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), beta-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 gL(-1). Treatment of acellular fractions with other modulators/activators namely LPS (1 gL(-1)), zymosan (0.6 gL(-1)) or laminarin (0.6 gL(-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.     

注射亲水性硅珠对棉铃虫血浆酚氧化酶活性的影响

昆虫通过细胞免疫和体液免疫的协同作用对入侵的异物做出防御反应。在不同时间向棉铃虫体内注射亲水性硅珠后,测定了血浆中酚氧化酶（PO）的活性,同时研究了不同抑制剂和激活剂对注射硅珠后的酚氧化酶活性的影响。结果表明,注射亲水性硅珠后,棉铃虫血浆中酚氧化酶的活性明显升高。分别以牛胰蛋白酶和昆布多糖作为酚氧化酶原（proPO）的激活剂,发现两者都可激活注射硅珠后血浆中的proPO。以牛胰蛋白酶激活时,随着注射硅珠后时间的延长,PO活性逐渐增高;而用昆布多糖激活后PO活性也明显升高,但注射硅珠后不同时间proPO被昆布多糖激活的情况基本相似。这些结果表明,在异物入侵后酚氧化酶原有很大程度的积累,并能被激活,协同细胞免疫抵御异物入侵。P-NPGB和PTU几乎能完全抑制酚氧化酶的活性。     

褐飞虱和桃蚜粗提液中酚氧化酶原激活系统的激活特点

采用研磨法制备褐飞虱Nilaparvata lugens (Stål)和桃蚜Myzus persicae (Sulzer)粗提液,用于研究几种化学因子对粗提液中酚氧化酶原激活系统(proPO-AS)的激活特点。结果表明, 在0.1～100 mmol/L的Ca²⁺浓度范围内,虱液和蚜液的酚氧化酶(PO)活性随Ca²⁺浓度升高而增强,并在30 mmol/L处PO活性达到最高,在此限之上PO活性反而下降。当昆布多糖浓度从10^-4 mg/mL升至10 mg/mL时,虱液和蚜液的PO活性也随之升高,但再提高昆布多糖浓度却未见PO活性的峰顶出现。在6种葡聚糖对PO的激活作用中,昆布多糖和酵母聚糖能显著增强PO活性,但curdlan对虱液和蚜液的PO无明显激活作用,而甘露聚糖、右旋糖苷和纤维素则降低PO活性。这些结果显示β-1,3-葡聚糖能有效激活proPO-AS。Ca²⁺和丝氨酸蛋白酶抑制剂PMSF加样顺序的不同也会影响PO活性。     

Ontogenetic variations of hydrolytic enzymes in the Pacific oyster Crassostrea gigas

Occurrence and level of hydrolytic enzymatic activity (proteases, glycosidases, phosphatases, lipases, and esterases) were studied in oocytes, larvae, juveniles, and adult haemolymph of the Pacific oyster Crassostrea gigas. Samples were obtained as oocyte lysate supernatant, larval homogenate supernatant, juvenile homogenate supernatant, haemocyte lysate supernatant, and plasma. The presence of enzymes was demonstrated by colorimetric and lysoplate assay techniques. Between stages, significant differences in enzymatic activity determined by the colorimetric technique were found. Higher levels of enzymatic activity were found in the adult stage. Lysozyme-like activity was not found in oocytes, but was present in larvae, juveniles, and adults. In larvae, the highest lysozyme-like activity was in 3-d larvae. Juveniles had a 48-fold higher level of lysozyme-like activity, compared with 20-h larvae and was six-fold higher compared with 3-d larvae. In adults, lysozyme-like activity had a five-fold higher level in haemocyte lysate supernatant compared with plasma and was 98-fold higher compared with 20-h larvae. As determined with the API ZYM kit, 19 hydrolytic enzymatic activities were present, in oocytes, larvae, juveniles, and adult haemolymph of C. gigas. The presence of important lysozyme-like activity was confirmed from trochophora larvae (20 h) to adult stages.     

