while the phenol-oxidase-dependent proPO system plays a comparatively minor role in antiviral defense of shrimp.

Immune depression induced by acanthocephalan parasites in their intermediate crustacean host: Consequences for the risk of super-infection and links with host behavioural manipulation

Parasite survival in hosts mainly depends on the capacity to circumvent the host immune response. Acanthocephalan infections in gammarids are linked with decreased activity of the prophenoloxidase (ProPO) system, suggesting an active immunosuppression process. Nevertheless, experimental evidence for this hypothesis is lacking: whether these parasites affect several immune pathways is unknown and the consequences of such immune change have not been investigated. In particular, the consequences for other pathogens are not known; neither are the links with other parasite-induced manipulations of the host. Firstly, using experimental infections of Pomphorhynchus laevis we confirmed that the lower immune activity in parasitised Gammarus pulex is induced by the parasite infection. Second, using natural infections of three different parasites, P. laevis, Pomphorhynchus tereticollis and Polymorphus minutus, we showed that acanthocephalan infection was associated with reduction of the activity of the ProPO system and the haemocyte concentration (two major parameters of crustacean immunity) suggesting that immune depression is a phenomenon affecting several immunological activities. This was confirmed by the fact that acanthocephalan infection (whatever the parasite species) was linked to a lower efficiency to eliminate a bacterial infection. The result suggests a cost of parasite immune depression. Finally, acanthocephalans are also known to induce behavioural alterations in the intermediate host which favour their transmission to definitive hosts. We did not find any correlation between behavioural and immunological alterations in both experimentally and naturally-infected gammarids. Overall, this study suggests that whilst immune depression might be beneficial to acanthocephalan survival within the intermediate gammarid host, it might also be costly if it increases host mortality to additional infections before transmission of the parasite.

Identification of a C-type lectin from the bay scallop Argopecten irradians

C-type lectins are Ca(2+) dependent carbohydrate-recognition proteins that play crucial roles in the invertebrate innate immunity, such as nonself recognition, activation of proPO system, antibacterial activity, promotion of phagocytosis and nodule formation. In this study, a novel C-type lectin of bay scallops Argopecten irradians (Ai Lec) was identified using expressed sequence tag (EST) and RACE techniques. The Ai Lec cDNA encoded a polypeptide of 171 amino acids with a putative signal peptide of 21 amino acid residues and a mature protein of 150 amino acids. The deduced amino acid sequence of Ai Lec was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 131 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. The expression of Ai Lec transcript was dominantly detected in the hepatopancreas and slightly detected in the haemocytes of normal scallops. 6 h after Vibrio anguillarum-challenge and 8 h after Micrococcus luteus-challenge, the temporal expression of Ai Lec mRNA in hemocytes was increased by 4.4- and 3.6-folds, respectively. The results suggested that Ai Lec was a constitutive and inducible acute-phase protein and might be involved in immune response to Gram-negative and Gram-positive microbial infection in bay scallop A. irradians.

The prophenoloxidase system is activated during the tunic inflammatory reaction of Ciona intestinalis

Phenoloxidase (PO) activity was examined in the tunic tissue of Ciona intestinalis following lipopolysaccharide (LPS) intratunic injection. Tunic homogenate supernatant (THS), assayed with the Dopa-MBTH reaction, displayed Ca(2+)-independent PO activity that was raised by LPS and further enhanced by proteases. Specific inhibitors (tropolone, phenylthiourea, diethylthiocarbamate) supported the specificity of the reaction. Assay with soybean trypsin inhibitor showed that, in the tunic, PO activation with trypsin was not significantly inhibited suggesting that proteases diverse from serine proteases were involved. In vivo experiments were carried out by injecting isosmotic medium or LPS, and THS was assayed for its PO activity. Analysis of variance of the time-course profiles showed that LPS was more effective in activating proPO. To disclose the PO response at the injured site, an assay with Dopa-MBTH was performed in vitro. Quinones were mainly contained in the tunic matrix enriched with inflammatory cells around the injection site. Microscopic observations and immunohistochemistry with anti-CinPO-2 antibodies showed granulocytes and unilocular refractile granulocytes containing PO, whereas few morula cells were stained. In THS zymograms (SDS-polyacrylamide gel electrophoresis), PO activity linked to 90-kDa and 120-kDa bands was observed as an effect of LPS injection, whereas the density of 170-kDa PO was weak. A third presumptive PO enzyme (CinPO-3) containing the CinPO-2 peptide was identified in the recent Ciona genome version. Presumably, LPS stimulated the production and dimerization (120 kDa) of CinPO-3 (66 kDa). Thus, the activated proPO system includes several POs that are distinguishable by size and that are contained and presumably released by tunic inflammatory cells and hemocytes of the pharynx bars.

