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The growth and role of Microplitis demolitor teratocytes in parasitism of Pseudoplusia includens
Vol.37, No. 7, pp. 503-515, 1991 Printed in Gnat Britain. All rights reserved
0022-1910/91 $3.00 + 0.00 Copyright 0 1991 Pergamon Press plc
J. Insect Physiol.
THE GROWTH AND ROLE OF MZCZtOPLZZ’Z~ DEMOLZTOZ? TERATOCYTES IN PARASITISM OF PSEUDOPLUSZA ZNCLUDENS M. R. STRAND and E. A. WONG Department of Entomology, 237 Russell Laboratories, University of Wisconsin-Madison, Madison, WI 53706, U.S.A.
(Received 4 April 1991)
Abstract-The growth and function of Microplitis demolitor teratocytes in Pseudoplusia in&dens larvae was investigated. An average of 542 teratocytes was produced per M. demolitor egg. The number of teratocytes present per host declined during the course of parasitoid development, but the size of individual cells increased. The ploidy levels of teratocytes ranged from 2 to 8C at hatching, but had increased to 128-256C at the completion of parasitoid development. Injection of in vitro cultured teratocytes into fourth stadium P. incluahs larvae had an inconsistent effect on development. Most larvae pupated without delay when injected with a physiological dose of 600 teratocytes, but 54% of larvae failed to pupate when injected with a super-physiological dose of 2400 teratocytes. Examination of teratocytes indicated most cells were encapsulated by P. includens haemocytes within 24 h of injection. However, prior injection of larvae with M. demolitor calyx fluid or polydnavirus plus venom suppressed the encapsulation response. Further, 87% of larvae injected with polydnavirus plus venom and 600 teratocytes exhibited alterations in growth that were very similar to parasitism by M. akmolitor. Key Word Index: Parasitoid; development;
haemocytes; immunity; encapsulation;
calyx fluid;
polydnavirus
INTRODUCTION Many hosts exhibit physiological alterations after parasitism by endoparasitic Hymenoptera (Vinson and Iwantsch, 1980; Lawrence, 1986). In certain braconids and ichneumonids, some of these alterations are attributed to the polydnaviruses and venom the adult wasp injects into the host at oviposition (Stoltz, 19g6). Other changes, however, may be due to factors associated with the developing parasitoid embryo or larva (Fisher, 1971). Of particular interest is the role of teratocytes (Dahlman, 1990). These unusual cells are produced by many of the polydnavirus carrying braconids. Teratocytes are derived from the serosa that envelops the developing parasitoid embryo. At hatching, the serosa dissociates into individual cells (teratocytes) that circulate in the haemolymph and become distributed throughout the host. Teratocytes persist during the course of parasitoid larval development, but their role in parasitism is unclear. Some studies suggest teratocytes serve a trophic function (Sluss, 1968) while others report they affect the development and immune response of hosts (Salt, 1968; Joiner et al., 1973; Strand et al., 1986; Zhang and Dahlman, 1989; Tanaka and Wago, 1990). Their interaction with other wasp factors such as polydnavirus and venom has not been investigated.
In the present study, we examined the teratocytes of the solitary, braconid wasp, Microplitis demolitor Wilkinson in its host, Pseudoplusia includens (Walker). Here we report the size and ploidy values of teratocytes increased greatly during development, but the number of cells per host declined. Doseresponse experiments indicated teratocytes caused some developmental alterations in P. incluhs larvae, but more pronounced effects occurred when larvae were also injected with M. demolitor polydnavirus and venom. MATERIALSAND METHODS Insects P. includens were reared as described by Strand (1990) in 30 ml plastic cups with paper lids at 27 f 1°C and a 16 h light-8 h dark photoperiod. Moths were fed a 20% sucrose solution. M. akmolitor were reared at 27 + 1°C and a 16 h light-8 h dark photoperiod as outlined by Strand et al. (1988). The M. akmolitor egg hatched between 26 and 28 h after oviposition (day 2). The parasitoid larva emerged from the host on day 7, spun a cocoon, and emerged as an adult on day 13. Hosts used in experiments were 6-18 h-old fourth stadium larvae, and all 503
504
M. R. STRANDand E. A. WONG
wasp ovipositions were observed to assure no superparasitism occurred. Development of M. demolitor and P. in&dens was monitored by previously established staging criteria (Strand et al., 1988; Strand, 1990). Teratocyte growth in parasitized hosts
To determine the number and size of teratocytes present during parasitoid development, hosts were dissected on day 2, and every 24 h subsequently through day 7. Hosts were dissected individually in 60 mm Falcon tissue culture plates (Be&on Dickenson, Lincoln Park, N.J.) filled with 1 ml of Pringle’s saline (Pringle, 1938). After settling on the bottom of the dish, the number of teratocytes per host was counted using a Nikon inverted, phase-contrast microscope. In order to estimate cell size during development, 50 teratocytes per host were selected randomly and their diameter measured using an ocular micrometer. For eliptic cells both short and long diameters were measured and the average was estimated as the cell diameter. To estimate the ploidy value of teratocytes, relative DNA content was measured by fluorometric analysis. Ploidy values were determined using male teratocytes only. This was done because M. demolitor is arrhenotokous, and the sex of the parasitoid could influence ploidy values of the teratocytes. Male progeny were selected by using unfertilized M. demolitor females to parasitize hosts. Cohorts of 10 hosts were dissected in saline every 24 h, and the teratocytes transferred to glass slides. Cells were fixed for 5 min in 100 ~1 of 70% ethanol containing 5% EGTA. The cells were resuspended in 25 ~1 of Hoechst 33342 dye (1 pgg/ml in water), and overlaid with a cover slip. Fifty teratocytes were selected randomly and the diameter of their nuclei was measured using an ocular micrometer. Cells were then analysed for relative DNA content using an Olympus BH-2 epifluorescent microscope interfaced with a Microscience image analysis fluorometry system (Federal Way, Wash.) (Weissman and Kiviat, 1985). The 1 and 2C standards were estimated using brain cells from male (1C) and female (2C) M. demolitor adults. As a second control, measurements were also taken on chicken erythrocytes which are reported to contain 2.5 pg of DNA per nucleus (Rasch et al., 1971). Data were expressed as the log, of the total fluorescence value. In vitro culture of teratocytes and injection into unparasitized hosts
To determine whether teratocytes affected host development, cells were injected into larvae. Collecting teratocytes from the haemolymph of parasitized hosts proved unsatisfactory, because of the inability to remove all haemocytes from a preparation. Thus, to eliminate these and other sources of host contamination, teratocytes were collected from M. demolitor eggs cultured in vitro (Strand et al., 1985). Eggs were
collected by first allowing wasps to parasitize hosts. The hosts were held on diet for 22 h under the rearing conditions described previously, and then dissected in saline. At this time, the M. demolitor embryos were in the segmented stage of development. The eggs were rinsed in 500 ~1 of saline (5 times), and then examined under an inverted microscope to confirm no host cells were present. The saline was removed, and varying numbers of eggs were resuspended in lo-20 ~1 drops of Ex-cell400 culture medium (J. R. Scientific, Woodland, Calif.). Under these conditions, hatching rates were consistently >95%, and the number of teratocytes produced per egg did not differ significantly from the number of teratocytes produced in vivo (see Results). The teratocytes were 12 f 2 h old when injected into hosts. Viability of the cells was tested by staining with carboxyfluorescein diacetate succinimidyl ester (CFSE) or propidium iodide (PI) (Molecular Probes Inc., Junction City, Ore.). CFSE is taken up readily by living cells, but PI binds to DNA in dead cells only (Haugland, 1989). A 10 mM stock solution of CFSE was prepared in dimethyl sulphoxide, and stored at 4°C. Working solutions were prepared by diluting the stock 1: 200 in medium. A 1 pg/ml solution of PI was prepared in water and stored at 4°C. Working solutions were prepared by diluting the stock 1: 100 in medium. Cells were stained at 7°C for 20 min, rinsed in medium and examined by epifluorescent microscopy. Teratocytes were drawn into a glass micropipette mounted on a micromanipulator. Larvae were anaesthetized with carbon dioxide and injected through a proleg. With approx. 600 teratocytes produced per M. demolitor egg, hosts were injected with 300 (0.5), 600 (l), 1200 (2) and 2400 (four egg equivalents) cells in l-2 ~1 of culture medium. The hosts were then reared individually in 30ml plastic cups half filled with diet. Controls consisted of larvae injected with Ex-cell 400 medium only or larvae parasitized by M. demolitor. All larvae were inspected and weighed daily, and the terminal stage formed was recorded. Larvae were assigned to the following terminal stages: (1) larva, individual died in the larval stage and exhibited no characters associated with metamorphosis; (2) larval-pupal intermediate, individual initiated metamorphosis but after moulting retained some larval characters and (3) pupa, individual completed metamorphosis successfully. Because teratocyte injections had an inconsistent effect on P. in&dens development, the possibility that injected cells were being eliminated by the host immune system was also examined. Unambiguous identification of encapsulated teratocytes proved very difficult. However, by fluorescently labelling cells in vitro with CFSE prior to injection, it was possible to monitor their fate in vivo. CFSE is useful in long-term tracing of cells, and has no apparent deleterious function on cell activity (Bronner-Fraser, 1985). Teratocytes were stained for 1 h in a I:200 working solution of CFSE as described previously.
