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Spodoptera litura multicapsid nucleopolyhedrovirus inhibits Microplitis bicoloratus polydnavirus-induced host granulocytes apoptosis
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Journal of Insect Physiology 52 (2006) 795–806 www.elsevier.com/locate/jinsphys
Spodoptera litura multicapsid nucleopolyhedrovirus inhibits Microplitis bicoloratus polydnavirus-induced host granulocytes apoptosis Kaijun Luoa,b, Yi Panga, a
State Key Laboratory of Biocontrol and Institute of Entomology, Sun Yat-Sen (Zhongshan) University, Guangzhou 510275, P.R. China b Agriculture Environment and Resource Research Institute, Yunnan Academy of Agriculture Sciences, Kunming 650205, P.R. China Received 9 February 2006; received in revised form 10 April 2006; accepted 10 April 2006
Abstract Baculoviruses and parasitoids are critically important biological control agents in integrated pest management (IPM). They have been simultaneously and sequentially used to target insect pests. In this study, we examined the impacts of both baculovirus and polydnavirus (PDV) infection on the host cellular immune response. Larvae of the lepidopteran Spodoptera litura were infected by Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) and then the animals were parasitized by the braconid wasp Microplitis bicoloratus. The fate of the parasitoids in the dually infected hosts was followed and encapsulation of M. bicoloratus first instar larvae was observed. Hemocytes of S. litura larvae underwent apoptosis in naturally parasitized hosts and in non-parasitized larvae after injection of M. bicoloratus ovarian calyx fluid (containing MbPDV) plus venom (CFPV). However, assessments of the percentages of cells undergoing apoptosis under different treatments indicated that SpltMNPV could inhibit MbPDV-induced apoptosis in hemocytes when hosts were first injected with SpltMNPV budded virus (BV) followed by injection with M. bicoloratus CFPV. As the time of injection with SpltMNPV BV increased, the percentages of apoptosis in hemocytes population declined. Furthermore, in vitro, the percentages of apoptosis showed that SpltMNPV BV could inhibit MbPDV-induced granulocytes apoptosis. The occurrence of MbPDV-induced host granulocytes apoptosis was inhibited in the dually infected hosts. As hemocytes apoptosis causes host immunosuppression, the parasitoids are normally protected from the host immune system. However, in larvae infected with both baculovirus and PDV, the parasitoids underwent encapsulation in the host hemocoel. r 2006 Elsevier Ltd. All rights reserved. Keywords: Insect immunity; Hemocyte; Apoptosis; Polydnavirus; Baculovirus; Microplitis bicoloratus
1. Introduction Apoptosis, or programmed cell death, is an energyrequiring programmed physiological process that eliminates superfluous, altered or malignant cells, characterized by a genetically controlled autodigestion of the cell activated via endogenous proteases. Symptoms of apoptosis include cytoskeletal disruption, cell shrinkage, membrane blebbing, nuclear condensation, and internucleosomal DNA fragmentation. Finally, the apoptotic cells fragment into membrane-enclosed bodies that are rapidly phagocytosed by neighboring cells (Savill et al., 2002; Wyllie et al., 1980; Kerr et al., 1972). Corresponding author. Tel.: +86 20 84113860; fax: +86 20 84037472.
E-mail address: [email protected] (Y. Pang). 0022-1910/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2006.04.007
The apoptotic process can be initiated by a variety of regulatory stimuli, including baculoviruses (Clem, 1997, 2001, 2005), which are known to regulate apoptosis of host cells during infection (O’Brien, 1998; Teodoro and Branton, 1997; Razvi and Welsh, 1995). The host apoptosis response to virus infection reduces baculovirus spread at the organismal level (Clarke and Clem, 2003) and may contribute towards limiting host-range and efficiency for baculovirus infections (Zhang et al., 2002a). To suppress this host defense response, baculoviruses have evolved two families of anti-apoptotic genes, i.e. p35 and iap (inhibitor of apoptosis) in order to replicate themselves. These two genes use different mechanisms in the apoptotic pathway to functionally block this cell death reaction (Zoog et al., 1999; Manji et al., 1997; Seshagiri and Miller, 1997; Clem and Miller, 1994; Clem, 2001, 2005).
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Other insect viruses such as polydnaviruses (PDVs) are active inducers of host cell apoptosis. PDVs are obligate symbionts of many endoparasitic wasps in the families Braconidae and Ichneumonidae. Viral DNA is integrated into the wasp’s genome (Xu and Stoltz, 1991; Fleming and Summers, 1991; Belle et al., 2002) and transmitted vertically to the wasp’s offspring in a proviral form. PDVs replicate in ovarian calyx cells in the female wasp. Following replication of the integrated proviral forms, the viral DNA is excised as segments to be incorporated into the virion and the flanking DNAs are rejoined (Gruber et al., 1996; Savary et al., 1997, 1999; Wyder et al., 2002; Wyder and Lanzrein, 2003; Webb, 1998). Virions are stored in the lumen of the calyx and oviduct and the suspension of virus and protein is called calyx fluid. When a female wasp finds a host, she injects calyx fluid, venom produced by the venom gland, and one or more eggs into the hemocoel of the host caterpillar. The virus enters different host tissues and viral genes are transcribed, in the apparent absence of viral replication in the host larva. Both early and/or late viral gene transcripts may be produced while the wasp’s progeny develop to maturity in the host hemocoel. Virus gene expression causes several physiological alternations in parasitized hosts including suppression of the host insect’s immune system. When parasitoid eggs are injected into a non-parasitized larva in the absence of PDV, the parasitoid’s progeny are encapsulated and killed by host immune cells (hemocytes), whereas progeny survive when virus is injected together with the egg (Webb, 1998; Asgari et al., 1997; Strand and Pech, 1995b; Lavine and Beckage, 1995; Strand and Noda, 1991). In several host–parasitoid systems, host hemocyte apoptosis occurs during the initial stages of parasitism, rendering those hemocytes incapable of mounting an encapsulation response (Lavine and Beckage, 1995; Amaya et al., 2005). Encapsulation is the primary cellular immune response of host insects directed against invading metazoan parasites or parasitoids. Encapsulation begins when host granulocytes attach to the surface of a foreign target such as a parasitoid egg. Following attachment, they lyse or degranulate, releasing the contents of their granules over the egg surface, which promotes attachment of plasmatocytes. This is followed by attachment of multiple layers of plasmatocytes to form the capsule. Termination of capsule formation occurs when a subpopulation of granulocytes adheres in a monolayer around the periphery of the capsule. Neither granulocytes nor plasmatocytes are capable of forming a capsule independently (Pech and Strand, 1996; Strand and Pech, 1995a; Schmit and Ratcliffe, 1978, 1977). In Pseudoplusia includens parasitized by Microplitis demolitor, infection with PDV induces host granulocytes to undergo apoptosis while plasmatocytes lose their capacity to adhere to foreign surfaces (Strand and Pech, 1995b; Strand and Noda, 1991). Both baculoviruses and parasitoids are important biological control agents in integrated pest management. Baculovirus cannot directly infect parasitoids and also
cannot replicate in the larvae of the parasitoid (Irabagon and Brooks, 1974; Hotchkin and Kaya, 1983). Hotchkin and Kaya (1983) also found that baculoviruses might affect the host’s immune system, because parasitoid larvae were encapsulated in the host infected with baculovirus. But, no supporting data could demonstrate the effect of baculoviruses on the host’s immune system in parasitized larvae infected with the viruses. Sequence analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) genome reveal the presence of two anti-apoptosis genes, p49 (p35 homologue) and iap in the genome (Pang et al., 2001). We have demonstrated that SpltMNPV can block AcMNPV-induced apoptosis in the S. litura cell line Sl-zsu-1 (Zhang et al., 2002b). SpltMNPV p49 is able to suppress apoptosis induced by infection of a mutant AcMNPV deficient in p35 and rescue the mutant virus replication from apoptosis in Sf-9 cells (Yu et al., 2005). In order to test this hypothesis that baculovirus infection blocks parasitoid PDV-induced host granulocytes apoptosis, resulting in parasitoid larval encapsulation by host hemocytes, we selected the braconid wasp Microplitis bicoloratus, a solitary larval parasitoid of S. litura, which carries a PDV, and SpltMNPV as a model system to study the combined effects of PDV and baculovirus infection on the host insect immune system. 2. Methods and material 2.1. Insects rearing and experimental animals S. litura colony was reared on artificial diet (formulated after Li et al., 1998) at 2771 1C, RH 60–80%, and under a 12:12 h LD photoperiod regime. M. bicoloratus was originally collected from parasitized S. litura larvae in Guangdong Province, China, in 2003. The parasitoid colony was maintained on S. litura larvae reared in the laboratory; adults were also provided honey as a dietary supplement. In all experiments, 2–3 d old mated female wasps were used. Newly ecdysed third instar S. litura larvae were parasitized by M. bicoloratus and screened for the occurrence of apoptotic changes in the host hemocyte population following parasitization. For experimental procedures requiring the injection of calyx fluid plus venom (CFPV)/BV into naive non-parasitized larvae, newly ecdysed fifth instar S. litura larvae were used as subjects for the injection experiments. In these injection experiments, control larvae (newly ecdysed fifth instar S. litura larvae) were mock-injected with Pringle’s solution and these samples were taken at 2 h after injection. 2.2. Baculovirus purification and cell lines SpltMNPV occluded virions (OV) containing polyhedra and budded virus (BV) were produced in S. litura larvae in vivo and purified as previously described (Wang et al., 2002). S. litura Sl-zsu-1 cells (Xie et al., 1998) were
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maintained in TC-100 medium (Gibco) supplemented with 10% fetal bovine serum. Titers of SpltMNPV were determined by TCID50 assay with infected Sl-zsu-1 cells. SpltMNPV BV (2 105 p.f.u. per larvae) was injected into newly ecdysed fifth instar S. litura larvae. 2.3. CFPV preparations Aliquots of CFPV were collected from dissected M. bicoloratus females under a dissecting microscope. The quantities of CFPV used in experiments were expressed in wasp equivalents (Albrecht et al., 1994; Strand and Noda, 1991). A volume of 5 ml CFPV solution (0.5 wasp equivalents) in Pringle’s solution (154.1 mM NaCl, 2.7 mM KCl, 14 mM CaCl2, 22.2 mM dextrose) (Pringle, 1938) was injected into the abdominal cavity of the nonparasitized larvae, then the larvae were screened for the occurrence of hemocyte apoptosis. 2.4. Encapsulation of M. bicoloratus larvae in host larvae co-infected with SpltMNPV polyhedra Development of M. bicoloratus in S. litura larvae required seven days: eggs hatched within 24 h, the first instar larva required two days, the second instar larva needed three days, and the third instar larvae exited the host and pupated in one day at 2771 1C, RH 60–80% and a 12:12 h photoperiod (unpublished data). Newly ecdysed second instar S. litura larvae were divided into two groups. One group was fed with SpltMNPV polyhedra applied to the surface of the artificial diet. Forty eight hours after ingestion of polyhedra, 100 S. litura larvae were exposed to five female wasps’ ovipositions. Thirty parasitized S. litura larvae were dissected after 72 h postparasitization (p.p.) (when immature parasitoid in host has developed from egg into the first larvae) in Pringle’s solution under a binocular microscope. The second group was fed on artificial diet without polyhedra as a control and was parasitized and dissected as above. The experiments were replicated three times. 2.5. DNA fragmentation DNA fragmentation in nuclei of S. litura hemocytes was assessed (Zhang et al., 2002a). S. litura larval hemocytes were collected, washed with PBS buffer (pH 7.2) with 0.1% DTT and centrifuged for 1 min at 1000g. The pellet was then incubated in lysis buffer (20mM Tris–HCl, pH 8.0, 10 mM EDTA and 1% NP-40) for 5 min and the supernatant collected after being centrifuged at 1600g for 5 min. SDS and RNase A were added to a final concentration of 1% and 5 mg/ml, respectively. After 2 h of incubation at 56 1C, the supernatant was digested with proteinase K at 37 1C for 2 h to a final concentration of 2.5 mg/ml. The DNA was precipitated with ethanol, dissolved in TE buffer and separated by electrophoresis on 1% agarose gels.
