Ultrastructural and functional maturation of teratocytes of Apanteles kariyai.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 13:187-197 (1990) Ultrastructural and Functional Maturation of Teratocytes of Apanteles kariyai Toshiharu Tanaka and Haruhisa Wag0 1-39-4 Tsurumaki, Setaguya, Tokyo, Japan (T.T.); Department of Bacteriology, Saifarna Medical School, Morqyama, Irurna, Saitama, Japan (H.W.) Eggs and larvae of Apanteles kariyai avoid the host defense reactions of Pseudaletia separata due to the action of calyx and venom fluids injected by females during oviposition and the teratocytes originated from the embryonic serosal cells 3.5 day postoviposition. Phenoloxidase (PO) activity in host larvae was unaffected during early stages of parasitization (4-6 days postoviposition), relative t o unparasitized larvae, but was greatly reduced to 25% during the late stage of parasitization (days 7-10). Hemolymph PO activity was not affected, i n vitro, by calyx and venom fluids but was reduced in the presence of teratocytes. An apparent PO inhibitor was detected i n older teratocyte cells. First instar parasitoid larvae implanted into unparasitized hosts, following transfer of either young teratocytes (4 day postoviposition) or old teratocytes (8 day post-oviposition) with calyx and venom fluids resulted i n avoidance of encapsulation only when calyx and venom fluids with young (4 day) teratocytes were injected. These results indicated that during early parasitization of the host, teratocytes just released from the embryonic serosal cells (4 day) function in conjunction with calyx and venom fluids injected into the host with the parasitoid egg to prevent its encapsulation by host hemocytes. During late parasitization, the older teratocytes (8 day) may also function in preventing host encapsulation by producing an PO inhibitor suppresses host hemolymph PO activity at the time of parasitoid egression. Key words: braconid parasitoid, phenoloxidase, calyx fluid, venom, encapsulation INTRODUCTION When large foreign objects invade the hemocoel of an insect, the common immune response is formation of a multicellular capsule foreign objects [1,2]. Acknowledgments: We thank S.B. Vinson and B.A. Webb of Texas A&M University for their critical reading of the manuscript. Received July21,1988; accepted October 16,1989. Address reprint requests to T. Tanaka, 1-39-4Tsurumaki, Setagaya, Tokyo 154,japan. 0 1990 Wiley-Liss, Inc. 188 Tanaka and Wag0 Melanin is formed due to the activation of PO* system . Furthermore, Leonard et al. suggested that the activated PO tends to stick to foreign surface and may act as an opsonin. Living eggs and larvae of endoparasitoids are able to evade the immune reaction without receiving recognition as foreign objects in their habitual hosts. There seems to be some difference in the avoiding system of the host's immune response between the larval stage and the egg stage of parasitoid. Tanaka reported that the calyx plus venom fluids were involved in the eggs of Apunteles kariyui being able to avoid the h o d s defense reaction. However, the mechanism allowing parasitoid larvae after hatching to avoid the encapsulation reaction of the host during later stages of the parasitoid's development was not examined. Stoltz and Cook  reported that an inhibition of the PO activity occurred in the hemolymph of parasitized host larvae of Tvichoplusia ni. The PO activity in the hemolymph was completely inhibited by purified calyx virus within 24 h after oviposition and the inhibition or reduction in PO activity was relevant to successful parasitism. However, in the case of A. kariyui, the reduction of PO activity does not occur until the later stages of development of the parasitoid from day 7 to emergence. It should be considered that some other factor that appears later in parasitoid development besides calyx and venom fluids may inhibit PO activity. We examined the teratocytes, embryonicserosal cells released from larvae of most braconid parasitoids at the time of hatching, as a source of this factor that may be involved in the PO reduction and thereby aid the parasitoid in avoiding the host encapsulation reaction in the later stages of parasitization. MATERIALS AND METHODS Insect Culture The braconid parasitoid A. kariyui was laboratory reared on Pseudaletiu separutu larvae