Fig. 1. Phase contrast micrographs of M. demolitor teratocytes. (A) Serosa from a newly hatched M. dtw olitor egg fragmenting into individual teratocytes (day 2). Many of the cells are liberated but others attached to one another within the egg chorion (EC). (B) Large teratocyte dissected from a teratocytes from M. demolitor eggs cultured in vitro in Ex-cell400 medium.
P. inch dens larva on day 6. (C) Eight-hour-old
505
M. abnolitor teratocytes
507
P. incluakns larvae were then injected with approx. 600 labelled teratocytes and held for varying periods. Larvae were anaesthetized with carbon dioxide, and bled onto Parafilm from a cut proleg. A 1~1 aliquot of haemolymph was transferred to a glass slide and diluted 1: 100 in saline. The slides were held in humidified boxes for 1 h to allow the cells to settle, and were then examined by phase-contrast and epifluorescent microscopy. P. incluakns haemocytes were identified by the morphological criteria of Gupta (1979). Previous study identified five types of haemocytes in P. includens (plasmatocytes, granulocytes, spherulocytes, oenocytoids and prohaemocytes), and indicated plasmatocytes were the primary cell type involved in encapsulation and nodulation responses (Strand and Noda, unpublished).
A 0.02 wasp equivalent of calyx fluid or 0.10 equivalent of purified M. demolitor polydnavirus was combined with a 0.02 equivalent of venom, and injected into hosts through a proleg. Previous studies indicated a 0.02 equivalent of calyx fluid was a physiological dose (Strand and Dover, 1991). A slightly higher dose of the virus was used to compensate for material lost during purification. After 18 h, the same hosts were injected with 600 CFSE labelled teratocytes, and bled at various times as described previously. Other larvae injected with calyx fluid or the virus plus venom and teratocytes were monitored until death or pupation. Larvae injected with medium or 0.02 wasp equivalents of calyx fluid plus venom served as controls.
Injection of calyx fluid or virus plus venom and teratocytes
RICXJLTS
To determine if the loss of teratocytes could be prevented, hosts were injected with M. demolitor calyx fluid or purified polydnavirus plus venom. The reproductive tracts of female wasps were excised under saline, and the calyces and venom gland were removed. The calyces and venom gland were punctured using forceps, and the contents allowed to diffuse into the drop. M. demolitor polydnavirus was purified on a 25-50% sucrose gradient made up in Pringle’s saline (Strand and Dover, 199 1). Calyx fluid from SO-100 wasps was collected and layered onto the surface of the gradient. The gradient was spun at 42,000 g for 20 min in a Beckman TLA-100.2 rotor using a Beckman TL-100 ultracentrifuge. The virus fraction forms a single discrete band that was collected by side puncture. The virus was suspended in 2 ml of saline and centrifuged at 25,000 g for 1 h. The resulting pellet was then resuspended in saline and diluted appropriately. To further substantiate that virus was responsible for enhancing the persistence of teratocytes, virus was inactivated before injection by treatment with trioxsalen (4,5,8 trimethylpsoralen) (Sigma, St Louis, MO.), and exposure to long-wave U.V. light (Cook et al., 1984).
Teratocyte growth and DNA content
The serosa that enveloped the M. demolitor embryo began to dissociate into individual teratocytes immediately prior to hatching. As the parasitoid larva freed itself from the chorion, the teratocytes were liberated individually and in small clumps that sometimes adhered to the larva (Fig. 1). However, within 8 h of hatching an average of 542 individual teratocytes were present per host (Fig. 2). The number of cells per host was similar on day 3, but had declined to 270 cells on day 4. The number of teratocytes then remained constant until the parasitoid larva emerged on day 7. Teratocyte and nuclear diameter increased throughout development (Fig. 3). By day 7 the cells were 45-105 pm in diameter, and were spherical or spheroidal in shape (Fig. 1). By day 7 the nuclei of teratocytes ranged from 41-83 pm in diameter. No mitotic activity was observed during the course of the study. Dissections indicated teratocytes were present in the guts of approx. 30% of 4-7 day old M. demolitor larvae. From 1-12 intact teratocytes were present per larva, and were easily identified by their colour and morphology.
100
g 3.
700
0 +I
600
80 t 60-
2
1002 1
2
3
4
5
6
7
Age (days post-oviposition) Fig. 2. Mean number of teratocytes present in P. in&dens larvae parasitized by M. demolitor. Ten larvae were dissected per time point.
T
Teratocytes Teratocyte nuclei
J
E 3
n
3
4
5
6
7
Age (days post-oviposition) Fig. 3. Mean diameter of teratocytes and their nuclei from P. in&dens larvae parasitized by M. demolitor.Ten larvae were dissected per time point, and 50 cells were selected at random from each host for measurement.
M. R. STRANDand E. A.
508
1C
2C
4C
6C
WONG 16C
32C
64C 126C 256C
~~~~V~V~V
0
3
12
4
DNA content
6
6
6
7
9
10
(log2 of total fluorescence)
Fig. 4. Frequency distribution of DNA ploidy classes for brain cell nuclei from male (A) and female (B) M. demoliloradults (n = 50 each). Standard values for DNA measurements were obtained for male and female brain tissue of M. akmolitor adults. Male and female brain cells had mean fluorescence values of 5.45 + 1.32 (1C) and 10.96 f 2.15 (2C) respectively (Fig. 4). Chicken erythrocytes had a median fluorescence value of 14.4. Based on these standards, the ploidy levels of teratocytes from newly hatched male eggs ranged from 2 to 8C (Fig. 5). Based on an estimate of 2.5 pg of DNA per nucleus for chicken erythrocytes (Rasch et al., 1971), newly released male teratocytes contained 1.9-7.6 pg of DNA. Ploidy levels were predominantly 16-32C on day 3 and 32-&K on day 4 (Fig. 5).
Ploidy levels continued to increase on days 5-7, but the values became progressively more variable (Fig. 6). By day 7 a large number of cells had DNA contents between 128 and 256C.
EJGectsof teratocytes on host development
Teratocytes from eggs hatched in vitro were similar in size, number and general appearance to teratocytes observed in vivo (Fig. 1). The viability of the teratocytes prior to injection was confirmed by uptake of CFSE and exclusion of PI. Greater than 98% of the cells fluoresced bright green when stained with CFSE while less than 1% fluoresced orange when stained with PI. P. in&dens larvae exhibited a dose-dependent response when injected with teratocytes although superphysiological numbers of teratocytes had to be used (Table 1). Ninety per cent of the larvae injected with 300-600 teratocytes attained a normal final weight and formed a pupa without developmental delay. The remaining individuals moulted to the fifth stadium, but gained less weight and died as larvae.
Table 1. Effect of teratocytes on the develooment of P. includens larvae Terminal stage
Larvae Final weight (mgf SD)
Number of teratocytes injected
n
%
300 600 1200 2400
24 30 30 26
12 114.3 + 22.3 10 143.0 + 24.8 26 121.8 +29.8 54 96.2* 19.1
Medium Parasitized
30 36
0 100
38.3 rt 6.3
Pupae
Intermediates Final weight (mgf SD)
Development time (days* SD)*
Development time (daysfSD)+
%
9.6k2.3 12.2k1.4 9.5k2.8 9.2 k 1.6
0 0 3 0
-
-
244 -
8 -
7.2 f 0.4
0 0
-
*Development time is measured from the time of injection or parasitism.
%
Final weight (mgf SD) 21.9 32.4 24.0 24.9
7.1 *0.2 1.3 f 0.3 7.3 f 0.4 7.5 f 0.4
100 268.3 f 14.4 0
7.3 f 0.3 -
88 90 71 46
264.2 + 292.3 f 286.2 + 275.6 +
Development time (daysfSD)*
M. demolitorteratocytes
time (Table 2), and that many cells were killed by host haemocytes (Fig. 7). Haemocytic capsules or nodules surrounded some teratocytes within 2 h of injection. By 18 h, some teratocytes had been phagocytized, and by 48 h almost no free teratocytes remained in circulation. Throughout the 48 h sampling period, unencapsulated teratocytes fluoresced bright green. The fluorescence of encapsulated teratocytes, however, was less intense, and it appeared the cells were in varying states of decomposition. Injection of teratocytes had no distinct effect on the spreading behaviour of P. in&dens plasmatocytes or granulocytes (Fig. 8). Plasmatocytes spread rapidly on contact with the slide and assumed their characteristic fibroblastic morphology, while the granulocytes spread and assumed a circular profile. Plasmatocytes were locomotory in vitro and were observed to migrate out of the capsules or nodules surrounding teratocytes. In contrast, the number of teratocytes present in larvae injected 18 h earlier with calyx fluid plus
The majority of larvae injected with 1200 teratocytes also formed normal pupae, but the number of individuals dying as larvae or larval-pupal intermediates increased to 29%. The most pronounced developmental alterations occurred in larvae injected with 2400 teratocytes. Fifty-four per cent died as fifth (n = 9) or sixth (n = 5) stadium larvae that attained smaller final weights and exhibited developmental delays when compared to individuals that pupated (Table 1). All control larvae injected with Ex-cell400 medium pupated without developmental delay, while larvae parasitized by M. demolitor moulted to the fifth stadium, but exhibited greatly reduced final weights and died as larvae after parasitoid emergence (Table 1). Teratocyte persistence
Examination of P. in&dens haemolymph after injection of CFSE labelled teratocytes indicated the number of teratocytes in circulation declined with 1c
2C
4C
6C
16C
32C
64C
126C
v
v
v
v
v
v
v
v
20 A
16
509
256C v
16 14 12
20. 16.