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2.6. Measurement of percentages of hemocytes undergoing apoptosis In vivo the percentages of apoptosis in hemocytes were measured with flow cytometry. Hochest 33258 (1 mg/ml) was added to a 100 ml hemocyte suspension (1–2 105 cells). After 5–8 min of incubation at 37 1C, the solution in the microfuge tube was cooled over ice and propidium iodide (PI) (5 mg/ml) was added to it. After filtering with 400-mesh net, dual-color flow cytometry was performed. A total of 10,000 hemocytes were counted per treatment. Experiments were replicated three times. For in vitro experiments using purified populations of granulocytes, the percentages of apoptosis in granulocytes were analyzed by quantifying phosphatidylserine residues exposed on the external cell membrane. One microliter of human recombinant fluorescein isothiocyanate (FITC)– conjugated annexin V (Jingmei, China) and PI (2 mg/ml) was added to a 100 ml cell suspension (1 105) of cells in binding buffer (10 mM Hepes/NaOH, pH 7.4; 140 mM NaCl; 5 mMCaCl2). After 15 min of incubation in the dark, dual-color flow cytometry were performed. A total of 10,000 cells were counted in each treatment, and experiments were replicated three times. 2.7. Granulocyte purification and culture The hemocytes of fifth instar S. litura larvae were classified into six different cell types: prohemocytes, plasmatocytes, granulocytes, spherule cells, coagulocytes and oenocytoids (Deng and Lee, 1995a). Hemocytes were collected from larvae by the procedure of Pech et al. (1994) with slight modification. Fifth instar S. litura larvae were rinsed with ddH2O to eliminate surface impurity, then placed on filter paper to absorb water. The larval surface was sterilized with 75% ethanol, and then the animals were bled from the proleg directly into 500 ml of ice-cold anticoagulant buffer (0.098 M NaOH, 0.186 M NaCl, 0.017 M EDTA, 0.041 M citric acid, pH 4.5) (Mead et al., 1986) to constitute the total hemocyte population. Percoll (Amersham Biosciences) gradient separations of the different hemocyte types were performed as outlined by Gardiner and Strand (1999) with slight modification. The gradient consisted of 2 ml of 55% Percoll, layered over 2 ml of 70% Percoll, and 0.5 ml 90% Percoll. One milliliter hemocytes (ca. 1 107 hemocytes total) from the total hemocyte population was layered onto a Percoll gradient. Gradients were centrifuged for 20 min at 480g in 4 1C in an Eppendorf 5810R centrifuge with a JS-7.5 swinging bucker rotor. The bands were transferred into microfuge tubes and washed twice with TC-100 culture medium in an Eppendorf 5415D centrifuge for 5 min at 800g, and the final pellet for each fraction was resuspended in 50 ml TC-100 medium plus 10% fetal bovine serum (FBS) and counted. The band at the top of the gradient contained on average 2.4 106 hemocytes (24% of the starting population), the majority of which were plasmatocytes (19% of the starting
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population) and granulocytes (5% of the starting population) as identified using morphological characters. The cells at the interface of the 55% and 70% Percoll were primarily granulocytes (2.2 106, 22% of the staring population) and no cells at the interface of the 70% and 90% Percoll were collected. For in vitro experiments, 1 105 granulocytes were infected with SpltMNPV BV (MOI ¼ 2) (Zhang et al., 2002a) or with 1 wasp equivalent of M. bicoloratus CFPV (Strand and Pech, 1995b) and then screened for apoptosis. 2.8. Statistical analysis All proportional data were transformed by arcsin square root before analysis of variance, one-way ANOVA, po0:05. Corresponding treatment means were separated using the Student–Newman–Keuls (SNK) test (SPSS Inc., 2003). 3. Results 3.1. Encapsulation of M. bicoloratus larvae in host larvae co-infected with SpltMNPV polyhedra In the control group, in which larvae were only parasitized, the percentages of encapsulation of M. bicoloratus larvae were all zero, and the larvae were all alive and developed normally as first instar larvae. In baculovirus infection, following ingestion of occlusion bodies (polyhedra) by the larva, the protective polyhedron coat dissolved in the midgut, releasing nucleocapsids that fuse with the midgut cells. Initial rounds of viral replication within the nucleus of the infected cell produced a second viral phenotype, the BV, which spreads the infection to other tissues (Pang, 1994). In S. litura larvae, large numbers of BVs are found in the hemolymph at 48 h after SpltMNPV polyhedra ingestion, leading to death at
around 120 h after inoculation. To explore the effect of baculovirus infection on parasitism, second instar S. litura larvae previously fed SpltMNPV polyhedra were parasitized by M. bicoloratus at 48 h after polyhedra ingestion. The larvae were then dissected at 72 h p.p. The fate of the parasitoids in the dually infected hosts was followed, and the percentages of fully encapsulated first instar larvae (Fig. 1aA), partly encapsulated first instar larvae (Fig. 1aB), non-encapsulated first instar larvae (Fig. 1aC) and dead embryos (Fig. 1aD) were 63.3%76.9, 17%70, 9%74.9 and 11.3%75.7, respectively. On fully encapsulated first instar larvae the body surface was covered by hemocytes and the larvae had died. The percentage of fully encapsulated larvae was significantly higher than any of the other three (F3,8 ¼ 9.339, p ¼ 0:005) (Fig. 1b). In partly encapsulated larvae, only part of the body surface was covered by hemocytes and larvae were still alive. In nonencapsulated larvae, the body surface of the larva was not covered by hemocytes and the larvae were still alive. Death was confirmed in suspected dead embryos using trypan blue stain. The percentages between partly encapsulated, non-encapsulated and dead embryos were not significantly different (p40:05) in the dually infected hosts (Fig. 1b). 3.2. Hemocytes of S. litura larvae undergo apoptosis in naturally parasitized hosts To analyze the direct effects of MbPDV on hemocytes in a naturally parasitized host, an oligonucleosome-size ladder was observed after electrophoresis of total genomic DNA extracted from hemocytes of S. litura larvae parasitized by M. bicoloratus. The DNA ladder patterns were detected in hemocytes of the parasitized larvae from 2 to 7 d p.p. (Fig. 2a). To further identify early apoptosis in hemocyte of naturally parasitized host, the flow cytometry analysis was performed in the whole population of hemocytes. Apoptotic cells were detected from 1 to 7 d
Fig. 1. Encapsulation of M. bicoloratus larvae in dually infected S. litura host larvae. (a) Light microscopy of (A) fully encapsulated first instar larva, (B) partly encapsulated first instar larva, (C) non-encapsulated first instar larva, and (D) dead embryo stained with trypan blue. (A, B and C: Bar ¼ 50 mm; D: Bar ¼ 20 mm). (b) The percentages (mean7SE) of non-, partly- or fully-encapsulated M. bicoloratus larvae and dead embryo. Means with the same letter are not significantly different (p40:05). The percentages of encapsulation of M. bicoloratus larvae in control groups were zero and the larvae were all normal and alive.
