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Ultrastructural and functional maturation of teratocytes of Apanteles kariyai.

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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 [3]. Furthermore, Leonard
et al. [4]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 [5]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 [6] 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 [5]. 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. [4]. 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 [5]. 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 [5], and the pro-PO activating system not only generates melanin but may also have opsonic activity [7], 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 [6] 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 [5], 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 [12] 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 [13]. 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 [7]. 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. [14] 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 [14].
~
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.
LITERATURE CITED
1. Salt G: Experimental studies in insect parasitism IX.The reaction of a stick insect to an alien
parasite. Proc R S o c BiolZ46,93 (1956).
2. Nappi AJ: The role of melanization in the immune reaction of larvae of Drusuphila algunquin
against Pseudocoila bochei. Parasitology 6 , 2 3 (1973).
3. Wyatt GR, Pan ML: Insect plasma proteins. Annu Rev Biochem 47,77 (1978).
4. Leonard C, Soderhall K, Ratcliffe NA: Studies on prophenoloxidase and protease activity of
Blaberus cranifer haemocytes. Insect Biochem 25,803 (1985).
5. Tanaka T: Effect of the venom of the endoparasitoid, Apanteles hriyai Watanabe, on the cellular defence reaction of the host, Pseudafetia separata Walker. J Insect Physiol33,413 (1987).
6. Stoltz DB, Cook DI: Inhibition of host phenoloxidase activity by parasitoid hymenoptera.
Experientia 39,1022 (1983).
7. Rowley AF, Ratcliffe NA, Leonard CM, Richards EH, Renwrantz L: Humoral recognition
factors in insect, with particular reference to agglutinins and the prophenoloxidase system.
In: Hemocytic and Humoral Immunity in Arthoropods. Gupta AP, ed. Wiley-Interscience
Publication, New York, pp 381-406 (1986).
8. Pye AE: Microbial activation of prophenoloxidase from immune insect larvae. Nature 251,
610 (1974).
9. Unestam T, Soderhall K: Soluble fragments from fungal cell walls elicit defence reactions in
crayfish. Nature 267,45 (1977).
10. Tanaka T: Morphological changes in haemocytes of the host, Pseudaletia separata, parasitized
by Microplitis mediator or Apanteles hriyai. Dev Comp Immunol12, 57 (1987).
11. Sroka P, Vinson SB: Phenoloxidase activity in the haemolymph of parasitized and unparasitized Heliuthis uirescens. Insect Biochem 8,399 (1978).
Teratocytes of Apanteleskariyai
197
12. Vinson SB: Development and possible functions of teratocytes in the host parasite association. J Invertebr Pathol16,93 (1970).
13. Vinson SB, Scott JR: Ultrastructure of teratocytes of Curdiochiles nigriceps Viereck (Hymenoptera: Braconidae). Int J Insect Morphol Embryo1 3,293 (1974).
14. Leonard CL, Ratcliffe NA, Rowley AF: The role of prophenoloxidase activation in non-self
recognition and phagocytosis by insect blood cells. J Insect Physiol31, 789 (1985).
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