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Mechanism of reduction in the number of the circulating hemocytes in the Pseudaletia separata host parasitized by Cotesia kariyai
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Journal of Insect Physiology 50 (2004) 1103–1111 www.elsevier.com/locate/jinsphys
Mechanism of reduction in the number of the circulating hemocytes in the Pseudaletia separata host parasitized by Cotesia kariyai Tokiyasu Teramoto, Toshiharu Tanaka Laboratory of Applied Entomology, Graduate School of Bio-Agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan Received 28 January 2004; received in revised form 26 August 2004; accepted 30 August 2004
Abstract Larval endoparasitoids can avoid the immune response of the host by the function of polydnavirus (PDV) and venom. PDV infects hemocytes and affects the hemocyte function of the host. In this paper, we investigated how PDV and venom affect the hemocyte population of the host. Cotesia kariyai, the larval endoparasitoid, lowers the hemocyte population of the noctuid host larvae soon after parasitization. The reduction in the number of circulating hemocytes is caused by the breakdown of the circulating hemocytes and of the hematopoietic organ which generates the circulating hemocytes. The decrease in the number of hemocytes shortly after parasitization is a response to the venom. However, the decrease in hemocyte population on and after 6 h postparasitization appears to be caused by the PDV. Apoptosis in circulating hemocytes was observed on and after 6 h post-injection of PDV plus venom. It was revealed through cytometry that mitosis of circulating hemocytes was halted within 24 h after the injection of PDV plus venom. Apoptosis in the hematopoietic organ was induced 12 h after the injection of PDV plus venom. Furthermore, the plasma from the hosts injected with PDV plus venom depressed the number of hemocytes released from the hemotopoiteic organs. r 2004 Elsevier Ltd. All rights reserved. Keywords: Polydnavirus; Venom; Hemocytes; Hematopoiesis; Apoptosis
1. Introduction Larval endoparasitoids must avoid the host immune response to develop in the host larvae. The response to large foreign objects like parasitoid eggs or larvae is a cellular defense reaction like encapsulation (Salt, 1970; Vinson, 1977; Ratcliffe and Rowley, 1979; Gupta, 1989; Engstro¨m, 1992; Pathak, 1993; Pech and Strand, 1995; Ribeiro et al., 1996; Lavine and Strand, 2002). For successful parasitization, endoparasitoids need polydnavirus (PDV) and venom, which are injected with their eggs into the host larva at the time of oviposition. PDV infects the hemocytes and causes much change in the host hemocytes; for example, inhibition of plasmatocyte Corresponding author. Tel.: +81 52 789 4033; fax: +81 52 789 4032. E-mail address: [email protected] (T. Teramoto).
0022-1910/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2004.08.005
spreading (Strand, 1994), apoptosis in granular cells (Strand and Pech, 1995), damage to the cytoskelton of the hemocytes (Webb and Luckhart, 1996), and blebs on hemocytes (Lavine and Beckage, 1996, Luckhart and Webb, 1996). Additionally, the total hemocyte count (THC) in parasitized hosts is altered (Stettler et al., 1998), being increased in four cases and decreased in two, and the adhesive ability of the hemocytes is reduced in almost all cases (Stettler et al., 1998). The decrease in THC seems to be the result of the induction of apoptosis by PDV. It has been reported that PDV induces apoptosis of granular cells in Pseudoplusia includens parasitized by Microplitis demolitor (Strand and Pech, 1995) and that hemocytes are blebbed and collapsed by PDV in the Manduca sexta–Cotesia congregata system (Lavine and Beckage, 1995). However, it has not been comprehensively clarified how the hemocyte population is altered
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by PDV and venom. Notably, it has not been examined whether the circulating hemocytes are supplied from hematopoietic organs in the host, parasitized or injected with the PDV and venom. It is known that the population of circulating hemocytes is supplied by the mitosis of circulating hemocytes itself and from hematopoietic organs. Hemocyte mitosis in circulating hemocytes and in hematopoietic organs has been observed by the incorporation of bromodeoxyuridine (BrdU) in vivo in Pseudoplusia includens and Spodoptera frugiperda (Gardiner and Strand, 2000). Mitoses in circulating hemocytes has been observed in Bombyx mori, Galleria mellonella and Euxoa declarata; it was verified that 1–8% of the population of circulating hemocytes are in the mitotic phase (Shapiro 1968; Jones 1970; Arnold and Hinks, 1976; Beaulaton, 1979). In arthropod shrimp Penaeus japonicus, the cell cycle of circulating hemocytes was reported by measuring the DNA content of the circulating hemocytes (Sequeira et al., 1996). Recruitment of hemocytes from hematopoietic organs has been reported in B. mori (Nakahara et al., 2003) and Pseudoplusia includens and S. frugiperda (Gardiner and Strand, 2000). In this study, we examine whether the decrease of circulating hemocytes is caused by the CkyPDV and/or venom, and then observe how the CkyPDV and venom affect the hematopoietic organ of the host in vitro, under several conditions.
venom is a physiologically effective dose (Tanaka, 1986). Venom was adjusted to a 1/3 female equivalent with PBS (0.067 M phosphate buffer, pH 7.4, 0.87% NaCl) after centrifuging at 12,000g for 10 min to remove cell debris. CkyPDV was adjusted to a 1/3 female equivalent in the prepared venom solution. A 1 ml mixture of a 1/3 female equivalent of CkyPDV plus venom was injected into the second proleg of the host larva with a fine glass needle. 2.3. Counting the THC To investigate the effects on the number of circulating hemocytes by parasitization or by CkyPDV plus venom injection, hemolymph was bled onto parafilm (American Can) on ice. Host larvae were narcotized with CO2, immersed in 70% alcohol for 5–10 s, washed with sterilized distilled water, and remained in an ice bath, allowing the host larvae to cool down for a few minutes. Ten microliters of hemolymph on parafilm were put in 490 ml of ACS (Anticoagulant solution; 0.098 M NaOH, 0.186 M NaCl, 0.017 M EDTA, 0.041 M Citric acid, pH 4.5) to prevent an aggregation of hemocytes. No aggregation of hemocytes was observed under these conditions. The total number of hemocytes was counted by a hemocyte counting chamber (Fuchs–Rosenthal, Hirschmann EM Techcolor, Germany). 2.4. Detection of fragmented DNA on electrophoresis
Cotesia kariyai colonies have been established in our laboratory since 1990 and are supplied from parasitized larvae of Pseudaletia separata collected in Kanoya, Kagoshima Prefecture every year. Adult wasps were kept for mating under long day conditions at 2571 1C with a 20–30% of sugar solution as food in a glass tube. Parasitization was performed individually to avoid superparasitism (Nakamatu et al., 2001). A laboratory colony of Pseudaletia separata was established using larvae collected in cornfields at the Agricultural Field Station of Nagoya University. The lepidopteran host was maintained on an artificial diet (INSECTA-LFs, Nihon Nohsan Co.) under a long day (16 h light:8 h dark) photoregime at 2571 1C.