Differential tissue distribution and specificity of phenoloxidases from the Pacific oyster Crassostrea gigas

Phenoloxidases (POs) play a key role in melanin production, are involved in invertebrate immune mechanisms, and have been detected in different bivalves. Recently, we identified catecholase- and laccase-like PO activities in plasma and haemocyte lysate supernatant (HLS) of the Pacific oyster Crassostrea gigas. To go further in our investigations, the aims of this study were (i) to determine the tissue distribution of PO activities in C. gigas, and (ii) to identify and characterise the different sub-classes of POs (i.e. tyrosinase, catecholase and/or laccase) involved in these oxido-reductase activities. With dopamine and p-phenylenediamine (PPD) but not with l-tyrosine used as substrates, PO-activities were detected by spectrophotometry in the gills, digestive gland, mantle, and muscle. These results suggest the presence of catecholase and laccase but not of tyrosinase activities in oyster tissues. The highest activity was recovered in the digestive gland. PO-like activities were all inhibited by 1-phenyl-2-thiourea (PTU) and by the specific laccase inhibitor, cethyltrimethylammonium bromide (CTAB). With dopamine as substrate, the catecholase inhibitor 4-hexylresorcinol (4-HR) only inhibited PO in the muscle. SDS-PAGE zymographic assays with dopamine and PPD elicited a unique ~40kDa protein band in the muscle. In the other tissues, laccase-like activities could be related to ~10kDa and/or ~200kDa protein bands. The ~10kDa protein band was also detected in plasma and HLS, confirming the presence of a laccase in the later compartments, and probably in most of the tissues of C. gigas. This is the first time to our knowledge that a ~10kDa protein band is associated to a laccase-like activity in a mollusc species, contributing to the characterisation of phenoloxidase activities in marine bivalves.     

First evidence of a potential antibacterial activity involving a laccase-type enzyme of the phenoloxidase system in Pacific oyster Crassostrea gigas haemocytes

Phenoloxidases (POs) are a group of copper proteins including tyrosinase, catecholase and laccase. In several insects and crustaceans, antibacterial substances are produced through the PO cascade, participating in the direct killing of invading microorganisms. However, although POs are widely recognised as an integral part of the invertebrate immune defence system, experimental evidence is lacking that these properties are conserved in molluscs, and more particularly in the Pacific oyster Crassostrea gigas. In the present study, Vibrio splendidus LGP32 and Vibrio aestuarianus 02/041 growths were affected, after being treated with C. gigas haemocyte lysate supernatant (HLS), and either a common substrate of POs, l-3,4-dihydroxyphenylalanine (L-DOPA), to detect catecholase-type PO activity, or a specific substrate of laccase, p-phenylenediamine (PPD), to detect laccase-type PO activity. Interestingly, a higher bacterial growth inhibition was observed in the presence of PPD than in the presence of L-DOPA. These effects were suppressed when the specific PO inhibitor, phenylthiourea (PTU), was added to the medium. Results of the present study suggest, for the first time in a mollusc species, that antibacterial activities of HLS from C. gigas potentially involve POs, and more particularly laccase catalysed reactions.     

几种化学因子对三种赤眼蜂离体酚氧化酶活性的影响

为比较不同赤眼蜂酚氧化酶（PO）活性的大小, 明确赤眼蜂种间免疫防御能力强弱, 本研究通过研磨、离心提取松毛虫赤眼蜂Trichogramma dendrolimi、螟黄赤眼蜂T. chilonis、玉米螟赤眼蜂T. ostriniae匀浆液, 首次测定了几种化学因子（Ca²⁺、昆布多糖、纤维素、右旋糖酐、脂多糖和丝氨酸蛋白酶抑制剂）对3种供试赤眼蜂匀浆液中酚氧化酶原激活系统（proPO-AS）的激活程度。结果表明: 玉米螟赤眼蜂匀浆液PO活性最高, 螟黄赤眼蜂次之, 松毛虫赤眼蜂最低。Ca²⁺是激活供试赤眼蜂proPO的必需因子, 且在0.03 mol/L浓度下激活作用最强; 昆布多糖和脂多糖也能有效激活proPO, 0.01及0.05 mg/mL昆布多糖能强烈激活PO活性; 纤维素和右旋糖酐对PO活性存在显著抑制作用。结果证明了赤眼蜂种间存在明显的免疫能力差异。本研究建立了小型昆虫proPO-AS研究体系, 为研究Wolbachia-小型昆虫的免疫互作体系奠定了基础。     

Recognition of microbial molecular patterns and stimulation of prophenoloxidase activation by a β-1,3-glucanase-related protein in Manduca sexta larval plasma

