Cotesia kariyai larvae need an anchor to emerge from the host Pseudaletia separata.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 66:1–8 (2007) Cotesia kariyai Larvae Need an Anchor to Emerge From the Host Pseudaletia separata Y. Nakamatsu,1* T. Tanaka,1 and J. A. Harvey2 Mature larvae of the gregarious endoparasitoid Cotesia kariyai construct cocoons for pupation approximately 10 days after parasitization and emerge from their host Pseudaletia separata under a long day photo-regime (16L8D) at 25 ± 1°C. The parasitoid larvae make capsules in the host hemocoel just prior to their emergence. These capsules function as “anchors,” which enable them to press against the host integument from inside the host. It was predicted that this anchor might be composed of silk proteins secreted from the parasitoid larvae, because a previous study showed that the anchor was made up of a glycoprotein and that the silk gland of parasitoid larvae developed from 2nd larval stage. Fibroin-like proteins in C. kariyai larva mainly consist of two proteins with molecular masses of the 300.6 and 46.7 kDa estimated by SDS-PAGE. The fibroin-like proteins with the same molecular mass were detected from the anchor proteins just prior to parasitoid emergence. These results indicate that the anchor was assembled with fibrion-like proteins and was formed just before parasitoid emergence while in the host body cavity. Injection of bovine pancreatic trypsin inhibited the emergence of parasitoid larvae from the host because the anchor was decomposed by trypsin. Trypsin activity in the parasitized host hemolymph increased only after parasitoid emergence. Arch. Insect Biochem. Physiol. 66:1–8, 2007. © 2007 Wiley-Liss, Inc. KEYWORDS: anchor; silk; fibroin; parasitoid emergence; parasitoid INTRODUCTION Mature endoparasitoid larvae within several subfamilies of the large family Braconidae (e.g., Microgastrinae) emerge from a still-living host to construct their cocoons and pupate (Askew, 1971; Godfray, 1994). Approximately 10 days after parasitization, third instar larvae of Cotesia kariyai emerge from their host and pupate (Tanaka et al., 1992; Nakamatsu et al., 2001). The mature endoparsitoid larvae make holes in the host integument just prior to their emergence (Askew, 1971). For example, larvae of C. kariyai perforate the host cuticle with their mandibles and enlarge the holes by physically moving their head capsules from side to side (Nakamatsu et al., 2006). In order to emerge from the host, some basal pressure (from terminal body segments) needs to be applied by the wasp larvae from within the host. Larvae of C. kariyai construct capsules for this purpose by using their own exuviae. A silk gland in the body cavity of second instar C. kariyai larvae is well developed (Nakamatsu et al., 2002). The viscosity in the parasitized host hemolymph is higher in proportion to the parasitoid development because a glycoprotein is secreted into the host by the parasitoid larvae as they mature (Nakamatsu et al., 2006). Mature larvae of the silkworm Bombyx mori secrete silk proteins (Akai, 1983; Magoshi et al., 1985). Two proteins, fibroin and sericin, are the main components of silk in B. mori (Voegeli et al., 1993; Kato et al., 1998). Seri- 1 Applied Entomology, Graduate School of Bio-Agricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan 2 NIOO-KNAW, Centre for Terrestrial Ecology, Heteren, The Netherlands *Correspondence to: Yutaka Nakamatsu, Applied Entomology, Graduate School of Bio-Agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan. E-mail: firstname.lastname@example.org Received 13 June 2006; Accepted 7 February 2007 © 2007 Wiley-Liss, Inc. DOI: 10.1002/arch.20189 Published online in Wiley InterScience (www.interscience.wiley.com) 2 Nakamatsu et al. cin is a protein that covers fibroin fibers and functions as an adhesive material in the cocoon of silkworms (Takahashi et al., 2003). The high viscosity in the hemolymph of parasitized Pseudaletia separata larvae also appears to be based on adhesive proteins that function like sericin. If parasitoid larvae need to make an anchor around their body, a large amount of protein would be required for this purpose. It is conceivable that the well-developed silk glands of parasitoid larvae are the leading candidate as an organ that can provide enough proteins