B
16. 14. 12.
20. 16.
c
16. 14. 12. 10,
ij, 0
,
,
,
.
,
1
2
3
4
5
DNA
.J&.. 6
7
6
9
10
content (log2 of total fluorescence)
Fig. 5. Frequency distribution of DNA ploidy classes for day 2 (A), day 3 (B) and day 4 (C) teratocytes (n = 50 each).
510
M. R.
and E. A.
STRAND
Table 2. Persistence of teratocytes in P.
WONG
includens in the presence and absence of calyx fluid plus venom
Calyx fluid plus venom and teratocytes*
Teratocytes only Bleeding time (h) 2 6 18 36 48
Cells/p 1 haemolymph (*SD) 50.3 * 39.5 f 22.4 + 9.4 k 4.3 f
Cells/p 1 haemolymph (&SD)
Encapsulated, nodulated or phagocytized (%)
5.9 12.7 16.4 9.6 4.4
9.0 19.8 56.5 34.2 5.0
61.5 * 54.5 + 74.8 + 97.2 k 75.0 +
Encapsulated, nodulated or phagocytimd (%)
16.8 14.6t 11.7t 24.2t 15.6t
0.5 0 0 0 0
*Larvae were injected with a 0.02 wasp equivalent of calyx fluid plus venom 18 h prior to injection with teratocytes (see Materials and Methods). Six-hundred teratocytes (1 egg equivalent) were injected per host. Haemolymph from 10 larvae was examined at the periods designated. ‘/Significantly different (P Q 0.05) from hosts injected with teratocytes only (Mann-Whitney U-Test).
1C
2c
4c
6C
16C
32C
64C
120C
256C
~VVV~~V~V
20 16
I 1 6.
A
14. 12. 1 0. a. 6. 4. 2.
20. 16.
B
1 6. 14. 1 2. I 0. 6. 6. 4. 2.
20. 16.
c
16. 14. 12.
‘;I*, . ,.,.A, 0
1
2
3
DNA content
4
5
6
7
6
9
10
(log2 of total fluorescence)
Fig. 6. Frequency distribution of DNA ploidy classes for day 5 (A), day 6 (B) and day 7 (C) teratocytes (n = 50 each).
Fig. 7. Epifluorescent micrographs of CIFC labelled teratocytes injected into P. inch&m larvae. Cells were obtained by adding haemolymph to Pringle’s saline on a glass slide and photographing after 30min. Unlabelled haemocytes were visualized by photographing under fluorescent and low level phase contrast lighting. All figures printed to the same scale. (A) Encapsulated teratocyte (CA) from a larva injected 18 h earlier with teratocytes only. The teratocyte in the upper left is within a phagocytic plasmatocyte (PH). (B) Teratocyte within a small haemocytic nodule (N) from a larva injected 6 h earlier with teratocytes only. (C) Unencapsulated teratocytes from a larva injected with 0.02 equivalents of calyx fluid plus venom. Larva was bled 18 h after injection of teratocytes.
511
Fig. 8. Phase contrast micrographs of P. in&dens haemocytes in vitro. Cells were obtained by adding haemolymph to saline in a culture dish and photographing after 1 h. Both figures are printed to the same scale. (A) Fully spread plasmatocytes (PL) and granulocytes (GR) from larvae injected with teratocytes only. (B) Spindle and veriform shaped plasmatocytes and unspread granulocytes from larvae injected with 0.02 equivalents of calyx fluid plus venom and teratocytes. In both figures larvae were bled 18 h after injection of teratocytes.
512
M. demolitor teratocytes
513
Table 3. Persistence of teratocytes in P. includens in the presence of purified virus plus venom and trioxsalen treated virus plus venom Virus plus venom and teratocytes’ Bleeding time (h) 6 18
Cells/l, 1 haemolymph (&SD)
Trioxsalen treated virus plus venom and teratocytes*
Encapsulated, nodulated or phagocytixed (%)
Cells//l 1 haemolymph (*SD)
Encapsulated, nodulated or phagocytixed (%)
0 .O
38.0 f 10.4t 14.4 + 19.7t
46.8 56.5
62.3 f 10.4 74.8 f 15.4
*Larvae were injected with a 0.10 wasp equivalent of purified virus or trioxsalen treated virus plus 0.02 equivalents of venom 18 h prior to injection with teratocytes (see Materials and Methods). Six-hundred teratocytes (1 egg equivalent) were injected per host. Haemolymph from 10 larvae was examined at the periods designated. tSianificantlv different (P < 0.05) from hosts injected with purified virus plus venom (Mann-kitney U-Test). ’
fluid and venom had a pronounced effect on plasmatocyte spreading behaviour (Fig. 8). Most of the plasmatocytes were spindle shaped or appeared as veriform cells, and almost none attached or spread on glass slides. Most granulocytes also remained unspread and spherical in shape. A few hosts were held for 10 days before bleeding, and they too still contained teratocytes. Although the number of cells per host had declined greatly by this time (14.2 f 7.4/~1 haemolymph, n = 8 hosts), the cells were larger (180.4 + 65.8 pm, n = 140 cells) than the maximum size observed in parasitized hosts (Fig. 3). Larvae injected with 0.10 equivalents of M. demolitor polydnavirus plus venom and teratocytes were examined at only 6 and 18 h post-injection, but the results were similar to the effects of calyx fluid plus venom (Table 3). However, teratocytes were encapsulated and plasmatocytes spread normally when larvae were injected with virus that had been inactivated with trioxsalen and U.V. light (Table 3).
with 600 teratocytes exhibited developmental delays, reduced weights and died as larvae. The number of stadia larvae underwent before dying varied. The majority died during the fifth stadium (67%), but others died after moulting to a supernumerary sixth (25%) or seventh (8%) stadium. Most of the remaining individuals pupated without developmental delay. Injecting 2400 teratocytes per host had a similar effect on larvae (Table 4). Few larvae injected with only calyx fluid plus venom died during the larval stage, but 87% died as intermediates (Table 4). Larvae moulted to a fifth (18%), sixth (47%), seventh (36%) or eighth (3%) stadium before initiating metamorphosis, and thus exhibited delays in development time and weight gain relative to control larvae injected with only medium. However, the larvae also attained final weights that were consistently higher than larvae injected with calyx fluid plus venom and teratocytes. All larvae injected with Ex-cell400 medium pupated normally (Table 4). Out of 15 larvae injected with 0.10 equivalent of polydnavirus plus venom and 600 teratocytes, 80% died as fifth or sixth stadium larvae that attained a final weight of 88.2 + 24.3 mg.
Eflects of calyx fluid or virus plus venom and teratocytes on host development
DISCUSSION
Hosts exhibited pronounced alterations when injected with calyx fluid plus venom and teratocytes (Table 4). Eighty-seven per cent of larvae injected
The number and size of M. akmolitor teratocytes are similar to reports for other microgastrine braconids (Salt, 1968; Vinson and Iwantsch, 1980). For example,
venom declined only slightly (Table 2). The teratocytes persisted free in the haemolymph, and almost no cells were encapsulated (Fig. 7). Injection of calyx
Table 4. Effect of teratocytes on the development of P. includens larvae that were injected with calyx fluid plus venom* Terminal stage Larvae
Intermediates Development time (daysfSD)t
62.4 + 34.6 71.6k29.4
11.2* 1.4 12.7 k2.3
56.0 f 27.9 -
6.8 f 2.2 -
Number of teratocytes injected
n
%
Final weight (mgf SD)
600 2400
30 38
87 87
Calyx fluid plus venom Medium
30 30
13 0
Pupae
%
Final weight (mgf SD)
Development time (days+SD)t
0 3
258.2
87 227.2 f 49.5 0 -
%
Final weight (mgk SD)
Development time (days *SD)t
7.0
13 10
273.8 rf: 7.2 261.3 k 9.6
6.0 & 0.8 6.5 + 0.6
12.3 f 1.4 -
0 100
268.3 f 16.4
7.0 * 1.1
*Larvae were injected with a 0.02 wasp equivalent of calyx fluid plus venom 18 h prior to injection with teratocytes (see Materials and Methods). TDevelopment time is measured from the time of teratocyte injection.