The effect of different acclimation temperatures on the prophenoloxidase system and other defence parameters in Litopenaeus vannamei

Water temperature changes (higher and lower than 24 °C) were shown to have a significant effect on dopamine (DA) concentration, haemocyte count and the proPO system in the white shrimp Litopenaeus vannamei. No significant difference in any of the parameters was observed in the control group. DA concentration in haemolymph in the experimental groups increased to a peak value at 0.5 days; meanwhile serine protease (SP) activity and proteinase inhibitor (PI) activity decreased. Total haemocyte count (THC), differential haemocyte count (DHC) and PO activity were lowest at 1 day. All defence parameters became stable after 1–3 days, while the total haemocyte and large granular cell count stabilized after 6 days. After these stabilized, there was no significant difference in DA concentration and PI activity between the control and experimental groups, as was the case for the THC, DHC, PO and SP activities of shrimp held at higher temperatures. However these latter four parameters in the lower temperature groups were distinctly lower than the control group. α 2-Macroglobulin activity in the experimental groups increased to a peak value after 1 day compared with the control and then stabilized after 6 days when the activity levels in higher temperature groups were higher than the control, while the lower temperature groups had no significant difference from the control. It was therefore concluded that water temperature changes modulated the immune system of L. vannamei.

Expression of immune-related genes in larval stages of the giant tiger shrimp, Penaeus monodon

Shrimp undergo several morphologically different stages during development and therefore the expression of some immune-related genes such as prophenoloxidase ( proPO), peroxinectin ( Prx), crustin ( Crus), penaeidin ( Pen), transglutaminase ( TGase), haemocyanin ( Hc) and astakine ( Ak) were determined during larval development of the shrimp ( Penaeus monodon), i.e. nauplius 4 (N4), protozoea 1 and 3 (Z1 and 3), mysis 3 (My 3), post-larvae 3 (PL3) and also in haemocytes of juveniles. Semi-quantitative RT-PCR analysis showed that all transcripts were already present in the early larval stage of N4 but at different levels. The transcript of proPO was found to be extremely low or even absent at N4, whereas Prx, Crus, Pen, TGase, Hc and Ak were significantly expressed at all larval stages. Up to now expression of proPO and Prx has only been reported from haemocytes in crustaceans and in this study Prx also appeared to be expressed in stages which appear to lack haemocytes. Thus, this may suggest that Prx is expressed in other cells than haemocytes. It is well known among invertebrates that the proPO system plays a crucial role as an immune effector molecule against microbes. However, in this study, the transcript of proPO was low during the larval stages and hardly present at all at N4. This might indicate that the development of immune-competent haemocytes during the larval stages is not completed and as a consequence they are likely to be more susceptible to infectious diseases during these stages.

Demonstration of a true phenoloxidase activity and activation of a ProPO cascade in Pacific oyster, Crassostrea gigas (Thunberg) in vitro

The prophenoloxidase (ProPO) system is the origin of melanin production and is considered to be an innate defence mechanism in invertebrates. In different bivalve species, phenoloxidase (PO) is present in the haemolymph as an inactive form of ProPO. The present study focuses on the Pacific adult oyster, Crassostrea gigas, an economically important bivalve species along French coasts. The results indicate that many factors may inhibit the PO-like activity. These include: phenylthiourea (PTU), sodium diethylthiocarbamate (DETC), β -mercaptoethanol and tropolone, which repressed the spontaneous PO activity. The activation of PO-like activity in C. gigas acellular fraction by lipopolysaccharide (LPS) involved participation of other factors, including at least one serine protease. PO was present as proPO in the acellular fraction of haemolymph and haemocytes of C. gigas and could be activated by an exogenous protease (trypsin- N-tosyl- l-phenylalanine chloromethyl ketone) when used at 1 g L −1. Treatment of acellular fractions with other modulators/activators namely LPS (1 g L −1), zymosan (0.6 g L −1) or laminarin (0.6 g L −1) also increased PO-like activity but to a less important way. The study demonstrated the evidence of a true phenoloxidase activity in Pacific oyster, C. gigas (Thunberg). The activation of a proPO system by non-self molecules suggests the role played by PO in vivo in the internal defence mechanisms. Understanding the activation of the ProPO system could enable the evaluation of the health of oyster stocks.

