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calyx fluid from the endoparasitic wasp Cotesia plutellae on the hemocytes of its host Plutella xylostella in vitro
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Journal of Insect Physiology 53 (2007) 22–29 www.elsevier.com/locate/jinsphys
Effects of venom/calyx fluid from the endoparasitic wasp Cotesia plutellae on the hemocytes of its host Plutella xylostella in vitro Rui-xian Yu, Ya-Feng Chen, Xue-xin Chen, Fang Huang, Yong-gen Lou, Shu-sheng Liu Institute of Insect Sciences, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, China Received 9 April 2006; received in revised form 22 September 2006; accepted 29 September 2006
Abstract Crude venom and calyx fluid from Cotesia plutellae (Hymenoptera Braconidae) were assayed for biological activity toward hemocytes of Plutella xylostella (Lepidoptera Plutellidae). Venom from C. plutellae displayed high activity toward the spreading of plasmatocytes of P. xylostella early in the incubation period, and the inhibition was more severe as the concentration of venom increased. However, most inhibited hemocytes spread normally after being incubated for 4 h. No effects were found toward granular cells from the host. Additionally, the venom from C. plutellae had some lethal effects on hemocytes of P. xylostella at high concentrations. In contrast, when incubated with different concentrations of calyx fluid, the spreading of some hemocytes was inhibited, some began to disintegrate, and some were badly damaged with only the nucleus left. After 4 h, the majority of hemocytes died. The same results were observed when hemocytes were incubated in calyx fluid together with venom. These results show that calyx fluid from C. plutellae may play a major role in the suppression of the host immune system, whereas venom from C. plutellae has a limited effect on hemocytes and probably synergizes the effect of calyx fluid or polydnavirus. r 2006 Elsevier Ltd. All rights reserved. Keywords: Cotesia plutellae; Plutella xylostella; Venom; Calyx fluid; Hemocytes; Immunosuppression
1. Introduction The insect immune system is classified into humoral and cellular defense responses, in which granular cells and plasmatocytes are the main hemocyte types reported to be active (Lavine and Strand, 2002). To develop successfully in the hemocoel of their insect hosts, endoparasitoids escape or suppress the host immune system by maternal secretions from the adult female wasp introduced into the host during oviposition. These secretions include polydnaviruses (PDVs) or virus-like particles (VLPs), soluble ovarian proteins, and venom (Strand and Pech, 1995; Beckage, 1998; Shelby and Webb, 1999). Great progress has been made in the elucidation of the importance of PDVs, VLPs, and ovarian proteins in suppressing the host immune defenses, while much less is known about venom proteins, especially from endoparasitoid venom (Beckage et al., 1990; Beckage, 1998; Stettler et al., 1998; Rizki and Corresponding author. Tel.: +86 571 86971219.
E-mail address: [email protected] (X.-x. Chen). 0022-1910/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2006.09.011
Rizki, 1994; Russo et al., 2001; Luckhart and Webb, 1996; Webb and Luckhart, 1994; Glatz et al., 2004a, b; Zhang et al., 2004; Morales et al., 2005). Many ectoparasitoids that have been studied induce permanent paralysis, host development arrest, host metabolic regulation, and inhibition of host immunity (Nakamatsu and Tanaka, 2003), whereas the role of endoparasitoid venom in suppressing host immune defense has not been clearly determined. In many braconid species, action of the venom components is necessary to enhance the effects of PDV or calyx fluid (Tanaka, 1987; Davies et al., 1987; Strand and Dover, 1991; Gupta and Ferkovich, 1998; Beckage and Gelman, 2004). However, a limited number of studies suggest that the venom from endoparasitoids devoid of symbiotic viruses may alone perturb host immune defenses. For example, venoms from Pteromalus puparum (Hymenoptera: Pteromalidae) and Nasonia vitripennis (Hymenoptera: Pteromalidae) suppress cellular immune responses in their hosts, Pieris rapae and Papilio xuthus (Cai et al., 2004; Zhang et al., 2005). In addition, venom from Pimpla hypochondriaca has been shown to be
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cytotoxic to hemocytes from host species and has a potent anti-hemocyte action that can impair hemocyte-mediated immune responses (Richards and Parkinson, 2000). Here we investigated the influence of the venom/calyx fluid on host Plutella xylostella hemocyte morphology and viability, in order to determine whether venom alone can suppress the host immune defense in this endoparasitoid that contains PDVs. 2. Materials and methods 2.1. Insect collection and rearing Initially, pupae and parasitized larvae of P. xylostella were collected from cabbage fields in the suburbs of Hangzhou, Zhejiang province, China. Subsequently, both P. xylostella and its endoparasitoid, Cotesia plutellae colony were raised on cabbage grown at 2571 1C, 65% relative humidity, and 14 h light:10 h dark. Once emerged, wasps were fed a 20% (v/v) honey solution and propagated using P. xylostella larvae. 2.2. Preparation of crude venom and calyx fluid About 3- or 4-day-old female adults were used for the collection of venom, as described by Parkinson and Weaver (1999). Prepared wasps were individually swabbed with 95% ethanol (v/v), dried and then the venom reservoirs were carefully removed from the abdomen of female wasps with the aid of a dissecting microscope (Leica MZ 12.5) and placed into 40 ml sterile phosphate buffered saline (PBS) (pH 7.4, 0.138 M NaCl, 0.0027 M KCl, 0.0073 M Na2HPO4, 0.00147 M KH2PO4) in a 0.5 ml eppendorf tube on ice. The reservoirs were then torn open with forceps to release the venom and then centrifuged at 12,000g for 10 min at 4 1C. The venom supernatant was transferred to a clean eppendorf tube and stored frozen at 70 1C. Routinely, 40 venom glands were dissected into 20 ml PBS. Prior to formal tests, the venom was diluted in PBS to concentrations of 2, 1, 0.5, and 0.25 venom reservoir equivalents (VRE) (one venom reservoir equivalent being defined as the supernatant from one torn venom reservoir in 1 ml PBS). To obtain calyx fluid, ovaries from female adults were removed in PBS, the calyces of oviduct were punctured with forceps and the contents allowed to diffuse into PBS. The calyx fluid solution was collected in a 0.5 ml eppendorf tube on ice. To remove eggs and cell debris, the suspension was centrifuged three times at 800g for 10 min each and stored at 70 1C. Prior to formal tests, the calyx fluid was diluted in PBS to concentrations of 2, 1, 0.5, and 0.25 female equivalents (FE) (one female equivalent being defined as the supernatant from one pair of torn ovary in 1 ml PBS). For the cocktail of calyx fluid and venom, 10 ml crude venom and 10 ml calyx fluid were mixed and diluted in PBS to concentrations of 2, 1, 0.5, and 0.25 female equivalents (FE).
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2.3. Analysis of proteins from venom and calyx fluid The venom and calyx fluid proteins from 3- or 4-day-old females were analyzed using sodium dodecyl sulfatepolyacrylimide gel electrophoresis (SDS-PAGE). Gels containing 12% acrylamide were run on a DYCZ-24D electrophoresis tank at 10 mA for 3 h, then visualized with 1% coomassie brilliant blue R250 in a solution of methanol, acetic acid, and water (5:1:4). Samples were diluted with sample buffer (0.125 M Tris pH 6.8, 12% (v/v) glycerol, 0.002% (w/v) bromophenol blue, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol), boiled for 5 min and run. The protein profile was analyzed by Quantity One 1D analysis software version 4.6 (Bio-Rad). Venom protein was quantified spectrophotometrically by the method of Bradford (1976).