514
M. R. STRAND and E. A. WONG
Cardiochiles nigriceps produces approx. 200 teratocytes per egg that attain a diameter of more than 3OOpm (Vinson, 1970) while M. croceipes produces 750 teratocytes that attain a maximum diameter of 140pm (Vinson and Lewis, 1973). Although ploidy levels have not been reported previously for teratocytes, the large increase in size and nuclear diameter would suggest these cells become highly polyploid (Strand et al., 1985). Indeed, our data indicate the ploidy levels for male teratocytes were between 2 and 8C at hatching, and progressively shifted upward during development. Some cells attained ploidy levels of 128-256C, but in the latter stages of development (day 6-7) both ploidy levels and cell size were quite variable. We are uncertain why some teratocytes increased in size during this period while others did not, but similar size variation has been reported in the literature (Kitano, 1965; Vinson, 1970; Vinson and Lewis, 1971). Some studies suggest teratocytes are secretory early in development (Strand et al., 1986; Tanaka and Wago, 1990), but assume a storage function as they age (Sluss, 1968; Sluss and Leutenegger, 1968; Cohen and Debolt, 1984). Interestingly, the teratocytes of C. nigriceps diverge into two morphologically distinct forms in the latter stages of development (Vinson and Scott, 1974). Type I cells contain extensive profiles of rough endoplasmic reticulum and appear to maintain a secretory function while type II cells contain an extensively vaculated endoplasmic reticulum and numerous inclusions that are suggestive of a storage function. Thus, the variation in M. demolitor teratocytes may reflect the possibility that the teratocytes of some braconids diverge into subpopulations with age. Although M. demolitor larvae consumed some teratocytes, our data do not support the suggestion these cells have primarily a trophic function (Sluss, 1968). This is based on the observation that teratocyte consumption was variable, and a large number of cells remained in the host throughout development. However, our data do indicate M. demoiitor teratocytes affect the development of P. in&dens, and that teratocyte survival is enhanced by the presence of calyx fluid or M. demolitor polydnavirus. Recently completed dose-response studies indicated calyx fluid and the virus inhibit filipodial elongation of P. includens plasmatocytes and suppress encapsulation of M. demolitor eggs and other foreign targets such as glass rods (Strand and Noda, unpublished). Venom alone has no effect on plasmatocyte spreading or encapsulation, but appears to synergize the effects of M. demolitor polydnavirus. A synergistic function is suggested on the basis that calyx fluid or M. demolitor polydnavirus plus venom inhibits cell spreading at lower dosages and for a longer period than calyx fluid or this virus alone. In the current study, most teratocytes were encapsulated within 24 h of injection in the absence of M. demolitor polydnavirus plus venom, but almost no teratocytes were encapsulated in its presence.
Injection of trioxsalen treated virus plus venom did not protect teratocytes from encapsulation suggesting transcriptionally active virus is necessary to prevent encapsulation. Collectively, our results suggest that in the absence of M. demolitor polydnavirus, most hosts were unaffected by a physiological dose of teratocytes (600 cells per host) because the cells were rapidly eliminated by the immune system. However, injecting superphysiological doses of cells overwhelmed the immune capability of an increasing proportion of hosts, and in some individuals enough teratocytes survived to disrupt development. In contrast, when calyx fluid or the virus plus venom was present, the host immune system was compromised, and a physiological dose of teratocytes was sufficient to induce developmental alterations. Hosts injected with calyx fluid or M. demolitor polydnavirus plus venom and teratocytes exhibited developmental alterations that were strikingly similar to the developmental alterations observed during natural parasitism (Strand et al., 1988). However, we are uncertain whether these effects reflect the activity of only teratocytes or are due to the cumulative effects of teratocytes, virus and venom. In addition to affecting the immune system, control experiments of the current study (Table 4) and rigorous doseresponse experiments conducted previously (Strand and Dover, 1991) indicated calyx fluid and purified virus alone cause P. includens to develop into larvalpupal intermediates. Virus expression is detectable in P. in&dens within 4 h of oviposition and persists for at least 8 days (Strand et al., unpublished), suggesting the activity of both teratocytes and virus may contribute to the developmental alterations reported here. Alterations in host development due to teratocytes have been reported for other micrograstrine braconids. Vinson (1970) reported C. nigriceps teratocytes caused Heliothis virescens larvae to form abnormal pupae. Zhang and Dahhnan (1989) reported the teratocytes of M. croceipes had a dose-dependent effect on H. virescens fifth stadium larvae. The majority of larvae injected with a physiological dose of M. croceipes teratocytes died as larvae while hosts injected with fewer teratocytes died as intermediates or pupated. Neither study reported the encapsulation of injected teratocytes, but unlike our in vitro produced teratocytes, the cells were collected from parasitized larvae. Since polydnaviruses enter a variety of host tissues (Stoltz and Vinson, 1979) it is possible other components besides teratocytes were injected into hosts and contributed to the developmental alterations reported. Our results and those for other species of microgastrine braconids suggest teratocytes and/or polydnaviruses have a juvenilizing effect on host development. However, the specific factors responsible for these effects remain largely undefined. Juvenile hormone activity was reported in the teratocytes of C. nigriceps (Joiner et al., 1973) and more
M. demolitorteratocytes
recently juvenile hormone esterase activity was shown to be reduced in hosts parasitized by M. croceipes and Aputtteles kuriyai (Dahlman et al., 1990; Hayakawa, 1990). In the case of A. kariyui, a 4500 Da peptide was purified from host haemolymph that repressed juvenile hormone esterase activity, but the source of the peptide was not identified. Undoubtedly, future studies will help clarify the role of specific wasp components in parasitism. Acknowledgements-The authors wish to thank T. Noda, V. Grass1 and K. Kervista for their assistance during various phases of the study. The authors also thank J. Aiken for sharing equipment in his laboratory and B. Dover for comments on the manuscript. This study was funded in part by USDA Grant No. 90-37250-5481, Wisconsin Hatch Project 3200 and a Hilldale Research Award from the University of Wisconsin-Madison Graduate School.
REFERENCES
515
lation on their biological significance. Zool. Mag., Tokyo 74, 192-197. Lawrence P. 0. (1986) Host-parasite hormonal interactions: an overview. J. Insect Phvsiol. 32. 295-298. Pringle J. H. W. (1938) Proprioception in insects. J. exp. Biol. 15, 101-103. Rasch E. M., Barr H. J. and Rasch R. W. (1971) The DNA content of sperm of Drosophila melanogaster. Chromosoma 33, 1-18.
Salt G. (1968) The resistance of insect parasitoids to the defence reactions of their hosts. Biol. Rev. 13, 381-495. Sluss R. R. (1968) Behavioral and anatomical responses of the convergent lady beetle to parasitism by Perilitus Braconidae). cocclnellae (Schronk) (Hymenoptera: J. Invertebr. path. 10, 9-27. Sluss R. R. and Leutenegger R. (1968) The fine structure of the trophic cells of Perilitus coccinellae (Hymenoptera: Braconidae). J. Ultrastruc. Res. 26, 44-451. Stoltz D. B. (1986) Interactions between parasitoid-derived products and host insects. J. Insect Physiol. 32, 347-350. Stoltz D. B. and Vinson S. B. (1979) Viruses and parasitism in insects. Ado. Virus Res. 24, 125-171. Strand M. R. (1990) Characterization of larval development in Pseudoplusia includens (Lepidoptera: Noctuidae). Ann ent. Sot. Am. 83, 5388544.
Bronner-Fraser M. (1985) Alterations in neural crest migration by a monoclonal antibody that affects cell adhesion. J. Cell Biol. 101, 610-617. Cohen A. C. and Debolt J. W. (1984) Fatty acid and amino acid composition of teratocytes from LJ+ra.r hesperus (Miridae: Hemiptera) parasitized by two species of parasites, Leiophron untformis (Braconidae: Hymenoptera) and Peristenus stygicus (Braconidae: Hymenoptera).
Strand M. R., Quarles J. M., Meola S. M. and Vinson S. B. (1985) Cultivation of teratocytes of the egg parasitoid Telenomus heliothidis (Hymenoptera: Scelionidae).
Comp. Biochem. Physiol. 79B, 335-337. Cook D. J., Stoltz D. B. and Vinson
Strand M. R., Johnson J. A. and Culin J. D. (1988) Developmental interactions between the parasitoid Microplitis demolitor and its host Heliothis virescens.
S. B. (1984) Induction of a new hemolymph glycoprotein in larvae of permissive hosts parasitized by Campoletis sonorensis.
Insect Biochem. 14, 45-50.
Dahlman D. L. (1990) Evaluation of teratocyte functions: an overview. Archs Insect Biochem. Physiol. 13, 159-166. Dahlman D. L., Coar, D. L., Koller N. C. and Neary T. J. (1990) Contributing factors to reduced ecdysteroid titers in Heliothis virescens parasitized by Microplitis croceipes. Archs Insect Biochem. Physiol. 13, 29-40.