Circulating hemocytes were collected from each larva injected with CkyPDV plus venom or from an untreated control. Hemolymph with hemocytes was flushed into a 1.5 ml tube while 500 ml ACS was injected into the second proleg with a 1 ml syringe; the sample was centrifuged at 760 g for 10 min to remove plasma. Hemocyte pellets were re-suspended in TNE (10 mM Tris-HCl, pH7.5, 100 mM NaCl, 1 mM EDTA) and treated with proteinase K digestion (10 mg/ml) to extract genomic DNA with phenol–chloroform, according to the method of Sambrook and Russell (2001). The supernatant was incubated for 1 h at 37 1C after adding DNase-free RNaseI (50 mg/ml, Sigma), then re-extracted with phenol–chloroform. The DNA was ethanol-precipitated, washed with 70% ethanol and re-suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA). Thirty micrograms of undigested hemocyte genomic DNA was separated on a 2% agarose gel.
2.2. Injection of CkyPDV and venom
2.5. Cytometory by laser scanning cytometer
CkyPDV and venom were collected separately from 2d-old females as previously described (Nakamatu et al., 2001). A female wasp injects approximately 18% of her total venom reservoir into the host hemocoel at the time of oviposition, and a 1/3 female equivalent of PDV or
To examine the effects of CkyPDV and venom on DNA ploidy, the amount of DNA was measured. Hemocytes in hemolymph flushed from the hosts by ACS injection were centrifuged at 760g for 10 min, washed once with ACS, fixed on ice with 2.5%
2. Materials and methods 2.1. Insect rearing
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2.6. The number of hemocytes released from hematopoietic organ in vitro We clarified the effects of CkyPDV plus venom on hematopoiesis by counting the hemocytes released from the hematopoietic organ in vitro. The effect on the hematopoietic organ was examined from the following two viewpoints. If CkyPDV plus venom affects the hematopoietic organ directly, fewer hemocytes might be released from the hematopoietic organ influenced by PDV plus venom, even under normal hemolymph conditions. Another consideration was that the humoral factor, which affects the hematopoiesis, exists in the hemolymph of the host injected with CkyPDV plus venom. The hematopoietic organs were dissected from the host larvae 1, 2, and 3 d after the injection of CkyPDV plus venom, as mentioned above. The hemolymph from the control or the injected larvae 1, 2, and 3 d after the injection of CkyPDV plus venom was added to 0.1 mM 1-phenyl-2-thiourea (PTU) and heated at 60 1C for 10 min to inhibit melanization after collection. Then, the treated hemolymph was used as plasma after centrifugation at 760g for 5 min and filteration with a 0.22 mm filter (Millipore). Hematopoietic organs were taken out along with a wing disc, washed in MGM450 medium (Mitsuhashi and Inoue, 1998) three times, and then incubated in 100 ml MGM450 containing 30% plasma for 24 h. Hemocytes released from the hematopoietic organ in each medium were collected to be counted by the hemocyte counting chamber.
2.7. Morphological observation of hematopoietic organ To clarify whether CkyPDV plus venom affects hematopoietic organs that supply circulating hemocytes, CkyPDV plus venom was injected into Day-0 sixth instar hosts. The hematopoietic organs taken out with wing-disc from larvae 1, 2, and 3 d after the injection of CkyPDV plus venom and non-treated Day-1 sixth instars as a control were fixed with 2.5% glutaraldehyde, postfixed with Bouin’s solution for 24 h, then dehydrated and embedded in paraffin. Eight mm sections were stained with Mayer’s hematoxylin and 1% Eosin Y solution (Sano, 1965).
2.8. Detection of fragmented DNA in hematopoietic organs Injection of CkyPDV plus venom was performed on Day-0 sixth instar hosts. Hematopoietic organs were collected 1, 2, and 3 d after injection to detect apoptosis. Day-1 sixth instar hosts were used as controls. Immediately after dissection, hematopoietic organs were fixed with 4% paraformaldehyde, post-fixed with Bouin’s solution for 24 h, dehydrated and embedded in paraffin, then cut into 8 mm sections. The fragmented DNA was labeled with an In Situ Cell Death Detection kit (Roche) and detected with BM Purple AP substrate (Roche). 2.9. Statistical analysis The Kruskal–Wallis (po0.01) test was performed for comparison among three or more results. Mann–Whitney’s U-test (po0.05) was used to determine significant differences between two sets of data.
3. Results 3.1. Effect of CkyPDV plus venom on hemocyte count The number of hemocytes in parasitized hosts was drastically decreased within 24 h post-parasitization and remained at a lower level than that of the control (Fig. 1A) through the time of the emergence of n=10
(X107) 8
Number of total hemocytes count (/ml)
glutaraldehyde in ACS for 4 h, re-suspended in 70% alcohol after centrifugalizing, then stored at 20 1C until measured. After washing once with PBS, the hemocyte samples were stained with PI (Pyropigium Iodite, Sigma) containing RNase H (50 mg/ml, Sigma) and were measured by LSC101 (Olympus, Tokyo). Typical data are shown graphically in Fig. 3. Five replications were performed in each treatment.
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Fig. 1. Parasitization of Cotesia kariyai reduces the number of circulating hemocytes in the host. Parasitization occurred on Day-0, sixth instar of Psudaletia separata host. Counting the hemocytes of each point was performed in ten replications. The vertical bar at each point shows the standard deviation.
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parasitoid larvae (data not shown). It was predicted that PDV and venom, injected along with the eggs at oviposition, would be involved in the rapid decrease in hemocytes count soon after oviposition. Injection with CkyPDV plus venom made the hemocytes decrease in number rapidly within 1 h post-injection, compared to that of saline injection (Fig. 2A). Although the hemocytes in the host injected with venom alone decreased only 1 h after injection, THC reached the same level as that of the control 12 and 24 h after venom injection, suggesting that the venom was responsible for
the reduction of THC within 1 h. The CkyPDV alone had no affect (Fig. 2B). 3.2. DNA ladder in hemocytes caused by CkyPDV plus venom To investigate if the CkyPDV plus venom caused apoptosis in the circulating hemocytes, genomic DNA from the hemocytes of the host injected with CkyPDV plus venom was analyzed by agarose gel electrophoresis. The DNA ladder was not detected 1–3 h after injection with the CkyPDV plus venom, but was weakly detected at 6 h and strongly detected at 10 h after injection (Fig. 3). 3.3. CkyPDV affects DNA ploidy of hemocyte Three peaks of 2C, 4C and 8C in the DNA ploidy of the circulating hemocytes were observed in the control larva (Fig. 4). The CkyPDV plus venom affected 4C and 8C peaks 8 h after injection, and the 8C peak disappeared 12 h after the injection. An injection of venom alone and saline had no effect on the peak value. 3.4. CkyPDV affects the hematopoietic organ
Fig. 2. Effects of CkyPDV and/or venom on the number of circulating hemocytes. (A) Hemocyte population after injection with CkyPDV plus venom and of saline into Day-0 sixth instar of the host Psudaletia separata. (B) Hemocyte population after each injection of saline, PDV plus venom, venom, and PDV alone into Day-0 sixth instar hosts. Hemocyte counting was repeated with each of the 10 host larvae on each point after injection. The vertical bar at each point shows the standard deviation.