2.4. Venom/calyx fluid activity to insect hemocytes Late third instar P. xylostella larvae were individually swabbed with 95% ethanol (v/v), dried and a small cut was made in a mid-proleg. Hemolymph was collected into a sterile microcentrifuge tube containing 1 ml anticoagulant solution (98 mM NaOH, 145 mM NaCl, 17 mM EDTA, 41 mM citric acid, pH 4.5; Mead et al., 1986), and approximately 50 ml of hemolymph was added to the tube as quickly as possible to minimize exposure to air. After mixing by gently inverting the tube, the hemocyte suspension was centrifuged at 250g for 2.5 min at room temperature, the supernatant removed, and the hemocytes were resuspended in 1 ml TC-100 insect tissue culture (Sigma, USA) (PH 6.3), consisting of 2.02 g TC-100 medium powder, 0.1 mg streptomycin sulfate and 10,000 units of ampicillin per 100 ml medium. The centrifugation step was repeated and the hemocyte pellet resuspended in 10 ml TC-100 containing 6 mM phenyl thiocarbamide (PTC, a phenoloxidase inhibitor) and 100 mg/ml ampicillin. Two microliters of this hemocyte suspension was then added into each well of 96-well plates containing 50 ml TC100 insect tissue culture medium with 10% fetal bovine serum per well. Then, a 2 ml aliquot of each dilution of venom or PBS as the control was applied to each well of the plate to reach the final venom doses of 0, 0.005, 0.01, 0.02, 0.04 VRE/ml, respectively. Hemocytes with or without venom treatment were incubated at 27 1C until observations were made. After incubation for 1 and 4 h, the spreading of hemocytes was observed using an inverted phase contrast microscope (Leica DM IRB). Finally, counting all hemocytes in three randomly chosen fields of view at 200 magnification gave the counts of spreading plasmatocytes and granular cells, which were identified according to the criteria of Gupta (1979). The spreading percentage of plasmatocytes and granular cells were calculated as follows: % spreading ¼ (number of spreading plasmatocytes or granular cells observed)/(total number of spreading and non-spreading plasmatocytes or granular
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cells observed) 100. Five wells were evaluated for each venom concentration in three replicates. In experiments examining the calyx fluid, the calyx fluid or the cocktail of calyx fluid and venom was added to the hemocytes as above. Hemocyte viability was determined by vital staining using trypan blue, as described by Rivers et al. (1993). First, the tissue culture medium was removed and then the wells were washed three times with PBS. About 50 ml TC100 and 50 ml 0.04% (w/v) trypan blue were added and incubated at 27 1C for 15 min. Finally, trypan blue was removed and TC-100 was added and the cells were observed with an inverted phase contrast microscope (Leica DM IRB). Then, counts of viable and dead hemocytes, determined by the criteria that viable cells exclude the dye and dead ones appear blue, were scored in a manner similar to counting the spreading or nonspreading hemocytes.
of venom protein from 20 parasitoids indicated an average value of 0.16 mg of protein per venom sac. Seventeen protein bands were found in the calyx fluid, with molecular weights ranging from 15 to 172 kDa; five bands including 84.3, 71.7, 54.8, 47.8, 27.4, and 21.4 kDa, were relatively abundant (Fig. 1B). A comparison of the two types of gels showed that venom proteins shared molecular weight similarities with calyx fluid in seven bands, 112.3, 70.5, 60.6, 46.1, 43.3, 27.4, and 21.4 kDa. 3.2. Effects of venom on hemocyte morphology in vitro When incubated in TC-100 without venom or calyx fluid in sterile 96-well plates, P. xylostella hemocytes exhibit high viability; approximately 94% engaged in spreading (Figs. 2A, 4A). For the first 30 min, the majority of hemocytes remained in a rounded configuration (Fig. 3A),
2.5. Data analysis
0 VRE/µl 0.005 VRE/µl
Mean results for different treatments for the same time were compared using one-way analysis of variance (ANOVA) and Duncan’s multiple range tests. All calculations were performed using the DPSr package (Tang and Feng, 2002). All tests were considered significant at Pp0.05.
0.01 VRE/µl 0.02 VRE/µl 0.04 VRE/µl 100
3. Results
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3.1. Venom and calyx fluid protein components of C. plutellae To clarify the protein components in the venom and calyx fluid of C. plutellae, SDS-PAGE electrophoresis was performed. Analysis of the 12% SDS-PAGE profile revealed the presence of 20 protein bands in the venom with molecular weights ranging from 8 to 300 kDa; nine protein bands including 112.3, 82.3, 63.8, 60.6, 45.1, 27.7, 26.4, 24.2, and 10.2 kDa, were relatively abundant (Fig. 1A). Quantification
Spreading (%)
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0 1h
4h Time after treatment
(A) 100
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Spreading (%)
80 60 40 20 0 1h (B)
Fig. 1. SDS-PAGE profiles of venom (A) and calyx fluid (B) from Cotesia plutellae. M: marker, Cv: venom; Cf: calyx fluid.
4h Time after treatment
Fig. 2. Effects of venoms from Cotesia plutellae at various concentrations on the spreading ability of plasmatocytes and granular cells of Plutella xylostella, respectively (A and B). Data are expressed as the mean7standard deviation (n ¼ 15). For each treatment time, values followed by different letters are significantly different (P%p0.05) according to ANOVA and Duncan’s multiple range tests.