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Fig. 2. Hemocytes of S. litura larvae underwent apoptosis in naturally parasitized hosts. (a) Detection of fragmented DNA by electrophoresis. Total cellular DNA was extracted from hemocytes collected from 500 ml hemolymph per sample from 2 to 7 days p.p. (lane U: non-parasitized larvae). (b) Flow cytometry analysis of hemocytes indicating the percentages of apoptotic cells. Hemocytes were stained with PI/Hochest 33258. The data is a representation of 3 individual experiments. Newly ecdysed third instar larvae were used in this experiment. Non-parasitized larvae were used as control. (c) Statistical analysis of the percentages (mean7SE) of apoptotic hemocytes from 1 to 7 days p.p.. Means with the same letter are not significantly different (P40:05).
p.p.. Early apoptotic cells can just be stained by Hochest 33258 alone and results appear in the upper left quadrant of the FACS data shown. Then necrotic cells and possible late apoptotic cells can be stained by both Hochest 33258 and PI and appear in the upper right quadrant of the FACS data shown. One representative dot plots from three individual experiments indicated that the percentages of apoptosis in hemocytes population from 1 to 7 d p.p. (11.99%, 15.02%, 25.49%, 20.76%, 21.75%, 20.39% and 24.56%, respectively) were higher than those in the control (non-parasitized larvae) (7.35%) (Fig. 2b). In order to further understand early apoptotic cells at different times during parasitoid development in its host, three time points were analyzed using one-way ANOVA statistical analysis,
and the results indicated that there were no significant differences between the percentages of apoptosis in hemocytes from 1 to 7 d p.p. (F6,14 ¼ 2.499, p ¼ 0:074) (Fig. 2c). The levels of MbPDV-induced host hemocytes in early apoptosis in naturally parasitized hosts were persistent and stable during parasitoid development. 3.3. Hemocytes of S. litura larvae undergo apoptosis in nonparasitized larvae after injection of M. bicoloratus ovarian calyx fluid (containing PDV) plus venom Injection of CFPV induced apoptosis in hemocytes of S. litura larvae (Fig. 3). One representative dot plot from three individual experiments indicated that the percentages
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Fig. 3. Hemocytes of S. litura larvae underwent apoptosis in non-parasitized larvae after injection of M. bicoloratus ovarian calyx fluid (containing PDV) plus venom (CFPV) (0.5 wasp equivalents). (a) Flow cytometry analysis of hemocytes indicating the percentages of apoptotic cells. Hemocytes were stained with PI/Hochest 33258. The data is a representation of 3 individual experiments. Newly ecdysed fifth instar larvae were used in this experiment. (b) Statistical analysis of the percentage of apoptosis (mean7SE) in hemocyte population from 2 to 72 h p.i.. Means with the same letter are not significantly different (P40:05).
of apoptosis in hemocyte populations collected from 2 to 72 h p.i. were all higher than controls (mock-injected larvae) (7.75%) (Fig. 3a). In order to further understand early apoptotic cells changes in hemocytes of host larvae injected with CFPV from 2 to 72 h p.i., these data were analyzed using one-way ANOVA statistical analysis and the results indicated that the percentage of apoptosis was significantly higher 72 h p.i. as compared to 2 h p.i. (F 4;10 ¼ 3:412, p ¼ 0:053) (Fig. 3b). There were no significant differences in the percentages of apoptosis between 12, 24 and 48 h p.i. They were lower compared to 72 h p.i. and higher compared to 2 h p.i. As shown above, occurrence of apoptosis was the highest at 72 h after injection of CFPV. The results suggested that levels of hemocytes in early apoptosis gradually increased in vivo. 3.4. Hemocytes of S. litura larvae underwent transient apoptosis after injection of SpltMNPV BV The flow cytometry analysis was performed on hemocytes following injection of SpltMNPV BV from 2 to 72 h p.i., and we found that injection of SpltMNPV BV induced
transient apoptosis in hemocytes of S. litura larvae at an early period p.i. (Fig. 4a). After 2 h of injection with SpltMNPV BV, 38.68% of hemocytes underwent apoptosis (9.51% in control conditions). As the time of injection with SpltMNPV BV increased, the percentages of apoptosis in hemocytes population declined. After 48 and 72 h p.i., the percentages of apoptosis (8.29% and 1.76%, respectively) were lower than controls. Further one-way ANOVA statistical analysis of the percentages of apoptosis in hemocytes from 2 to 72 h p.i., indicated that the percentages of apoptosis in hemocyte populations were significantly higher at 2 and 12 h p.i. as compared to 48 and 72 h p.i. (F 4;10 ¼ 8:242, p ¼ 0:003) (Fig. 4b). The percentages of apoptosis between 2 and 12 h p.i. were not significantly different. Similarly, there were no significant differences between the percentages of apoptosis at 48 and 72 h p.i. To further verify that the hemocytes underwent transient apoptosis after injection of SpltMNPV BV, DNA ladder were performed with hemocytes from 2 to 24 h p.i SpltMNPV BV. An oligonucleosome-size ladder was observed in DNA isolated at 2 h p.i (Fig. 4c). The results suggested that the level of hemocyte apoptosis
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Fig. 4. Hemocytes of S. litura larvae underwent transient apoptosis after injection of SpltMNPV BV (2 105 p.f.u. per larvae). (a) Flow cytometry analysis of hemocytes indicating the percentages of apoptotic cells. Hemocytes were stained with PI/Hochest 33258. The data is a representation of 3 individual experiments. Newly ecdysed fifth instar larvae were used in this experiment. (b) Statistical analysis of the percentage of apoptosis (mean7SE) in hemocyte population from 2 to 72 h p.i.. Means with the same letters are not significantly different (P40:05). (c) Detection of fragmented DNA by electrophoresis. Total cellular DNA was extracted from hemocytes collected from 500 ml hemolymph per sample at 2, 12, 24 h p.i. and from mock-injected (Mi) larvae injected with Pringle’s solution, respectively.
in hosts injected with SpltMNPV BV rapidly declined in vivo. 3.5. Inhibition of apoptosis in hemocytes of parasitized S. litura larvae by SpltMNPV To find out whether SpltMNPV could inhibit MbPDVinduced apoptosis in hemocytes, hosts were first injected with SpltMNPV BV followed by injection of M. bicoloratus CFPV 24 h after BV injection. Then, the percentage of apoptosis was measured by flow cytometry at various times after CFPV injection. Apoptosis was significantly reduced from 24 h after injection of CFPV (48 h BV p.i.) (12.49%) and decreased even further at 48 and 72 h after CFPV injection (9.42% and 4.71%) (Fig. 5a). One-way ANOVA statistical analysis of the percentages of apoptosis in
hemocytes from 2 to 72 h p.i. indicated that the percentage of apoptosis in hemocytes population was significantly lower at 72 h CFPV p.i. as compared to 2, 12, 24 and 48 h p.i. (F 4;10 ¼ 28:454, po0:001) (Fig. 5b). The percentages of apoptosis in hemocytes were significantly lower between 24 and 48 h CFPV p.i. as compared to 2 and 12 h CFPV p.i. (po0:05). The percentages of apoptosis at 2 and 12 h CFPV p.i. were not significantly different. Similarly, there were no significant differences between the percentages of apoptosis at 48 and 72 h p.i. CFPV (p40:05). The results suggested that the levels of hemocyte apoptosis in hosts first injected with SpltMNPV BV followed by injection of M. bicoloratus CFPV 24 h after BV injection gradually declined in vivo. The occurrence of MbPDVinduced host hemocytes apoptosis was inhibited in dually infected hosts.
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Fig. 5. Inhibition of apoptosis in hemocytes of S. litura larvae by SpltMNPV in dually, SpltMNPV BV (2 105 p.f.u. per larvae) and M. bicoloratus CFPV (0.5 wasp equivalents per larvae), injected hosts. (a) Flow cytometry analysis of hemocytes indicating the percentages of apoptotic cells. Hemocytes were stained with PI/Hochest 33258. The data is a representation of 3 individual experiments. Newly ecdysed fifth instar larvae were used in this experiment. (b) Statistical analysis of the percentage (mean7SE) of apoptotic hemocytes from 2 to 72 h p.i.. Means with the same letters are not significantly different (P40:05).
3.6. Flow cytometry analysis of hemocytes comparision between M. bicloratus CFPV injected larvae, SpltMNPV BV injected larvae, and M. bicloratus CFPV injected larvae 12 h after SpltMNPV BV injection
This clearly showed that SpltMNPV BV infection could inhibit PDV-induced apoptosis and consequently the immune suppression leading to encapsulation of the developing parasitoids (Fig. 1).
Based on own previous results, we further compared the percentages of apoptosis in hemocytes of hosts between injection of CFPV 12 h after SpltMNPV BV injection, injection of CFPV alone and injection SpltMNPV BV alone after various times to focus on apoptotic induction (by MbPDV) and blocking (by baculovirus) (Fig. 6). The percentage of apoptosis was measured by flow cytometry at various times after CFPV injection. At 24 h after CFPV injection, the percentage of apoptosis in hemocytes of hosts that were first injected with SpltMNPV BV followed by injection of M. bicoloratus CFPV at 12 h after BV injection were 11.04% (15.75% in CFPV injection alone, 7.16% in BV injection alone). At 48 h after CFPV injection, the percentages of apoptosis declined to 4.27% (10.49% in CFPV injection alone, 3.65% in BV injection alone) and decreased even further at 72 h p.i. to 1.61% (17.63% in CFPV injection alone, 1.16% in BV injection alone).