To clarify the depression of hematopoiesis as a function of CkyPDV, we examined damage to the hematopoietic organ and the humoral factor that influences hematopoiesis by counting the number of hemocytes released from hematopoietic organs in vitro. The number of hemocytes released from the hematopoietic organ in the control larva was approximately 4000 in 24 h (Fig. 5A). When both the hematopoietic organ and the plasma as a humoral factor were prepared from the host 24 h after the injection of CkyPDV plus venom, about 600 cells had been released into the incubation medium for 24 h (Fig. 5A). On the other hand, when the hematopoietic organ from the control larva was incubated with plasma from hosts 1, 2, or 3 d after the injection of CkyPDV plus venom, the number of released cells decreased to about 2000 for 24 h (Fig. 5B). This was a significant difference (po0.01) from the number of hemocytes released in the incubation medium that contained the control hemolymph. Furthermore, the first 24 h of each hematopoietic organ’s incubation 1, 2, or 3 d after injection of CkyPDV plus venom with normal plasma, depressed hemocyte release to approximately 1000 (Fig. 5C). However, during the next 24 h of incubation with normal plasma, the hematopoietic organs 1 and 2 d post-injection released 1600 cells, but hematopoietic organs 3 d after the injection released 1000 cells, suggesting that the hematopoietic organ was changed within 2 days after CkyPDV infection, and was not refreshed when supplied with normal plasma.
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Fig. 3. DNA ploidy of hemocytes in hosts injected with CkyPDV plus venom or venom alone. The amount of DNA in hemocytes was measured with a laser scanning cytometer (Olympus LSC101). The horizontal bar shows the relative DNA amount. Injection was performed in Day-0 of the sixth instar host. Five replications were performed in each treatment.
3.5. Morphological changes in hematopoietic organs caused by CkyPDV plus venom The hematopoietic cells from non-treated sixth instar hosts were large, had large nuclei and the cytoplasm stained well with Eosin Y solution (Fig. 6). However, the nucleus started to atrophy 12 h after injection of CkyPDV plus venom and was strongly atrophied 24 h post-injection. 3.6. Apoptosis in hematopoietic organs To confirm that apoptosis in the hematopoietic organs was caused by CkyPDV plus venom, DNA fragments were detected in the hematopoietic organs by TUNEL methods. Many cells in the hematopoietic organs from the host injected with CkyPDV plus venom had fragmented DNA in their nucleus 24 h after injection (Fig. 7). No DNA fragments were observed in hematopoietic organs of the control host.
4. Discussion In host Pseudaletia separata parasitized by Cotesia kariyai, the hemocytes decreased in number during early
stages of parasitization and remained at a low density. It was similarly reported that the number of hemocytes in the host decreased during the early stages of parasitization of C. melanoscela (Guzo and Stoltz, 1987), Campoletis sonorensis (Davis et al., 1987) and Cardiochiles nigriceps (Vinson, 1971). Injection of venom alone decreased the number of hemocytes rapidly within 1 h after injection, although CkyPDV and venom also caused a decrease in the number of hemocytes. This suggests that the venom causes the first decrease in hemocytes. However, the mechanism that caused the decrease of circulating hemocytes soon after the injection was not clarified. The hemocyte decrease on and after 4 h post-parasitization seemed to be caused by CkyPDV. Venom seems to cause the temporal decrease of the circulating hemocytes until the PDV function begins (Kitano, 1986; Tanaka, 1987b). The CkyPDV genes were expressed in the hemocytes 4 h after parasitization (Yamanaka et al., 1996). Furthermore, the CkyPDV induced apoptosis in the circulating hemocytes; the DNA ladder in the hemocytes was detected 6 h after injection of CkyPDV plus venom. Microplitis demolitor PDV induces apoptosis in host hemocytes (Strand and Pech, 1995). Cotesia congregata PDV makes blebs on the host hemocytes as a sign of
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Fig. 4. DNA profile of circulating hemocytes from hosts injected with CkyPDV plus venom. Day-0 sixth instars of Pseudaletia host were injected with CkyPDV plus venom; the genomic DNA of circulating hemocytes was analyzed on a 2% agarose gel. The right lane is a DNA marker. Numbers on each lane represent hours after injection.
apoptosis (Lavine and Beckage, 1996). Apoptosis in circulating hemocytes seems to be induced by CkyPDV and decreases the number of hemocytes in the parasitized host larva 4 h post-parasitization. Mitosis in circulating hemocytes of lepidopteran larvae is observed in B. mori, G. mellonella, and E. declarata (Shapiro, 1968; Jones, 1970; Arnold and Hinks, 1976; Beaulaton, 1979). Hyposoter fugitivus PDV inhibits the cell-proliferation of the Ld-652Y cell line that originates from the ovary of Lymantria dispar (Kim et al., 1996). CkyPDV plus venom may also inhibit the cell cycle, suppressing the increase of the hemocyte population because the injection of CkyPDV plus venom caused the disappearance of the 4C and 8C ploidies. Disappearance of DNA ploidy has been observed in spermatogenesis of the parasitized host in the Cotesia kariyai–Pseudaletia separata system (Tagashira and Tanaka, 1998). These results suggest that CkyPDV regulates cell proliferation related to the cell cycle. The hematopoietic organ from the host injected with CkyPDV plus venom released the lowest number of hemocytes when combined with the plasma from a host injected with PDV plus venom. This result suggests the possibility that CkyPDV directly affects hematopoiesis in the hematopoietic organs and produces the humoral plasma factor(s) that suppresses the cell cycle. When the
Fig. 5. Number of hemocytes released from hematopoietic organs. Hemocytes released from hematopoietic organs were counted 24 h after incubation in MGM450 medium containing 30% plasma. Hematopoietic organs from hosts injected with PDV plus venom, as well as from untreated hosts were dissected. Plasma was prepared from both sets of hosts. Eight replications were performed in each experiment. Each set of data was analyzed with the Mann–Whitney test (po0.01). (A) Both hematopoietic organs and plasma were prepared from CkyPDV plus venom injected hosts. (B) Hematopoietic organs from normal hosts were incubated with plasma from hosts injected with CkyPDV plus venom. (C) Hematopoietic organs from hosts injected with CkyPDV plus venom were incubated with normal plasma.