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after which granular cells and plasmatocytes began to spread (Fig. 3B). After 4 h, the majority of plasmatocytes spread extensively with a flat elliptoid or angular shape, and a great number of granular cells had extended large pseudopods (Fig. 3C). Morphological examination of plasmatocytes exposed for 1 h to serially diluted venom revealed inhibition of spreading. This became more severe as the concentration of venom increased (Fig. 2A,3D–F). Venom concentrations of 0.04 VRE/ml displayed significantly inhibitory effects on the spreading of plasmatocytes (approximately 86%) (Fig. 2A), and almost all of the hemocytes remained round (Fig. 3F). At venom concentrations below 0.04 VRE/ml, the venom was less potent in blocking the spreading of plasmatocytes and application of venom at a concentration of 0.005 VRE/ml had virtually no effect on hemocyte spreading when compared with non-venom-treated controls. When incubated for 4 h, a large number of plasmatocytes spread extensively (Fig. 2B, 3G–I), although the spreading of most of these plasmatocytes was severely inhibited, perhaps it was due to recovery of the spreading ability. However, no effects were observed on granular cells, which spread normally after treatment with serially diluted venom (Fig. 3D–I). 3.3. Effects of venom on hemocyte viability in vitro When exposed to venom at high concentrations, especially at 0.02 and 0.04 VRE/ml venom, viability of
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plasmocytes, and granular cells decreased. Although the decease was not so obviously visually, it was statistically significant. When hemocytes were incubated with 0.04 VRE/ml venom for more than 4 h, viability of plasmocytes decreased from 93.6972.56% in the control to 74.1371.91%, and mortality of granular cells increased from 5.8571.75% (control) to 16.3573.26% (Fig. 4A). 3.4. Effects of calyx fluid on hemocyte morphology in vitro The calyx fluid from C. plutellae displayed significant inhibitory effects on the spreading of plasmatocytes and granular cells of P. xylostella. At higher calyx fluid dilutions, almost all of the hemocytes remained rounded, while the hemocytes in the control spread extensively (Fig. 5A, 5B). At concentrations of 0.04 FE/ml most hemocytes appeared to be multivesicular bodies (Fig. 5C) and others were observed with intracellular vaculoles (Fig. 5E). After 4 h, hemocytes were visibly damaged, and damage became more severe as the concentration of calyx fluid increased. After that time period, concentrations of 0.01 FE/ml caused disintegration of the plasma membrane (Fig. 5D), and at concentrations of 0.04 FE/ml, granular cells were badly damaged (Fig. 5F). Plasmocytes appeared to undergo lysis or deterioration (Fig. 5(H), and only remnants of the nucleus were left (Fig. 5I). In addition, large amounts of cell debris were present in the tissue culture medium with calyx fluid (Fig. 5D, 5H, 5I), while
Fig. 3. Phase contrast micrographs of Plutella xylostella hemocytes in vitro with different concentrations of venom from Cotesia plutellae. A, B, C: hemocytes without venom treatment after 0 h (A), 1 h (B) and 4 h (C); D, E, F: hemocytes at venom concentrations of 0.01 VRE/ml (D), 0.02 VRE/ml (F), and 0.04 VRE/ml (H) for 1 h. The spreading of plasmatocytes was inhibited and this became more severe as the venom concentration increased. G, H, I: hemocytes at venom concentrations of 0.01 VRE/ml (D), 0.02 VRE/ml (F), and 0.04 VRE/ml (H) for 4 h. Most hemocytes spread extensively. Scale bar ¼ 20 mm.
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Fig. 4. Effects of venom and calyx fluid from Cotesia plutellae at various concentrations on the viability of hemocytes of Plutella xylostella, respectively (A and B). For each treatment time, values followed by different letters are significantly different (P%p0.05) according to ANOVA and Duncan’s multiple range tests.
that of controls were clear. The same results were observed when the cells were incubated in calyx fluid together with venom. 3.5. Effects of calyx fluid on hemocyte viability in vitro The calyx fluid of C. plutellae had damaging effects on plasmocytes and granular cells of P. xylostella (Fig. 4B). Overall viability of plasmocytes and granular cells was less than 40% after 4 h. As calyx fluid (0.04 FE/ml) was added, most of the hemocytes began to disintegrate into multivesicular bodies (Fig. 5C) and appeared to be inactive. Subsequently many cells were extensively damaged (Fig. 5F, 5I), and viability decreased from 95.3771.95% in the control to 21.6373.20% after 4 h. Again, the same results were observed when the cells were incubated in calyx fluid together with venom. 4. Discussion Until recently venom was perceived to play a minor role in mediating host immune suppression in PDV-producing endoparasitoids because this function was thought to be mediated primarily by the calyx-derived virus (Beckage, 1998; Asgari, 2006). For example, venom from Campoletis sonorensi that contains PDVs had no visible function in
parasitism of Heliothis virescens (Webb and Luckhart, 1994). Venom synergizes the effect of calyx fluid or polydnavirus in several systems, such as C. kariyai/ Pseudaletia separate, in which neither venom nor calyx fluid alone can affect granular cells, while venom together with calyx fluid can inhibit the spreading ability and eventually cause disintegration (Wago and Tanaka, 1989). Only in the C. glomeratus/P. rapae system was venom shown to prevent encapsulation of eggs on its own (Kitano, 1986). On the contrary, in endoparasitoids devoid of symbiotic viruses, such as P. puparum, N. vitripennis, and P. hypochondriaca, venom can suppress the immune system as a result of a compensatory adaptation to the lack of other immune suppressive factors in the calyx fluid (Richards and Parkinson, 2000; Cai et al., 2004; Zhang et al., 2005). Here we have demonstrated that C. plutellae venom has subtle direct effects on hemocytes from P. xylostella. Microscopic examination of hemocytes revealed that C. plutellae venom can suppress the spreading of plasmatocytes markedly at the early phase of incubation. When venom concentrations increased, the inhibition of spreading became more severe. Spreading of plasmatocytes was almost absent at venom concentration of 0.04 VRE/ml, but abundant at venom concentrations of 0.005 VRE/ml and control. The viability of plasmocytes and granular cells was also decreased at high venom concentrations. This suggests that increased concentrations of venom proteins not only affected spreading but also viability after exposure over extended time periods. However, almost all of the plasmatocytes recovered after 4 h, and most of hemocytes appeared active in spreading with bright phase. Perhaps it was due to the loss of the suppression function of venom, or to the recovery of immune functions of hemocytes or to the function of different immune suppressors at different times. For example, the observation of reversible effects of venom probably corroborates observations in C. rubecula, where transient expression of a polydnavirus gene causes transient inactivation (Asgari et al., 1996), although it is not clear yet if the same mechanism exists in venom proteins. Stoltz and Guzo (1986) reported the recovery of immunocompetence in the Hyposoter fugitives/Malacosoma disstria system. Webb and Summers (1990) suggested that venom might protect the egg from the host immune reaction in the period between oviposition and the expression of PDV genes. As summarized in Fig. 4B, the calyx fluid from C. plutellae had highly adverse effects on hemocytes of P. xylostella. For example, the mortality of hemocytes was reduced by 50% or more (Fig. 4B). In each dose of calyx fluid, some hemocytes were damaged while others were unaffected (Fig. 5H), which suggests a more selective action of the calyx fluid. Additionally, we demonstrated that the calyx fluid caused extensive damage to hemocytes, with large amounts of cell debris appearing in the culture medium, including disintegration, damaged plasma membranes (Fig. 5D), the formation of multivesicular bodies,
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Fig. 5. Phase contrast micrographs of Plutella xylostella hemocytes in vitro with different concentrations of calyx fluid from Cotesia plutellae. A, D: Most of hemocyte remained rounded after incubated with 0.01 FE/ml calyx fluids for 1 h (A), the disintegration of the plasma membrane occurred after 4 h (D, arrows); B, H: The majority of hemocytes remained rounded after incubation with 0.02 FE/ml calyx fluids for 1 h (A), and many plasmatocytes began to be damaged after 4 h (H, arrows). Cell debris was noticeable (black dots); C, E, F, I: Multivesicular bodies (C) and intracellular vaculoles (E), appeared after incubation with 0.04 FE/ml calyx fluids for 1 h. Many granular cells and plasmatocytes were badly damaged (F, arrows) and so only remnants of the nucleus remained after 4 h (I, arrows). Scale bar ¼ 20 mm.
