Prophenoloxidase from Pieris rapae: gene cloning, activity, and transcription in response to venom/calyx fluid from the endoparasitoid wasp Cotesia glomerata

Prophenoloxidase from Pieris rapae : gene cloning, activity, and transcription in response to venom/calyx fluid from the endoparasitoid wasp Cotesia glomerata 1 first-author †E-mail:jiayingzhu001@yahoo.com.cn †E-mail:jiayingzhu001@yahoo.com.cn jiayingzhu001@yahoo.com.cn 2 3 Key Laboratory of Forest Disaster Warning and Control of Yunnan Province and Key Laboratory of Southwest Mountain Forest Resources Conservation and Utilization of Ministry of Education, Southwest Forestry University, Kunming 650224, China Key Laboratory of Forest Disaster Warning and Control of Yunnan Province and Key Laboratory of Southwest Mountain Forest Resources Conservation and Utilization of Ministry of Education, Southwest Forestry University, Kunming 650224, China Research Institute of Resource Insects, Chinese Academy of Forestry, Kunming 650224, China Research Institute of Resource Insects, Chinese Academy of Forestry, Kunming 650224, China College of Plant Protection, Yunnan Agricultural University, Kunming 650201, China College of Plant Protection, Yunnan Agricultural University, Kunming 650201, China Prophenoloxidase (PPO) plays an important role in melanization, necessary for defense against intruding parasitoids. Parasitoids have evolved to inject maternal virulence factors into the host hemocoel to suppress hemolymph melanization for the successful development of their progeny. In this study, the full-length complementary DNA (cDNA) of a Pieris rapae PPO was cloned. Its cDNA contained a 2 076-base pair (bp) open reading frame (ORF) encoding 691 amino acids (aa). Two putative copper-binding sites, a proteolytic activation site, three conserved hemocyanin domains, and a thiol ester motif were found in the deduced amino acid sequence. According to both multiple alignment and phylogenetic analysis, P. rapae PPO gene cloned here is a member of the lepidopteran PPO-2 family. Injection of Cotesia glomerata venom or calyx fluid resulted in reduction of P. rapae hemolymph phenoloxidase activity, demonstrating the ability to inhibit the host′s melanization. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) showed that transcripts of P. rapae PPO-2 in the haemocytes from larvae had not significantly changed following venom injection, suggesting that the regulation of PPO messenger RNA (mRNA) expression by venom was not employed by C. glomerata to cause failure of melanization in parasitized host. While decreased P. rapae PPO-2 gene expression was observed in the haemocytes after calyx fluid injection, no detectable transcriptional change was induced by parasitization, indicating that transcriptional down-regulation of PPO by calyx fluid might play a minor role involved in inhibiting the host′s melanization. Prophenoloxidase Cloning Venom Calyx fluid Gene expression Parasitoid Introduction 1 Insects, lacking an acquired immune system, have an efficient innate immune system including an array of humoral and cellular immune responses to combat invading pathogens and parasites. Melanization is an important part of the immune defense involved in the process of sclerotization, pigmentation, and wound healing of the cuticle as well as in defence reactions (nodule formation, phagocytosis, and/or encapsulation) against invaders entering the insect’s hemocoel (Ratcliffe et al., 1985 1985 ; Sritunyalucksana and Söderhäll, 2000 2000 ; Cerenius et al., 2008 2008 ). The key enzyme involved in melanin formation is phenoloxidase (PO). PO generally exists in hemolymph, haemocytes, and/or cuticle of insects in its inactive form as a proenzyme, prophenoloxidase (PPO), which is converted to the active form via a serine protease cascade (Ashida, 1971 1971 ; Ashida and Brey, 1995 1995 ; Kopácek et al., 1995 1995 ; Cerenius and Söderhäll, 2004 2004 ). PPO activation cascade leads to the formation of cytotoxic molecules including quinones from phenolic compounds and reactive oxygen intermediates, which are important immune effectors vital against foreign intruders (Nappi and Christensen, 2005 2005 ). In