Fisher R. C. (1971) Aspects of the physiology of endoparasitic Hymenoptera. Biol. Rev. 46, 243-278. Gupta A. P. (1979) Hemocyte types: their structures, synonymies interrelationships and taxonomic significance. In Insect Hemocytes (Ed. Gupta A. P.), pp. 86-127. Cambridae Universitv Press. Cambridae. Mass. Haugland R. P. (1989) Vital’dyes and-polar tracers. In Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, pp. 105-l 13. Molecular Probes Inc.,
Eugene, Ore. Hayakawa Y. (1990) Juvenile hormone esterase activity repressive factor in the plasma of parasitized insect larvae. J. biol. Chem. 265. 10813-10816. Joiner R. L., Vinson S. B. and Benskin J. B. (1973) Teratocytes as source of juvenile hormone activity in a parasitoid-host relationship. Nature New Biol. 246, 120-121.
Kitano H. (1965) Studies on the origin of giant cells in the body fluid of Pieris rapae crucivora attacked by Apanteles glomeratus L. II. Determination of their origin and specu-
In Vitro Cell Dev. Biol. 21, 361-367.
Strand M. R., Meola S. M. and Vinson S. B. (1986) Correlating pathological symptoms in Heliothis virescens eggs with development of the parasitoid Telenomus heliothidis. J. Insect Physiol. 32, 389-402.
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Strand M. R. and Dover B. A. (1991) Developmental disruption of Pseudoplasma includens and Heliothis virestens larvae by the calyx fluid and venom of Microplitis demolitor. Archs Insect Biochem. Physiol. In press. Tanaka T. and Wago H. (1990) Ultrastructural and functional maturation of teratocytes of Apanteles kariyai. Archs Insect Biochem. Physiol. 13, 187-197. Vinson S. B. (1970) Development and possible functions of teratocytes in the host-parasite association. J. Invertebr. path. 16, 93-101.
Vinson S. B. and Lewis W. J. (1973) Teratocytes: growth and numbers in the hemocoel of Heliothis virescens attacked by Microplitis croceipes. J. Invertebr. Path. 22, 351-355.
Vinson S. B. and Iwantsch G. F. (1980) Host regulation by . parasitoids. Q. Rev. Biol. 55, 143-165. Vinson S. B. and Scott J. R. (1974) Ultrastructure of teratocytes of Cardiochiles nigriceps Vierick. J. Insect Morph. Embryol. 3, 293-304.
Weissman M. A. and Kiviat N. (1985) Measurements of nuclear DNA on an IBM-PC-tvoe cornouter. In Proc. 7th Annu. Conf IEEEIEngeng Meh: Biol. ioc. (Eds Lin J. C. and Feinberg B. N.), pp. 976-979. Institute of Electrical and Electronic Engineers, New York. Zhang D. and Dahlman D. L. (1989) Microplitis croceipes teratocytes cause developmental arrest of Heliothis virescens larvae. Archs Insect Biochem. Physiol. 12,51--61.
0022-1910/91 $3.00 + 0.00 Copyright 0 1991 Pergamon Press plc
J. Insect Physiol.
THE GROWTH AND ROLE OF MZCZtOPLZZ’Z~ DEMOLZTOZ? TERATOCYTES IN PARASITISM OF PSEUDOPLUSZA ZNCLUDENS M. R. STRAND and E. A. WONG Department of Entomology, 237 Russell Laboratories, University of Wisconsin-Madison, Madison, WI 53706, U.S.A.
(Received 4 April 1991)
Abstract-The growth and function of Microplitis demolitor teratocytes in Pseudoplusia in&dens larvae was investigated. An average of 542 teratocytes was produced per M. demolitor egg. The number of teratocytes present per host declined during the course of parasitoid development, but the size of individual cells increased. The ploidy levels of teratocytes ranged from 2 to 8C at hatching, but had increased to 128-256C at the completion of parasitoid development. Injection of in vitro cultured teratocytes into fourth stadium P. incluahs larvae had an inconsistent effect on development. Most larvae pupated without delay when injected with a physiological dose of 600 teratocytes, but 54% of larvae failed to pupate when injected with a super-physiological dose of 2400 teratocytes. Examination of teratocytes indicated most cells were encapsulated by P. includens haemocytes within 24 h of injection. However, prior injection of larvae with M. demolitor calyx fluid or polydnavirus plus venom suppressed the encapsulation response. Further, 87% of larvae injected with polydnavirus plus venom and 600 teratocytes exhibited alterations in growth that were very similar to parasitism by M. akmolitor. Key Word Index: Parasitoid; development;
haemocytes; immunity; encapsulation;
calyx fluid;
polydnavirus
INTRODUCTION Many hosts exhibit physiological alterations after parasitism by endoparasitic Hymenoptera (Vinson and Iwantsch, 1980; Lawrence, 1986). In certain braconids and ichneumonids, some of these alterations are attributed to the polydnaviruses and venom the adult wasp injects into the host at oviposition (Stoltz, 19g6). Other changes, however, may be due to factors associated with the developing parasitoid embryo or larva (Fisher, 1971). Of particular interest is the role of teratocytes (Dahlman, 1990). These unusual cells are produced by many of the polydnavirus carrying braconids. Teratocytes are derived from the serosa that envelops the developing parasitoid embryo. At hatching, the serosa dissociates into individual cells (teratocytes) that circulate in the haemolymph and become distributed throughout the host. Teratocytes persist during the course of parasitoid larval development, but their role in parasitism is unclear. Some studies suggest teratocytes serve a trophic function (Sluss, 1968) while others report they affect the development and immune response of hosts (Salt, 1968; Joiner et al., 1973; Strand et al., 1986; Zhang and Dahlman, 1989; Tanaka and Wago, 1990). Their interaction with other wasp factors such as polydnavirus and venom has not been investigated.
In the present study, we examined the teratocytes of the solitary, braconid wasp, Microplitis demolitor Wilkinson in its host, Pseudoplusia includens (Walker). Here we report the size and ploidy values of teratocytes increased greatly during development, but the number of cells per host declined. Doseresponse experiments indicated teratocytes caused some developmental alterations in P. incluhs larvae, but more pronounced effects occurred when larvae were also injected with M. demolitor polydnavirus and venom. MATERIALSAND METHODS Insects P. includens were reared as described by Strand (1990) in 30 ml plastic cups with paper lids at 27 f 1°C and a 16 h light-8 h dark photoperiod. Moths were fed a 20% sucrose solution. M. akmolitor were reared at 27 + 1°C and a 16 h light-8 h dark photoperiod as outlined by Strand et al. (1988). The M. akmolitor egg hatched between 26 and 28 h after oviposition (day 2). The parasitoid larva emerged from the host on day 7, spun a cocoon, and emerged as an adult on day 13. Hosts used in experiments were 6-18 h-old fourth stadium larvae, and all 503
504
M. R. STRANDand E. A. WONG
wasp ovipositions were observed to assure no superparasitism occurred. Development of M. demolitor and P. in&dens was monitored by previously established staging criteria (Strand et al., 1988; Strand, 1990). Teratocyte growth in parasitized hosts
To determine the number and size of teratocytes present during parasitoid development, hosts were dissected on day 2, and every 24 h subsequently through day 7. Hosts were dissected individually in 60 mm Falcon tissue culture plates (Be&on Dickenson, Lincoln Park, N.J.) filled with 1 ml of Pringle’s saline (Pringle, 1938). After settling on the bottom of the dish, the number of teratocytes per host was counted using a Nikon inverted, phase-contrast microscope. In order to estimate cell size during development, 50 teratocytes per host were selected randomly and their diameter measured using an ocular micrometer. For eliptic cells both short and long diameters were measured and the average was estimated as the cell diameter. To estimate the ploidy value of teratocytes, relative DNA content was measured by fluorometric analysis. Ploidy values were determined using male teratocytes only. This was done because M. demolitor is arrhenotokous, and the sex of the parasitoid could influence ploidy values of the teratocytes. Male progeny were selected by using unfertilized M. demolitor females to parasitize hosts. Cohorts of 10 hosts were dissected in saline every 24 h, and the teratocytes transferred to glass slides. Cells were fixed for 5 min in 100 ~1 of 70% ethanol containing 5% EGTA. The cells were resuspended in 25 ~1 of Hoechst 33342 dye (1 pgg/ml in water), and overlaid with a cover slip. Fifty teratocytes were selected randomly and the diameter of their nuclei was measured using an ocular micrometer. Cells were then analysed for relative DNA content using an Olympus BH-2 epifluorescent microscope interfaced with a Microscience image analysis fluorometry system (Federal Way, Wash.) (Weissman and Kiviat, 1985). The 1 and 2C standards were estimated using brain cells from male (1C) and female (2C) M. demolitor adults. As a second control, measurements were also taken on chicken erythrocytes which are reported to contain 2.5 pg of DNA per nucleus (Rasch et al., 1971). Data were expressed as the log, of the total fluorescence value. In vitro culture of teratocytes and injection into unparasitized hosts