to enclose their entire bodies. In this study, we investigate the structure and functions of the anchor. MATERIALS AND METHODS Insect Rearing The endoparasitoid Cotesia kariyai (Watanabe) emerged from Pseudaletia separata (Walker) larvae that had been collected in cornfields in Kanoya, Kagoshima Prefecture. They were thereafter maintained as a colony in our laboratory. Adult wasps were kept for mating under a long-day condition at 25 ± 1°C with a 20–30% sugar solution as food in a glass tube (12 cm long × 5 cm in diameter) for 2 days. Fifth instars of P. separata were individually presented to single female parasitoids that were allowed to sting once to avoid superparasitism. In addition, a laboratory colony of P. separata was established using larvae collected in cornfields of the agricultural field station at Nagoya University. Pseudaletia separata larvae were reared on an artificial diet (Insecta LF®, Nihon Nosan, Kanagawa) under a long-day photo-regime (16 h light:8 h dark) at 25 ± 1°C. It was designated as day 0 when each larva ecdysed to the next stadium. The P. separata larvae parasitized by C. kariyai were also maintained under the conditions described above. Extract of Fibroin-Like Proteins Initially, a cocoon protein was prepared by the method of Takasu et al. (2002) but with a slight modification. A combined total of 25 mg of para- sitoid cocoons was put into 1 ml of 60% lithium thiocyanate (LiSCN pH 7.0) to obtain the whole cocoon protein solution. Dissolved protein solution was dialyzed at room temperature against 20 mM Tris-HCl buffer, pH 8.0, containing 5M urea for 3 h with exchanges of the buffer at 1-h intervals. It was then kept in the dark at room temperature. Solid fibroin-like proteins and liquid fibroinlike proteins were prepared by the method of Sumida et al. (1993) but also with a slight modification. Second instar larvae of C. kariyai were dissected in ice-cold 1.15% KCl solution and the silk glands were isolated. The glands were washed in ice-cold fresh 1.15% KCl solution to remove hemolymph, and immersed in ice-cold 30% ethanol. After being kept in 4°C, the surrounding fixed silk gland tissues were separated by stirring the solution in a small beaker with a magnetic stirrer. Coagulated fibroin-like proteins were then collected. They were washed several times in ice-cold distilled water and immersed consecutively in 50%, 99.5% ethanol and ethyl ether for desiccation to give solid fibroin-like proteins. Solid fibroin-like proteins were kept in desiccated dark conditions at room temperature. Liquid fibroin-like proteins were prepared as follows. Briefly, 0.2 ml of 60% LiSCN, pH 7.0, was added to 0.01 g of solid fibroin-like proteins. This was kept for 6 h at room temperature to dissolve the solid fibroin-like proteins. Dissolved solid fibroin-like proteins were dialyzed at room temperature against 20 mM Tris-HCl buffer, pH 8.0, containing 5M urea for 3h with exchanges of the buffer at 1-h intervals. This was then kept in the dark at room temperature. Collecting Hemolymph from the Host Day 0–L6 instars of P. separata were prepared for parasitization. The hemolymph from Day 10 host larvae after parasitization was collected. The proleg of the host was cut with scissors, and from the wound 100 µl of hemolymph per host was collected on parafilm (American Can Co. LTD) and put on ice. A saturated phenylthiourea solution added 8% to the hemolymph volume. Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch. C. kariyai Larvae Need Anchor to Emerge From Host 3 Fibroin-Like Protein Extract From Anchor Observation of Anchors in the Host A cluster of mature parasitoid larvae was removed from the host just before emergence by incising the dorsal skin with scissors after CO 2 anesthesia. A 0.1-ml solution of 60% lithium thiocyanate (LiSCN, pH 7.0) was added to the mass of parasitoid larvae. The solution containing the clusters was mixed with a stirrer, until the parasitoid larvae were physically separated from each other. The dissolved solution was dialyzed at room temperature against 20 mM of Tris-HCl buffer (pH 8.0) containing 5M Urea for 3 h with exchanges of the buffer at 1-h intervals. The dialyzed sample solution was diluted with 2 volumes of the fresh buffer used for dialysis. Morphological observations in the body cavity of the parasitized hosts were