order to overcome insect defensive melanization, microorganisms and parasites depend to a large extent on regulations of PO activity and gene expressions that are associated with the PPO activating system (Nappi et al., 2009 2009 ; Vazquez et al., 2009 2009 ). Endoparasitic wasps exhibit a unique form of immature development. They lay eggs into the body cavities of their hosts, consuming them during their own development, thereby almost ultimately causing the host′s death (Kraaijeveld and Godfray, 2009 2009 ). To ensure successfully development to maturity in the host hemocoel, parasitoids use a diversified range of virulence factors including polydnaviruses (PDVs), venom, virus-like particles (VLPs), teratocytes, ovarian proteins, and parasitoid larval secretions to deactivate their host′s immune responses (Beckage and Gelman, 2004 2004 ; Brodeur and Boivin, 2004 2004 ; Moreau and Guillot, 2005 2005 ; Pennacchio and Strand, 2006 2006 ; Zhu et al., 2008 2008 ; 2009a 2009a ; 2009b 2009b ; Kraaijeveld and Godfray, 2009 2009 ). Inhibition of melanization and PO activity by parasitization has been frequently observed (Lavine and Beckage, 1995 1995 ; Asgari et al., 2003 2003 ; Colinet et al., 2009 2009 ; Nappi et al., 2009 2009 ). However, relatively little is known about the molecular mechanisms of the host′s melanization disrupted by the parasitoid. Recently, several virulence proteins notably known to inhibit melanization have been characterized from parasitoid venom and PDVs (Asgari et al., 2003 2003 ; Beck and Strand, 2007 2007 ; Colinet et al., 2009 2009 ; Zhu et al., 2011 2011 ). These findings made a large contribution for explaining how parasitoids inhibit the host′s melanization, and demonstrated that these virulence factors target the PPO activation cascade associated with the reduction in PO activity. Expression of PPO reduced by parasitization might be one of the strategies ascribed to decrease PO activity (Shelby et al., 2000 2000 ), which is only investigated in Tranosema rostrale - Choristoneura fumiferana system (Doucet et al., 2008 2008 ). Cotesia glomerata is a common gregarious endoparasitoid attacking many species of lepidopterous young larvae as the host. It is an important larval parasitoid of the small white butterfly, Pieris rapae , which is heavily parasitized in fields of cruciferous vegetables (Laing and Levin, 1982 1982 ). Its venom and calyx fluid impair the host′s cellular and humoral immune defences, and its teratocytes possibly suppress the host′s larval hemolymph PO activity (Kitano, 1974 1974 ; 1982 1982 ; Kitano and Nakatsuji, 1978 1978 ; Kitano et al., 1990 1990 ; Ockroy et al., 2002 2002 ; Madanagopal and Kim, 2006 2006 ). However, there is no report focused on the effect of C . glomerata venom or calyx fluid on P . rapae haemolymph PO activity. Here, complementary DNA (cDNA) of P . rapae PPO was cloned, and P . rapae PO activity and PPO transcription were investigated in response to C . glomerata venom or calyx fluid. Materials and methods 1 Insects 2 C . glomerata colony was established by collecting cocoons or parasitized P . rapae larvae from cabbage fields in the suburbs of Kunming, Yunnan Province, China. Once emerged, wasps were held in a glass tube (20 cm×5 cm) and fed a 20% (v/v) honey solution. Its colony was maintained continuously on P . rapae larvae. Eggs of P . rapae on cabbage in the fields were collected and held in a growth chamber under a regime of 12-h light:12-h dark, (25±1) °C, and 60% relative humidity. Newly emerged larvae were reared on cabbage grown at the same conditions. cDNA cloning of PPO 2 P . rapae larvae were surface sterilized in 95% ethanol and a small cut was made in a proleg. Hemolymph was collected in a 1.5-ml sterilized Eppendorf tube. After mixing by gently inverting the tube, haemocytes were collected by centrifuging the hemolymph at 250× g for 5 min at room temperature. The resulting haemocyte pellets were used for total RNA isolation. Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. 