WEB 2086, a platelet-activating factor antagonist, inhibits prophenoloxidase-activating system and hemocyte microaggregation reactions induced by Trypanosoma rangeli infection in Rhodnius prolixus hemolymph

The effects of the triazolodiazepine WEB 2086, a platelet-activating factor (PAF) antagonist, on hemocyte microaggregation and prophenoloxidase (proPO)-activating system in the hemolymph, hemocoelic infection and mortality in fifth-instar larvae of Rhodnius prolixus inoculated with Trypanosoma rangeli were investigated. Hemocoelic injection of short T. rangeli epimastigotes (1×10 4 parasites/insect) in R. prolixus that were previously fed with blood containing 1 μM of WEB 2086 resulted in (i) reduced hemocyte microaggregations as well as an attenuated proPO system in the hemolymph and (ii) greater parasitemia and mortality among the insects. In vitro assays using hemolymph from insects previously fed with blood containing WEB 2086 exhibited attenuated hemocyte microaggregations when T. rangeli was employed as the inducer of the reaction, and this effect was not counteracted by PAF treatment. In vitro assays using hemolymph from insects previously fed with blood, regardless of WEB 2086 presence increased the PO activity when incubated with the parasites. However, the PO activity was drastically inhibited when hemolymph from insects fed with blood, whether or not it contained WEB 2086, was incubated with fat body homogenates from insects fed with blood containing WEB 2086. The addition of PAF did not enhance the PO activity. These analyses did not reveal any PAF influence on WEB 2086 effects in the two defense reactions.

Effects of lipopolysaccharide on gene expression of antimicrobial peptides (penaeidins and crustin), serine proteinase and prophenoloxidase in haemocytes of the Pacific white shrimp, Litopenaeus vannamei

For shrimp immune defences, prophenoloxidase (proPO) activating system and antimicrobial peptides in circulating haemocytes play important roles. In the present study, the effects of lipopolysaccharide (LPS) injection on gene expression of penaeidins, crustin, serine proteinase and proPO in haemocytes were determined using real-time reverse transcription–polymerase chain reaction (PCR) in the Pacific white shrimp Litopenaeus vannamei. After injection of LPS, mRNA levels of antimicrobial peptides, penaeidin 2 (PEN2), penaeidin 3 (PEN3), penaeidin 4 (PEN4) and crustin decreased in a dose-dependent manner, while mRNA levels of serine proteinase and proPO did not change significantly. In a time-course experiment, injection of LPS caused significant depression in mRNA levels of PEN2, PEN3, PEN4 and crustin at 4 h post-injection, and the depressed mRNA levels returned to initial levels by 72 h post-injection. On the other hand, mRNA levels of serine proteinase and proPO did not show a significant change after injection. These results suggest that the antimicrobial peptide system responds to LPS injection at a gene expression level while the proPO system does not respond at a gene expression level.

A Synthetic Peptidoglycan Fragment as a Competitive Inhibitor of the Melanization Cascade