(Fig. 5C) and intracellular vaculoles (Fig. 5E), which were also observed in hemocytes incubated with P. hypochondriaca venom (Richards and Parkinson, 2000). Thus, the calyx fluid from C. plutellae appears to be able to exert cytotoxic effects on P. xylostella hemocytes. The observation corroborates the phenomenon that the continued expression of a related immune suppressing agent in C. congregata causes apoptosis of hemocytes (Le et al., 2003). Several PDV proteins have been directly linked to haemocytic effects (Glatz et al., 2004a), including disruption of encapsulation (Zhang and Wang, 2003; Turnbull et al., 2004), disruption of hemocyte activity (Beckage, 1998; Le et al., 2003; Turnbull et al., 2004; Glatz et al., 2004b) and hemocyte cytoskeleton degradation (Li and Webb, 1994; Asgari and Schmidt, 2002; Zhang and Wang, 2003). Our findings reveal that venom of C. plutellae alone may not be sufficient to suppress the host immune system, although it can inhibit the spreading of plasmocytes of P. xylostella during the early phase of incubation. By comparison with the effect of calyx fluid on the viability of hemocytes, the effect of venom appeared to be much less. In addition, venom had no effect on granular cells, which spread normally all the time. On the contrary, the calyx fluid had significant
effects on host hemocytes, even at low concentrations. Obviously venom of C. plutellae might also synergize the effects of calyx fluid or polydnavirus like in other systems. Venom from C. melanoscela was reported to promote the release of virions into the cytoplasm after uptake by host cells facilitating the uncoating of PDVs at nuclear pores in host hemocytes (Stoltz et al., 1988). Venom proteins may mediate short-term immune suppression before PDV gene expression becomes effective, as in C. sonorensis (Webb and Luckhart, 1994). In some parasitoid systems, venom proteins are essential for polydnavirus infection and gene expression, such as C. glomeratus (Kitano, 1986) and C. kariyai (Tanaka, 1987). In C. rubecula, a 1598-Da peptide (Vn1.5) isolated from venom was required for the expression of C. rubecula bracoviruses (CrBVs) in host hemocytes (P. rapae) (Zhang et al., 2004) and two other proteins (Vn4.6 and Vn50) from venom can synergize with an immune suppressive protein from C. rubecula PDVs (Asgari et al., 1996, 1997) and a calyx protein (Asgari et al., 1998, 2003b) to inhibit the host immune response (Asgari et al., 2003a). In addition, venom proteins enhance virus persistence in the host. However, the mechanism of action of these peptides in promoting expression of PDV genes is not known.
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Although we have not isolated a peptide of similar size to these proteins in the present study, specific peptides may exist in the venom of C. plutellae. SDS analyses revealed that several peptides of similar size were present in the two resources (Fig. 1), which may imply a close structural and functional relationship (Asgari, 2006). Webb and Summers (1990) showed that venom proteins from C. sonorensis share epitopes with PDV structural proteins. Asgari et al. (2003a) found the venom gene homologs in the viral genome and revealed that viral genes show tissue-specific coordination of expression with venom proteins. These results could indicate that proteins from venom and calyx tissues interact but have to be apart before oviposition (Asgari, 2006). It was interesting that application of an isolated C. rubecula immune suppressor to hemocytes from Pieris rapae did not show any effects unless the protein was injected into the hemocoel. Recent experiments suggested that the immune suppressor has to interact with lipophorin particles first to exert its effects (Asgari and Schmidt, 2002), and Schmidt et al. (2005) proposed some dimeric and oligomeric adhesion molecules which are able to cross-link receptors on the cell surface and depolymerise actin by leverage-mediated clearance reactions in the hemolymph. Obviously the immune suppression mechanisms are remarkably diverse in different host-parasitoid systems. The possible functional mechanism of the immune suppressors in C. plutellae/P. xylostella system is still largely unknown and needs further research work. Acknowledgments We are grateful to Professor Chen-zhu Wang (Beijing) for his critical review of an early draft of the manuscript. We also thank two anonymous reviewers for their valuable comments. Funding for this study was provided jointly by national natural science foundation of China (30370959), 973 Program (2006CB102005), program for new century excellent talents in University (NCET-04-0521) and innovation research team program of the ministry of education of China (IRT0355). References Asgari, S., 2006. Venom proteins from polydnavirus-producing endoparasitoids: their role in host-parasite interactions. Archives of Insect Biochemistry and Physiology 61, 146–156. Asgari, S., Schmidt, O., 2002. A coiled-coil region of an insect immune suppressor protein is involved in binding and uptake by hemocytes. Insect Biochemistry and Molecular Biology 32, 497–504. Asgari, S., Hellers, M., Schmidt, O., 1996. Host hemocyte inactivation by an insect parasitoid: transient expression of a polydnavirus gene. Journal of General Virology 77, 2653–2662. Asgari, S., Schmidt, O., Theopold, U., 1997. A polydnavirus-encoded protein of an endoparasitoid wasp is an immune suppressor. Journal of General Virology 78, 3061–3070. Asgari, S., Theopold, U., Wellby, C., Schmidt, O., 1998. A protein with protective properties against the cellular defence reactions in insects. Proceedings of the National Academy of Sciences of the USA 95, 3690–3695.
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ARTICLE IN PRESS R.-x. Yu et al. / Journal of Insect Physiology 53 (2007) 22–29 physiological state of Pseudaletia separate (Lepidoptera: Noctuidae) host as food resource. Journal of Insect Physiology 49, 149–159. Richards, E.H., Parkinson, N.M., 2000. Venom from the endoparasitic wasp Pimpla hypochondriaca adversely affects the morphology, viability, and immune function of hemocytes from larvae of the tomato moth, Lacanobia oleracea. Journal of Invertebrate Pathology 76, 33–42. Rivers, D.B., Hink, W.F., Denlinger, D.L., 1993. Toxicity of the venom from Nasonia vitripennis (Hymenoptera: Pteromalidae) toward fly hosts, nontarget insects, different developmental stage, and cultured insect cells. Toxicon 31, 755–765. Rizki, T.M., Rizki, R.M., 1994. Parasitoid-induced cellular immune deficiency in Drosophila. Annals of the New York Academy of Sciences 712, 178–194. Russo, J., Brehelin, M., Carton, Y., 2001. Haemocyte changes in resistant and susceptible strains of D melanogaster caused by virulent and avirulent strains of the parasitic wasp Leptopilina boulardi. Journal of Insect Physiology 47, 167–172. Schmidt, O., Glatz, R.V., Asgari, S., Roberts, H.S.L., 2005. Archives of Insect Biochemistry and Physiology 60, 153–158. Shelby, K.S., Webb, B.A., 1999. Polydnavirus-mediated suppression of insect immunity. Journal of Insect Physiology 45, 507–514. Stettler, P., Trenczek, T., Wyler, T., Wilhelm, P.R., Lanzrein, B., Pennacchio, F., Vinson, B.S., 1998. Overview of parasitism associated effects on host haemocytes in larval parasitoids and comparison with effects of the egg-larval parasitoid Chelonus inanitus on its host Spodoptera littoralis. Journal of Insect Physiology 44, 817–831. Stoltz, D.B., Guzo, D., 1986. Apparent haemocytic transformations associated with parasitoid-induced inhibition of immunity in Malacosoma disstria larvae. Journal of Insect Physiology 32, 377–388. Stoltz, D.B., Guzo, D., Belland, E.R., Lucarotti, C.J., Mackinnon, E.A., 1988. Venom promotes uncoating in vitro and persistence in vivo of DNA from a braconid polydnavirus. Journal of General Virology 69, 903–907. Strand, M.R., Dover, B.A., 1991. Developmental disruption of Pseudoplusia includens and Heliothis virescens larvae by the calyx fluid and