normal hematopoietic organ was incubated with plasma from each host 24, 48, and 72 h after the injection of PDV plus venom, the number of released hemocytes was
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Fig. 6. Histological analysis of hematopoietic organs from Pseudaletia hosts. The bar in the figure is 50 mm in length. Histological samples were stained with hematoxylin and Eosin. (A) Hematopoietic organs of untreated hosts, (B) 12 h, (C) 24 h, (D) 48 h, and (E) 72 h after the injection of CkyPDV plus venom. WD: wing disc, HO: hematopoietic organ, T: tracheae.
decreased to half of that of the control. The hemolymph of the parasitized host is somehow changed (Beckage et al., 1987; Beckage and Kanost, 1993). Possibly humoral factors such as the hematopoiesis inhibiting factor (HIF) may be produced in the hemolymph of the Pseudaletia host by CkyPDV. Another possibility is that the cytokine like growth blocking peptide (GBP) is changed in the host hemolymph by Cotesia kariyai parasitization (Ohnishi et al., 1995). In B. mori, hematopoiesis ability depends on the concentration of B. mori parasitic peptide (BmPP), which is similar to GBP (Nakahara et al., 2003). Hematopoiesis ability in parasitized Pseudaletia separata may be depressed by different concentrations of cytokine. Apoptosis was observed in the hematopoietic organ of the host injected with CkyPDV plus venom. Furthermore, the many segmented-DNA in the nuclei
of hematopoietic cells 24 h after the injection of PDV plus venom indicates that CkyPDV induced apoptosis of the hematopoietic organs. It has been reported that the behavior of the hemocytes is changed by parasitization in several species (Stettler et al., 1998). In some species, an immune response against foreign substances is lowered during the early stages of parasitism, although it recovered in later stages. In Cotesia kariyai, the encapsulation reaction to Sephadex beads injected into the parasitized host occurs normally but not in parasitoid eggs 24 h post-parasitization (Tanaka, 1987a). If some type of hemocyte that recognizes specifically the parasitoid eggs disappears from the hemocyte population and is not supplied by the hematopoietic organ, parasitoid eggs can avoid the encapsulation reaction of the host.
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Fig. 7. Apoptosis in hematopoietic organs. Nick-ends of DNA in hematopoietic cells were detected in situ by the TUNEL method. Fragmented DNA stained light blue is indicated by an arrowhead. (A) Twenty-four hours after the injection of CkyPDV plus venom. (B) Magnified hematopoietic cells. (C) Hematopoietic organ of control at the same stage as (A).
Acknowledgments We thank T. Nishioka and colleagues of the Kagoshima Agricultural Experiment Station for their help in collecting Pseudaletia separata larvae. We also express our thanks to Yuichi Nakahara for methods of culturing the hematopoietic organs. This work was supported in a part by a Grant-in-Aid for Scientific Research, Work no. (B)-(2) 15380041 of the Ministry of Education, Science and Culture, Japan. References Arnold, J.W., Hinks, C.F., 1976. Haemopoiesis in Lepidoptera. I. A note on the multiplication of spherule cells and granular haemocytes. Canadian Journal of Zoolology 61, 275–277. Beaulaton, J., 1979. Hemocytes and hemocytopoiesis in silkworms. Biochimie 61, 157–164. Beckage, N.E., Kanost, M.R., 1993. Effects of parasitism by the braconid wasp Cotesia congregata on host hemolymph proteins of the tobacco hornworm, Manduca sexta. Insect Biochemistry and Molecular Biology 23, 643–653. Beckage, N.E., Templeton, T.J., Nielsen, B.D., Cook, D.I., Stolz, D.B., 1987. Parasitism-induced haemolymph polypeptides in Manduca sexta (L.) larval parasitized by the braconid wasp Cotesia congregata (Say). Insect Biochemistry 17, 439–455. Davis, D.H., Strand, M.R., Vinson, S.B., 1987. Changes in differential haemocyte count and in vitro behaviour of plasmatocytes from host Heliothis virescens caused by Campoletis sonorensis polydnavirus. Journal of Insect Physiology 33, 143–153.
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ARTICLE IN PRESS T. Teramoto, T. Tanaka / Journal of Insect Physiology 50 (2004) 1103–1111 Luckhart, S., Webb, B.A., 1996. Interaction of a wasp ovarian protein and polydnavirus in host immune suppression. Developmental and Comparative Immunology 20, 1–21. Mitsuhashi, J., Inoue, H., 1998. Obtainment of continuous cell line from the larval fat bodies of the mulberry tiger moth, Spilosoma imparilis (Lepidoptera: Arctiidae). Applied Entomology and Zoology 23, 488–490. Nakahara, Y., Kanamori, Y., Kiuchi, M., Kamimura, M., 2003. Effects of silkworm paralytic peptide on in vitro hematopoiesis plasmatocyte spreading. Archives of Insect Biochemistry and Physiology 52, 163–174. Nakamatu, Y., Gyotoku, Y., Tanaka, T., 2001. The endoparasitoid Cotesia kariyai (CK) regulates the growth and metabolic efficiency of Pseudaletia separata larvae by venom and CK polydnavirus. Journal of Insect Physiology 47, 573–584. Ohnishi, A., Hayakawa, Y., Matsuda, Y., Kwon, K.W., Takahashi, T.A., Sekiguchi, S., 1995. Growth-blocking peptide titer during larval development of parasitized and cold-stressed armyworm. Insect Biochemistry and Molecular Biology 25, 1121–1127. Pathak, J.P.N., 1993. Cell mediated defense reactions in insects. In: Pathak, J.P.N. (Ed.), Insect Immunity. Kluwer Academic Publishers, London, pp. 47–58. Pech, L.L., Strand, M.R., 1995. Encapsulation of foreign targets by hemocytes of the moth Pseudoplusia includens (Lepidoptera, Noctuidae) Involves an RGD-dependent cell adhesion mechanism. Journal of Insect Physiology 41, 481–488. Ratcliffe, N.A., Rowley, A.F., 1979. Role of hemocytes in defense against biological agents. In: Gupta, A.P. (Ed.), Insect hemocytes. Cambridge University Press, London, pp. 331–414. Ribeiro, C., Simoes, N., Brehelin, M., 1996. Insect immunity: the haemocytes of the armyworm Mythimna unipuncta (Lepidoptera, Noctuidae) and their role in defense reactions. in vitro and in vivo studies. Journal of Insect Physiology 42, 815–822. Salt, G., 1970. The Cellular Defence Reactions of Insects. Cambridge University Press, Cambridge, UK. Sambrook, J., Russell, D.W., 2001. Molecular Cloning a Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Sano, Y., 1965. Histological Techniques. Nanzando Company, Tokyo. Sequeira, T., Tavares, D., Arala-Chaves, M., 1996. Evidence for curculating hemocyte proliferation in the shrimp Penaeus japonicus. Developmental and Comparative Immunology 20, 97–104.