Detection of pathogenic invaders is the essential first step of a successful defense response in multicellular organisms. In this study, we have identified a new member of the ??-1,3-glucanase-related protein superfamily from the tobacco hornworm Manduca sexta. This protein, designated microbe binding protein (MBP), is 61% identical in sequence to Bombyx mori Gram-negative bacteria binding protein, but only 34-36% identical to M. sexta ??-1,3-glucan recognition protein-1 and 2. Its mRNA levels were strongly up-regulated in hemocytes and fat body of immune challenged larvae, along with an increase in concentration of the plasma protein. We expressed M. sexta MBP in a baculovirus-insect cell system. The purified protein associated with intact bacteria and fungi. It specifically bound to lipoteichoic acid, lipopolysaccharide, diaminopimelic acid-type peptidoglycans (DAP-PGs) from Escherichia coli and Bacillus subtilis, but less so to laminarin or Lys-type PG from Staphylococcus aureus. The complex binding pattern was influenced by other plasma factors and additional microbial surface molecules. After different amounts of MBP had been incubated with larval plasma on ice, a concentration-dependent increase in phenoloxidase (PO) activity occurred in the absence of any microbial elicitor. The activity increase was also observed in the mixture of plasma and a bacterial or fungal cell wall component. The prophenoloxidase (proPO) activation became more prominent when DAP-PGs, Micrococcus luteus Lys-PG, or lipoteichoic acid was included in the mixture of MBP and plasma. Statistic analysis suggested that a synergistic enhancement of proPO activation was caused by an interaction between MBP and these elicitors, but not S. aureus Lys-PG, lipopolysaccharide, curdlan, or laminarin. These data indicate that M. sexta MBP is a component of the surveillance mechanism and, by working together with other pattern recognition molecules and serine proteinases, triggers the proPO activation system.     

根虫瘟霉不同菌株对小菜蛾幼虫血淋巴酚氧化酶原的激活作用

在对小菜蛾Plutella xylostella幼虫血淋巴酚氧化酶原的存在部位及免疫激活作用特点研究的基础上,比较了根虫瘟霉Zoophthora radicans不同菌株对酚氧化酶原激活系统的免疫激化及防御作用的差异。研究发现, 酚氧化酶原主要位于小菜蛾幼虫血细胞膜及血细胞裂解液中,极少存在于血浆中。在免疫激活剂昆布多糖存在下,分别测得小菜蛾幼虫血细胞碎片、血细胞裂解液和血浆的酚氧化酶活性为26.80 U,16.68 U和2.53 U。酚氧化酶原显著地受血浆和昆布多糖同时存在的激活,但两者单独存在时对酚氧化酶原的激活作用较弱。根虫瘟霉菌丝裂解液对酚氧化酶原有不同程度的激活作用,其激活作用在有血浆存在时显著增强,其酚氧化酶活性可提高2.9～3.4倍。各菌株间对酚氧化酶原的激活作用则以ARSEF1342菌株最强,ARSEF2699和F99101菌株次之,ARSEF1100菌株最弱。被激活的酚氧化酶可粘附于根虫瘟霉菌丝上并能产生黑化反应,各菌株间酚氧化酶粘附于ARSEF1342菌株的能力最强,粘附于ARSEF2699和F99101菌株的次之,粘附于ARSEF1100菌株的最弱。但酚氧化酶粘附于昆布多糖的能力显著强于各虫霉菌株,表明各菌株在一定程度上能逃避寄主的免疫识别;各菌株激活酚氧化酶原及酚氧化酶粘附于菌株强弱,与对小菜蛾毒力呈负相关性,表明高毒力菌株具有易逃避寄主免疫识别的趋向。     

Short‐term larval food stress and associated compensatory growth reduce adult immune function in a damselfly

Abstract 1. In animals with a complex life cycle, larval stressors may carry over to the adult stage. Carry‐over effects not mediated through age and size at metamorphosis have rarely been studied. The present study focuses on the poorly documented immune costs of short‐term food stress both in the larval stage and after metamorphosis in the adult stage. 2. The present study quantified immune function [number of haemocytes, activity of prophenoloxidase (proPO) and phenoloxidase (PO)] in an experiment where larvae of the damselfly Lestes viridis were exposed to a transient starvation period. 3. Directly after starvation, immune variables were reduced in starved larvae. Levels of proPO and PO remained low after starvation, even after metamorphosis. In contrast, haemocyte numbers were fully compensated by the end of the larval stage, yet were lower in previously starved animals after metamorphosis. This can be explained as a cost of the observed compensatory growth after starvation. Focusing only on potential costs of larval stressors within the larval stage may therefore be misleading. 4. The here‐identified immunological cost in the adult stage of larval short‐term food stress and associated compensatory growth strongly indicates that physiological costs may explain hidden carry‐over effects bridging metamorphosis. This adds to the increasing awareness that the larval and adult stages in animals with a complex life cycle should be jointly studied, as trade‐offs may span metamorphosis.     