maintained on an artificial diet at 25 f 1°Cunder a 16:8 (light: dark) photoperiod and fed a 40% sugar solution in the glass tube as described previously . Host larvae were individually parasitized to avoid superparasitism. Preparation of Host Plasma for the Assay of PO Activity After centrifuging hemolymph of unparasitized larvae at 40g for 10 min at 4"C, supernatant was diluted 1:9 with ice-cold Ca2+-freephysiological saline (150 mM NaC1, 5 mM KC1, pH 6.8). PO activity was determined spectrophotometrically by measuring the formation of dopachrome at 490 nm as described by Leonard et al. . Additionally, PO activity in plasma obtained from parasitized larvae was similarly measured with time after oviposition. *Abbreviations used: AV = autophagic vacuoles; ER = endoplasmic reticulum; G = Colgi; L-Dopa = L-dihydroxyphenylalanine; MF = myelin-like figures; M V = microvilli; N = nucleus; PO = phenoloxidase; pro-PO = prophenoloxidase. Teratocytesof Apanteles kariyai 189 Collection of Venom and Calyx Fluids and Teratocytes Venom and calyx fluids were separately collected, mixed, and injected into unparasitized host as described previously . Hemolymph of parasitized larvae was collected into a plastic petri dish (9 cm in diameter) containing saline with 0.1% 1-phenyl-2-thiourea. As both granular cells and plasmatocytes attached to the surface of the dish after 40 min incubation at 22-24"C, it was easy to remove these cells from the teratocyte suspension, which also included the other hemocytes. Five repetitions of this procedure reduced the hemocyte contamination to 1 x lo3 celldm1 in the teratocyte suspension. The teratocytes suspension was then diluted to 2.2 x lo4 cells/ml with Grace's medium (Gibco) and cultured in a well of a Lab-Tek eight-chamber slides for 48 h at 24 ? 1°C. The viability of teratocytes in this preparation was over 90% as determined by neutral red staining. After centrifugation, teratocyte cell components were diluted with 0.2 ml CaZf-free saline. The teratocyte suspension after lysis by ultrasonic treatment and the supernatant were mixed 1:9 with the normal plasma to assay for the presence of PO inhibitory factors. RESULTS Reduction of PO Activity in the Hemolymph of Parasitized Larvae Although PO activity in the hemolymph of parasitized hosts showed the same level as that of unparasitized control on days 2 and 3 after parasitization, it gradually decreased with parasitoid development following oviposition and was especially depressed on days 8 and 9 (Fig. 1). Effect of Calyx and Venom Fluids on the PO Activity of Normal Plasma Hemolymph melanization occurs due to the activation of a PO system present in insect plasma [ 3 ] .Since the calyx plus venom fluids have the ability to inhibit encapsulation , and the pro-PO activating system not only generates melanin but may also have opsonic activity , it was necessary to examine the effects of calyx and venom fluids on melanization. However, after a 60 min incubation, the calyx plus venom fluids did not inhibit PO activity (Fig. 2A). Inhibition of PO Activity in Hemolymph by Teratocytes The reduction of PO activity in parasitized host occurred at a later stage of parasitization (Fig. 1). Using the L-Dopa system without the PO-forming system from hemolymph, which is a more simplified system than normal plasma, the relation between teratocytes and reduction of PO activity was examined. Cell lysates of older teratocytes (8 days after oviposition) depressed melanin formation more than those of younger teratocytes (4days after oviposition; Fig. 28). Tanaka and Wag0 190 PARAS1 TE STAGE 1st instar Egg 2nd instar 2.4 day 4 g day 5 00 0' 1.2 G 0.8 iD 0.4 20 40 20 40 Fig. I . Reduction of phenoloxidase activity in hernolymph of the Pseudaletia separata host larvae parasitized by Apanteles kariyai. PO activity was measured spectrophotometrically at 490 nrn. Host larvae parasitized in the late fifth instar ecdysed to the sixth (last) instar on the next day. Control larvae pupate on day 6 after ecdysis. 