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venom of Microplitis demolitor. Archives of Insect Biochemistry and Physiology 18, 131–145. Strand, M.R., Pech, L.L., 1995. Immunological basis for compatibility in parasitoid-host relationships. Annual Review of Entomology 40, 31–56. Tanaka, T., 1987. Effect of the venom of the endoparasitoid Apanteles kariyai Watanabe, on the cellular defence reaction of the host, Pseudaletia separata Walker. Journal of Insect Physiology 33, 413–420. Tang, Q.Y., Feng, M.G., 2002. DPSr Data Processing System for Practical Statistics. Science Press, Beijing, China. Turnbull, M.W., Martin, S.B., Webb, B.A., 2004. Quantitative analysis of hemocyte morphological abnormalities associated with Campoletis sonorensis parasitization. Journal of Insect Science 4, 1–15. Wago, H., Tanaka, T., 1989. Synergistic effects of calyx fluid and venom of Apanteles kariyai Watanabe (Hymenoptera: Braconidae) on the granular cells of Pseudaletia separata Walker (Lepidoptera: Noctuidae). Zoological Science 6, 691–696. Webb, B.A., Luckhart, S., 1994. Evidence for an early immunosuppressive role for related Campoletis sonorensis venom and ovarian proteins in Heliothis virescens. Archives of Insect Biochemistry and Physiology 26, 147–163. Webb, B.A., Summers, M.D., 1990. Venom and viral expression products of the endoparasitic wasp Campoletis sonorensis share epitopes and related sequences. Proceedings of the National Academy of Sciences of the USA 87, 4961–4965. Zhang, C., Wang, C.Z., 2003. cDNA cloning and molecular characterization of a cysteine-rich gene from Campoletis chlorideae polydnavirus. DNA Sequence 14, 413–419. Zhang, G.M., Schmidt, O., Asgari, S., 2004. A novel venom peptide from an endoparasitoid wasp is required for expression of polydnavirus genes in host hemocytes. Journal of Biological Chemistry 279, 41580–41585. Zhang, Z., Ye, G.Y., Cai, J., Hu, C., 2005. Comparative venom toxicity between Pteromalus puparum and Nasonia vitripennis (Hymenoptera: Pteromalidae) toward the hemocytes of their natural hosts, non-target insects and cultured insect cells. Toxicon 46, 337–349.
Journal of Insect Physiology 53 (2007) 22–29 www.elsevier.com/locate/jinsphys
Effects of venom/calyx fluid from the endoparasitic wasp Cotesia plutellae on the hemocytes of its host Plutella xylostella in vitro Rui-xian Yu, Ya-Feng Chen, Xue-xin Chen, Fang Huang, Yong-gen Lou, Shu-sheng Liu Institute of Insect Sciences, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, China Received 9 April 2006; received in revised form 22 September 2006; accepted 29 September 2006
Abstract Crude venom and calyx fluid from Cotesia plutellae (Hymenoptera Braconidae) were assayed for biological activity toward hemocytes of Plutella xylostella (Lepidoptera Plutellidae). Venom from C. plutellae displayed high activity toward the spreading of plasmatocytes of P. xylostella early in the incubation period, and the inhibition was more severe as the concentration of venom increased. However, most inhibited hemocytes spread normally after being incubated for 4 h. No effects were found toward granular cells from the host. Additionally, the venom from C. plutellae had some lethal effects on hemocytes of P. xylostella at high concentrations. In contrast, when incubated with different concentrations of calyx fluid, the spreading of some hemocytes was inhibited, some began to disintegrate, and some were badly damaged with only the nucleus left. After 4 h, the majority of hemocytes died. The same results were observed when hemocytes were incubated in calyx fluid together with venom. These results show that calyx fluid from C. plutellae may play a major role in the suppression of the host immune system, whereas venom from C. plutellae has a limited effect on hemocytes and probably synergizes the effect of calyx fluid or polydnavirus. r 2006 Elsevier Ltd. All rights reserved. Keywords: Cotesia plutellae; Plutella xylostella; Venom; Calyx fluid; Hemocytes; Immunosuppression
1. Introduction The insect immune system is classified into humoral and cellular defense responses, in which granular cells and plasmatocytes are the main hemocyte types reported to be active (Lavine and Strand, 2002). To develop successfully in the hemocoel of their insect hosts, endoparasitoids escape or suppress the host immune system by maternal secretions from the adult female wasp introduced into the host during oviposition. These secretions include polydnaviruses (PDVs) or virus-like particles (VLPs), soluble ovarian proteins, and venom (Strand and Pech, 1995; Beckage, 1998; Shelby and Webb, 1999). Great progress has been made in the elucidation of the importance of PDVs, VLPs, and ovarian proteins in suppressing the host immune defenses, while much less is known about venom proteins, especially from endoparasitoid venom (Beckage et al., 1990; Beckage, 1998; Stettler et al., 1998; Rizki and Corresponding author. Tel.: +86 571 86971219.
E-mail address: [email protected] (X.-x. Chen). 0022-1910/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2006.09.011
Rizki, 1994; Russo et al., 2001; Luckhart and Webb, 1996; Webb and Luckhart, 1994; Glatz et al., 2004a, b; Zhang et al., 2004; Morales et al., 2005). Many ectoparasitoids that have been studied induce permanent paralysis, host development arrest, host metabolic regulation, and inhibition of host immunity (Nakamatsu and Tanaka, 2003), whereas the role of endoparasitoid venom in suppressing host immune defense has not been clearly determined. In many braconid species, action of the venom components is necessary to enhance the effects of PDV or calyx fluid (Tanaka, 1987; Davies et al., 1987; Strand and Dover, 1991; Gupta and Ferkovich, 1998; Beckage and Gelman, 2004). However, a limited number of studies suggest that the venom from endoparasitoids devoid of symbiotic viruses may alone perturb host immune defenses. For example, venoms from Pteromalus puparum (Hymenoptera: Pteromalidae) and Nasonia vitripennis (Hymenoptera: Pteromalidae) suppress cellular immune responses in their hosts, Pieris rapae and Papilio xuthus (Cai et al., 2004; Zhang et al., 2005). In addition, venom from Pimpla hypochondriaca has been shown to be
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cytotoxic to hemocytes from host species and has a potent anti-hemocyte action that can impair hemocyte-mediated immune responses (Richards and Parkinson, 2000). Here we investigated the influence of the venom/calyx fluid on host Plutella xylostella hemocyte morphology and viability, in order to determine whether venom alone can suppress the host immune defense in this endoparasitoid that contains PDVs. 2. Materials and methods 2.1. Insect collection and rearing Initially, pupae and parasitized larvae of P. xylostella were collected from cabbage fields in the suburbs of Hangzhou, Zhejiang province, China. Subsequently, both P. xylostella and its endoparasitoid, Cotesia plutellae colony were raised on cabbage grown at 2571 1C, 65% relative humidity, and 14 h light:10 h dark. Once emerged, wasps were fed a 20% (v/v) honey solution and propagated using P. xylostella larvae. 2.2. Preparation of crude venom and calyx fluid About 3- or 4-day-old female adults were used for the collection of venom, as described by Parkinson and Weaver (1999). Prepared wasps were individually swabbed with 95% ethanol (v/v), dried and then the venom reservoirs were carefully removed from the abdomen of female wasps with the aid of a dissecting microscope (Leica MZ 12.5) and placed into 40 ml sterile phosphate buffered saline (PBS) (pH 7.4, 0.138 M NaCl, 0.0027 M KCl, 0.0073 M Na2HPO4, 0.00147 M KH2PO4) in a 0.5 ml eppendorf tube on ice. The reservoirs were then torn open with forceps to release the venom and then centrifuged at 12,000g for 10 min at 4 1C. The venom supernatant was transferred to a clean eppendorf tube and stored frozen at 70 1C. Routinely, 40 venom glands were dissected into 20 ml PBS. Prior to formal tests, the venom was diluted in PBS to concentrations of 2, 1, 0.5, and 0.25 venom reservoir equivalents (VRE) (one venom reservoir equivalent being defined as the supernatant from one torn venom reservoir in 1 ml PBS). To obtain calyx fluid, ovaries from female adults were removed in PBS, the calyces of oviduct were punctured with forceps and the contents allowed to diffuse into PBS. The calyx fluid solution was collected in a 0.5 ml eppendorf tube on ice. To remove eggs and cell debris, the suspension was centrifuged three times at 800g for 10 min each and stored at 70 1C. Prior to formal tests, the calyx fluid was diluted in PBS to concentrations of 2, 1, 0.5, and 0.25 female equivalents (FE) (one female equivalent being defined as the supernatant from one pair of torn ovary in 1 ml PBS). For the cocktail of calyx fluid and venom, 10 ml crude venom and 10 ml calyx fluid were mixed and diluted in PBS to concentrations of 2, 1, 0.5, and 0.25 female equivalents (FE).