performed to investigate the structure of the anchor. Parasitization was performed on day 0–L5 host larvae. Coincident with larval wasp emergence, parasitized hosts were fixed with 2.5% glutaraldehyde and postfixed with Bouin’s solution for 24 h, dehydrated and then embedded in paraffin. Eight-micrometer sections were stained with Mayer’s hematoxylin and 1% eosin Y solution (Sano, 1965). SDS-PAGE Analysis Samples for SDS-PAGE were prepared by adding the same volume of sample buffer (0.1M sodium phosphate buffer of pH7, containing 6M urea, 1% SDS, 2% 2-mercaptoethanol, and 0.025% bromophenol blue). Samples were incubated at 60°C for 15 min and put on 8 to 15% gel. The gels were stained after the run with Simply Blue (Invitrogen). Trypsin Treatment The host moved away from the parasitoid cocoons soon after parasitoid emergence. If the anchor is not disrupted, the host would cease to function. In the Euplectrus separatae–C. kariyai system, before pupating the matured parasitoid larvae secrete trypsin-like enzyme into the host body cavity (Nakamatsu and Tanaka, 2004). It is possible that the C. kariyai larvae also secrete the same enzyme. Fifty microliters of trypsin solution (635 U/µl Tris-Saline buffer; 50 mM Tris-HCl, pH 8.0, containing 0.15 M NaCl, 1 mM CaCl2, 0.1 mg/ml BSA) was added to the 0.2 ml of the liquid fibroin-like proteins solution. The mixed solution was incubated for 1 h at 37°C. One unit activity was hydrolyzed in 1.0 µmol of substrate/min at 37°C. Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch. Effect of Collapsed Anchor on the Parasitoid Larvae and Their Hosts To clarify the relationship between the destruction of the anchor and the success/failure of parasitoid emergence, we examined the effect of bovine pancreatic trypsin on parasitoid emergence behavior. A trypsin solution (10 µl at 635 U/µl) was injected into day-10 parasitized larvae just prior to parasitoid emergence. The trypsin-injected larvae were incubated at 37°C for 30 min and were maintained until parasitoid emergence at 25°C. We counted the number of parasitoid larvae that failed to emerge from hosts that had been injected with trypsin. The number of parasitoid larvae that remained in the host were also counted when these larvae were injected with Tris-saline buffer as a control after parasitoid emergence. The total number of eggs laid was calculated by combining the number of parasitoid larvae that remained in the host with the number of parasitoid pupae in cocoons. A 10-µl trypsin solution (635 U/µl) was injected into non-parasitized larva to check for effects of toxicity. One activity unit shows the ability to hydrolyze 1.0 µmol of substrate per min at 37°C. Measurement of Trypsin Activity on Fluorescence Spectrophotometer The parasitized host larva was fixed as the parasitoid emerged and was thus not moved. This immobility may be caused by a solid anchor formed 4 Nakamatsu et al. in the host hemocoel. After parasitoid larvae emerge from the host, the host caterpillar usually crawls up to several centimeters away from the cocoon cluster. If the activity of trypsin-like enzymes increases after the completion of parasitoid larval emergence, the destruction of the anchor might facilitate host movement. One hundred microliters of hemolymph from day 4–L6 control and parasitized larvae were collected immediately prior to and after parasitoid emergence in a 1.5-ml plastic tube on ice. The saturated 1-phenyl-2-thiourea (PTU) solution was added immediately to adjust to 8% of the final volume. Twenty microliters of 0.05 mM MCA-substrate (BocGln-Ala-Arg-MCA, Peptide Institute Inc.) and 0.4 ml of each buffer were put into a cuvette, and then 8 µl of hemolymph from healthy and parasitized larvae was added in the cuvette. A fluorescence spectrophotometer was used to record emission fluorescence at 440 nm at 37°C for 10 min. RESULTS Figure 1. Protein Extracts from Parasitoid Cocoon Polypeptides with various molecular masses from parasitoid cocoons were detected on SDSPAGE analysis (Fig. 1). The silk protein-extract consisted of nine proteins. The estimated molecular masses of the nine major proteins were 300.6 kDa (F1), 46.7 kDa (F2), 17.5 kDa (S1), 16.2 kDa (S2), 15.3 kDa (S3), 14.8 kDa (S4), 14.2 kDa (S5), 13.0 Fig. 1. Electrophoretic profile of silk protein (S), fibroinlike (F) proteins extracted from the cocoons of the Cotesia kariyai. Thirty micrograms total protein was run on 15% for silk protein and 10% SDS-PAGE gels for fibroin-like proteins, and stained with Simply Blue (Invitrogen). Numbers on the right and left indicate molecular masses (kDa) of standards. F: fibroin-like proteins. Fig. 2. Electrophoretic profile of the fibroin-like proteins (F) extracted from the cocoons of the C. kariyai and of the hemolymph of day-10 parasitized host larvae (P10). Protein (30 µg total) was run on 10% SDS-PAGE gel and stained with Simply Blue (Invitrogen). Numbers on the right and left indicate estimated molecular masses (kDa). Figure 2. Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch. C. kariyai Larvae Need Anchor to Emerge From Host Fig. 3. The parasitized larva (P) is just beginning to emerge from the host. Each parasitoid larva is enclosed by an anchor (FH). The parasitized host was pre-fixed by injection of 2.5% glutaraldehyde solution and post-fixed Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch. 5 with Bouin’s solution for 24 h. An 8-µm section was stained with Hematoxylin and Eosin dyes. I: Host integument. Scale bar = 500 µm 6 Nakamatsu et al. kDa (S6), and 9.5 kDa (S7). The fibroin-like protein-extract consisted of two proteins, 300.6 kDa (F1) and 46.7 kDa (F2). Proteins in the Parasitized Host Hemolymph We could not detect the proteins with the same molecular mass as fibroin-like proteins (300.6 kDa, 46.7 kDa) in the day-10 parasitized host hemolymph (Fig. 2). Structure and Proteins of Anchor Morphological observations with paraffin sections revealed that the parasitoid larvae were only able to emerge from hosts by using the anchor. The parasitoid larvae attained a stable anchor by gluing the remaining exuvium on the parasitoid larval body just before emergence (Fig. 3). The protein with the same molecular masses as 300.6 kDa (F1) in fibroin-like protein was detected from the extract of the anchor (Fig. 4). Fig. 4. Electrophoretic profile of the fibroin-like proteins (F) extracted from the cocoons of the C. kariyai, the anchor extract (Anchor), and the hemolymph of day-10 parasitized host larvae (P10). The arrows show the bands of fibroin-like proteins (300.6kDa) in the F and the anchor. Thirty micrograms total protein was run on 15% SDSPAGE gel and stained with Simply Blue (Invitrogen). Numbers on the right indicate molecular masses (kDa) of standard marker. Treatment of Trypsin Treatment with trypsin reduced the fibroin-like protein of 300.6 kDa on the gel (Fig. 5). Injection of trypsin solution inhibited the emergence of almost all parasitoid larvae (Table 1). All of the nonparasitized larvae injected with the same amount of trypsin solution survived and pupated (n = 20). The emergence of parasitoid larvae from the saline-injected parasitized hosts as control was also not affected. Trypsin Activity in the Parasitized Host Hemolymph Although we have not characterized the enzyme detected in host hemolymph as trypsin in this study, this enzyme in the hemolymph shows an Fig. 5. Electrophoretic profile of the fibroin-like proteins (F) extracted from the cocoons of the C. kariyai and the fibroin-like proteins, which were treated by 50 µl of trypsin solution (Bovine pancreatic trypsin 3 mg/0.2 ml Tris-saline buffer) (T). There are no fibroin-like proteins in the trypsin-treated. Total protein (30 µg per lane) was stained with Simply Blue (Invitrogen) after running on 15% SDSPAGE gels. Numbers on the right and left indicate molecular masses (kDa) of standard marker. Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch. C. kariyai Larvae Need Anchor to Emerge From Host TABLE 1. Number of Parasitoid Larvae That Could Not Emerge From Trypsin Solution (10 µl at 635 units/µl)-Injected Host† Treatment No. of host larvae used No. of eggs oviposited* (Mean ± S.D.) No. of parasitoid larvae not emerged* (Mean ± S.D.) 20 14 148.6 ± 58.8 a 144.7 ± 49.1 a 142.9 ± 63.0 a 0.7 ± 1.1 b Trypsin solution-injected Saline-injected control † All parasitoid larvae involved not emerged parasitoids in the trypsin solution-injected hosts were living. Non-parasitized hosts which were injected with trypsin solution did not die (n =15). *Means within a column followed by the same letter are not significantly different (P < 0.01; one-way ANOVA). activity similar to bovine