To determine whether teratocytes affected host development, cells were injected into larvae. Collecting teratocytes from the haemolymph of parasitized hosts proved unsatisfactory, because of the inability to remove all haemocytes from a preparation. Thus, to eliminate these and other sources of host contamination, teratocytes were collected from M. demolitor eggs cultured in vitro (Strand et al., 1985). Eggs were
collected by first allowing wasps to parasitize hosts. The hosts were held on diet for 22 h under the rearing conditions described previously, and then dissected in saline. At this time, the M. demolitor embryos were in the segmented stage of development. The eggs were rinsed in 500 ~1 of saline (5 times), and then examined under an inverted microscope to confirm no host cells were present. The saline was removed, and varying numbers of eggs were resuspended in lo-20 ~1 drops of Ex-cell400 culture medium (J. R. Scientific, Woodland, Calif.). Under these conditions, hatching rates were consistently >95%, and the number of teratocytes produced per egg did not differ significantly from the number of teratocytes produced in vivo (see Results). The teratocytes were 12 f 2 h old when injected into hosts. Viability of the cells was tested by staining with carboxyfluorescein diacetate succinimidyl ester (CFSE) or propidium iodide (PI) (Molecular Probes Inc., Junction City, Ore.). CFSE is taken up readily by living cells, but PI binds to DNA in dead cells only (Haugland, 1989). A 10 mM stock solution of CFSE was prepared in dimethyl sulphoxide, and stored at 4°C. Working solutions were prepared by diluting the stock 1: 200 in medium. A 1 pg/ml solution of PI was prepared in water and stored at 4°C. Working solutions were prepared by diluting the stock 1: 100 in medium. Cells were stained at 7°C for 20 min, rinsed in medium and examined by epifluorescent microscopy. Teratocytes were drawn into a glass micropipette mounted on a micromanipulator. Larvae were anaesthetized with carbon dioxide and injected through a proleg. With approx. 600 teratocytes produced per M. demolitor egg, hosts were injected with 300 (0.5), 600 (l), 1200 (2) and 2400 (four egg equivalents) cells in l-2 ~1 of culture medium. The hosts were then reared individually in 30ml plastic cups half filled with diet. Controls consisted of larvae injected with Ex-cell 400 medium only or larvae parasitized by M. demolitor. All larvae were inspected and weighed daily, and the terminal stage formed was recorded. Larvae were assigned to the following terminal stages: (1) larva, individual died in the larval stage and exhibited no characters associated with metamorphosis; (2) larval-pupal intermediate, individual initiated metamorphosis but after moulting retained some larval characters and (3) pupa, individual completed metamorphosis successfully. Because teratocyte injections had an inconsistent effect on P. in&dens development, the possibility that injected cells were being eliminated by the host immune system was also examined. Unambiguous identification of encapsulated teratocytes proved very difficult. However, by fluorescently labelling cells in vitro with CFSE prior to injection, it was possible to monitor their fate in vivo. CFSE is useful in long-term tracing of cells, and has no apparent deleterious function on cell activity (Bronner-Fraser, 1985). Teratocytes were stained for 1 h in a I:200 working solution of CFSE as described previously.
Fig. 1. Phase contrast micrographs of M. demolitor teratocytes. (A) Serosa from a newly hatched M. dtw olitor egg fragmenting into individual teratocytes (day 2). Many of the cells are liberated but others attached to one another within the egg chorion (EC). (B) Large teratocyte dissected from a teratocytes from M. demolitor eggs cultured in vitro in Ex-cell400 medium.
P. inch dens larva on day 6. (C) Eight-hour-old
505
M. abnolitor teratocytes
507
P. incluakns larvae were then injected with approx. 600 labelled teratocytes and held for varying periods. Larvae were anaesthetized with carbon dioxide, and bled onto Parafilm from a cut proleg. A 1~1 aliquot of haemolymph was transferred to a glass slide and diluted 1: 100 in saline. The slides were held in humidified boxes for 1 h to allow the cells to settle, and were then examined by phase-contrast and epifluorescent microscopy. P. incluakns haemocytes were identified by the morphological criteria of Gupta (1979). Previous study identified five types of haemocytes in P. includens (plasmatocytes, granulocytes, spherulocytes, oenocytoids and prohaemocytes), and indicated plasmatocytes were the primary cell type involved in encapsulation and nodulation responses (Strand and Noda, unpublished).
A 0.02 wasp equivalent of calyx fluid or 0.10 equivalent of purified M. demolitor polydnavirus was combined with a 0.02 equivalent of venom, and injected into hosts through a proleg. Previous studies indicated a 0.02 equivalent of calyx fluid was a physiological dose (Strand and Dover, 1991). A slightly higher dose of the virus was used to compensate for material lost during purification. After 18 h, the same hosts were injected with 600 CFSE labelled teratocytes, and bled at various times as described previously. Other larvae injected with calyx fluid or the virus plus venom and teratocytes were monitored until death or pupation. Larvae injected with medium or 0.02 wasp equivalents of calyx fluid plus venom served as controls.
Injection of calyx fluid or virus plus venom and teratocytes
RICXJLTS
To determine if the loss of teratocytes could be prevented, hosts were injected with M. demolitor calyx fluid or purified polydnavirus plus venom. The reproductive tracts of female wasps were excised under saline, and the calyces and venom gland were removed. The calyces and venom gland were punctured using forceps, and the contents allowed to diffuse into the drop. M. demolitor polydnavirus was purified on a 25-50% sucrose gradient made up in Pringle’s saline (Strand and Dover, 199 1). Calyx fluid from SO-100 wasps was collected and layered onto the surface of the gradient. The gradient was spun at 42,000 g for 20 min in a Beckman TLA-100.2 rotor using a Beckman TL-100 ultracentrifuge. The virus fraction forms a single discrete band that was collected by side puncture. The virus was suspended in 2 ml of saline and centrifuged at 25,000 g for 1 h. The resulting pellet was then resuspended in saline and diluted appropriately. To further substantiate that virus was responsible for enhancing the persistence of teratocytes, virus was inactivated before injection by treatment with trioxsalen (4,5,8 trimethylpsoralen) (Sigma, St Louis, MO.), and exposure to long-wave U.V. light (Cook et al., 1984).
Teratocyte growth and DNA content
The serosa that enveloped the M. demolitor embryo began to dissociate into individual teratocytes immediately prior to hatching. As the parasitoid larva freed itself from the chorion, the teratocytes were liberated individually and in small clumps that sometimes adhered to the larva (Fig. 1). However, within 8 h of hatching an average of 542 individual teratocytes were present per host (Fig. 2). The number of cells per host was similar on day 3, but had declined to 270 cells on day 4. The number of teratocytes then remained constant until the parasitoid larva emerged on day 7. Teratocyte and nuclear diameter increased throughout development (Fig. 3). By day 7 the cells were 45-105 pm in diameter, and were spherical or spheroidal in shape (Fig. 1). By day 7 the nuclei of teratocytes ranged from 41-83 pm in diameter. No mitotic activity was observed during the course of the study. Dissections indicated teratocytes were present in the guts of approx. 30% of 4-7 day old M. demolitor larvae. From 1-12 intact teratocytes were present per larva, and were easily identified by their colour and morphology.
100
g 3.
700
0 +I
600
80 t 60-
2
1002 1
2
3
4
5
6
7
Age (days post-oviposition) Fig. 2. Mean number of teratocytes present in P. in&dens larvae parasitized by M. demolitor. Ten larvae were dissected per time point.
T
Teratocytes Teratocyte nuclei
J
E 3
n
3
4
5
6
7
Age (days post-oviposition) Fig. 3. Mean diameter of teratocytes and their nuclei from P. in&dens larvae parasitized by M. demolitor.Ten larvae were dissected per time point, and 50 cells were selected at random from each host for measurement.
M. R. STRANDand E. A.
508
1C
2C
4C
6C
WONG 16C
32C
64C 126C 256C
~~~~V~V~V
0
3
12
4
DNA content
6
6
6
7
9
10
(log2 of total fluorescence)
Fig. 4. Frequency distribution of DNA ploidy classes for brain cell nuclei from male (A) and female (B) M. demoliloradults (n = 50 each). Standard values for DNA measurements were obtained for male and female brain tissue of M. akmolitor adults. Male and female brain cells had mean fluorescence values of 5.45 + 1.32 (1C) and 10.96 f 2.15 (2C) respectively (Fig. 4). Chicken erythrocytes had a median fluorescence value of 14.4. Based on these standards, the ploidy levels of teratocytes from newly hatched male eggs ranged from 2 to 8C (Fig. 5). Based on an estimate of 2.5 pg of DNA per nucleus for chicken erythrocytes (Rasch et al., 1971), newly released male teratocytes contained 1.9-7.6 pg of DNA. Ploidy levels were predominantly 16-32C on day 3 and 32-&K on day 4 (Fig. 5).