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Shapiro, M., 1968. Changes in the hemocyte population of the wax moth, Galleria mellonella, during wound healing. Journal of Insect Physiology 14, 1725–1733. Stettler, P., Trenczek, T., Wyler, T., Pfister-Wilhelm, R., Lanzrein, B., 1998. Overview of parasitism associated effects on host haemocytes in larval parasitoids and compariosn with effects of the egg-larval parasitoid Chelonus inanitus on its host Spodoptera littoralis. Journal of Insect Physiology 44, 817–831. Strand, M.R., 1994. Microplitis demolitor polydnavirus infects and expresses in specific morphotypes of Pseudoplusia includens haemocytes. Journal of General Virology 75, 3007–3020. Strand, M.R., Pech, L.L., 1995. Microplitis demolitor polydnavirus induces apoptosis of a specific haemocyte morphotype in Pseudoplusia includens. Journal of Virology 76, 283–291. Tagashira, E., Tanaka, T., 1998. Parasitic castration of Pseudaletia separata by Cotesia kariyai and its association with polydnavirus gene expression. Journal of Insect Physiology 44, 733–744. Tanaka, T., 1986. Physiological interactions between a host, Pseudaletia separata and its parasitoids Microplitis mediator and Apanteles kariyai. Memoirs of the Faculty of Science, Kyoto University Series of Biology 11, 1–27. Tanaka, T., 1987a. 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. Tanaka, T., 1987b. Morphological changes in haemocytes of the host, Pseudaletia separata, parasitized by Microplitis mediator or Apanteles kariyai. Developmental and Comparative Immunology 11, 57–67. Vinson, S.B., 1971. Defence reaction and hemocytic changes in Heliothis virescens in response to its habitual parasitoid Cardiochiles nigriceps. Journal of Invertebrate Pathology 18, 94–100. Vinson, S.B., 1977. Insect host responses against parasitoids and the parasitoid’s resistance: with emphasis on the Lepidoptera–Hymenoptera association. In: Bulla, Jr., L.A., Cheng, T.C. (Eds.), Comparative Pathology, vol. 3. Plenum, New York, pp. 103–125. Webb, B.A., Luckhart, S., 1996. Factors mediating short- and longterm immune suppression in a parasitized insect. Journal of Insect Physiology 42, 33–40. Yamanaka, A., Hayakawa, Y., Noda, H., Nakashima, N., Watanabe, H., 1996. Characterization of polydnavirus-encoded mRNA in parasitized armyworm larvae. Insect Biochemistry and Molecular Biology 26, 529–536.
Journal of Insect Physiology 50 (2004) 1103–1111 www.elsevier.com/locate/jinsphys
Mechanism of reduction in the number of the circulating hemocytes in the Pseudaletia separata host parasitized by Cotesia kariyai Tokiyasu Teramoto, Toshiharu Tanaka Laboratory of Applied Entomology, Graduate School of Bio-Agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan Received 28 January 2004; received in revised form 26 August 2004; accepted 30 August 2004
Abstract Larval endoparasitoids can avoid the immune response of the host by the function of polydnavirus (PDV) and venom. PDV infects hemocytes and affects the hemocyte function of the host. In this paper, we investigated how PDV and venom affect the hemocyte population of the host. Cotesia kariyai, the larval endoparasitoid, lowers the hemocyte population of the noctuid host larvae soon after parasitization. The reduction in the number of circulating hemocytes is caused by the breakdown of the circulating hemocytes and of the hematopoietic organ which generates the circulating hemocytes. The decrease in the number of hemocytes shortly after parasitization is a response to the venom. However, the decrease in hemocyte population on and after 6 h postparasitization appears to be caused by the PDV. Apoptosis in circulating hemocytes was observed on and after 6 h post-injection of PDV plus venom. It was revealed through cytometry that mitosis of circulating hemocytes was halted within 24 h after the injection of PDV plus venom. Apoptosis in the hematopoietic organ was induced 12 h after the injection of PDV plus venom. Furthermore, the plasma from the hosts injected with PDV plus venom depressed the number of hemocytes released from the hemotopoiteic organs. r 2004 Elsevier Ltd. All rights reserved. Keywords: Polydnavirus; Venom; Hemocytes; Hematopoiesis; Apoptosis
1. Introduction Larval endoparasitoids must avoid the host immune response to develop in the host larvae. The response to large foreign objects like parasitoid eggs or larvae is a cellular defense reaction like encapsulation (Salt, 1970; Vinson, 1977; Ratcliffe and Rowley, 1979; Gupta, 1989; Engstro¨m, 1992; Pathak, 1993; Pech and Strand, 1995; Ribeiro et al., 1996; Lavine and Strand, 2002). For successful parasitization, endoparasitoids need polydnavirus (PDV) and venom, which are injected with their eggs into the host larva at the time of oviposition. PDV infects the hemocytes and causes much change in the host hemocytes; for example, inhibition of plasmatocyte Corresponding author. Tel.: +81 52 789 4033; fax: +81 52 789 4032. E-mail address: [email protected] (T. Teramoto).
0022-1910/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2004.08.005
spreading (Strand, 1994), apoptosis in granular cells (Strand and Pech, 1995), damage to the cytoskelton of the hemocytes (Webb and Luckhart, 1996), and blebs on hemocytes (Lavine and Beckage, 1996, Luckhart and Webb, 1996). Additionally, the total hemocyte count (THC) in parasitized hosts is altered (Stettler et al., 1998), being increased in four cases and decreased in two, and the adhesive ability of the hemocytes is reduced in almost all cases (Stettler et al., 1998). The decrease in THC seems to be the result of the induction of apoptosis by PDV. It has been reported that PDV induces apoptosis of granular cells in Pseudoplusia includens parasitized by Microplitis demolitor (Strand and Pech, 1995) and that hemocytes are blebbed and collapsed by PDV in the Manduca sexta–Cotesia congregata system (Lavine and Beckage, 1995). However, it has not been comprehensively clarified how the hemocyte population is altered
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by PDV and venom. Notably, it has not been examined whether the circulating hemocytes are supplied from hematopoietic organs in the host, parasitized or injected with the PDV and venom. It is known that the population of circulating hemocytes is supplied by the mitosis of circulating hemocytes itself and from hematopoietic organs. Hemocyte mitosis in circulating hemocytes and in hematopoietic organs has been observed by the incorporation of bromodeoxyuridine (BrdU) in vivo in Pseudoplusia includens and Spodoptera frugiperda (Gardiner and Strand, 2000). Mitoses in circulating hemocytes has been observed in Bombyx mori, Galleria mellonella and Euxoa declarata; it was verified that 1–8% of the population of circulating hemocytes are in the mitotic phase (Shapiro 1968; Jones 1970; Arnold and Hinks, 1976; Beaulaton, 1979). In arthropod shrimp Penaeus japonicus, the cell cycle of circulating hemocytes was reported by measuring the DNA content of the circulating hemocytes (Sequeira et al., 1996). Recruitment of hemocytes from hematopoietic organs has been reported in B. mori (Nakahara et al., 2003) and Pseudoplusia includens and S. frugiperda (Gardiner and Strand, 2000). In this study, we examine whether the decrease of circulating hemocytes is caused by the CkyPDV and/or venom, and then observe how the CkyPDV and venom affect the hematopoietic organ of the host in vitro, under several conditions.