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2.3. Analysis of proteins from venom and calyx fluid The venom and calyx fluid proteins from 3- or 4-day-old females were analyzed using sodium dodecyl sulfatepolyacrylimide gel electrophoresis (SDS-PAGE). Gels containing 12% acrylamide were run on a DYCZ-24D electrophoresis tank at 10 mA for 3 h, then visualized with 1% coomassie brilliant blue R250 in a solution of methanol, acetic acid, and water (5:1:4). Samples were diluted with sample buffer (0.125 M Tris pH 6.8, 12% (v/v) glycerol, 0.002% (w/v) bromophenol blue, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol), boiled for 5 min and run. The protein profile was analyzed by Quantity One 1D analysis software version 4.6 (Bio-Rad). Venom protein was quantified spectrophotometrically by the method of Bradford (1976).
2.4. Venom/calyx fluid activity to insect hemocytes Late third instar P. xylostella larvae were individually swabbed with 95% ethanol (v/v), dried and a small cut was made in a mid-proleg. Hemolymph was collected into a sterile microcentrifuge tube containing 1 ml anticoagulant solution (98 mM NaOH, 145 mM NaCl, 17 mM EDTA, 41 mM citric acid, pH 4.5; Mead et al., 1986), and approximately 50 ml of hemolymph was added to the tube as quickly as possible to minimize exposure to air. After mixing by gently inverting the tube, the hemocyte suspension was centrifuged at 250g for 2.5 min at room temperature, the supernatant removed, and the hemocytes were resuspended in 1 ml TC-100 insect tissue culture (Sigma, USA) (PH 6.3), consisting of 2.02 g TC-100 medium powder, 0.1 mg streptomycin sulfate and 10,000 units of ampicillin per 100 ml medium. The centrifugation step was repeated and the hemocyte pellet resuspended in 10 ml TC-100 containing 6 mM phenyl thiocarbamide (PTC, a phenoloxidase inhibitor) and 100 mg/ml ampicillin. Two microliters of this hemocyte suspension was then added into each well of 96-well plates containing 50 ml TC100 insect tissue culture medium with 10% fetal bovine serum per well. Then, a 2 ml aliquot of each dilution of venom or PBS as the control was applied to each well of the plate to reach the final venom doses of 0, 0.005, 0.01, 0.02, 0.04 VRE/ml, respectively. Hemocytes with or without venom treatment were incubated at 27 1C until observations were made. After incubation for 1 and 4 h, the spreading of hemocytes was observed using an inverted phase contrast microscope (Leica DM IRB). Finally, counting all hemocytes in three randomly chosen fields of view at 200 magnification gave the counts of spreading plasmatocytes and granular cells, which were identified according to the criteria of Gupta (1979). The spreading percentage of plasmatocytes and granular cells were calculated as follows: % spreading ¼ (number of spreading plasmatocytes or granular cells observed)/(total number of spreading and non-spreading plasmatocytes or granular
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cells observed) 100. Five wells were evaluated for each venom concentration in three replicates. In experiments examining the calyx fluid, the calyx fluid or the cocktail of calyx fluid and venom was added to the hemocytes as above. Hemocyte viability was determined by vital staining using trypan blue, as described by Rivers et al. (1993). First, the tissue culture medium was removed and then the wells were washed three times with PBS. About 50 ml TC100 and 50 ml 0.04% (w/v) trypan blue were added and incubated at 27 1C for 15 min. Finally, trypan blue was removed and TC-100 was added and the cells were observed with an inverted phase contrast microscope (Leica DM IRB). Then, counts of viable and dead hemocytes, determined by the criteria that viable cells exclude the dye and dead ones appear blue, were scored in a manner similar to counting the spreading or nonspreading hemocytes.
of venom protein from 20 parasitoids indicated an average value of 0.16 mg of protein per venom sac. Seventeen protein bands were found in the calyx fluid, with molecular weights ranging from 15 to 172 kDa; five bands including 84.3, 71.7, 54.8, 47.8, 27.4, and 21.4 kDa, were relatively abundant (Fig. 1B). A comparison of the two types of gels showed that venom proteins shared molecular weight similarities with calyx fluid in seven bands, 112.3, 70.5, 60.6, 46.1, 43.3, 27.4, and 21.4 kDa. 3.2. Effects of venom on hemocyte morphology in vitro When incubated in TC-100 without venom or calyx fluid in sterile 96-well plates, P. xylostella hemocytes exhibit high viability; approximately 94% engaged in spreading (Figs. 2A, 4A). For the first 30 min, the majority of hemocytes remained in a rounded configuration (Fig. 3A),
2.5. Data analysis
0 VRE/µl 0.005 VRE/µl
Mean results for different treatments for the same time were compared using one-way analysis of variance (ANOVA) and Duncan’s multiple range tests. All calculations were performed using the DPSr package (Tang and Feng, 2002). All tests were considered significant at Pp0.05.
0.01 VRE/µl 0.02 VRE/µl 0.04 VRE/µl 100
3. Results
a
a
a
a
a
a
a
3.1. Venom and calyx fluid protein components of C. plutellae To clarify the protein components in the venom and calyx fluid of C. plutellae, SDS-PAGE electrophoresis was performed. Analysis of the 12% SDS-PAGE profile revealed the presence of 20 protein bands in the venom with molecular weights ranging from 8 to 300 kDa; nine protein bands including 112.3, 82.3, 63.8, 60.6, 45.1, 27.7, 26.4, 24.2, and 10.2 kDa, were relatively abundant (Fig. 1A). Quantification
Spreading (%)
80 60 40
b
b
20
c
0 1h
4h Time after treatment
(A) 100
a
a
a
a
a
a
a
a
a
a
Spreading (%)
80 60 40 20 0 1h (B)
Fig. 1. SDS-PAGE profiles of venom (A) and calyx fluid (B) from Cotesia plutellae. M: marker, Cv: venom; Cf: calyx fluid.