Ploidy levels continued to increase on days 5-7, but the values became progressively more variable (Fig. 6). By day 7 a large number of cells had DNA contents between 128 and 256C.
EJGectsof teratocytes on host development
Teratocytes from eggs hatched in vitro were similar in size, number and general appearance to teratocytes observed in vivo (Fig. 1). The viability of the teratocytes prior to injection was confirmed by uptake of CFSE and exclusion of PI. Greater than 98% of the cells fluoresced bright green when stained with CFSE while less than 1% fluoresced orange when stained with PI. P. in&dens larvae exhibited a dose-dependent response when injected with teratocytes although superphysiological numbers of teratocytes had to be used (Table 1). Ninety per cent of the larvae injected with 300-600 teratocytes attained a normal final weight and formed a pupa without developmental delay. The remaining individuals moulted to the fifth stadium, but gained less weight and died as larvae.
Table 1. Effect of teratocytes on the develooment of P. includens larvae Terminal stage
Larvae Final weight (mgf SD)
Number of teratocytes injected
n
%
300 600 1200 2400
24 30 30 26
12 114.3 + 22.3 10 143.0 + 24.8 26 121.8 +29.8 54 96.2* 19.1
Medium Parasitized
30 36
0 100
38.3 rt 6.3
Pupae
Intermediates Final weight (mgf SD)
Development time (days* SD)*
Development time (daysfSD)+
%
9.6k2.3 12.2k1.4 9.5k2.8 9.2 k 1.6
0 0 3 0
-
-
244 -
8 -
7.2 f 0.4
0 0
-
*Development time is measured from the time of injection or parasitism.
%
Final weight (mgf SD) 21.9 32.4 24.0 24.9
7.1 *0.2 1.3 f 0.3 7.3 f 0.4 7.5 f 0.4
100 268.3 f 14.4 0
7.3 f 0.3 -
88 90 71 46
264.2 + 292.3 f 286.2 + 275.6 +
Development time (daysfSD)*
M. demolitorteratocytes
time (Table 2), and that many cells were killed by host haemocytes (Fig. 7). Haemocytic capsules or nodules surrounded some teratocytes within 2 h of injection. By 18 h, some teratocytes had been phagocytized, and by 48 h almost no free teratocytes remained in circulation. Throughout the 48 h sampling period, unencapsulated teratocytes fluoresced bright green. The fluorescence of encapsulated teratocytes, however, was less intense, and it appeared the cells were in varying states of decomposition. Injection of teratocytes had no distinct effect on the spreading behaviour of P. in&dens plasmatocytes or granulocytes (Fig. 8). Plasmatocytes spread rapidly on contact with the slide and assumed their characteristic fibroblastic morphology, while the granulocytes spread and assumed a circular profile. Plasmatocytes were locomotory in vitro and were observed to migrate out of the capsules or nodules surrounding teratocytes. In contrast, the number of teratocytes present in larvae injected 18 h earlier with calyx fluid plus
The majority of larvae injected with 1200 teratocytes also formed normal pupae, but the number of individuals dying as larvae or larval-pupal intermediates increased to 29%. The most pronounced developmental alterations occurred in larvae injected with 2400 teratocytes. Fifty-four per cent died as fifth (n = 9) or sixth (n = 5) stadium larvae that attained smaller final weights and exhibited developmental delays when compared to individuals that pupated (Table 1). All control larvae injected with Ex-cell400 medium pupated without developmental delay, while larvae parasitized by M. demolitor moulted to the fifth stadium, but exhibited greatly reduced final weights and died as larvae after parasitoid emergence (Table 1). Teratocyte persistence
Examination of P. in&dens haemolymph after injection of CFSE labelled teratocytes indicated the number of teratocytes in circulation declined with 1c
2C
4C
6C
16C
32C
64C
126C
v
v
v
v
v
v
v
v
20 A
16
509
256C v
16 14 12
20. 16.
B
16. 14. 12.
20. 16.
c
16. 14. 12. 10,
ij, 0
,
,
,
.
,
1
2
3
4
5
DNA
.J&.. 6
7
6
9
10
content (log2 of total fluorescence)
Fig. 5. Frequency distribution of DNA ploidy classes for day 2 (A), day 3 (B) and day 4 (C) teratocytes (n = 50 each).
510
M. R.
and E. A.
STRAND
Table 2. Persistence of teratocytes in P.
WONG
includens in the presence and absence of calyx fluid plus venom
Calyx fluid plus venom and teratocytes*
Teratocytes only Bleeding time (h) 2 6 18 36 48
Cells/p 1 haemolymph (*SD) 50.3 * 39.5 f 22.4 + 9.4 k 4.3 f
Cells/p 1 haemolymph (&SD)
Encapsulated, nodulated or phagocytized (%)
5.9 12.7 16.4 9.6 4.4
9.0 19.8 56.5 34.2 5.0
61.5 * 54.5 + 74.8 + 97.2 k 75.0 +
Encapsulated, nodulated or phagocytimd (%)
16.8 14.6t 11.7t 24.2t 15.6t
0.5 0 0 0 0
*Larvae were injected with a 0.02 wasp equivalent of calyx fluid plus venom 18 h prior to injection with teratocytes (see Materials and Methods). Six-hundred teratocytes (1 egg equivalent) were injected per host. Haemolymph from 10 larvae was examined at the periods designated. ‘/Significantly different (P Q 0.05) from hosts injected with teratocytes only (Mann-Whitney U-Test).
1C
2c
4c
6C
16C
32C
64C
120C
256C
~VVV~~V~V
20 16
I 1 6.
A
14. 12. 1 0. a. 6. 4. 2.
20. 16.
B
1 6. 14. 1 2. I 0. 6. 6. 4. 2.
20. 16.
c
16. 14. 12.
‘;I*, . ,.,.A, 0
1
2
3
DNA content
4
5
6
7
6
9
10
(log2 of total fluorescence)
Fig. 6. Frequency distribution of DNA ploidy classes for day 5 (A), day 6 (B) and day 7 (C) teratocytes (n = 50 each).
Fig. 7. Epifluorescent micrographs of CIFC labelled teratocytes injected into P. inch&m larvae. Cells were obtained by adding haemolymph to Pringle’s saline on a glass slide and photographing after 30min. Unlabelled haemocytes were visualized by photographing under fluorescent and low level phase contrast lighting. All figures printed to the same scale. (A) Encapsulated teratocyte (CA) from a larva injected 18 h earlier with teratocytes only. The teratocyte in the upper left is within a phagocytic plasmatocyte (PH). (B) Teratocyte within a small haemocytic nodule (N) from a larva injected 6 h earlier with teratocytes only. (C) Unencapsulated teratocytes from a larva injected with 0.02 equivalents of calyx fluid plus venom. Larva was bled 18 h after injection of teratocytes.
511
Fig. 8. Phase contrast micrographs of P. in&dens haemocytes in vitro. Cells were obtained by adding haemolymph to saline in a culture dish and photographing after 1 h. Both figures are printed to the same scale. (A) Fully spread plasmatocytes (PL) and granulocytes (GR) from larvae injected with teratocytes only. (B) Spindle and veriform shaped plasmatocytes and unspread granulocytes from larvae injected with 0.02 equivalents of calyx fluid plus venom and teratocytes. In both figures larvae were bled 18 h after injection of teratocytes.
512
M. demolitor teratocytes
513
Table 3. Persistence of teratocytes in P. includens in the presence of purified virus plus venom and trioxsalen treated virus plus venom Virus plus venom and teratocytes’ Bleeding time (h) 6 18
Cells/l, 1 haemolymph (&SD)
Trioxsalen treated virus plus venom and teratocytes*
Encapsulated, nodulated or phagocytixed (%)
Cells//l 1 haemolymph (*SD)
Encapsulated, nodulated or phagocytixed (%)
0 .O
38.0 f 10.4t 14.4 + 19.7t
46.8 56.5
62.3 f 10.4 74.8 f 15.4
*Larvae were injected with a 0.10 wasp equivalent of purified virus or trioxsalen treated virus plus 0.02 equivalents of venom 18 h prior to injection with teratocytes (see Materials and Methods). Six-hundred teratocytes (1 egg equivalent) were injected per host. Haemolymph from 10 larvae was examined at the periods designated. tSianificantlv different (P < 0.05) from hosts injected with purified virus plus venom (Mann-kitney U-Test). ’
fluid and venom had a pronounced effect on plasmatocyte spreading behaviour (Fig. 8). Most of the plasmatocytes were spindle shaped or appeared as veriform cells, and almost none attached or spread on glass slides. Most granulocytes also remained unspread and spherical in shape. A few hosts were held for 10 days before bleeding, and they too still contained teratocytes. Although the number of cells per host had declined greatly by this time (14.2 f 7.4/~1 haemolymph, n = 8 hosts), the cells were larger (180.4 + 65.8 pm, n = 140 cells) than the maximum size observed in parasitized hosts (Fig. 3). Larvae injected with 0.10 equivalents of M. demolitor polydnavirus plus venom and teratocytes were examined at only 6 and 18 h post-injection, but the results were similar to the effects of calyx fluid plus venom (Table 3). However, teratocytes were encapsulated and plasmatocytes spread normally when larvae were injected with virus that had been inactivated with trioxsalen and U.V. light (Table 3).