3′ and 5′ RACE (rapid amplification of cDNA ends) cDNAs were synthesized with an SMART™ RACE cDNA amplification kit (Clontech, Mountain View, CA, USA). Degenerated primer pairs (5′-GARCTGTTYTAYTAYATGC-3′ and 5′-CACRTGNCCCATRTTRTG-3′) were designed based on the amino acid sequences of the two conserved copper-binding sites (QIFYYMH and HNMGHV) of other insect PPOs (Park et al., 1998 1998 ). They were used to amplify part of the coding sequence of PPO from 3′ RACE cDNA. Polymerase chain reaction (PCR) was preceded by denaturation at 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min, and finishing with chain extension at 72 °C for 10 min. Based on this result, 3′ and 5′ gene-specific primers (5′-CGACCGTTACCATCCCATTCGAGCAGAC-3′ and 5′-TGGTTGGCAGTAAATGGGTAGACGAAAT-3′) were designed, respectively. They were used to clone the 3′ and 5′ ends in conjunction with adapter primers for both ends provided in an SMART™ RACE cDNA amplification kit (Clontech). The RACE-PCR was carried as follows: 94 °C for 1 min; 30 s at 94 °C, 6 min at 68 °C, and 2 min at 72 °C for 30 cycles; extension at 72 °C for 10 min. PCR products were subjected to 0.01 g/ml agarose gel electrophoresis, purified, and cloned to pGEM-T® easy vector (Promega, San Luis Obispo, CA, USA) for sequencing. Sequence analysis 2 The nucleotide and deduced amino acid sequences were analyzed using Genetyx Version 8.0 (Software Development, Tokyo, Japan). The signal peptide was analyzed by SigalP 3.0 Server (Dyrløv Bendtsen et al., 2004 2004 ). The gene characteristic structures were predicted by Motif Scan (Hulo et al., 2008 2008 ). The multiple sequence alignment was created with ClustalX (Version 1.83) program (Thompson et al., 1997 1997 ). The phylogenetic analysis was conducted using molecular evolutionary genetics analysis 4 (MEGA4) with the neighbor-joining method and bootstrapping sampled 1000 times (Tamura et al., 2007 2007 ). Venom and calyx fluid preparation 2 Venom apparatus and ovaries were dissected from 4-d-old C . glomerata female wasps according to the method described by Rivers et al. ( 1993 1993 ) and Yu et al. ( 2007 2007 ). After several sequential washes, they were transferred to 0.5 ml sterilized Eppendorf tube containing 50 μl sterile phosphate buffered saline (PBS, pH 7.4). The samples were homogenized manually, and the extract was centrifuged at 12 000× g at 4 °C for 10 min to discard cellular debris. The supernatant was collected and diluted in PBS to the concentration of 0.5 female equivalents of venom or calyx fluid (one female equivalent being defined as the supernatant from one venom reservoir or ovary in 1 μl PBS). PO activity analysis 2 Each P . rapae larva of the 3rd instar was kept in a glass tube (5 cm×3 cm). One 4-d-old mated C . glomerata female wasp was introduced to each tube. Immediately after being attacked, the parasitoid wasp was removed from the tube. Similar aged host larvae were also injected with 0.5 female equivalents of venom or calyx fluid as described by Béliveau et al. ( 2000 2000 ). The larvae were reared using cabbage leaves. Hemolymph collected from five larvae of each treatment was centrifuged at 10 000× g for 5 min at 4 °C to precipitate insoluble material, and then used in PO activity assays. PO activity was assayed following the protocol of Fisher and Brady ( 1980 1980 ) and Parkinson and Weaver ( 1999 1999 ). Briefly, 5 μl hemolymph was added to 5 mmol/L L-3,4-dihydroxyphenylalanine (L-DOPA) in cacodylate buffer (10 mmol/L sodium cacodylate, 5 mmol/L CaCl 2 ; pH 7.0) and absorbance (490 nm) measured using a microplate reader (Bio-Rad, Hercules, CA, USA) for 30 min at room temperature. PO activity assays were performed on three separate collections of hemolymph at each of the time points. All data analysis was carried out by one-way analysis of variance (ANOVA). The acceptance level of statistical significance was P < 0.05 in all instances. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) 2 Parasitization and injection of venom or calyx fluid were conducted as described above. Controls consisted of larvae non-parasitized and injected with equal volume of PBS. After 0, 12, 24, 48, 72, and 96 h treatments, haemocytes were collected. Their total RNA was isolated using TRIzol Reagent (Invitrogen). There were three replicates for each time point. Residual genomic DNA was removed using RNase-free DNase I (Promega). cDNAs were synthesized using a RevertAid™ first strand cDNA synthesis kit (Fermentas, Burlington, Canada), and then subjected to real-time RT-PCR analysis. RT-PCR was performed in the SYBR® Premix (TaKaRa, Shiga, Japan) according to the manufacture’s standard protocol. The PPO transcript was determined using specific primers (5′-CTTGACAGTCTTACATCCTCCCG-3′ and 5′-CCTCATCGCCTATTACGCCAAAC-3′). The 18S rRNA profile was used as internal control for normalization of input cDNA. The total RNA following DNase treatment was subjected to RT-PCR to assure that DNA removal has been successful. PCR conditions were as follows: 1 cycle of 94 °C for 3 min, followed by 40 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s. The relative mRNA level of PPO was normalized relative to the control using the 2 −ΔΔ C t method (Livak and Schmittgen, 2001 2001 ). Results 1 cDNA cloning of P. rapae PPO 2 The cDNA of P . rapae PPO was 2 397 base pairs (bp) in length, containing a 2076-bp of open reading frame (ORF), a 72-bp of 5′ untranslated region (UTR), and a 250-bp of 3′ UTR with polyadenylation signal sequences AATAAA and a poly(A) tail (GenBank accession No. HM210573) (Fig. 1 1 ). The deduced amino acid (aa) sequence revealed a protein of 691 aa with a predicted molecular weight of 79.15 kDa and a theoretical pI of 6.13. No putative signal peptide was predicted at the N-terminus. The proteolytic cleavage site forming PPO into PO was predicted to be between Arg51 and Leu52. Two conserved copper-binding sites, namely coppers A and B, were preserved at aa 201–245 and aa 346–413, which contained six histidine residues (aa 213, 217, 243, 366, 370, and 406). Three conserved hemocyanin domains were encoded by the polypeptide sequence: hemocyanin-N at aa 47–147, hemocyanin-M at aa 152–415, and hemocyanin-C at aa 423–679. A common central domain of tyrosinase was detected at aa 233–419. A highly conserved thiol ester motif (GCGWPQHM) was present at aa 584–591. Other possible functional sites were predicted, including 6 N -glycosylation sites, 2 cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-dependent protein kinase phosphorylation sites, 8 casein kinase II phosphorylation sites, 9 N -myristoylation sites, 12 protein kinase C phosphorylation sites, and 1 tyrosine kinase phosphorylation site. Amino acids are represented by one letter abbreviations. Double asterisks (**) indicate the putative cleavage site. Copper-binding sites A and B are underlined and double underlined, respectively. The conserved histidine (H) residues of two copper-binding sites are shown in bold letter. The thiol ester-like motif is shaded in grey. The asterisk (*) indicates the stop codon. The potential polyadenylation signal (AATAAA) is boxed Sequence comparison showed that the identity between the amino acid sequence of P . rapae PPO and those of Plutella xylostella PPO 1 and PPO 2, Tenebrio molitor PPO, Musca domestica PPO, and Apis mellifera PPO were 47%, 74%, 51%, 53%, and 50%, respectively (Fig. 2 2 ). The copper-binding regions were highly conserved among these species, and the histidine residues and the thiol ester motifs were present in all sequences analyzed. The sequences compared are from PraPPO2, Pieris rapae PPO-2 (HM210573); PxylPPO1, Plutella xylostella PPO-1 (ADA60206); PxylPPO2, P . xylostella PPO-2 (ADA60207); TmolPPO, Tenebrio molitor PPO (BAA75470); MdomPPO, Musca domestica PPO (AAR84669); AmelPPO, Apis mellifera PPO (AAO72539). Black and grey shadings indicate different degrees of overall conservation for each site (black, identical; grey, conservative). Six conserved histidines are marked with asterisks (*). Thiol ester-like motif is boxed A phylogeny tree was constructed by neighbour-joining distance method using amino acid sequences of PPO of P . rapae and PPOs of other Insecta and Crustacea (Fig. 3 3 ). The PPO of P . rapae together with PPOs of other insects placed far away from that of Crustacea. Seven main well-supported groups in the PPO sequences of Insecta analysis were identified. Dipteran and hymenopteran PPOs were separated into two distinct groups, respectively. PPOs from Coleoptera