4h Time after treatment
Fig. 2. Effects of venoms from Cotesia plutellae at various concentrations on the spreading ability of plasmatocytes and granular cells of Plutella xylostella, respectively (A and B). Data are expressed as the mean7standard deviation (n ¼ 15). For each treatment time, values followed by different letters are significantly different (P%p0.05) according to ANOVA and Duncan’s multiple range tests.
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after which granular cells and plasmatocytes began to spread (Fig. 3B). After 4 h, the majority of plasmatocytes spread extensively with a flat elliptoid or angular shape, and a great number of granular cells had extended large pseudopods (Fig. 3C). Morphological examination of plasmatocytes exposed for 1 h to serially diluted venom revealed inhibition of spreading. This became more severe as the concentration of venom increased (Fig. 2A,3D–F). Venom concentrations of 0.04 VRE/ml displayed significantly inhibitory effects on the spreading of plasmatocytes (approximately 86%) (Fig. 2A), and almost all of the hemocytes remained round (Fig. 3F). At venom concentrations below 0.04 VRE/ml, the venom was less potent in blocking the spreading of plasmatocytes and application of venom at a concentration of 0.005 VRE/ml had virtually no effect on hemocyte spreading when compared with non-venom-treated controls. When incubated for 4 h, a large number of plasmatocytes spread extensively (Fig. 2B, 3G–I), although the spreading of most of these plasmatocytes was severely inhibited, perhaps it was due to recovery of the spreading ability. However, no effects were observed on granular cells, which spread normally after treatment with serially diluted venom (Fig. 3D–I). 3.3. Effects of venom on hemocyte viability in vitro When exposed to venom at high concentrations, especially at 0.02 and 0.04 VRE/ml venom, viability of
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plasmocytes, and granular cells decreased. Although the decease was not so obviously visually, it was statistically significant. When hemocytes were incubated with 0.04 VRE/ml venom for more than 4 h, viability of plasmocytes decreased from 93.6972.56% in the control to 74.1371.91%, and mortality of granular cells increased from 5.8571.75% (control) to 16.3573.26% (Fig. 4A). 3.4. Effects of calyx fluid on hemocyte morphology in vitro The calyx fluid from C. plutellae displayed significant inhibitory effects on the spreading of plasmatocytes and granular cells of P. xylostella. At higher calyx fluid dilutions, almost all of the hemocytes remained rounded, while the hemocytes in the control spread extensively (Fig. 5A, 5B). At concentrations of 0.04 FE/ml most hemocytes appeared to be multivesicular bodies (Fig. 5C) and others were observed with intracellular vaculoles (Fig. 5E). After 4 h, hemocytes were visibly damaged, and damage became more severe as the concentration of calyx fluid increased. After that time period, concentrations of 0.01 FE/ml caused disintegration of the plasma membrane (Fig. 5D), and at concentrations of 0.04 FE/ml, granular cells were badly damaged (Fig. 5F). Plasmocytes appeared to undergo lysis or deterioration (Fig. 5(H), and only remnants of the nucleus were left (Fig. 5I). In addition, large amounts of cell debris were present in the tissue culture medium with calyx fluid (Fig. 5D, 5H, 5I), while
Fig. 3. Phase contrast micrographs of Plutella xylostella hemocytes in vitro with different concentrations of venom from Cotesia plutellae. A, B, C: hemocytes without venom treatment after 0 h (A), 1 h (B) and 4 h (C); D, E, F: hemocytes at venom concentrations of 0.01 VRE/ml (D), 0.02 VRE/ml (F), and 0.04 VRE/ml (H) for 1 h. The spreading of plasmatocytes was inhibited and this became more severe as the venom concentration increased. G, H, I: hemocytes at venom concentrations of 0.01 VRE/ml (D), 0.02 VRE/ml (F), and 0.04 VRE/ml (H) for 4 h. Most hemocytes spread extensively. Scale bar ¼ 20 mm.
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Fig. 4. Effects of venom and calyx fluid from Cotesia plutellae at various concentrations on the viability of hemocytes of Plutella xylostella, respectively (A and B). For each treatment time, values followed by different letters are significantly different (P%p0.05) according to ANOVA and Duncan’s multiple range tests.
that of controls were clear. The same results were observed when the cells were incubated in calyx fluid together with venom. 3.5. Effects of calyx fluid on hemocyte viability in vitro The calyx fluid of C. plutellae had damaging effects on plasmocytes and granular cells of P. xylostella (Fig. 4B). Overall viability of plasmocytes and granular cells was less than 40% after 4 h. As calyx fluid (0.04 FE/ml) was added, most of the hemocytes began to disintegrate into multivesicular bodies (Fig. 5C) and appeared to be inactive. Subsequently many cells were extensively damaged (Fig. 5F, 5I), and viability decreased from 95.3771.95% in the control to 21.6373.20% after 4 h. Again, the same results were observed when the cells were incubated in calyx fluid together with venom. 4. Discussion Until recently venom was perceived to play a minor role in mediating host immune suppression in PDV-producing endoparasitoids because this function was thought to be mediated primarily by the calyx-derived virus (Beckage, 1998; Asgari, 2006). For example, venom from Campoletis sonorensi that contains PDVs had no visible function in
parasitism of Heliothis virescens (Webb and Luckhart, 1994). Venom synergizes the effect of calyx fluid or polydnavirus in several systems, such as C. kariyai/ Pseudaletia separate, in which neither venom nor calyx fluid alone can affect granular cells, while venom together with calyx fluid can inhibit the spreading ability and eventually cause disintegration (Wago and Tanaka, 1989). Only in the C. glomeratus/P. rapae system was venom shown to prevent encapsulation of eggs on its own (Kitano, 1986). On the contrary, in endoparasitoids devoid of symbiotic viruses, such as P. puparum, N. vitripennis, and P. hypochondriaca, venom can suppress the immune system as a result of a compensatory adaptation to the lack of other immune suppressive factors in the calyx fluid (Richards and Parkinson, 2000; Cai et al., 2004; Zhang et al., 2005). Here we have demonstrated that C. plutellae venom has subtle direct effects on hemocytes from P. xylostella. Microscopic examination of hemocytes revealed that C. plutellae venom can suppress the spreading of plasmatocytes markedly at the early phase of incubation. When venom concentrations increased, the inhibition of spreading became more severe. Spreading of plasmatocytes was almost absent at venom concentration of 0.04 VRE/ml, but abundant at venom concentrations of 0.005 VRE/ml and control. The viability of plasmocytes and granular cells was also decreased at high venom concentrations. This suggests that increased concentrations of venom proteins not only affected spreading but also viability after exposure over extended time periods. However, almost all of the plasmatocytes recovered after 4 h, and most of hemocytes appeared active in spreading with bright phase. Perhaps it was due to the loss of the suppression function of venom, or to the recovery of immune functions of hemocytes or to the function of different immune suppressors at different times. For example, the observation of reversible effects of venom probably corroborates observations in C. rubecula, where transient expression of a polydnavirus gene causes transient inactivation (Asgari et al., 1996), although it is not clear yet if the same mechanism exists in venom proteins. Stoltz and Guzo (1986) reported the recovery of immunocompetence in the Hyposoter fugitives/Malacosoma disstria system. Webb and Summers (1990) suggested that venom might protect the egg from the host immune reaction in the period between oviposition and the expression of PDV genes. As summarized in Fig. 4B, the calyx fluid from C. plutellae had highly adverse effects on hemocytes of P. xylostella. For example, the mortality of hemocytes was reduced by 50% or more (Fig. 4B). In each dose of calyx fluid, some hemocytes were damaged while others were unaffected (Fig. 5H), which suggests a more selective action of the calyx fluid. Additionally, we demonstrated that the calyx fluid caused extensive damage to hemocytes, with large amounts of cell debris appearing in the culture medium, including disintegration, damaged plasma membranes (Fig. 5D), the formation of multivesicular bodies,
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Fig. 5. Phase contrast micrographs of Plutella xylostella hemocytes in vitro with different concentrations of calyx fluid from Cotesia plutellae. A, D: Most of hemocyte remained rounded after incubated with 0.01 FE/ml calyx fluids for 1 h (A), the disintegration of the plasma membrane occurred after 4 h (D, arrows); B, H: The majority of hemocytes remained rounded after incubation with 0.02 FE/ml calyx fluids for 1 h (A), and many plasmatocytes began to be damaged after 4 h (H, arrows). Cell debris was noticeable (black dots); C, E, F, I: Multivesicular bodies (C) and intracellular vaculoles (E), appeared after incubation with 0.04 FE/ml calyx fluids for 1 h. Many granular cells and plasmatocytes were badly damaged (F, arrows) and so only remnants of the nucleus remained after 4 h (I, arrows). Scale bar ¼ 20 mm.