6L1,day 1 of sixth larval stage. A,Paraunparasitized control. sitized; 0, PO Inhibitorb) Released From Teratocytes If PO inhibitory factors are released from teratocytes, then inhibitory activity may be detected in teratocyte culture medium. Both teratocyte cell components and culture supernatant showed the PO inhibitory activity (Fig. 2C), indicating that older teratocytes produce and release the PO inhibitory factor(s). Comparative Morphology of Early- and Late-Stage Teratocytes Young teratocytes in the first 24 h following their liberation (4 days after oviposition) had a diameter of approximately 42 pm, and the N were extensively ramified. h4V were also abundant on the cell surface.The ER consisted of widely separated, rough-surfaced cistemae of variable diameter, which were abundant throughout the cytoplasm. Many organized G were visible between rough ER (Fig. 3). By 8 days after oviposition, the old teratocytes were -85 pm in diameter and had developed rough ER adjusted to the extensive ramified nucleus. The cisternae of the vesiculated rough ER were enlarged and appeared as vacuoles. In some regions, near the cell membrane, small vacuoles developed and increased in size and appeared to fuse to each other and to give rise to larger Teratocytes of Apanteles kariyai 191 A contr. B venom calyx calyx plus venom I DAY 8 0 Q) B AAAA A A 0 0.5 1 3 24 0.5 1 3 24 INCUBATION TIME, HRS ( IN DOPA) C cells 0 20 40 60 I1 supernatant 0 20 40 60 INCUBATION TIME, M I N ( I N HEMOLYMPH Fig. 2. A: Effect of the calyx plus venom fluids of Apanteles kariyai on the phenoloxidase activity in the normal plasmaof fseodaletia host. Three microliters of calyxandlorvenom fluids were added to 1.5 ml of normal plasma. The same volume of saline was added in control. B: Melanin formation in the L-Dopa system was depressed when incubated with old teratocytes (A, 8 days postoviposition) as well as in normal plasma. Incubation with young teratocytes (0,4 days postoviposition) in L-Dopa system has no effect on melanin formation. C: Phenoloxidase inhibitory factors existed in both cell components and culture media of old teratocytesfollowing the 48 h culture in Grace's medium. Teratocytes and culture supernatant after 48 h of culture were mixed 1:9 with normal plasma of day 3 unparasitized last instar larvae and were incubated at25"C for measuringan absorbance at 490 nm at intervals of 10 min. The control was treatedthesame. 0, Control; A, teratocytes; A,supernatant.Thestarsmeanasignificantdifference compared to control at <0.01 level by one way ANOVA (N = 5). 192 Tanaka and Wag0 Fig. 3. Teratocytes of Apanteles kariyai 193 vacuolar structures. In addition, some AV and MF were observed. The number of MV also had increased relative to young teratocytes (Fig. 4). Can Teratocytes Inhibit Encapsulation of the First Instar Parasitoid? Injection of young or old teratocytes alone did not inhibit encapsulation of first instar parasitoids. However, when young teratocytes were injected along with calyx plus venom fluids, all first instar parasitoids developed and possessed normal caudal vesicles. In contrast, transfer of old teratocytes with calyx plus venom fluids did not entirely prevent encapsulation; first instar larvae were partially encapsulated (Table 1). The caudal vesicles of the first instar larvae was often encapsulated even when the other parts of the body were not. Injection of only calyx plus venom fluids resulted in a similar, slight encapsulation of the first instar parasitoid larvae (Table 1). Injection of Zymosan Into the Parasitized Larvae The activation reaction of pro-PO is known to be elicited in the plasma fraction of hemolymph by zymosan or laminarin with P-1,S-glycosidic linkages [8,9]. Therefore, zymosan was injected into parasitized larvae in which PO activity was greatly depressed. Although the injection of 200 pg zymosan in 10 pl saline caused extensive melanin formation in the hemolymph of parasitized larvae, no parasitoid larvae were encapsulated 24 h after injection and eventually egressed from the host normally (Fig. 5). DISCUSSION In larvae parasitized by A. kariyui, a reduction of PO activity was observed in the late stages of parasitization. The reduction of PO activity was not caused by calyx and venom fluids but by PO inhibitor in old teratocyte cells. Stoltz and Cook  reported that the complete inhibition of PO activity occurred immediately after oviposition in Hyposoter exiguue-Trichoplusia ni system and that this inhibition was caused by the calyx virus. However, in their report, PO activity in a different host species was only partially reduced. Apparently, the degree of inhibition of PO activity seems to