venom is a physiologically effective dose (Tanaka, 1986). Venom was adjusted to a 1/3 female equivalent with PBS (0.067 M phosphate buffer, pH 7.4, 0.87% NaCl) after centrifuging at 12,000g for 10 min to remove cell debris. CkyPDV was adjusted to a 1/3 female equivalent in the prepared venom solution. A 1 ml mixture of a 1/3 female equivalent of CkyPDV plus venom was injected into the second proleg of the host larva with a fine glass needle. 2.3. Counting the THC To investigate the effects on the number of circulating hemocytes by parasitization or by CkyPDV plus venom injection, hemolymph was bled onto parafilm (American Can) on ice. Host larvae were narcotized with CO2, immersed in 70% alcohol for 5–10 s, washed with sterilized distilled water, and remained in an ice bath, allowing the host larvae to cool down for a few minutes. Ten microliters of hemolymph on parafilm were put in 490 ml of ACS (Anticoagulant solution; 0.098 M NaOH, 0.186 M NaCl, 0.017 M EDTA, 0.041 M Citric acid, pH 4.5) to prevent an aggregation of hemocytes. No aggregation of hemocytes was observed under these conditions. The total number of hemocytes was counted by a hemocyte counting chamber (Fuchs–Rosenthal, Hirschmann EM Techcolor, Germany). 2.4. Detection of fragmented DNA on electrophoresis
Cotesia kariyai colonies have been established in our laboratory since 1990 and are supplied from parasitized larvae of Pseudaletia separata collected in Kanoya, Kagoshima Prefecture every year. Adult wasps were kept for mating under long day conditions at 2571 1C with a 20–30% of sugar solution as food in a glass tube. Parasitization was performed individually to avoid superparasitism (Nakamatu et al., 2001). A laboratory colony of Pseudaletia separata was established using larvae collected in cornfields at the Agricultural Field Station of Nagoya University. The lepidopteran host was maintained on an artificial diet (INSECTA-LFs, Nihon Nohsan Co.) under a long day (16 h light:8 h dark) photoregime at 2571 1C.
Circulating hemocytes were collected from each larva injected with CkyPDV plus venom or from an untreated control. Hemolymph with hemocytes was flushed into a 1.5 ml tube while 500 ml ACS was injected into the second proleg with a 1 ml syringe; the sample was centrifuged at 760 g for 10 min to remove plasma. Hemocyte pellets were re-suspended in TNE (10 mM Tris-HCl, pH7.5, 100 mM NaCl, 1 mM EDTA) and treated with proteinase K digestion (10 mg/ml) to extract genomic DNA with phenol–chloroform, according to the method of Sambrook and Russell (2001). The supernatant was incubated for 1 h at 37 1C after adding DNase-free RNaseI (50 mg/ml, Sigma), then re-extracted with phenol–chloroform. The DNA was ethanol-precipitated, washed with 70% ethanol and re-suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA). Thirty micrograms of undigested hemocyte genomic DNA was separated on a 2% agarose gel.
2.2. Injection of CkyPDV and venom
2.5. Cytometory by laser scanning cytometer
CkyPDV and venom were collected separately from 2d-old females as previously described (Nakamatu et al., 2001). A female wasp injects approximately 18% of her total venom reservoir into the host hemocoel at the time of oviposition, and a 1/3 female equivalent of PDV or
To examine the effects of CkyPDV and venom on DNA ploidy, the amount of DNA was measured. Hemocytes in hemolymph flushed from the hosts by ACS injection were centrifuged at 760g for 10 min, washed once with ACS, fixed on ice with 2.5%
2. Materials and methods 2.1. Insect rearing
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2.6. The number of hemocytes released from hematopoietic organ in vitro We clarified the effects of CkyPDV plus venom on hematopoiesis by counting the hemocytes released from the hematopoietic organ in vitro. The effect on the hematopoietic organ was examined from the following two viewpoints. If CkyPDV plus venom affects the hematopoietic organ directly, fewer hemocytes might be released from the hematopoietic organ influenced by PDV plus venom, even under normal hemolymph conditions. Another consideration was that the humoral factor, which affects the hematopoiesis, exists in the hemolymph of the host injected with CkyPDV plus venom. The hematopoietic organs were dissected from the host larvae 1, 2, and 3 d after the injection of CkyPDV plus venom, as mentioned above. The hemolymph from the control or the injected larvae 1, 2, and 3 d after the injection of CkyPDV plus venom was added to 0.1 mM 1-phenyl-2-thiourea (PTU) and heated at 60 1C for 10 min to inhibit melanization after collection. Then, the treated hemolymph was used as plasma after centrifugation at 760g for 5 min and filteration with a 0.22 mm filter (Millipore). Hematopoietic organs were taken out along with a wing disc, washed in MGM450 medium (Mitsuhashi and Inoue, 1998) three times, and then incubated in 100 ml MGM450 containing 30% plasma for 24 h. Hemocytes released from the hematopoietic organ in each medium were collected to be counted by the hemocyte counting chamber.
2.7. Morphological observation of hematopoietic organ To clarify whether CkyPDV plus venom affects hematopoietic organs that supply circulating hemocytes, CkyPDV plus venom was injected into Day-0 sixth instar hosts. The hematopoietic organs taken out with wing-disc from larvae 1, 2, and 3 d after the injection of CkyPDV plus venom and non-treated Day-1 sixth instars as a control were fixed with 2.5% glutaraldehyde, postfixed with Bouin’s solution for 24 h, then dehydrated and embedded in paraffin. Eight mm sections were stained with Mayer’s hematoxylin and 1% Eosin Y solution (Sano, 1965).
2.8. Detection of fragmented DNA in hematopoietic organs Injection of CkyPDV plus venom was performed on Day-0 sixth instar hosts. Hematopoietic organs were collected 1, 2, and 3 d after injection to detect apoptosis. Day-1 sixth instar hosts were used as controls. Immediately after dissection, hematopoietic organs were fixed with 4% paraformaldehyde, post-fixed with Bouin’s solution for 24 h, dehydrated and embedded in paraffin, then cut into 8 mm sections. The fragmented DNA was labeled with an In Situ Cell Death Detection kit (Roche) and detected with BM Purple AP substrate (Roche). 2.9. Statistical analysis The Kruskal–Wallis (po0.01) test was performed for comparison among three or more results. Mann–Whitney’s U-test (po0.05) was used to determine significant differences between two sets of data.