Melanin synthesis is essential for defense and development but must be tightly controlled because systemic hyperactivation of the prophenoloxidase and excessive melanin synthesis are deleterious to the hosts. The melanization cascade of the arthropods can be activated by bacterial lysine-peptidoglycan (PGN), diaminopimelic acid (DAP)-PGN, or fungal beta-1,3-glucan. The molecular mechanism of how DAP- or Lys-PGN induces melanin synthesis and which molecules are involved in distinguishing these PGNs are not known. The identification of PGN derivatives that can work as inhibitors of the melanization cascade and the characterization of PGN recognition molecules will provide important information to clarify how the melanization is regulated and controlled. Here, we report that a novel synthetic Lys-PGN fragment ((GlcNAc-MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala)2, T-4P2) functions as a competitive inhibitor of the natural PGN-induced melanization reaction. By using a T-4P2-coupled column, we purified the Tenebrio molitor PGN recognition protein (Tm-PGRP) without causing activation of the prophenoloxidase. The purified Tm-PGRP recognized both Lys- and DAP-PGN. In vitro reconstitution experiments showed that Tm-PGRP functions as a common recognition molecule of Lys- and DAP-PGN-dependent melanization cascades.

White spot syndrome virus (WSSV) interaction with crayfish haemocytes

WSSV particles were detected in separated granular cells (GCs) and semigranular cells (SGCs) by in situ hybridisation from WSSV-infected crayfish and the prevalence of WSSV-infected GCs was 5%, whereas it was 22% in SGCs. This indicates that SGCs are more susceptible to WSSV and that this virus replicated more rapidly in SGCs than in GCs and as a result the number of SGCs gradually decreased from the blood circulation. The effect of haemocyte lysate supernatant (HLS), containing the degranulation factor (peroxinectin), phorbol 12-myristate 13-acetate (PMA), the Ca 2+ ionophore A23187 on GCs from WSSV-infected and sham-injected crayfish was studied. The results showed that the percentage of degranulated GCs of WSSV-infected crayfish treated with HLS or PMA was significantly lower than that in the control, whereas no significant difference was observed when treated with the Ca 2+ ionophore. It was previously shown that peroxinectin and PMA have a degranulation effect via intracellular signalling involving protein kinase C (PKC), whereas the Ca 2+ ionophore uses an alternative pathway. HLS treatment of GCs and SGCs from WSSV-infected crayfish results in three different morphological types: non-spread, spread and degranulated cells. The non-spread cell group from both GCs and SGCs after treatment with HLS had more WSSV positive cells than degranulated cells, when detected by in situ hybridisation. Taken together, it is reasonable to speculate that the PKC pathway might be affected during WSSV infection. Another interesting phenomenon was that GCs from non-infected crayfish exhibited melanisation, when incubated in L-15 medium, while no melanisation was found in GCs of WSSV-infected crayfish. However, the phenoloxidase activities of both sham- and WSSV-injected crayfish in HLS were the same as well as proPO expression as detected by RT-PCR. This suggests that the WSSV inhibits the proPO system upstream of phenoloxidase or simply consumes the native substrate for the enzyme so that no activity is shown. The percentage of apoptotic haemocytes in WSSV-infected crayfish was very low, but it was significantly higher than that in the sham-injected crayfish on day 3 or 5 post-infection. The TEM observation in haematopoietic cells (hpt cells) suggests that WSSV infect specific cell types in haematopoietic tissue and non-granular hpt cells seem more favourable to WSSV infection.

Cloning the prophenoloxidase cDNA and monitoring the expression of proPO mRNA in prawns ( Macrobrachium rosenbergii) stimulated in vivo by CpG oligodeoxynucleotides

The prophenoloxidase (proPO) system forms the basis of the non-specific defence system in crustaceans. The aim of this study was to develop an RT-PCR procedure to determine proPO gene expression. We used several degenerate primers designed from the conserved regions in amino acid sequences of proPOs from other species to clone the possible cDNA(s) from the haemocytes of the prawn Macrobrachium rosenbergii. One DNA fragment, 2016 bp long, was cloned by a combination of reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (3′/5′-RACE). This fragment could encode a putative polypeptide with 671 amino acids. Further analysis showed that this putative polypeptide contained six histidine residues and a thiol ester-like motif (GCGWPRHM) just like the structural features of proPOs from the shrimp Penaeus monodon. A partial fragment of the sequence with 934 bp containing three histidine residues and the thiol ester-like motif was used as a target to monitor the activation of the proPO gene by the semi-quantitative RT-PCR analysis. The result showed that the highest level of proPO mRNA was detected at 1 h after in vivo injection with 5 μg of CpG oligodeoxynucleotide 2006 per prawn; in the same experiment, the highest PO activity was detected at 6 h after injection. In the control, a continuous and slow elevation of PO activity was observed during the experimental period, but such elevation of proPO mRNA was not observed. From our previous study and the time course of gene expression of proPO and enzymatic activity of PO in this study, it can be concluded that the enhancement effect was through its transcriptional level, its translational level and then its post-translational level sequentially. These results suggest that, both for reliability, sensitivity and for the timing of sampling, the change in proPO mRNA is more useful than the PO activity in monitoring the activation of the proPO non-specific defence system of prawns after treatment with stimulants.