3.7. In vitro inhibition of M. bicoloratus CFPV-induced apoptosis in purified granulocytes by SpltMNPV BV To identify whether granulocytes underwent apoptosis and to further confirm inhibition of MbPDV-induced apoptosis by SpltMNPV, in vitro assays were carried out using purified S. litura granulocytes. When cells were inoculated with CFPV alone considerable apoptosis was noted, apoptosis was detected at 12 h after inoculation by flow cytometry using Annexin-V conjugated with FITC; the early apoptotic cells can be stained by Annexin-V alone and appear in the lower right quadrant of the FACS data shown; the necrotic cells can be stained by PI alone and appear in the upper left quadrant of the FACS data shown (Fig. 7). However, when cells were inoculated with CFPV 2 h after BV infection, the percentage of apoptosis was significantly reduced to 6.7% compared to 23.7% when
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Fig. 6. Flow cytometry analysis of hemocytes comparision between M. bicoloratus CFPV (0.5 wasp equivalents per larvae) injected larvae, SpltMNPV BV (2 105 p.f.u. per larvae) injected larvae and M. bicoloratus CFPV injected larvae 12 h after SpltMNPV BV injected. Hemocytes were stained with PI/ Hochest 33258. The data is a representation of 3 individual experiments. Newly ecdysed fifth instar larvae were used in this experiment.
CFPV was used alone (Fig. 7). Mock infected or 2 h BV infected cells showed low levels of apoptosis (Fig. 7).
4. Discussion Baculoviruses and parasitoids are critically important biological control agents in integrated pest management (IPM). They have been simultaneously and sequentially used to target insect pests. In this study, we examined the impacts of both baculovirus and PDV infection on the host cellular immune response. Our results demonstrated that a baculovirus expressing anti-apoptotic gene(s) inhibited parasitoid PDV-induced host granulocyte apoptosis, which resulted in the parasitoid larvae becoming encapsulated by host blood cells. To our knowledge, this is the first report describing the effect of an anti-apoptosis mechanism of
baculoviruses on development of parasitoid larvae that acts on PDV-induced apoptosis in host hemocytes. There are few identified baculovirus caspase inhibitors, namely P35, IAPs and P49. Baculovirus protein P35 is a wide-ranging caspase inhibitor, inhibiting mammalian caspases 1–4 and 7 (Barry and McFadden, 1998; Miller, 1997). Caspase-dependent autophagy has been documented in Drosophila melanogaster salivary gland tissues, in which baculovirus p35 expression inhibits cell death (Kimura et al., 2004). The other anti-apoptotic baculovirus protein is IAP, which has been shown to have an inhibitory effect on apoptosis involving X IAP, c-IAP, c-IAP2 and survivin (Deveraux and Reed, 1999). These proteins have been shown to interact with and suppress the activity of caspase-3 (Nogal et al., 2001). SpltMNPV P49 protein suppresses apoptosis by inhibiting protein death caspases3-like (Yu et al., 2005).
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Fig. 7. Flow cytometry analysis of purified S. litura granulocytes (1 105/ well) in dually infected in vitro with SpltMNPV BV (MOI ¼ 2) and M. bicoloratus CFPV (1 wasp equivalents). Granulocytes were stained with Annexin-V-FITC/PI. The data is a representation of 3 individual experiments.
PDVs have been shown to induce apoptosis in lepidopteran host granulocytes in P. includens/M. demolitor (Strand and Pech, 1995b) and in Manduca sexta/Cotesia congregate systems, although host hemocyte apoptosis is transient in the later system (Amaya et al., 2005). Although PDV-induced apoptotic genes have not been reported, the expression of a Toxoneuron nigriceps PDV-encoded TnBV1 protein can increase caspase-3-like activity and cause apoptosis-like programmed cell death in cultured lepidopteran insect cells (Lapointe et al., 2005). This result indicated that the induction of apoptosis by TnPDV involves a caspase-dependent pathway. In the present study, we showed that MbPDV could induce apoptosis in S. litura hemocytes both in vivo and in vitro; SpltMNPV was able to inhibit PDV-induced granulocytes apoptosis. In vivo dual infection experiments demonstrated that as time passed following injection of SpltMNPV BV, the percentages of PDV-induced apoptosis in the total
hemocyte population declined. Under these conditions, most parasitoid larvae were fully encapsulated by host hemocytes and some were partly encapsulated. However, at earlier hours after SpltMNPV infection, when apoptosis of hemocytes by PDV was occurring in a preponderance in the hosts, parasitoid larvae could not be encapsulated. Irabagon and Brooks (1974) reported that the longer the interval between host parasitism and NPV infection, the higher the percentage of parasitoid cocoons. A more recent study by Nusawardani et al. (2005) showed that wild-type and recombinant AcMNPV (expressing a catepsin-like protease) decrease the survival of Cotesia marginiventris parasitoid emerging from Heliothis virescens if the host is infected with the virus less than 72 h after parasitization. However, at later stages of parasitism (120 h) the baculoviruses do not have detrimental effect on parasitoid survival. The percentage of apoptosis in hemocyte populations of the parasitized S. litura was stably persistent until M. bicoloratus larva finished development. Although we are unsuccessful in culturing plasmatocytes in vitro, PDVs have been shown to lose their capacity to adhere to foreign surface in lepidopteran host plasmatocytes (Strand and Pech, 1995b), and PDVs proteins are an immune suppressor caused by the destabilization of actin filaments (Asgari et al., 1997) and inhibition of spreading and adhesion, especially, plasmatocytes (Amaya et al., 2005). Thus, it will be worthwhile to perform additional experiments using Hi5 cells, a hemocyte-like cell line (substitute plasmatocytes) to confirm the effects of MbPDV on Hi5 in vitro. Interestingly, we found that injection of SpltMNPV BV into S. litura larvae also induced transient apoptosis in hemocytes in vivo. Generally, baculoviruses are host specific, and can only induce apoptosis in heterogenous hosts, but we found that AcMNPV can induce apoptosis in S. litura hemocytes (Zhang et al., 2002a). However, our results revealed that SpltMNPV BV could not induce apoptosis in purified granulocytes in vitro (data not shown). Hemocytes of S. litura larvae infected with SpltMNPV can form polyhedra in the nucleus (Deng and Lee, 1995b). In our experiments, we also found that embryos were never encapsulated. The egg surface of Macrocentrus cingulum has a fibrous layer which plays a role in protecting eggs from host’s immune attack (Hu et al., 2003). Thus, we presume that M. bicoloratus egg’s surface may have some factors protecting the egg or developing embryo from encapsulation in the early periods. Polydnavirus expression needs 6 h to be detected in some species (Strand, 1994), but only 1 h was needed for Sephadex G-25 beads to be avidly encapsulated by hemocytes in in vitro experiments performed in hemocytes of S. litura larvae (data not shown). In conclusion, our findings demonstrated that SpltMNPV could inhibit MbPDV-induced apoptosis in S. litura granulocytes. This might have implications for concurrent application of the virus and the parasitoid in the field. In natural parasitization, MbPDVs induce
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apoptosis in the host’s hemocytes to suppress host cellular immunity. However, the baculovirus inhibitors of apoptosis apparently defy PDV-induced apoptosis, making the developing parasitoid vulnerable to encapsulation. What MbPDV gene(s) are responsible for inducing apoptosis in host hemocytes remains to be discovered. This gene and the one caspase inhibitor encoded by SpltMNPV, p49, will require further research using Hi5 cells, a hemocyte-like cell line, and an in vitro approach to understand the mechanisms of anti-apoptosis and apoptosis in both baculovirus and polydnavirus. Acknowledgements The authors gratefully acknowledge Dr. Nancy E. Beckage (University of California-Riverside) and Dr. Sassan Asgari (University of Queensland) for their comments and suggestions on the manuscript. The research was supported by the National Major Basic Research Project (‘973’) of China (G2000016209), National Natural Science Foundation of China (30530540) and Open-funding of the State Key Laboratory of Biocontrol in Sun Yat-Sen University (0404). References Albrecht, U., Wyler, T., Pfister-Wilhelm, R., Gruber, A., Stettler, P., Heiniger, P., Kurt, E., Schu¨mperli, D., Lanzrein, B., 1994. Polydnavirus of the parasitic wasp Chelonus inanitus (Braconidae): characterization, genome organisation and time point of replication. Journal of General Virology 75, 3353–3363. Amaya, K.E., Asgari, S., Jung, R., Hongskula, M., Beckage, N.E., 2005. Parasitization of Manduca sexta larvae by the parasitoid wasp Cotesia congregate induces am impaired host immune response. Journal of Insect Physiology 51, 505–512. 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. Barry, M., McFadden, G., 1998. Apoptosis regulators from DNA viruses. Current Opinion in Immunology 10, 422–430. Belle, E., Beckage, N.E., Rousselet, J., Poirie, M., Lemeunier, F., Drezen, J-M., 2002. Visualization of polydnavirus sequences in a parasitoid wasp chromosome. Journal of Virology 76, 5793–5796. Clarke, T.E., Clem, R.J., 2003. In vivo induction of apoptosis correlating with reduced infectivity during baculovirus infection. Journal of Virology 77, 2227–2232. Clem, R.J., 1997. Regulation of programmed cell death by baculoviruses. In: Miller, L.K. (Ed.), The Baculoviruses. Plenum, New York, pp. 237–266. Clem, R.J., 2001. Baculoviruses and apoptosis: the good, the bad, and the ugly. Cell Death and Differentiation 8, 137–143. Clem, R.J., 2005. The role of apoptosis in defense against baculovirus infection in insects. Current Topics in Microbiology and Immunology 289, 113–239. Clem, R.J., Miller, L.K., 1994. Control of programmed cell death by the baculovirus genes p35 and iap. Molecular and Cellular Biology 14, 5212–5222. Deng, R.Q., Lee, C.Y., 1995a. A study on the types and in vitro changes of hemocytes of larval Spodoptera litura. Supplement to the Journal of Sun Yatsen University 2, 15–17. Deng, R.Q., Lee, C.Y., 1995b. A study on the cytophathology of larval Spodoptera litura infected with SlNPV. Supplement to the Journal of Sun Yatsen University 2, 18–20.