were clustered into the same group. Lepidopteran PPOs were subdivided into two distinct clades that separate PPO-1 from PPO-2. P . rapae PPO belonged to the PPO-2 clade, and was most closely related to P . xylostella and C . fumiferana PPO-2. The reliability of each branch was tested by 1 000 bootstrap replications. Bootstrap values for each node are shown. GenBank accession numbers are listed for each PPO sequence Effects of venom and calyx fluid on PO activity 2 Levels of PO activity of non-parasitized larvae increased as development progressed, while that in parasitized larvae only ascended gradually up to 48 h, followed by a gradual decrease during 72 to 96 h (Fig. 4 4 ). Parasitization had significant down-regulation effects on PO activity during 24 to 96 h. PBS-injected larvae were similar to non-parasitized larvae within the time observed. When compared to PBS injection, venom injection had significant effects on PO activity during 6–96 h. The PO activity of PBS-injected larvae was not statistically different from that of calyx fluid-injected larvae at 0–12 h. However, calyx fluid injection significantly reduced the PO activity throughout 24–96 h. From 48 to 96 h, calyx fluid injection induced a significantly higher decrease in PO activity than venom injection, and both venom and calyx fluid injections resulted in significantly lower levels of PO activity than parasitization during 24 to 96 h. Error bars represent standard deviation of three replications Effects of venom and calyx fluid on PPO transcription 2 The relative mRNA profiles showed continuous increase in transcript levels of PPO in non-parasitized larvae with a steep rise between 24 and 96 h (Fig. 5 5 ). In contrast to non-parasitized larvae, PPO transcription in parasitized larvae did not significantly increase until 72 h after parasitization, and then the transcription significantly dropped down at 96 h. The transcription of PPO in PBS-injected larvae gradually rose up to 24 h after injection, and then kept at similar levels at between 48–96 h. In venom or calyx fluid-injected larvae, the PPO mRNA did not show differential expression compared to that of PBS-injected, non-parasitized, and parasitized larvae 12 h before treatment, and there were no significant differences between PBS and venom injection throughout 24–96 h. Significantly lower transcription levels were detected at 24–96 h, however, in calyx fluid-injected larvae. During this interval, the transcription levels in calyx fluid-injected larvae gradually decreased and reached the lowest level at 96 h. The levels of PPO transcription were analyzed by real-time RT-PCR using the SYBR® Green fluorescent probe. Error bars represent standard deviation of three replications Discussion 1 Melanization reactions in insects as a common response to intruders occur due to changes in PO activity, an enzyme that is usually present in an inactive PPO form. As a potentially important immunity gene, PPO has been sequenced from a number of different insect species representative of Lepidoptera, Diptera, Coleoptera, and Hymenoptera (Park et al., 1998 1998 ; Cui et al., 2000 2000 ; Hartzer et al., 2005 2005 ; Lourenço et al., 2005 2005 ; Doucet et al., 2008 2008 ; Shelby and Popham, 2008 2008 ). Two PPO isoforms have been characterized from several Lepidoptera such as Manduca sexta , Bombyx mori , Galleria mellonella , Plodia interpunctella , Heliothis virescens , and Hyphantria cunea , and in the case of some Diptera, many more PPOs have been characterized (Hartzer et al., 2005 2005 ). In this study, only one PPO cDNA cloned from P . rapae , and the protein deduced from it has a higher identity to PPO-2s reported in other lepidopteran species compared to other lepidopteran PPO-1s. Like in other lepidopteran insects PPO-2 from any species has a higher similarity to PPO-2s from other species than it does with PPO-1s from other species and its own PPO-1. The result presented here indicates that the protein encoded by P . rapae PPO gene cloned here is a PPO-2 representative. Signal peptide sequence present in the putative N-terminus has been noted in the case of few insect PPOs, and may be related to zymogen