Molecular cloning and characterisation of prophenoloxidase from haemocytes of the white shrimp, Litopenaeus vannamei

cDNA encoding prophenoloxidase (proPO) of the white shrimp Litopenaeus vannamei was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA using oligonucleotide primers based on the proPO sequence of tiger shrimp Penaeus monodon, freshwater crayfish Pacifastacus leniusculus, green tiger shrimp Penaeus semisulcatus (accession no.: AF521949) and kuruma shrimp Marsupenaeus japonicus (accession no.: AB0733223). proPO of L. vannamei was constitutively expressed. The 2471-bp cDNA contained an open reading frame (ORF) of 2058 bp, a 96-bp 5′-untranslated region, and a 317-bp 3′-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (686 amino acids) was 78.1 kDa with an estimated pI of 6.02. It contained putative copper binding sites, a complement-like motif (GCGWPQHM), a proteolytic activation site, and a conserved C-terminal region common to all known proPOs. However, no signal peptide sequence was detected in white shrimp proPO. Comparison of amino acid sequences showed that white shrimp proPO is more closely related to the proPO of another penaeid than to that of a freshwater crayfish. White shrimp proPO mRNA was synthesized in haemocytes and not in the hepatopancreas or muscle. The activation responses of the proPO of the white shrimp to an exogenous protease (trypsin), a detergent (sodium dodecyl sulphate), and algal and microbial cell wall components (laminarin, sodium alginate, zymosan, and lipopolysaccharide), and its susceptibility to protease inhibitors in vitro resemble the proPO activation system of other crustaceans. These facts suggest that the proPO system in haemocytes of the white shrimp Litopenaeus vannamei serves an important function in non-self recognition and host immune reactions.

Signal transduction of the prophenoloxidase activating system of prawn haemocytes triggered by CpG oligodeoxynucleotides

Intracellular phenoloxidase (PO) activity in haemocyte lysate supernatant (HLS) of giant freshwater prawn ( Macrobrachium rosenbergii) was shown to be enhanced by CpG oligodeoxynucleotide (ODN) 2006, but not by so-ODN13. When haemocytes were treated in vitro with 50 μg/ml of ODN2006 for 30 min, the increases in both intra- and extracellular stimulated PO activity (PO S) and extracellular total PO activity (PO T) and the reduction of PO T suggest that the PO activity of haemocytes is enhanced by ODN2006 stimulation, but new prophenoloxidase (proPO) is not synthesised. In an attempt to determine which signal transduction pathway is involved in the activation of the proPO system, haemocytes were separately treated with activators or inhibitors of specific signalling components. The results show that there was an increase in both intra- and extracellular PO T of haemocytes treated with sodium fluoride (a G-protein activator); the addition of phosphokinase A (PKA)-activating 8-bromo-cAMP to haemocytes only increased intracellular PO T, and the addition of either phorbol-12-myristate-13-acetate (PMA; a phosphokinase C (PKC) activator) or caffeine (a phosphodiesterase inhibitor) only increased extracellular PO T. When PMA-stimulated haemocytes were treated with chelerythrine (a PKC inhibitor), the induced extracellular PO T was significantly reduced. Furthermore, the study of ODN2006-stimulated haemocytes treated with chelerythrine or palmitoyl- dl-carnitine (a PKC inhibitor) showed that the enhancement effects of ODN2006 on the intra- and extracellular PO S and extracellular PO T were significantly decreased. ODN-stimulated haemocytes treated with genistein (an inhibitor of protein tyrosine kinase) showed a further increase in extracellular PO T, but the other PO activities remained the same as those of the ODN-stimulated group. These results suggest that the activation of the proPO system of prawn haemocytes, including degranulation and PO activity, is induced by ODN2006 via a PKC-activating signalling pathway, but negatively regulated via the tyrosine kinase pathway.