(Fig. 5C) and intracellular vaculoles (Fig. 5E), which were also observed in hemocytes incubated with P. hypochondriaca venom (Richards and Parkinson, 2000). Thus, the calyx fluid from C. plutellae appears to be able to exert cytotoxic effects on P. xylostella hemocytes. The observation corroborates the phenomenon that the continued expression of a related immune suppressing agent in C. congregata causes apoptosis of hemocytes (Le et al., 2003). Several PDV proteins have been directly linked to haemocytic effects (Glatz et al., 2004a), including disruption of encapsulation (Zhang and Wang, 2003; Turnbull et al., 2004), disruption of hemocyte activity (Beckage, 1998; Le et al., 2003; Turnbull et al., 2004; Glatz et al., 2004b) and hemocyte cytoskeleton degradation (Li and Webb, 1994; Asgari and Schmidt, 2002; Zhang and Wang, 2003). Our findings reveal that venom of C. plutellae alone may not be sufficient to suppress the host immune system, although it can inhibit the spreading of plasmocytes of P. xylostella during the early phase of incubation. By comparison with the effect of calyx fluid on the viability of hemocytes, the effect of venom appeared to be much less. In addition, venom had no effect on granular cells, which spread normally all the time. On the contrary, the calyx fluid had significant
effects on host hemocytes, even at low concentrations. Obviously venom of C. plutellae might also synergize the effects of calyx fluid or polydnavirus like in other systems. Venom from C. melanoscela was reported to promote the release of virions into the cytoplasm after uptake by host cells facilitating the uncoating of PDVs at nuclear pores in host hemocytes (Stoltz et al., 1988). Venom proteins may mediate short-term immune suppression before PDV gene expression becomes effective, as in C. sonorensis (Webb and Luckhart, 1994). In some parasitoid systems, venom proteins are essential for polydnavirus infection and gene expression, such as C. glomeratus (Kitano, 1986) and C. kariyai (Tanaka, 1987). In C. rubecula, a 1598-Da peptide (Vn1.5) isolated from venom was required for the expression of C. rubecula bracoviruses (CrBVs) in host hemocytes (P. rapae) (Zhang et al., 2004) and two other proteins (Vn4.6 and Vn50) from venom can synergize with an immune suppressive protein from C. rubecula PDVs (Asgari et al., 1996, 1997) and a calyx protein (Asgari et al., 1998, 2003b) to inhibit the host immune response (Asgari et al., 2003a). In addition, venom proteins enhance virus persistence in the host. However, the mechanism of action of these peptides in promoting expression of PDV genes is not known.
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Although we have not isolated a peptide of similar size to these proteins in the present study, specific peptides may exist in the venom of C. plutellae. SDS analyses revealed that several peptides of similar size were present in the two resources (Fig. 1), which may imply a close structural and functional relationship (Asgari, 2006). Webb and Summers (1990) showed that venom proteins from C. sonorensis share epitopes with PDV structural proteins. Asgari et al. (2003a) found the venom gene homologs in the viral genome and revealed that viral genes show tissue-specific coordination of expression with venom proteins. These results could indicate that proteins from venom and calyx tissues interact but have to be apart before oviposition (Asgari, 2006). It was interesting that application of an isolated C. rubecula immune suppressor to hemocytes from Pieris rapae did not show any effects unless the protein was injected into the hemocoel. Recent experiments suggested that the immune suppressor has to interact with lipophorin particles first to exert its effects (Asgari and Schmidt, 2002), and Schmidt et al. (2005) proposed some dimeric and oligomeric adhesion molecules which are able to cross-link receptors on the cell surface and depolymerise actin by leverage-mediated clearance reactions in the hemolymph. Obviously the immune suppression mechanisms are remarkably diverse in different host-parasitoid systems. The possible functional mechanism of the immune suppressors in C. plutellae/P. xylostella system is still largely unknown and needs further research work. Acknowledgments We are grateful to Professor Chen-zhu Wang (Beijing) for his critical review of an early draft of the manuscript. We also thank two anonymous reviewers for their valuable comments. Funding for this study was provided jointly by national natural science foundation of China (30370959), 973 Program (2006CB102005), program for new century excellent talents in University (NCET-04-0521) and innovation research team program of the ministry of education of China (IRT0355). References Asgari, S., 2006. Venom proteins from polydnavirus-producing endoparasitoids: their role in host-parasite interactions. Archives of Insect Biochemistry and Physiology 61, 146–156. Asgari, S., Schmidt, O., 2002. A coiled-coil region of an insect immune suppressor protein is involved in binding and uptake by hemocytes. Insect Biochemistry and Molecular Biology 32, 497–504. Asgari, S., Hellers, M., Schmidt, O., 1996. Host hemocyte inactivation by an insect parasitoid: transient expression of a polydnavirus gene. Journal of General Virology 77, 2653–2662. Asgari, S., Schmidt, O., Theopold, U., 1997. A polydnavirus-encoded protein of an endoparasitoid wasp is an immune suppressor. Journal of General Virology 78, 3061–3070. Asgari, S., Theopold, U., Wellby, C., Schmidt, O., 1998. A protein with protective properties against the cellular defence reactions in insects. Proceedings of the National Academy of Sciences of the USA 95, 3690–3695.
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