trypsin. Trypsin-like enzyme activity was detected in the parasitized host (Table 2). Especially after parasitoid emergence, the trypsin-like enzyme activity in the host hemolymph became about 12-fold higher than that in the hemolymph before emergence. DISCUSSION Silk proteins are made up of fibroins and fibrous adhesives (Craig and Riekel, 2002). Although the silk proteins of moths (Lepidoptera) are used primarily for forming cocoons, predatory arthropods such as spiders use their silk for many functions, including the capture of prey, shelter construction or reproduction (Vollrath and Knight, 2001). One type of spider silk consists mainly of glycoproteins (Vollrath, 1999), which are able to maintain high levels of plasticity by retaining a high water content (Vollrath and Tillinghast, 1991). The anchor of C. kariyai was composed of glycoproteins (Nakamatsu et al., 2006) and also appeared to require some flexibility to enhance the successful emergence of the parasitoid larvae. TABLE 2. Trypsin Activities in the Parasitized Host Hemolymph Before and After Parasitoid Emergence on Day 10 After Parasitization† Host hemolymph collected No. of host larvae used Enzyme activity* Means ± S.D. (×10–3 units) 4 4 4 286.0 ± 18.7 b 22.2 ± 7.2 a 3.2 ± 0.2 c After parasitoid emergence Before parasitoid emergence Control † The day 4-6th instar hosts were used as control. *Means within a column followed by a different letter are significantly different (P < 0.01; one-way ANOVA). Archives of Insect Biochemistry and Physiology September 2007 doi: 10.1002/arch. 7 The fibroin in C. kariyai’s silk was composed of two proteins with a large molecular mass (300.6 kDa) and a small molecular mass (46.7 kDa). The fibroin of B. mori is also composed of two proteins with large (350 kDa) and small molecular masses (25 kDa) (Shimura, 1988). By contrast, Yamada et al. (2003) reported that fibroin-like protein extracted from cocoon of Cotesia glomerata was comprised of a single polypeptide with a molecular mass of over 500 kDa. This variation may reflect differences in the cocoon structure of the two Cotesia species, even though they are closely related. C. kariyai makes a cocoon cluster that is composed of two different layers: an inner layer that surrounds each individual pupa, and a thick outer layer that covers each cocoon entirely, whereas C. glomerata larvae only construct individual cocoons after emergence from the host (unpublished data). The 300.6-kDa protein, which has the same molecular mass as fibroin-like proteins in C. kariyai cocoons, was detected in the anchor extraction, suggesting that the fibroin-like protein was a component of the anchor. However, they were not detected in hemolymph of parasitized host larvae from day 0 to 10 after parasitization except just prior to parasitoid emergence at day 10. A fibroinase in the silk gland of B. mori larvae decompose the fibroin (Sumida et al., 1993). In this study, we used bovine trypsin, because the activities of trypsin-like enzymes in the host larvae increased just after parasitoid emergence and the anchor was left in the host hemocoal. The fibroin-like protein was not detected on SDS-PAGE gels after treatment with trypsin. Over 96% of the parasitoid larvae failed to emerge from the host when the trypsin was injected into the host 10 days after parasitization. The parasitoid larvae are unable to perforate the host cuticle probably because they cannot gain sufficient basal support to exert enough pressure on the host integument due to the collapse of the anchor. ACKNOWLEDGMENTS We thank M. Nishioka and colleagues at the Kagoshima Agricultural Experiment Station for their help in collecting Pseudaletia separata larvae. 8 Nakamatsu et al. LITERATURE CITED Akai H. 1983. The structure and ultrastructure of the silk gland. Experientia 39:443–449. Askew RR. 1971. Parasitic insect. London: Heinemann Educational Books. p 113–184. Craig CL, Riekel C. 2002. Comparative architecture of silks, fibrous proteins and their encoding genes in insects and spiders. Comp Biochem Physiol 133B:493–507. Sano Y. 1965. Histological techniques. Tokyo: Nanzando Company. Shimura K. 1988. The structure, synthesis and secretion of fibroin in the silkworm, Bombyx mori. Sericologia 28:457–479. Sumida M, Takimoto S, Matsubara F. 1993. 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