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Journal of Insect Physiology 52 (2006) 795–806 www.elsevier.com/locate/jinsphys
Spodoptera litura multicapsid nucleopolyhedrovirus inhibits Microplitis bicoloratus polydnavirus-induced host granulocytes apoptosis Kaijun Luoa,b, Yi Panga, a
State Key Laboratory of Biocontrol and Institute of Entomology, Sun Yat-Sen (Zhongshan) University, Guangzhou 510275, P.R. China b Agriculture Environment and Resource Research Institute, Yunnan Academy of Agriculture Sciences, Kunming 650205, P.R. China Received 9 February 2006; received in revised form 10 April 2006; accepted 10 April 2006
Abstract Baculoviruses and parasitoids are critically important biological control agents in integrated pest management (IPM). They have been simultaneously and sequentially used to target insect pests. In this study, we examined the impacts of both baculovirus and polydnavirus (PDV) infection on the host cellular immune response. Larvae of the lepidopteran Spodoptera litura were infected by Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) and then the animals were parasitized by the braconid wasp Microplitis bicoloratus. The fate of the parasitoids in the dually infected hosts was followed and encapsulation of M. bicoloratus first instar larvae was observed. Hemocytes of S. litura larvae underwent apoptosis in naturally parasitized hosts and in non-parasitized larvae after injection of M. bicoloratus ovarian calyx fluid (containing MbPDV) plus venom (CFPV). However, assessments of the percentages of cells undergoing apoptosis under different treatments indicated that SpltMNPV could inhibit MbPDV-induced apoptosis in hemocytes when hosts were first injected with SpltMNPV budded virus (BV) followed by injection with M. bicoloratus CFPV. As the time of injection with SpltMNPV BV increased, the percentages of apoptosis in hemocytes population declined. Furthermore, in vitro, the percentages of apoptosis showed that SpltMNPV BV could inhibit MbPDV-induced granulocytes apoptosis. The occurrence of MbPDV-induced host granulocytes apoptosis was inhibited in the dually infected hosts. As hemocytes apoptosis causes host immunosuppression, the parasitoids are normally protected from the host immune system. However, in larvae infected with both baculovirus and PDV, the parasitoids underwent encapsulation in the host hemocoel. r 2006 Elsevier Ltd. All rights reserved. Keywords: Insect immunity; Hemocyte; Apoptosis; Polydnavirus; Baculovirus; Microplitis bicoloratus
1. Introduction Apoptosis, or programmed cell death, is an energyrequiring programmed physiological process that eliminates superfluous, altered or malignant cells, characterized by a genetically controlled autodigestion of the cell activated via endogenous proteases. Symptoms of apoptosis include cytoskeletal disruption, cell shrinkage, membrane blebbing, nuclear condensation, and internucleosomal DNA fragmentation. Finally, the apoptotic cells fragment into membrane-enclosed bodies that are rapidly phagocytosed by neighboring cells (Savill et al., 2002; Wyllie et al., 1980; Kerr et al., 1972). Corresponding author. Tel.: +86 20 84113860; fax: +86 20 84037472.
E-mail address: [email protected] (Y. Pang). 0022-1910/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2006.04.007
The apoptotic process can be initiated by a variety of regulatory stimuli, including baculoviruses (Clem, 1997, 2001, 2005), which are known to regulate apoptosis of host cells during infection (O’Brien, 1998; Teodoro and Branton, 1997; Razvi and Welsh, 1995). The host apoptosis response to virus infection reduces baculovirus spread at the organismal level (Clarke and Clem, 2003) and may contribute towards limiting host-range and efficiency for baculovirus infections (Zhang et al., 2002a). To suppress this host defense response, baculoviruses have evolved two families of anti-apoptotic genes, i.e. p35 and iap (inhibitor of apoptosis) in order to replicate themselves. These two genes use different mechanisms in the apoptotic pathway to functionally block this cell death reaction (Zoog et al., 1999; Manji et al., 1997; Seshagiri and Miller, 1997; Clem and Miller, 1994; Clem, 2001, 2005).
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Other insect viruses such as polydnaviruses (PDVs) are active inducers of host cell apoptosis. PDVs are obligate symbionts of many endoparasitic wasps in the families Braconidae and Ichneumonidae. Viral DNA is integrated into the wasp’s genome (Xu and Stoltz, 1991; Fleming and Summers, 1991; Belle et al., 2002) and transmitted vertically to the wasp’s offspring in a proviral form. PDVs replicate in ovarian calyx cells in the female wasp. Following replication of the integrated proviral forms, the viral DNA is excised as segments to be incorporated into the virion and the flanking DNAs are rejoined (Gruber et al., 1996; Savary et al., 1997, 1999; Wyder et al., 2002; Wyder and Lanzrein, 2003; Webb, 1998). Virions are stored in the lumen of the calyx and oviduct and the suspension of virus and protein is called calyx fluid. When a female wasp finds a host, she injects calyx fluid, venom produced by the venom gland, and one or more eggs into the hemocoel of the host caterpillar. The virus enters different host tissues and viral genes are transcribed, in the apparent absence of viral replication in the host larva. Both early and/or late viral gene transcripts may be produced while the wasp’s progeny develop to maturity in the host hemocoel. Virus gene expression causes several physiological alternations in parasitized hosts including suppression of the host insect’s immune system. When parasitoid eggs are injected into a non-parasitized larva in the absence of PDV, the parasitoid’s progeny are encapsulated and killed by host immune cells (hemocytes), whereas progeny survive when virus is injected together with the egg (Webb, 1998; Asgari et al., 1997; Strand and Pech, 1995b; Lavine and Beckage, 1995; Strand and Noda, 1991). In several host–parasitoid systems, host hemocyte apoptosis occurs during the initial stages of parasitism, rendering those hemocytes incapable of mounting an encapsulation response (Lavine and Beckage, 1995; Amaya et al., 2005). Encapsulation is the primary cellular immune response of host insects directed against invading metazoan parasites or parasitoids. Encapsulation begins when host granulocytes attach to the surface of a foreign target such as a parasitoid egg. Following attachment, they lyse or degranulate, releasing the contents of their granules over the egg surface, which promotes attachment of plasmatocytes. This is followed by attachment of multiple layers of plasmatocytes to form the capsule. Termination of capsule formation occurs when a subpopulation of granulocytes adheres in a monolayer around the periphery of the capsule. Neither granulocytes nor plasmatocytes are capable of forming a capsule independently (Pech and Strand, 1996; Strand and Pech, 1995a; Schmit and Ratcliffe, 1978, 1977). In Pseudoplusia includens parasitized by Microplitis demolitor, infection with PDV induces host granulocytes to undergo apoptosis while plasmatocytes lose their capacity to adhere to foreign surfaces (Strand and Pech, 1995b; Strand and Noda, 1991). Both baculoviruses and parasitoids are important biological control agents in integrated pest management. Baculovirus cannot directly infect parasitoids and also
cannot replicate in the larvae of the parasitoid (Irabagon and Brooks, 1974; Hotchkin and Kaya, 1983). Hotchkin and Kaya (1983) also found that baculoviruses might affect the host’s immune system, because parasitoid larvae were encapsulated in the host infected with baculovirus. But, no supporting data could demonstrate the effect of baculoviruses on the host’s immune system in parasitized larvae infected with the viruses. Sequence analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) genome reveal the presence of two anti-apoptosis genes, p49 (p35 homologue) and iap in the genome (Pang et al., 2001). We have demonstrated that SpltMNPV can block AcMNPV-induced apoptosis in the S. litura cell line Sl-zsu-1 (Zhang et al., 2002b). SpltMNPV p49 is able to suppress apoptosis induced by infection of a mutant AcMNPV deficient in p35 and rescue the mutant virus replication from apoptosis in Sf-9 cells (Yu et al., 2005). In order to test this hypothesis that baculovirus infection blocks parasitoid PDV-induced host granulocytes apoptosis, resulting in parasitoid larval encapsulation by host hemocytes, we selected the braconid wasp Microplitis bicoloratus, a solitary larval parasitoid of S. litura, which carries a PDV, and SpltMNPV as a model system to study the combined effects of PDV and baculovirus infection on the host insect immune system. 2. Methods and material 2.1. Insects rearing and experimental animals S. litura colony was reared on artificial diet (formulated after Li et al., 1998) at 2771 1C, RH 60–80%, and under a 12:12 h LD photoperiod regime. M. bicoloratus was originally collected from parasitized S. litura larvae in Guangdong Province, China, in 2003. The parasitoid colony was maintained on S. litura larvae reared in the laboratory; adults were also provided honey as a dietary supplement. In all experiments, 2–3 d old mated female wasps were used. Newly ecdysed third instar S. litura larvae were parasitized by M. bicoloratus and screened for the occurrence of apoptotic changes in the host hemocyte population following parasitization. For experimental procedures requiring the injection of calyx fluid plus venom (CFPV)/BV into naive non-parasitized larvae, newly ecdysed fifth instar S. litura larvae were used as subjects for the injection experiments. In these injection experiments, control larvae (newly ecdysed fifth instar S. litura larvae) were mock-injected with Pringle’s solution and these samples were taken at 2 h after injection. 2.2. Baculovirus purification and cell lines SpltMNPV occluded virions (OV) containing polyhedra and budded virus (BV) were produced in S. litura larvae in vivo and purified as previously described (Wang et al., 2002). S. litura Sl-zsu-1 cells (Xie et al., 1998) were
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maintained in TC-100 medium (Gibco) supplemented with 10% fetal bovine serum. Titers of SpltMNPV were determined by TCID50 assay with infected Sl-zsu-1 cells. SpltMNPV BV (2 105 p.f.u. per larvae) was injected into newly ecdysed fifth instar S. litura larvae. 2.3. CFPV preparations Aliquots of CFPV were collected from dissected M. bicoloratus females under a dissecting microscope. The quantities of CFPV used in experiments were expressed in wasp equivalents (Albrecht et al., 1994; Strand and Noda, 1991). A volume of 5 ml CFPV solution (0.5 wasp equivalents) in Pringle’s solution (154.1 mM NaCl, 2.7 mM KCl, 14 mM CaCl2, 22.2 mM dextrose) (Pringle, 1938) was injected into the abdominal cavity of the nonparasitized larvae, then the larvae were screened for the occurrence of hemocyte apoptosis. 2.4. Encapsulation of M. bicoloratus larvae in host larvae co-infected with SpltMNPV polyhedra Development of M. bicoloratus in S. litura larvae required seven days: eggs hatched within 24 h, the first instar larva required two days, the second instar larva needed three days, and the third instar larvae exited the host and pupated in one day at 2771 1C, RH 60–80% and a 12:12 h photoperiod (unpublished data). Newly ecdysed second instar S. litura larvae were divided into two groups. One group was fed with SpltMNPV polyhedra applied to the surface of the artificial diet. Forty eight hours after ingestion of polyhedra, 100 S. litura larvae were exposed to five female wasps’ ovipositions. Thirty parasitized S. litura larvae were dissected after 72 h postparasitization (p.p.) (when immature parasitoid in host has developed from egg into the first larvae) in Pringle’s solution under a binocular microscope. The second group was fed on artificial diet without polyhedra as a control and was parasitized and dissected as above. The experiments were replicated three times. 2.5. DNA fragmentation DNA fragmentation in nuclei of S. litura hemocytes was assessed (Zhang et al., 2002a). S. litura larval hemocytes were collected, washed with PBS buffer (pH 7.2) with 0.1% DTT and centrifuged for 1 min at 1000g. The pellet was then incubated in lysis buffer (20mM Tris–HCl, pH 8.0, 10 mM EDTA and 1% NP-40) for 5 min and the supernatant collected after being centrifuged at 1600g for 5 min. SDS and RNase A were added to a final concentration of 1% and 5 mg/ml, respectively. After 2 h of incubation at 56 1C, the supernatant was digested with proteinase K at 37 1C for 2 h to a final concentration of 2.5 mg/ml. The DNA was precipitated with ethanol, dissolved in TE buffer and separated by electrophoresis on 1% agarose gels.