The prophenoloxidase-activating system in invertebrates.

A major innate defense system in invertebrates is the melanization of pathogens and damaged tissues. This important process is controlled by the enzyme phenoloxidase (PO) that in turn is regulated in a highly elaborate manner for avoiding unnecessary production of highly toxic and reactive compounds. Recent progress, especially in arthropods, in the elucidation of mechanisms controlling the activation of zymogenic proPO into active PO by a cascade of serine proteinases and other factors is reviewed. The proPO-activating system (proPO system) is triggered by the presence of minute amounts of compounds of microbial origins, such as beta-1,3-glucans, lipopolysaccharides, and peptidoglycans, which ensures that the system will become active in the presence of potential pathogens. The presence of specific proteinase inhibitors prevents superfluous activation. Concomitant with proPO activation, many other immune reactions will be produced, such as the generation of factors with anti-microbial, cytotoxic, opsonic, or encapsulation-promoting activities.

The role of Steinernema feltiae body-surface lipids in host-parasite immunological interactions.

Interactions between entomopathogenic nematodes (Steinernema feltiae) and insect host (Galleria mellonella) immune system were investigated. We focused on the immunosuppressive properties of the parasite cuticle and on its interaction with hemolymph humoral components. Effects of parasite cuticle against host proPO system enzymatic cascade were evaluated a short time after infection. The presence of parasite cuticles decreased both normal and LPS-elicited proPO system activity, suggesting that S. feltiae body surface plays a key role in the early parasitation phase, probably interfering with host proPO activation pathways. The data obtained showed that cuticle lipidic compounds are able to interact with host humoral components, removing them from the hemolymph. The depletion of these molecules, arbitrarily named host-interacting proteins (HIPs), seems to be responsible of the drastic decrease in proPO system activity. Moreover, hemolymph HIPs showed LPS-binding properties and parasite cuticle cross-reacted with anti-LPS antibodies. Finally, we also assessed the involvement of parasite body surface on immunoevasion strategies of S. feltiae against host cell-mediated encapsulation processes. We conclude that S. feltiae body surface is responsible for short-term immunosuppression and immunoevasion processes; since it is able to sequester host hemolymph compounds involved in proPO system activation and this process could be responsible for a molecular disguise strategy against cellular encapsulation.

Peptidoglycan Recognition Proteins Involved in 1,3-β-D-Glucan-dependent Prophenoloxidase Activation System of Insect

The prophenoloxidase (proPO) cascade is a major innate immune response in invertebrates, which is triggered into its active form by elicitors, such as lipopolysaccharide, peptidoglycan, and 1,3-beta-D-glucan. A key question of the proPO system is how pattern recognition proteins recognize pathogenic microbes and subsequently activate the system. To investigate the biological function of 1,3-beta-D-glucan pattern recognition protein in the proPO cascade system, we isolated eight different 1,3-beta-D-glucan-binding proteins from the hemolymph of large beetle (Holotrichia diomphalia) larvae by using 1,3-beta-D-glucan immobilized column. Among them, a 20- and 17-kDa protein (referred to as Hd-PGRP-1 and Hd-PGRP-2) show high sequence identity with the short forms of peptidoglycan recognition proteins (PGRPs-S) from human and Drosophila melanogaster. To be able to characterize the biochemical properties of these two proteins, we expressed them in Drosophila S2 cells. Hd-PGRP-1 and Hd-PGRP-2 were found to specifically bind both 1,3-beta-D-glucan and peptidoglycan. By BIAcore analysis, the minimal 1,3-beta-D-glucan structure required for binding to Hd-PGRP-1 was found to be laminaritetraose. Hd-PGRP-1 increased serine protease activity upon binding to 1,3-beta-D-glucan and subsequently induced the phenoloxidase activity in the presence of both 1,3-beta-D-glucan and Ca(2+), but no phenoloxidase activity was elicited under the same conditions in the presence of peptidoglycan and Ca(2+). These results demonstrate that Hd-PGRP-1 can serve as a receptor for 1,3-beta-D-glucan in the insect proPO activation system.