differ between every host parasitoid system [lo]. Sroka and Vinson [ l l ] also reported that host hemolymph melanization did not occur during the egg stage of the parasitoid. Although the calyx plus venom fluids of A . kuriyui had the ability to inhibit the encapsulation reaction against the egg , these fluids alone had no effect on PO activity of hemolymph (Fig. 2, Table 1)or of hemocytes (unpublished data). In A . Kariyui, young teratocytes inhibited the encapsulation of first instar Fig. 3. A: Four-day teratocytes from Pseudaletia separata, parasitized by Apanteles kariyai, showing ramified nucleus (N),a lot of microvilli (MV), and many organized Golgi (GI.X2,OOO. B: Magnification of microvilli and endoplasmic reticulum (ER). X 20,000. 194 Tanaka and Wag0 Fig. 4 Teratocytes of Apanteles kariyai 195 TABLE 1. Effect of Pretreatment With Young or Old Teratocytes and/or Calyx Venom plus Fluids on the Encapsulation of First Instar Parasitoids* Materials injected Young teratocytes + saline Young teratocytes + calyx venom plus fluids Old teratocytes + saline Old teratocytes + calyx venom plus fluids Calyx venom plus fluids Total no. of injections Degree of encapsulation of parasitoid larvae (%) Full Slightly Not encapsulated” encapsulated encapsulated 13 13 (100) 0 0 12 0 0 12 (100) 11 11(100) 0 0 11 0 11(100) 0 14 0 11(79) 3 (21) ‘Unparasitized hosts were conditioned by injection of teratocytes andor calyx venom plus fluids 48 h before implanting parasitoid larvae. Teratocytes were taken from hosts 4 days (young) and 8 days (old) after parasitization. Calyx venom plus fluids were collected individually from female Apanteles kariyui and mixed at a 1:l ratio of a total volume, then 3 pI per host was injected. One or two first instar parasitoid larvae were transferred 4 days after oviposition into the hemocoel of the host at 48 h after conditioning. Dissections were performed 48 h after transferring first instar larvae. aParasitoidlarvae that were slightly encapsulated had grown less and had a smaller caudal vesicle than control and were slightly or partly encapsulated. larvae only when injected with calyx plus venom fluids, although there was no change in PO activity compared to the control. Neither injection of old teratocytes with calyx plus venom fluids nor that of calyx plus venom fluids alone inhibited the encapsulation reaction. Old teratocytes seem not to have an inhibitory function. Vinson  suggested a trophic function, especially for older teratocytes. The extensive vesicular ER seems to show that teratocytes secrete some substances, as suggested by Vinson and Scott . Old teratocytes were thought to produce some inhibitors of the pro-PO-activating system in the plasma. It is believed that the pro-PO-activation in the plasma leads to the recognition of non-self materials . However, injection of zymosan, an elicitor of the pro-PO-activation, did not induce the encapsulation against the parasitoid larvae, although the formation of melanin was evident. Leonard et al.  reported that incubation of test bacteria with laminarin significantly increased the number of microorganisms attaching to both plasmatocytes and granular cells. However, old teratocytes injected together with calyx plus venom fluids could not completely inhibit encapsulation of first instar larvae. Therefore, encapsulation-avoiding mechanism cannot be explained only by the depression of PO activity in this species. There is a possibility that old teratocytes are unable to inhibit the secretion of an opsonin-like substance like a sticky protein from hemocytes . ~ Fig. 4. Eight day teratocytes showing extensive vacuolated endoplasmic reticulum (ER), a myelin-like figure (MF), ramified nucleus (N), and autophagicvacuoles (AV). Golgi ( G ) . A: ~ 2 , 0 0 0 B: . ~ 8 , 0 0 0 .C: x15,OOO. 196 Tanaka and Wag0 I DAY 4 DAY 8 ENCAPSULATION (24 H AFTER INJECTION) 0% EGRESSION 100 % 1.84 /-- 1.50 1.2- ;r" 0.9- Q, 0.6- 0.30 L-771 2 3 TIME 4 1 I I I 1 2 3 4 AFTER I N J E C T I O N , H Fig. 5. Effect of the artificial increase of the prophenoloxidase activity in hernolymph of parasitized host by zymosan o n successful parasitism. Injection of 200 pg zymosan in 10 p1 saline was performed into day4 and day 8 parasitized larvae. A,Ca2+-freesaline injection. 0, zymosan injection. 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