3. Results 3.1. Effect of CkyPDV plus venom on hemocyte count The number of hemocytes in parasitized hosts was drastically decreased within 24 h post-parasitization and remained at a lower level than that of the control (Fig. 1A) through the time of the emergence of n=10
(X107) 8
Number of total hemocytes count (/ml)
glutaraldehyde in ACS for 4 h, re-suspended in 70% alcohol after centrifugalizing, then stored at 20 1C until measured. After washing once with PBS, the hemocyte samples were stained with PI (Pyropigium Iodite, Sigma) containing RNase H (50 mg/ml, Sigma) and were measured by LSC101 (Olympus, Tokyo). Typical data are shown graphically in Fig. 3. Five replications were performed in each treatment.
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Fig. 1. Parasitization of Cotesia kariyai reduces the number of circulating hemocytes in the host. Parasitization occurred on Day-0, sixth instar of Psudaletia separata host. Counting the hemocytes of each point was performed in ten replications. The vertical bar at each point shows the standard deviation.
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parasitoid larvae (data not shown). It was predicted that PDV and venom, injected along with the eggs at oviposition, would be involved in the rapid decrease in hemocytes count soon after oviposition. Injection with CkyPDV plus venom made the hemocytes decrease in number rapidly within 1 h post-injection, compared to that of saline injection (Fig. 2A). Although the hemocytes in the host injected with venom alone decreased only 1 h after injection, THC reached the same level as that of the control 12 and 24 h after venom injection, suggesting that the venom was responsible for
the reduction of THC within 1 h. The CkyPDV alone had no affect (Fig. 2B). 3.2. DNA ladder in hemocytes caused by CkyPDV plus venom To investigate if the CkyPDV plus venom caused apoptosis in the circulating hemocytes, genomic DNA from the hemocytes of the host injected with CkyPDV plus venom was analyzed by agarose gel electrophoresis. The DNA ladder was not detected 1–3 h after injection with the CkyPDV plus venom, but was weakly detected at 6 h and strongly detected at 10 h after injection (Fig. 3). 3.3. CkyPDV affects DNA ploidy of hemocyte Three peaks of 2C, 4C and 8C in the DNA ploidy of the circulating hemocytes were observed in the control larva (Fig. 4). The CkyPDV plus venom affected 4C and 8C peaks 8 h after injection, and the 8C peak disappeared 12 h after the injection. An injection of venom alone and saline had no effect on the peak value. 3.4. CkyPDV affects the hematopoietic organ
Fig. 2. Effects of CkyPDV and/or venom on the number of circulating hemocytes. (A) Hemocyte population after injection with CkyPDV plus venom and of saline into Day-0 sixth instar of the host Psudaletia separata. (B) Hemocyte population after each injection of saline, PDV plus venom, venom, and PDV alone into Day-0 sixth instar hosts. Hemocyte counting was repeated with each of the 10 host larvae on each point after injection. The vertical bar at each point shows the standard deviation.
To clarify the depression of hematopoiesis as a function of CkyPDV, we examined damage to the hematopoietic organ and the humoral factor that influences hematopoiesis by counting the number of hemocytes released from hematopoietic organs in vitro. The number of hemocytes released from the hematopoietic organ in the control larva was approximately 4000 in 24 h (Fig. 5A). When both the hematopoietic organ and the plasma as a humoral factor were prepared from the host 24 h after the injection of CkyPDV plus venom, about 600 cells had been released into the incubation medium for 24 h (Fig. 5A). On the other hand, when the hematopoietic organ from the control larva was incubated with plasma from hosts 1, 2, or 3 d after the injection of CkyPDV plus venom, the number of released cells decreased to about 2000 for 24 h (Fig. 5B). This was a significant difference (po0.01) from the number of hemocytes released in the incubation medium that contained the control hemolymph. Furthermore, the first 24 h of each hematopoietic organ’s incubation 1, 2, or 3 d after injection of CkyPDV plus venom with normal plasma, depressed hemocyte release to approximately 1000 (Fig. 5C). However, during the next 24 h of incubation with normal plasma, the hematopoietic organs 1 and 2 d post-injection released 1600 cells, but hematopoietic organs 3 d after the injection released 1000 cells, suggesting that the hematopoietic organ was changed within 2 days after CkyPDV infection, and was not refreshed when supplied with normal plasma.
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Fig. 3. DNA ploidy of hemocytes in hosts injected with CkyPDV plus venom or venom alone. The amount of DNA in hemocytes was measured with a laser scanning cytometer (Olympus LSC101). The horizontal bar shows the relative DNA amount. Injection was performed in Day-0 of the sixth instar host. Five replications were performed in each treatment.
3.5. Morphological changes in hematopoietic organs caused by CkyPDV plus venom The hematopoietic cells from non-treated sixth instar hosts were large, had large nuclei and the cytoplasm stained well with Eosin Y solution (Fig. 6). However, the nucleus started to atrophy 12 h after injection of CkyPDV plus venom and was strongly atrophied 24 h post-injection. 3.6. Apoptosis in hematopoietic organs To confirm that apoptosis in the hematopoietic organs was caused by CkyPDV plus venom, DNA fragments were detected in the hematopoietic organs by TUNEL methods. Many cells in the hematopoietic organs from the host injected with CkyPDV plus venom had fragmented DNA in their nucleus 24 h after injection (Fig. 7). No DNA fragments were observed in hematopoietic organs of the control host.
4. Discussion In host Pseudaletia separata parasitized by Cotesia kariyai, the hemocytes decreased in number during early
stages of parasitization and remained at a low density. It was similarly reported that the number of hemocytes in the host decreased during the early stages of parasitization of C. melanoscela (Guzo and Stoltz, 1987), Campoletis sonorensis (Davis et al., 1987) and Cardiochiles nigriceps (Vinson, 1971). Injection of venom alone decreased the number of hemocytes rapidly within 1 h after injection, although CkyPDV and venom also caused a decrease in the number of hemocytes. This suggests that the venom causes the first decrease in hemocytes. However, the mechanism that caused the decrease of circulating hemocytes soon after the injection was not clarified. The hemocyte decrease on and after 4 h post-parasitization seemed to be caused by CkyPDV. Venom seems to cause the temporal decrease of the circulating hemocytes until the PDV function begins (Kitano, 1986; Tanaka, 1987b). The CkyPDV genes were expressed in the hemocytes 4 h after parasitization (Yamanaka et al., 1996). Furthermore, the CkyPDV induced apoptosis in the circulating hemocytes; the DNA ladder in the hemocytes was detected 6 h after injection of CkyPDV plus venom. Microplitis demolitor PDV induces apoptosis in host hemocytes (Strand and Pech, 1995). Cotesia congregata PDV makes blebs on the host hemocytes as a sign of
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Fig. 4. DNA profile of circulating hemocytes from hosts injected with CkyPDV plus venom. Day-0 sixth instars of Pseudaletia host were injected with CkyPDV plus venom; the genomic DNA of circulating hemocytes was analyzed on a 2% agarose gel. The right lane is a DNA marker. Numbers on each lane represent hours after injection.