A lipopolysaccharide- and beta-1,3-glucan-binding protein from hemocytes of the freshwater crayfish Pacifastacus leniusculus. Purification, characterization, and cDNA cloning

A lipopolysaccharide- and beta-1,3-glucan-binding protein (LGBP) was isolated and characterized from blood cells (hemocytes) of the freshwater crayfish Pacifastacus leniusculus. The LGBP was purified by chromatography on Blue-Sepharose and phenyl-Sepharose, followed by Sephacryl S-200. The LGBP has a molecular mass of 36 kDa and 40 kDa on 10% SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. The calculated mass of LGBP is 39,492 Da, which corresponds to the native size of LGBP; the estimated pI of the mature LGBP is 5.80. LGBP has binding activity to lipopolysaccharides as well as to beta-1,3-glucans such as laminarin and curdlan, but peptidoglycan could not bind to LGBP. Cloning and sequencing of LGBP showed significant homology with several putative Gram-negative bacteria-binding proteins and beta-1, 3-glucanases. Interestingly, LGBP also has a structure and functions similar to those of the coelomic cytolytic factor-1, a lipopolysaccharide- and glucan-binding protein from the earthworm Eisenia foetida. To evaluate the involvement of LGBP in the prophenoloxidase (proPO) activating system, a polyclonal antibody against LGBP was made and used for the inhibition of phenoloxidase (PO) activity triggered by the beta-1,3-glucan laminarin in the hemocyte lysate of crayfish. The PO activity was blocked completely by the anti-LGBP antibody. Moreover, the PO activity could be recovered by the addition of purified LGBP. These results suggest that the 36-kDa LGBP plays a role in the activation of the proPO activating system in crayfish and thus seems to play an important role in the innate immune system of crayfish.     

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 microg/ml of ODN2006 for 30 min, the increases in both intra- and extracellular stimulated PO activity (POS) and extracellular total PO activity (POT) and the reduction of POT 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 POT 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 POT, and the addition of either phorbol-12-myristate-13-acetate (PMA; a phosphokinase C (PKC) activator) or caffeine (a phosphodiesterase inhibitor) only increased extracellular POT. When PMA-stimulated haemocytes were treated with chelerythrine (a PKC inhibitor), the induced extracellular POT 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 POS and extracellular POT were significantly decreased. ODN-stimulated haemocytes treated with genistein (an inhibitor of protein tyrosine kinase) showed a further increase in extracellular POT, 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.     

香港巨牡蛎和长牡蛎幼虫及稚贝的表型性状

为了评估香港巨牡蛎和长牡蛎在北方沿海的早期表型性状,于2010年7月,以2009年6月在青岛繁育的两种牡蛎为材料,在大连研究了温度(Mt:(22±1.0)℃及Ht:(28±1.0)℃)、盐度(S₂₀:20±1.0及S₃₀:30±1.0)及中间育成环境(ID:室内及OD:室外)对两种牡蛎幼虫及稚贝表型性状的影响。结果表明:香港巨牡蛎壳宽显著大于长牡蛎(P<0.05),壳高及怀卵量显著小于长牡蛎(P<0.05),壳长、鲜重及壳重两者间无显著差异(P>0.05)。在温度和盐度相同情况下,长牡蛎卵径、受精率、孵化率及D形幼虫均大于香港巨牡蛎;香港巨牡蛎幼虫浮游前期生长较慢,而后快于长牡蛎。两种牡蛎幼虫存活能力在15日龄时高温组>中温组;相同温度下,香港巨牡蛎中盐组>高盐组,长牡蛎高盐组>中盐组。幼虫变态期间,较低的温度延迟了变态时间,降低了变态率,使得两种幼虫变态规格大型化。温度是影响幼虫生长、存活、变态的最主要因素,其次为盐度,交互作用几乎尚未起到作用。中间育成阶段,室外比室内培育效果更好,且香港巨牡蛎稚贝壳高在60日龄以后显著大于长牡蛎(P<0.05),环境是影响稚贝生长的最主要因素;无论室内还是室外两种牡蛎稚贝的存活率均在90%以上,且各实验组间无显著差异(P>0.05)。     

Purification and characterization of a small cationic protein from the tobacco hornworm Manduca sexta