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2.6. Measurement of percentages of hemocytes undergoing apoptosis In vivo the percentages of apoptosis in hemocytes were measured with flow cytometry. Hochest 33258 (1 mg/ml) was added to a 100 ml hemocyte suspension (1–2 105 cells). After 5–8 min of incubation at 37 1C, the solution in the microfuge tube was cooled over ice and propidium iodide (PI) (5 mg/ml) was added to it. After filtering with 400-mesh net, dual-color flow cytometry was performed. A total of 10,000 hemocytes were counted per treatment. Experiments were replicated three times. For in vitro experiments using purified populations of granulocytes, the percentages of apoptosis in granulocytes were analyzed by quantifying phosphatidylserine residues exposed on the external cell membrane. One microliter of human recombinant fluorescein isothiocyanate (FITC)– conjugated annexin V (Jingmei, China) and PI (2 mg/ml) was added to a 100 ml cell suspension (1 105) of cells in binding buffer (10 mM Hepes/NaOH, pH 7.4; 140 mM NaCl; 5 mMCaCl2). After 15 min of incubation in the dark, dual-color flow cytometry were performed. A total of 10,000 cells were counted in each treatment, and experiments were replicated three times. 2.7. Granulocyte purification and culture The hemocytes of fifth instar S. litura larvae were classified into six different cell types: prohemocytes, plasmatocytes, granulocytes, spherule cells, coagulocytes and oenocytoids (Deng and Lee, 1995a). Hemocytes were collected from larvae by the procedure of Pech et al. (1994) with slight modification. Fifth instar S. litura larvae were rinsed with ddH2O to eliminate surface impurity, then placed on filter paper to absorb water. The larval surface was sterilized with 75% ethanol, and then the animals were bled from the proleg directly into 500 ml of ice-cold anticoagulant buffer (0.098 M NaOH, 0.186 M NaCl, 0.017 M EDTA, 0.041 M citric acid, pH 4.5) (Mead et al., 1986) to constitute the total hemocyte population. Percoll (Amersham Biosciences) gradient separations of the different hemocyte types were performed as outlined by Gardiner and Strand (1999) with slight modification. The gradient consisted of 2 ml of 55% Percoll, layered over 2 ml of 70% Percoll, and 0.5 ml 90% Percoll. One milliliter hemocytes (ca. 1 107 hemocytes total) from the total hemocyte population was layered onto a Percoll gradient. Gradients were centrifuged for 20 min at 480g in 4 1C in an Eppendorf 5810R centrifuge with a JS-7.5 swinging bucker rotor. The bands were transferred into microfuge tubes and washed twice with TC-100 culture medium in an Eppendorf 5415D centrifuge for 5 min at 800g, and the final pellet for each fraction was resuspended in 50 ml TC-100 medium plus 10% fetal bovine serum (FBS) and counted. The band at the top of the gradient contained on average 2.4 106 hemocytes (24% of the starting population), the majority of which were plasmatocytes (19% of the starting
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population) and granulocytes (5% of the starting population) as identified using morphological characters. The cells at the interface of the 55% and 70% Percoll were primarily granulocytes (2.2 106, 22% of the staring population) and no cells at the interface of the 70% and 90% Percoll were collected. For in vitro experiments, 1 105 granulocytes were infected with SpltMNPV BV (MOI ¼ 2) (Zhang et al., 2002a) or with 1 wasp equivalent of M. bicoloratus CFPV (Strand and Pech, 1995b) and then screened for apoptosis. 2.8. Statistical analysis All proportional data were transformed by arcsin square root before analysis of variance, one-way ANOVA, po0:05. Corresponding treatment means were separated using the Student–Newman–Keuls (SNK) test (SPSS Inc., 2003). 3. Results 3.1. Encapsulation of M. bicoloratus larvae in host larvae co-infected with SpltMNPV polyhedra In the control group, in which larvae were only parasitized, the percentages of encapsulation of M. bicoloratus larvae were all zero, and the larvae were all alive and developed normally as first instar larvae. In baculovirus infection, following ingestion of occlusion bodies (polyhedra) by the larva, the protective polyhedron coat dissolved in the midgut, releasing nucleocapsids that fuse with the midgut cells. Initial rounds of viral replication within the nucleus of the infected cell produced a second viral phenotype, the BV, which spreads the infection to other tissues (Pang, 1994). In S. litura larvae, large numbers of BVs are found in the hemolymph at 48 h after SpltMNPV polyhedra ingestion, leading to death at
around 120 h after inoculation. To explore the effect of baculovirus infection on parasitism, second instar S. litura larvae previously fed SpltMNPV polyhedra were parasitized by M. bicoloratus at 48 h after polyhedra ingestion. The larvae were then dissected at 72 h p.p. The fate of the parasitoids in the dually infected hosts was followed, and the percentages of fully encapsulated first instar larvae (Fig. 1aA), partly encapsulated first instar larvae (Fig. 1aB), non-encapsulated first instar larvae (Fig. 1aC) and dead embryos (Fig. 1aD) were 63.3%76.9, 17%70, 9%74.9 and 11.3%75.7, respectively. On fully encapsulated first instar larvae the body surface was covered by hemocytes and the larvae had died. The percentage of fully encapsulated larvae was significantly higher than any of the other three (F3,8 ¼ 9.339, p ¼ 0:005) (Fig. 1b). In partly encapsulated larvae, only part of the body surface was covered by hemocytes and larvae were still alive. In nonencapsulated larvae, the body surface of the larva was not covered by hemocytes and the larvae were still alive. Death was confirmed in suspected dead embryos using trypan blue stain. The percentages between partly encapsulated, non-encapsulated and dead embryos were not significantly different (p40:05) in the dually infected hosts (Fig. 1b). 3.2. Hemocytes of S. litura larvae undergo apoptosis in naturally parasitized hosts To analyze the direct effects of MbPDV on hemocytes in a naturally parasitized host, an oligonucleosome-size ladder was observed after electrophoresis of total genomic DNA extracted from hemocytes of S. litura larvae parasitized by M. bicoloratus. The DNA ladder patterns were detected in hemocytes of the parasitized larvae from 2 to 7 d p.p. (Fig. 2a). To further identify early apoptosis in hemocyte of naturally parasitized host, the flow cytometry analysis was performed in the whole population of hemocytes. Apoptotic cells were detected from 1 to 7 d
Fig. 1. Encapsulation of M. bicoloratus larvae in dually infected S. litura host larvae. (a) Light microscopy of (A) fully encapsulated first instar larva, (B) partly encapsulated first instar larva, (C) non-encapsulated first instar larva, and (D) dead embryo stained with trypan blue. (A, B and C: Bar ¼ 50 mm; D: Bar ¼ 20 mm). (b) The percentages (mean7SE) of non-, partly- or fully-encapsulated M. bicoloratus larvae and dead embryo. Means with the same letter are not significantly different (p40:05). The percentages of encapsulation of M. bicoloratus larvae in control groups were zero and the larvae were all normal and alive.