release via haemocyte lysis (Shelby and Popham, 2008 2008 ). However, it was not detected in P . rapae PPO, as is the case in most arthropod PPOs (Olivares and Solano, 2009 2009 ). PPOs lacking a signal peptide necessary for secretion from endoplasmic reticulum are unlikely to have N -glycosylation associated with protein movement across the cell membrane (Hartzer et al., 2005 2005 ). N -glycosylation has been demonstrated in G . mellonella PPO, and P . interpunctella PPO was a possible glycoprotein (Kopácek et al., 1995 1995 ; Hartzer et al., 2005 2005 ). Six potential N-linked glycosylation sites and other putative post-translational modifications, such as phosphorylation and myristoylation, which are usually involved in regulating translation, co-translation, and post-translation reactions, were present in P . rapae PPO, thus suggesting that it is released into the hemolymph by haemocyte rupture similar to PPOs of other insects lacking a signal peptide (Taft et al., 2001 2001 ). A conserved N-terminal serine proteinase cleavage site for PPO activation by PPO-activating enzyme is predicted to locate at Arg50-Ser51 (Huang et al., 2001 2001 ). Once pathogens and parasites invade the host, PPO would be cleaved and converted to the active PO by a serine protease cascade response (Söderhäll et al., 1994 1994 ). Furthermore, as described for other insects, several highly conserved features, in particular two sites that code for copper-binding sites displaying the six canonical histidines involved in Cu 2+ and oxygen binding, and a thiol ester-like motif, were present in P . rapae PPO (Jaenicke and Decker, 2004 2004 ). These conserved features are common to PPO, suggesting a remarkable evolutionary conservation of the structure of this protein. Sequence comparison of PPOs suggests that copper-binding sites are present in a group of oxygen-transporting proteins of arthropods known as hemocyanins, suggesting that the origin of arthropod hemocyanins is ancient PPO-like proteins (Gaykema et al., 1984 1984 ; Fujimoto et al., 1995 1995 ; Burmester and Scheller, 1996 1996 ). The domains of hemocyanin-N, hemocyanin-M, and hemocyanin-C were found in P . rapae PPO, indicating that this gene is a potential member of the hemocyanin gene family (Hughes, 1999 1999 ). A thiol ester-like motif has been suggested to be responsible for binding to other macromolecules (Dodds and Day, 1996 1996 ). The presence of a thiol ester motif in PPO may be related to the function of immobilization of invading organisms during the process of insect defense reactions (Sritunyalucksana et al., 1999 1999 ; Lourenço et al., 2005 2005 ). The phylogenic tree of the lepidopteran PPOs showed that they were divided into two groups, namely PPO-1 and PPO-2. The result is in good agreement with the previous findings that lepidopterans have two PPO genes (Cui et al., 2000 2000 ; Hartzer et al., 2005 2005 ; Lourenço et al., 2005 2005 ; Shelby and Popham, 2008 2008 ). The P . rapae PPO gene cloned here is obviously grouped into the PPO-2 clad, demonstrating that this gene is classified as PPO-2 type. Partial or complete inhibition of hemolymph PO activity in host species attacked by parasitoid wasps has been reported from several systems (Stoltz and Cook, 1983 1983 ; Beckage et al., 1990 1990 ; Kitano et al., 1990 1990 ; Beck et al., 2000 2000 ; Shelby et al., 2000 2000 ; Hartzer et al., 2005 2005 ). The parasitoid associated virulence factors, such as PDVs, VLPs, venom, teratocytes, ovarian proteins, and parasitoid larval secretions, which are efficient to suppress host immune defense, are likely to do so with the regulation of host hemolymph melanization. For example, PDV of Cotesia congregate depressed hemolymph monophenoloxidase activity in Manduca sexta (Beckage et al., 1990 1990 ), venom of C . rubecula inhibited melanization of P . rapae hemolymph (Asgari et al., 2003 2003 ), and the feeding larvae produced factors of Eulophus pennicornis reduced host haemolymph PO activity (Richards and Edwards, 2003 2003 ). In the C . glomerata - P . rapae system, teratocytes might contribute to the suppression of PO activity in the host hemolymph (Kitano et al., 1990 1990 ). However, except for teratocyte, C . glomerata is also associated with venom and calyx fluid to knock out host′s immune responses (Kitano, 1974 1974 ; 1982 1982 ; Kitano and Nakatsuji, 1978 1978 ; Kitano et al., 1990 1990 ; Ockroy et al., 2002 2002 ; Madanagopal and Kim, 2006 2006 ). Thus, C . glomerata venom and calyx fluid together may be involved in inhibiting the host′s melanization. In order to test this hypothesis, it is important to investigate whether the PO activity is inhibited individually by C . glomerata venom and calyx fluid or not. In the present study, not only parasitism by C . glomerata was able to reduce the PO activity in P . rapae hemolymph, but also injection of C . glomerata venom or calyx fluid had the same effect, demonstrating that both venom and calyx fluid of this parasitoid are ascribed to the lack of melanization in the parasitized host. In addition, we found that both venom and calyx fluid injections induced significantly lower PO activity than parasitization. This may be the result of significantly higher amounts of venom and calyx fluid manually injected into the host than that delivered by female wasp during oviposition (Cusson et al., 2000 2000 ). Parasitoid venom often mediates short-term immune suppression before PDV gene expression becomes effective (Webb and Luckhart, 1994 1994 ). In accordance with this, at the first 6 h following injection of C . glomerata venom, P . rapae larvae showed a strong PO activity suppression. The venom induced a rapid decrease of PO activity, possibly due to its components exerting their action directly on the PPO activation cascade. In the same genus to C . glomerata , a venom protein (Vn50) from Cotesia rubecula inhibited melanization of P . rapae hemolymph by interfering with the PPO activation cascade (Asgari et al., 2003 2003 ). Serpin from the venom of Leptopilina boulardi disrupted the activation pathway of PO in the Drosophila host (Colinet et al., 2009 2009 ). Serine protease protein family involved in PPO signaling was best represented by Nasonia vitripennis venom constituents (de Graaf et al., 2010 2010 ). Thus, regulation of PPO transcription by C . glomerata venom might not be the reason for PO activity reduction in P . rapae larvae. In the present study, C . glomerata venom injection had no observed effect on P . rapae PPO transcription. In contrast to venom injection, PO activity in calyx fluid injected larvae was significantly lower than that detected in the controls until 24 h post treatment. Except for PDVs, the calyx fluid extract also contained non-viral proteins, ovarian proteins, which play a role in early and short-term immunosuppression (Webb and Luckhart, 1994 1994 ). Thus, the results suggest that viral gene expression was required to inhibit melanization. Similarly, the delay in PO inhibition induced by PDVs observed here has been reported for other parasitic wasps, e.g., inhibition of PO activity in the hemolymph of C . fumiferana injected with T . rostrale calyx fluid was not elicited immediately following treatment, with maximum inhibition at 20 h after injection (Doucet and Cusson, 1996 1996 ); inhibition of defensive melanization of Heliothis virescens increased after parasitization by Campoletis sonorensis in a time frame consistent with this wasp’s CsIV expression (Shelby et al., 2000 2000 ). The inhibitory effect of C . glomerata calyx fluid on P . rapae PO activity could be due to the down-regulation expression of PPO gene. A significant decrease in the transcript of PPO was found after 24 h injection with calyx fluid. However, there was no detectable PPO transcriptional difference between parasitized and non-parasitized larvae. Similarly, C . fumiferana injected with T . rostrale calyx fluid caused transient depressions in the titer of PPO mRNA levels, while C . fumiferana through natural parasitization by T . rostrale has no detectable impact on the abundance of PPO transcript (Doucet et al., 2008 2008 ). As suggested by Doucet et al. ( 2008 2008 ), significant reduction of PPO transcript resulting from calyx fluid injection could be due to the high PDV inoculum. 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