with 600 teratocytes exhibited developmental delays, reduced weights and died as larvae. The number of stadia larvae underwent before dying varied. The majority died during the fifth stadium (67%), but others died after moulting to a supernumerary sixth (25%) or seventh (8%) stadium. Most of the remaining individuals pupated without developmental delay. Injecting 2400 teratocytes per host had a similar effect on larvae (Table 4). Few larvae injected with only calyx fluid plus venom died during the larval stage, but 87% died as intermediates (Table 4). Larvae moulted to a fifth (18%), sixth (47%), seventh (36%) or eighth (3%) stadium before initiating metamorphosis, and thus exhibited delays in development time and weight gain relative to control larvae injected with only medium. However, the larvae also attained final weights that were consistently higher than larvae injected with calyx fluid plus venom and teratocytes. All larvae injected with Ex-cell400 medium pupated normally (Table 4). Out of 15 larvae injected with 0.10 equivalent of polydnavirus plus venom and 600 teratocytes, 80% died as fifth or sixth stadium larvae that attained a final weight of 88.2 + 24.3 mg.
Eflects of calyx fluid or virus plus venom and teratocytes on host development
DISCUSSION
Hosts exhibited pronounced alterations when injected with calyx fluid plus venom and teratocytes (Table 4). Eighty-seven per cent of larvae injected
The number and size of M. akmolitor teratocytes are similar to reports for other microgastrine braconids (Salt, 1968; Vinson and Iwantsch, 1980). For example,
venom declined only slightly (Table 2). The teratocytes persisted free in the haemolymph, and almost no cells were encapsulated (Fig. 7). Injection of calyx
Table 4. Effect of teratocytes on the development of P. includens larvae that were injected with calyx fluid plus venom* Terminal stage Larvae
Intermediates Development time (daysfSD)t
62.4 + 34.6 71.6k29.4
11.2* 1.4 12.7 k2.3
56.0 f 27.9 -
6.8 f 2.2 -
Number of teratocytes injected
n
%
Final weight (mgf SD)
600 2400
30 38
87 87
Calyx fluid plus venom Medium
30 30
13 0
Pupae
%
Final weight (mgf SD)
Development time (days+SD)t
0 3
258.2
87 227.2 f 49.5 0 -
%
Final weight (mgk SD)
Development time (days *SD)t
7.0
13 10
273.8 rf: 7.2 261.3 k 9.6
6.0 & 0.8 6.5 + 0.6
12.3 f 1.4 -
0 100
268.3 f 16.4
7.0 * 1.1
*Larvae were injected with a 0.02 wasp equivalent of calyx fluid plus venom 18 h prior to injection with teratocytes (see Materials and Methods). TDevelopment time is measured from the time of teratocyte injection.
514
M. R. STRAND and E. A. WONG
Cardiochiles nigriceps produces approx. 200 teratocytes per egg that attain a diameter of more than 3OOpm (Vinson, 1970) while M. croceipes produces 750 teratocytes that attain a maximum diameter of 140pm (Vinson and Lewis, 1973). Although ploidy levels have not been reported previously for teratocytes, the large increase in size and nuclear diameter would suggest these cells become highly polyploid (Strand et al., 1985). Indeed, our data indicate the ploidy levels for male teratocytes were between 2 and 8C at hatching, and progressively shifted upward during development. Some cells attained ploidy levels of 128-256C, but in the latter stages of development (day 6-7) both ploidy levels and cell size were quite variable. We are uncertain why some teratocytes increased in size during this period while others did not, but similar size variation has been reported in the literature (Kitano, 1965; Vinson, 1970; Vinson and Lewis, 1971). Some studies suggest teratocytes are secretory early in development (Strand et al., 1986; Tanaka and Wago, 1990), but assume a storage function as they age (Sluss, 1968; Sluss and Leutenegger, 1968; Cohen and Debolt, 1984). Interestingly, the teratocytes of C. nigriceps diverge into two morphologically distinct forms in the latter stages of development (Vinson and Scott, 1974). Type I cells contain extensive profiles of rough endoplasmic reticulum and appear to maintain a secretory function while type II cells contain an extensively vaculated endoplasmic reticulum and numerous inclusions that are suggestive of a storage function. Thus, the variation in M. demolitor teratocytes may reflect the possibility that the teratocytes of some braconids diverge into subpopulations with age. Although M. demolitor larvae consumed some teratocytes, our data do not support the suggestion these cells have primarily a trophic function (Sluss, 1968). This is based on the observation that teratocyte consumption was variable, and a large number of cells remained in the host throughout development. However, our data do indicate M. demoiitor teratocytes affect the development of P. in&dens, and that teratocyte survival is enhanced by the presence of calyx fluid or M. demolitor polydnavirus. Recently completed dose-response studies indicated calyx fluid and the virus inhibit filipodial elongation of P. includens plasmatocytes and suppress encapsulation of M. demolitor eggs and other foreign targets such as glass rods (Strand and Noda, unpublished). Venom alone has no effect on plasmatocyte spreading or encapsulation, but appears to synergize the effects of M. demolitor polydnavirus. A synergistic function is suggested on the basis that calyx fluid or M. demolitor polydnavirus plus venom inhibits cell spreading at lower dosages and for a longer period than calyx fluid or this virus alone. In the current study, most teratocytes were encapsulated within 24 h of injection in the absence of M. demolitor polydnavirus plus venom, but almost no teratocytes were encapsulated in its presence.
Injection of trioxsalen treated virus plus venom did not protect teratocytes from encapsulation suggesting transcriptionally active virus is necessary to prevent encapsulation. Collectively, our results suggest that in the absence of M. demolitor polydnavirus, most hosts were unaffected by a physiological dose of teratocytes (600 cells per host) because the cells were rapidly eliminated by the immune system. However, injecting superphysiological doses of cells overwhelmed the immune capability of an increasing proportion of hosts, and in some individuals enough teratocytes survived to disrupt development. In contrast, when calyx fluid or the virus plus venom was present, the host immune system was compromised, and a physiological dose of teratocytes was sufficient to induce developmental alterations. Hosts injected with calyx fluid or M. demolitor polydnavirus plus venom and teratocytes exhibited developmental alterations that were strikingly similar to the developmental alterations observed during natural parasitism (Strand et al., 1988). However, we are uncertain whether these effects reflect the activity of only teratocytes or are due to the cumulative effects of teratocytes, virus and venom. In addition to affecting the immune system, control experiments of the current study (Table 4) and rigorous doseresponse experiments conducted previously (Strand and Dover, 1991) indicated calyx fluid and purified virus alone cause P. includens to develop into larvalpupal intermediates. Virus expression is detectable in P. in&dens within 4 h of oviposition and persists for at least 8 days (Strand et al., unpublished), suggesting the activity of both teratocytes and virus may contribute to the developmental alterations reported here. Alterations in host development due to teratocytes have been reported for other micrograstrine braconids. Vinson (1970) reported C. nigriceps teratocytes caused Heliothis virescens larvae to form abnormal pupae. Zhang and Dahhnan (1989) reported the teratocytes of M. croceipes had a dose-dependent effect on H. virescens fifth stadium larvae. The majority of larvae injected with a physiological dose of M. croceipes teratocytes died as larvae while hosts injected with fewer teratocytes died as intermediates or pupated. Neither study reported the encapsulation of injected teratocytes, but unlike our in vitro produced teratocytes, the cells were collected from parasitized larvae. Since polydnaviruses enter a variety of host tissues (Stoltz and Vinson, 1979) it is possible other components besides teratocytes were injected into hosts and contributed to the developmental alterations reported. Our results and those for other species of microgastrine braconids suggest teratocytes and/or polydnaviruses have a juvenilizing effect on host development. However, the specific factors responsible for these effects remain largely undefined. Juvenile hormone activity was reported in the teratocytes of C. nigriceps (Joiner et al., 1973) and more
M. demolitorteratocytes
recently juvenile hormone esterase activity was shown to be reduced in hosts parasitized by M. croceipes and Aputtteles kuriyai (Dahlman et al., 1990; Hayakawa, 1990). In the case of A. kariyui, a 4500 Da peptide was purified from host haemolymph that repressed juvenile hormone esterase activity, but the source of the peptide was not identified. Undoubtedly, future studies will help clarify the role of specific wasp components in parasitism. Acknowledgements-The authors wish to thank T. Noda, V. Grass1 and K. Kervista for their assistance during various phases of the study. The authors also thank J. Aiken for sharing equipment in his laboratory and B. Dover for comments on the manuscript. This study was funded in part by USDA Grant No. 90-37250-5481, Wisconsin Hatch Project 3200 and a Hilldale Research Award from the University of Wisconsin-Madison Graduate School.
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515
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