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Fig. 2. Hemocytes of S. litura larvae underwent apoptosis in naturally parasitized hosts. (a) Detection of fragmented DNA by electrophoresis. Total cellular DNA was extracted from hemocytes collected from 500 ml hemolymph per sample from 2 to 7 days p.p. (lane U: non-parasitized larvae). (b) Flow cytometry analysis of hemocytes indicating the percentages of apoptotic cells. Hemocytes were stained with PI/Hochest 33258. The data is a representation of 3 individual experiments. Newly ecdysed third instar larvae were used in this experiment. Non-parasitized larvae were used as control. (c) Statistical analysis of the percentages (mean7SE) of apoptotic hemocytes from 1 to 7 days p.p.. Means with the same letter are not significantly different (P40:05).
p.p.. Early apoptotic cells can just be stained by Hochest 33258 alone and results appear in the upper left quadrant of the FACS data shown. Then necrotic cells and possible late apoptotic cells can be stained by both Hochest 33258 and PI and appear in the upper right quadrant of the FACS data shown. One representative dot plots from three individual experiments indicated that the percentages of apoptosis in hemocytes population from 1 to 7 d p.p. (11.99%, 15.02%, 25.49%, 20.76%, 21.75%, 20.39% and 24.56%, respectively) were higher than those in the control (non-parasitized larvae) (7.35%) (Fig. 2b). In order to further understand early apoptotic cells at different times during parasitoid development in its host, three time points were analyzed using one-way ANOVA statistical analysis,
and the results indicated that there were no significant differences between the percentages of apoptosis in hemocytes from 1 to 7 d p.p. (F6,14 ¼ 2.499, p ¼ 0:074) (Fig. 2c). The levels of MbPDV-induced host hemocytes in early apoptosis in naturally parasitized hosts were persistent and stable during parasitoid development. 3.3. Hemocytes of S. litura larvae undergo apoptosis in nonparasitized larvae after injection of M. bicoloratus ovarian calyx fluid (containing PDV) plus venom Injection of CFPV induced apoptosis in hemocytes of S. litura larvae (Fig. 3). One representative dot plot from three individual experiments indicated that the percentages
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Fig. 3. Hemocytes of S. litura larvae underwent apoptosis in non-parasitized larvae after injection of M. bicoloratus ovarian calyx fluid (containing PDV) plus venom (CFPV) (0.5 wasp equivalents). (a) Flow cytometry analysis of hemocytes indicating the percentages of apoptotic cells. Hemocytes were stained with PI/Hochest 33258. The data is a representation of 3 individual experiments. Newly ecdysed fifth instar larvae were used in this experiment. (b) Statistical analysis of the percentage of apoptosis (mean7SE) in hemocyte population from 2 to 72 h p.i.. Means with the same letter are not significantly different (P40:05).
of apoptosis in hemocyte populations collected from 2 to 72 h p.i. were all higher than controls (mock-injected larvae) (7.75%) (Fig. 3a). In order to further understand early apoptotic cells changes in hemocytes of host larvae injected with CFPV from 2 to 72 h p.i., these data were analyzed using one-way ANOVA statistical analysis and the results indicated that the percentage of apoptosis was significantly higher 72 h p.i. as compared to 2 h p.i. (F 4;10 ¼ 3:412, p ¼ 0:053) (Fig. 3b). There were no significant differences in the percentages of apoptosis between 12, 24 and 48 h p.i. They were lower compared to 72 h p.i. and higher compared to 2 h p.i. As shown above, occurrence of apoptosis was the highest at 72 h after injection of CFPV. The results suggested that levels of hemocytes in early apoptosis gradually increased in vivo. 3.4. Hemocytes of S. litura larvae underwent transient apoptosis after injection of SpltMNPV BV The flow cytometry analysis was performed on hemocytes following injection of SpltMNPV BV from 2 to 72 h p.i., and we found that injection of SpltMNPV BV induced
transient apoptosis in hemocytes of S. litura larvae at an early period p.i. (Fig. 4a). After 2 h of injection with SpltMNPV BV, 38.68% of hemocytes underwent apoptosis (9.51% in control conditions). As the time of injection with SpltMNPV BV increased, the percentages of apoptosis in hemocytes population declined. After 48 and 72 h p.i., the percentages of apoptosis (8.29% and 1.76%, respectively) were lower than controls. Further one-way ANOVA statistical analysis of the percentages of apoptosis in hemocytes from 2 to 72 h p.i., indicated that the percentages of apoptosis in hemocyte populations were significantly higher at 2 and 12 h p.i. as compared to 48 and 72 h p.i. (F 4;10 ¼ 8:242, p ¼ 0:003) (Fig. 4b). The percentages of apoptosis between 2 and 12 h p.i. were not significantly different. Similarly, there were no significant differences between the percentages of apoptosis at 48 and 72 h p.i. To further verify that the hemocytes underwent transient apoptosis after injection of SpltMNPV BV, DNA ladder were performed with hemocytes from 2 to 24 h p.i SpltMNPV BV. An oligonucleosome-size ladder was observed in DNA isolated at 2 h p.i (Fig. 4c). The results suggested that the level of hemocyte apoptosis
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Fig. 4. Hemocytes of S. litura larvae underwent transient apoptosis after injection of SpltMNPV BV (2 105 p.f.u. per larvae). (a) Flow cytometry analysis of hemocytes indicating the percentages of apoptotic cells. Hemocytes were stained with PI/Hochest 33258. The data is a representation of 3 individual experiments. Newly ecdysed fifth instar larvae were used in this experiment. (b) Statistical analysis of the percentage of apoptosis (mean7SE) in hemocyte population from 2 to 72 h p.i.. Means with the same letters are not significantly different (P40:05). (c) Detection of fragmented DNA by electrophoresis. Total cellular DNA was extracted from hemocytes collected from 500 ml hemolymph per sample at 2, 12, 24 h p.i. and from mock-injected (Mi) larvae injected with Pringle’s solution, respectively.
in hosts injected with SpltMNPV BV rapidly declined in vivo. 3.5. Inhibition of apoptosis in hemocytes of parasitized S. litura larvae by SpltMNPV To find out whether SpltMNPV could inhibit MbPDVinduced apoptosis in hemocytes, hosts were first injected with SpltMNPV BV followed by injection of M. bicoloratus CFPV 24 h after BV injection. Then, the percentage of apoptosis was measured by flow cytometry at various times after CFPV injection. Apoptosis was significantly reduced from 24 h after injection of CFPV (48 h BV p.i.) (12.49%) and decreased even further at 48 and 72 h after CFPV injection (9.42% and 4.71%) (Fig. 5a). One-way ANOVA statistical analysis of the percentages of apoptosis in
hemocytes from 2 to 72 h p.i. indicated that the percentage of apoptosis in hemocytes population was significantly lower at 72 h CFPV p.i. as compared to 2, 12, 24 and 48 h p.i. (F 4;10 ¼ 28:454, po0:001) (Fig. 5b). The percentages of apoptosis in hemocytes were significantly lower between 24 and 48 h CFPV p.i. as compared to 2 and 12 h CFPV p.i. (po0:05). The percentages of apoptosis at 2 and 12 h CFPV p.i. were not significantly different. Similarly, there were no significant differences between the percentages of apoptosis at 48 and 72 h p.i. CFPV (p40:05). The results suggested that the levels of hemocyte apoptosis in hosts first injected with SpltMNPV BV followed by injection of M. bicoloratus CFPV 24 h after BV injection gradually declined in vivo. The occurrence of MbPDVinduced host hemocytes apoptosis was inhibited in dually infected hosts.
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Fig. 5. Inhibition of apoptosis in hemocytes of S. litura larvae by SpltMNPV in dually, SpltMNPV BV (2 105 p.f.u. per larvae) and M. bicoloratus CFPV (0.5 wasp equivalents per larvae), injected hosts. (a) Flow cytometry analysis of hemocytes indicating the percentages of apoptotic cells. Hemocytes were stained with PI/Hochest 33258. The data is a representation of 3 individual experiments. Newly ecdysed fifth instar larvae were used in this experiment. (b) Statistical analysis of the percentage (mean7SE) of apoptotic hemocytes from 2 to 72 h p.i.. Means with the same letters are not significantly different (P40:05).
3.6. Flow cytometry analysis of hemocytes comparision between M. bicloratus CFPV injected larvae, SpltMNPV BV injected larvae, and M. bicloratus CFPV injected larvae 12 h after SpltMNPV BV injection
This clearly showed that SpltMNPV BV infection could inhibit PDV-induced apoptosis and consequently the immune suppression leading to encapsulation of the developing parasitoids (Fig. 1).
Based on own previous results, we further compared the percentages of apoptosis in hemocytes of hosts between injection of CFPV 12 h after SpltMNPV BV injection, injection of CFPV alone and injection SpltMNPV BV alone after various times to focus on apoptotic induction (by MbPDV) and blocking (by baculovirus) (Fig. 6). The percentage of apoptosis was measured by flow cytometry at various times after CFPV injection. At 24 h after CFPV injection, the percentage of apoptosis in hemocytes of hosts that were first injected with SpltMNPV BV followed by injection of M. bicoloratus CFPV at 12 h after BV injection were 11.04% (15.75% in CFPV injection alone, 7.16% in BV injection alone). At 48 h after CFPV injection, the percentages of apoptosis declined to 4.27% (10.49% in CFPV injection alone, 3.65% in BV injection alone) and decreased even further at 72 h p.i. to 1.61% (17.63% in CFPV injection alone, 1.16% in BV injection alone).
3.7. In vitro inhibition of M. bicoloratus CFPV-induced apoptosis in purified granulocytes by SpltMNPV BV To identify whether granulocytes underwent apoptosis and to further confirm inhibition of MbPDV-induced apoptosis by SpltMNPV, in vitro assays were carried out using purified S. litura granulocytes. When cells were inoculated with CFPV alone considerable apoptosis was noted, apoptosis was detected at 12 h after inoculation by flow cytometry using Annexin-V conjugated with FITC; the early apoptotic cells can be stained by Annexin-V alone and appear in the lower right quadrant of the FACS data shown; the necrotic cells can be stained by PI alone and appear in the upper left quadrant of the FACS data shown (Fig. 7). However, when cells were inoculated with CFPV 2 h after BV infection, the percentage of apoptosis was significantly reduced to 6.7% compared to 23.7% when
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Fig. 6. Flow cytometry analysis of hemocytes comparision between M. bicoloratus CFPV (0.5 wasp equivalents per larvae) injected larvae, SpltMNPV BV (2 105 p.f.u. per larvae) injected larvae and M. bicoloratus CFPV injected larvae 12 h after SpltMNPV BV injected. Hemocytes were stained with PI/ Hochest 33258. The data is a representation of 3 individual experiments. Newly ecdysed fifth instar larvae were used in this experiment.
CFPV was used alone (Fig. 7). Mock infected or 2 h BV infected cells showed low levels of apoptosis (Fig. 7).
4. Discussion Baculoviruses and parasitoids are critically important biological control agents in integrated pest management (IPM). They have been simultaneously and sequentially used to target insect pests. In this study, we examined the impacts of both baculovirus and PDV infection on the host cellular immune response. Our results demonstrated that a baculovirus expressing anti-apoptotic gene(s) inhibited parasitoid PDV-induced host granulocyte apoptosis, which resulted in the parasitoid larvae becoming encapsulated by host blood cells. To our knowledge, this is the first report describing the effect of an anti-apoptosis mechanism of
baculoviruses on development of parasitoid larvae that acts on PDV-induced apoptosis in host hemocytes. There are few identified baculovirus caspase inhibitors, namely P35, IAPs and P49. Baculovirus protein P35 is a wide-ranging caspase inhibitor, inhibiting mammalian caspases 1–4 and 7 (Barry and McFadden, 1998; Miller, 1997). Caspase-dependent autophagy has been documented in Drosophila melanogaster salivary gland tissues, in which baculovirus p35 expression inhibits cell death (Kimura et al., 2004). The other anti-apoptotic baculovirus protein is IAP, which has been shown to have an inhibitory effect on apoptosis involving X IAP, c-IAP, c-IAP2 and survivin (Deveraux and Reed, 1999). These proteins have been shown to interact with and suppress the activity of caspase-3 (Nogal et al., 2001). SpltMNPV P49 protein suppresses apoptosis by inhibiting protein death caspases3-like (Yu et al., 2005).
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Fig. 7. Flow cytometry analysis of purified S. litura granulocytes (1 105/ well) in dually infected in vitro with SpltMNPV BV (MOI ¼ 2) and M. bicoloratus CFPV (1 wasp equivalents). Granulocytes were stained with Annexin-V-FITC/PI. The data is a representation of 3 individual experiments.
PDVs have been shown to induce apoptosis in lepidopteran host granulocytes in P. includens/M. demolitor (Strand and Pech, 1995b) and in Manduca sexta/Cotesia congregate systems, although host hemocyte apoptosis is transient in the later system (Amaya et al., 2005). Although PDV-induced apoptotic genes have not been reported, the expression of a Toxoneuron nigriceps PDV-encoded TnBV1 protein can increase caspase-3-like activity and cause apoptosis-like programmed cell death in cultured lepidopteran insect cells (Lapointe et al., 2005). This result indicated that the induction of apoptosis by TnPDV involves a caspase-dependent pathway. In the present study, we showed that MbPDV could induce apoptosis in S. litura hemocytes both in vivo and in vitro; SpltMNPV was able to inhibit PDV-induced granulocytes apoptosis. In vivo dual infection experiments demonstrated that as time passed following injection of SpltMNPV BV, the percentages of PDV-induced apoptosis in the total
hemocyte population declined. Under these conditions, most parasitoid larvae were fully encapsulated by host hemocytes and some were partly encapsulated. However, at earlier hours after SpltMNPV infection, when apoptosis of hemocytes by PDV was occurring in a preponderance in the hosts, parasitoid larvae could not be encapsulated. Irabagon and Brooks (1974) reported that the longer the interval between host parasitism and NPV infection, the higher the percentage of parasitoid cocoons. A more recent study by Nusawardani et al. (2005) showed that wild-type and recombinant AcMNPV (expressing a catepsin-like protease) decrease the survival of Cotesia marginiventris parasitoid emerging from Heliothis virescens if the host is infected with the virus less than 72 h after parasitization. However, at later stages of parasitism (120 h) the baculoviruses do not have detrimental effect on parasitoid survival. The percentage of apoptosis in hemocyte populations of the parasitized S. litura was stably persistent until M. bicoloratus larva finished development. Although we are unsuccessful in culturing plasmatocytes in vitro, PDVs have been shown to lose their capacity to adhere to foreign surface in lepidopteran host plasmatocytes (Strand and Pech, 1995b), and PDVs proteins are an immune suppressor caused by the destabilization of actin filaments (Asgari et al., 1997) and inhibition of spreading and adhesion, especially, plasmatocytes (Amaya et al., 2005). Thus, it will be worthwhile to perform additional experiments using Hi5 cells, a hemocyte-like cell line (substitute plasmatocytes) to confirm the effects of MbPDV on Hi5 in vitro. Interestingly, we found that injection of SpltMNPV BV into S. litura larvae also induced transient apoptosis in hemocytes in vivo. Generally, baculoviruses are host specific, and can only induce apoptosis in heterogenous hosts, but we found that AcMNPV can induce apoptosis in S. litura hemocytes (Zhang et al., 2002a). However, our results revealed that SpltMNPV BV could not induce apoptosis in purified granulocytes in vitro (data not shown). Hemocytes of S. litura larvae infected with SpltMNPV can form polyhedra in the nucleus (Deng and Lee, 1995b). In our experiments, we also found that embryos were never encapsulated. The egg surface of Macrocentrus cingulum has a fibrous layer which plays a role in protecting eggs from host’s immune attack (Hu et al., 2003). Thus, we presume that M. bicoloratus egg’s surface may have some factors protecting the egg or developing embryo from encapsulation in the early periods. Polydnavirus expression needs 6 h to be detected in some species (Strand, 1994), but only 1 h was needed for Sephadex G-25 beads to be avidly encapsulated by hemocytes in in vitro experiments performed in hemocytes of S. litura larvae (data not shown). In conclusion, our findings demonstrated that SpltMNPV could inhibit MbPDV-induced apoptosis in S. litura granulocytes. This might have implications for concurrent application of the virus and the parasitoid in the field. In natural parasitization, MbPDVs induce
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apoptosis in the host’s hemocytes to suppress host cellular immunity. However, the baculovirus inhibitors of apoptosis apparently defy PDV-induced apoptosis, making the developing parasitoid vulnerable to encapsulation. What MbPDV gene(s) are responsible for inducing apoptosis in host hemocytes remains to be discovered. This gene and the one caspase inhibitor encoded by SpltMNPV, p49, will require further research using Hi5 cells, a hemocyte-like cell line, and an in vitro approach to understand the mechanisms of anti-apoptosis and apoptosis in both baculovirus and polydnavirus. Acknowledgements The authors gratefully acknowledge Dr. Nancy E. Beckage (University of California-Riverside) and Dr. Sassan Asgari (University of Queensland) for their comments and suggestions on the manuscript. The research was supported by the National Major Basic Research Project (‘973’) of China (G2000016209), National Natural Science Foundation of China (30530540) and Open-funding of the State Key Laboratory of Biocontrol in Sun Yat-Sen University (0404). References Albrecht, U., Wyler, T., Pfister-Wilhelm, R., Gruber, A., Stettler, P., Heiniger, P., Kurt, E., Schu¨mperli, D., Lanzrein, B., 1994. Polydnavirus of the parasitic wasp Chelonus inanitus (Braconidae): characterization, genome organisation and time point of replication. Journal of General Virology 75, 3353–3363. Amaya, K.E., Asgari, S., Jung, R., Hongskula, M., Beckage, N.E., 2005. Parasitization of Manduca sexta larvae by the parasitoid wasp Cotesia congregate induces am impaired host immune response. Journal of Insect Physiology 51, 505–512. 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. Barry, M., McFadden, G., 1998. Apoptosis regulators from DNA viruses. Current Opinion in Immunology 10, 422–430. Belle, E., Beckage, N.E., Rousselet, J., Poirie, M., Lemeunier, F., Drezen, J-M., 2002. Visualization of polydnavirus sequences in a parasitoid wasp chromosome. Journal of Virology 76, 5793–5796. Clarke, T.E., Clem, R.J., 2003. In vivo induction of apoptosis correlating with reduced infectivity during baculovirus infection. Journal of Virology 77, 2227–2232. Clem, R.J., 1997. Regulation of programmed cell death by baculoviruses. In: Miller, L.K. (Ed.), The Baculoviruses. Plenum, New York, pp. 237–266. Clem, R.J., 2001. Baculoviruses and apoptosis: the good, the bad, and the ugly. Cell Death and Differentiation 8, 137–143. Clem, R.J., 2005. The role of apoptosis in defense against baculovirus infection in insects. Current Topics in Microbiology and Immunology 289, 113–239. Clem, R.J., Miller, L.K., 1994. Control of programmed cell death by the baculovirus genes p35 and iap. Molecular and Cellular Biology 14, 5212–5222. Deng, R.Q., Lee, C.Y., 1995a. A study on the types and in vitro changes of hemocytes of larval Spodoptera litura. Supplement to the Journal of Sun Yatsen University 2, 15–17. Deng, R.Q., Lee, C.Y., 1995b. A study on the cytophathology of larval Spodoptera litura infected with SlNPV. Supplement to the Journal of Sun Yatsen University 2, 18–20.
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