apoptosis (Lavine and Beckage, 1996). Apoptosis in circulating hemocytes seems to be induced by CkyPDV and decreases the number of hemocytes in the parasitized host larva 4 h post-parasitization. Mitosis in circulating hemocytes of lepidopteran larvae is observed in B. mori, G. mellonella, and E. declarata (Shapiro, 1968; Jones, 1970; Arnold and Hinks, 1976; Beaulaton, 1979). Hyposoter fugitivus PDV inhibits the cell-proliferation of the Ld-652Y cell line that originates from the ovary of Lymantria dispar (Kim et al., 1996). CkyPDV plus venom may also inhibit the cell cycle, suppressing the increase of the hemocyte population because the injection of CkyPDV plus venom caused the disappearance of the 4C and 8C ploidies. Disappearance of DNA ploidy has been observed in spermatogenesis of the parasitized host in the Cotesia kariyai–Pseudaletia separata system (Tagashira and Tanaka, 1998). These results suggest that CkyPDV regulates cell proliferation related to the cell cycle. The hematopoietic organ from the host injected with CkyPDV plus venom released the lowest number of hemocytes when combined with the plasma from a host injected with PDV plus venom. This result suggests the possibility that CkyPDV directly affects hematopoiesis in the hematopoietic organs and produces the humoral plasma factor(s) that suppresses the cell cycle. When the
Fig. 5. Number of hemocytes released from hematopoietic organs. Hemocytes released from hematopoietic organs were counted 24 h after incubation in MGM450 medium containing 30% plasma. Hematopoietic organs from hosts injected with PDV plus venom, as well as from untreated hosts were dissected. Plasma was prepared from both sets of hosts. Eight replications were performed in each experiment. Each set of data was analyzed with the Mann–Whitney test (po0.01). (A) Both hematopoietic organs and plasma were prepared from CkyPDV plus venom injected hosts. (B) Hematopoietic organs from normal hosts were incubated with plasma from hosts injected with CkyPDV plus venom. (C) Hematopoietic organs from hosts injected with CkyPDV plus venom were incubated with normal plasma.
normal hematopoietic organ was incubated with plasma from each host 24, 48, and 72 h after the injection of PDV plus venom, the number of released hemocytes was
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Fig. 6. Histological analysis of hematopoietic organs from Pseudaletia hosts. The bar in the figure is 50 mm in length. Histological samples were stained with hematoxylin and Eosin. (A) Hematopoietic organs of untreated hosts, (B) 12 h, (C) 24 h, (D) 48 h, and (E) 72 h after the injection of CkyPDV plus venom. WD: wing disc, HO: hematopoietic organ, T: tracheae.
decreased to half of that of the control. The hemolymph of the parasitized host is somehow changed (Beckage et al., 1987; Beckage and Kanost, 1993). Possibly humoral factors such as the hematopoiesis inhibiting factor (HIF) may be produced in the hemolymph of the Pseudaletia host by CkyPDV. Another possibility is that the cytokine like growth blocking peptide (GBP) is changed in the host hemolymph by Cotesia kariyai parasitization (Ohnishi et al., 1995). In B. mori, hematopoiesis ability depends on the concentration of B. mori parasitic peptide (BmPP), which is similar to GBP (Nakahara et al., 2003). Hematopoiesis ability in parasitized Pseudaletia separata may be depressed by different concentrations of cytokine. Apoptosis was observed in the hematopoietic organ of the host injected with CkyPDV plus venom. Furthermore, the many segmented-DNA in the nuclei
of hematopoietic cells 24 h after the injection of PDV plus venom indicates that CkyPDV induced apoptosis of the hematopoietic organs. It has been reported that the behavior of the hemocytes is changed by parasitization in several species (Stettler et al., 1998). In some species, an immune response against foreign substances is lowered during the early stages of parasitism, although it recovered in later stages. In Cotesia kariyai, the encapsulation reaction to Sephadex beads injected into the parasitized host occurs normally but not in parasitoid eggs 24 h post-parasitization (Tanaka, 1987a). If some type of hemocyte that recognizes specifically the parasitoid eggs disappears from the hemocyte population and is not supplied by the hematopoietic organ, parasitoid eggs can avoid the encapsulation reaction of the host.
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Fig. 7. Apoptosis in hematopoietic organs. Nick-ends of DNA in hematopoietic cells were detected in situ by the TUNEL method. Fragmented DNA stained light blue is indicated by an arrowhead. (A) Twenty-four hours after the injection of CkyPDV plus venom. (B) Magnified hematopoietic cells. (C) Hematopoietic organ of control at the same stage as (A).
Acknowledgments We thank T. Nishioka and colleagues of the Kagoshima Agricultural Experiment Station for their help in collecting Pseudaletia separata larvae. We also express our thanks to Yuichi Nakahara for methods of culturing the hematopoietic organs. This work was supported in a part by a Grant-in-Aid for Scientific Research, Work no. (B)-(2) 15380041 of the Ministry of Education, Science and Culture, Japan. References Arnold, J.W., Hinks, C.F., 1976. Haemopoiesis in Lepidoptera. I. A note on the multiplication of spherule cells and granular haemocytes. Canadian Journal of Zoolology 61, 275–277. Beaulaton, J., 1979. Hemocytes and hemocytopoiesis in silkworms. Biochimie 61, 157–164. Beckage, N.E., Kanost, M.R., 1993. Effects of parasitism by the braconid wasp Cotesia congregata on host hemolymph proteins of the tobacco hornworm, Manduca sexta. Insect Biochemistry and Molecular Biology 23, 643–653. Beckage, N.E., Templeton, T.J., Nielsen, B.D., Cook, D.I., Stolz, D.B., 1987. Parasitism-induced haemolymph polypeptides in Manduca sexta (L.) larval parasitized by the braconid wasp Cotesia congregata (Say). Insect Biochemistry 17, 439–455. Davis, D.H., Strand, M.R., Vinson, S.B., 1987. Changes in differential haemocyte count and in vitro behaviour of plasmatocytes from host Heliothis virescens caused by Campoletis sonorensis polydnavirus. Journal of Insect Physiology 33, 143–153.
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