The prophenoloxidase (proPO) activation system is an important defense mechanism in arthropods, and activation of proPO to active phenoloxidase (PO) involves a serine proteinase cascade. Here, we report the purification and characterization of a small cationic protein CP8 from the tobacco hornworm, Manduca sexta, which can stimulate proPO activation. BLAST search showed that Manduca CP8 is similar to a fungal proteinase inhibitor-1 (AmFPI-1), an inducible serine proteinase inhibitor-1 (ISPI-1), and other small cationic proteins with unknown functions. However, we showed that Manduca CP8 did not inhibit proteinase activity, but stimulated proPO activation in plasma. When small amount (0.1 μg) of purified native CP8 or BSA was added to cell-free plasma samples and incubated for 20 min, low PO activity was observed in both groups. But significantly higher PO activity was observed in the CP8-group than in the BSA-group when more proteins (0.5 μg) were added and incubated for 20 min. However, when the plasma samples were incubated with proteins for 30 min, high PO activity was observed in both the CP8 and BSA groups regardless of the amount of proteins added. Moreover, when PO in the plasma was pre-activated with Micrococcus luteus, addition of CP8 did not have an effect on PO activity, and CP8/bacteria mixture did not stimulate PO activity to a higher level than did BSA/bacteria. These results suggest that CP8 helps activate proPO more rapidly at the initial stage. CP8 mRNA was specifically expressed in fat body and its mRNA level decreased when larvae were injected with saline or bacteria. However, CP8 protein concentration in hemolymph did not change significantly in larvae injected with saline or microorganisms.     

Manduca sexta prophenoloxidase (proPO) activation requires proPO-activating proteinase (PAP) and serine proteinase homologs (SPHs) simultaneously

In the tobacco hornworm Manduca sexta, proteolytic activation of prophenoloxidase (proPO) is mediated by three proPO-activating proteinases (PAPs) and two serine proteinase homologs (SPHs) (Proceedings of the National Academy of Sciences, USA 95 (1998) 12220-12225; J. Biol. Chem. 278 (2003a) 3552-3561; Insect Biochem. Mol. Biol. 33 (2003b) 1049-1060). While our current data are consistent with the hypothesis that the SPHs serve as a cofactor/anchor for PAPs (Insect Biochemistry and Molecular Biology 33 (2003) 197-208; Insect Biochemistry and Molecular Biology 34 (2004) 731-742), roles of these clip-domain proteins (i.e. PAPs and SPHs) in proPO activation are poorly defined. To better understand this process, we further characterized the activation reaction using proPO, PAP-1 and SPHs. PAP-1 itself cleaved nearly 1/3 of proPO at Arg51 without generating much phenoloxidase (PO) activity. In the presence of SPHs, the cleavage of proPO became more complete while the increase in PO activity was over 20-fold, indicating that the extent of cleavage does not directly correlate with PO activity. Since SPHs and p-amidinophenyl methanesulfonyl fluoride (APMSF)-treated PAP-1 did not generate active PO by interacting with proPO, proteolytic cleavage is critical for proPO activation. After 1/5 of proPO was processed by PAP-1 alone which was then inactivated by M. sexta serpin-1J or APMSF, further incubation of the reaction mixture with SPHs failed to generate active PO either. Thus, SPHs cannot generate PO activity by simply binding to cleaved proPO. M. sexta proPO activation requires active PAP-1 and SPHs at the same time-one for limited proteolysis and the other as a cofactor, perhaps. Gel filtration chromatography and native gel electrophoresis revealed the PAP-SPH, proPO-PAP, and SPH-proPO associations, essential for generating high Mr, active PO at the site of infection.     

Phenoloxidase activation in the embryo of the common cuttlefish Sepia officinalis and responses to the Ag and Cu exposure

The prophenoloxidase (proPO) system catalyzing the melanin production is considered as implicated in the innate immune system in invertebrates. The phenoloxidase (PO)-like activity was detected in the cuttlefish embryo sampled at the end of the organogenesis and few hours before hatching. Various modulators of the PO activity were used to assess the triggering of the proPO activating system. The results demonstrated the evidence of a true PO activity in the cuttlefish embryo. However, SDS and LPS granted contrasting effects on the PO-like activity between the developmental stages suggesting a progressive maturation of the proPO system from the embryonic to the juvenile stages. In eggs exposed to dissolved trace metals all along the embryonic development, Ag (1.2 μg L⁻¹) inhibited the PO-like activity in the cuttlefish embryo except at hatching time, suggesting the synthesis of a new “juvenile” form of the PO enzyme. In similar conditions as for Ag, Cu (230 μg L⁻¹) stimulated and then inhibited the PO-like activity according to a progressive metal accumulation within the egg and suggesting the occurrence of a threshold, above which the toxicity of the essential metal reduced the PO activity.     

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta

In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12 h post-injection, but dropped to the normal level at 24 h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules.