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Pergamon Int. J. Insect Morphol. & Embryol. Vol. 23, No. 3, pp. 173-187, 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 002~7322/94 $7.00 +0.00 ULTRASTR UCTURE OF MICROPLITIS CROCEIPES (CRESSON) (BRACONIDAE • HYMENOPTERA) TERATOCYTES DEQINGZHANG,* D. L. DAHLMAN,* U. E. J,'~RLFORS,t H. H. SOUTHGATE*and SCOTT P. WILEY* *Department of Entomology; tDepartment of Plant Pathology, University of Kentucky, Lexington, KY 40546, U.S.A. (Accepted 21 September 1993) Abstract--The developing embryo of the braconid, Microplitis croceipes (Braconidae : Hymenowera ), is encased in an extraembryonic serosal membrane. Hatching of the parasitoid within the larva of its habitual host, Heliothis virescens (Noctuidae : Lepidoptera), is initiated about 40 hr after oviposition when held at 25 + 2°C. At this time, the monolayered serosal membrane begins to dissociate into individual cells (teratocytes). After dissociation, teratocytes become dispersed in the hemotymph of the host. The average number of teratocytes released from each parasitoid embryo is 914 _+ 43. Teratocytes average 14.1 + 2.4/xm in diameter when first released, and reach a maximum average diameter of 68.1 + 4.6/xm 6 days after liberation. Newly released teratocyte:~ have ovoid nuclei, simple mitochondria and a limited number of profiles of the endoplasmic reticulum, all of which indicate relative metabolic inactivity. The ramified nuclei, extensive endoplasmic reticulum, polymorphic mitochondria and accu- mulation of glycogen granules and lipid droplets observed in older teratocytes provide circumstantial evidence that protein synthesis is occurring. Within hours after dissocia- tion, microvilli begin to cover the surface of the teratocytes. Anatomical deformation (blebs) that occurred on some older (8-day-old) teratocytes probably resulted from enlargement or expansion of microvilli. Index descriptors (in addition to those in title): Parasitoid, tobacco budworm, Hefiothis virescens, Braconidae, Noctuidae. INTRODUCTION When eggs of certain hymenopteran parasitoids hatch, the extraembryonic membrane (serosal membrane) that envelops the developing parasitoid embryo dissociates into individual or small clusters of cells which, shortly after release, acquire independent existence and usually become distributed throughout the host body cavity via hemolymph flow (Tawfik, 1961; Salt, 1968). These cells are commonly found in the hemolymph of hosts attacked by Braconidae but have also been found in hosts attacked by Platygasteridae (Hill and Emery, 1937) and Scelionidae (Gerling and Orion, 1973; Strand et al., 1985; Volkoff and Colazza, 1992). Teratocytes have been described from at least 30 parasitic species in these 3 families that attack either the egg, larval or adult stages of more than 30 known host species in 5 different insect orders. These cells have been described with several different terms including "trophoserosa cells" (Jackson, 1928), "giant cells" (Jackson, 1935; Huff, 1940; Kitano, 1965; Gerling and Orion, 1973), "trophic cells" (Sluss, 1968; Sluss and Leutenegger, 1968), "trophamnion cells" 173

Ultrastructure of Microplitis croceipes (Cresson) (Braconidae : Hymenoptera) teratocytes

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Pergamon Int. J. Insect Morphol. & Embryol. Vol. 23, No. 3, pp. 173-187, 1994

Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved

002~7322/94 $7.00 +0.00

U L T R A S T R U C T U R E O F MICROPLITIS CROCEIPES ( C R E S S O N ) ( B R A C O N I D A E • H Y M E N O P T E R A ) T E R A T O C Y T E S

DEQING ZHANG,* D. L. DAHLMAN,* U. E. J,'~RLFORS,t H. H. SOUTHGATE* and SCOTT P. WILEY*

*Department of Entomology; tDepartment of Plant Pathology, University of Kentucky, Lexington, KY 40546, U.S.A.

(Accepted 21 September 1993)

Abstract--The developing embryo of the braconid, Microplitis croceipes (Braconidae : Hymenowera ), is encased in an extraembryonic serosal membrane. Hatching of the parasitoid within the larva of its habitual host, Heliothis virescens (Noctuidae : Lepidoptera), is initiated about 40 hr after oviposition when held at 25 + 2°C. At this time, the monolayered serosal membrane begins to dissociate into individual cells (teratocytes). After dissociation, teratocytes become dispersed in the hemotymph of the host. The average number of teratocytes released from each parasitoid embryo is 914 _+ 43. Teratocytes average 14.1 + 2.4/xm in diameter when first released, and reach a maximum average diameter of 68.1 + 4.6/xm 6 days after liberation. Newly released teratocyte:~ have ovoid nuclei, simple mitochondria and a limited number of profiles of the endoplasmic reticulum, all of which indicate relative metabolic inactivity. The ramified nuclei, extensive endoplasmic reticulum, polymorphic mitochondria and accu- mulation of glycogen granules and lipid droplets observed in older teratocytes provide circumstantial evidence that protein synthesis is occurring. Within hours after dissocia- tion, microvilli begin to cover the surface of the teratocytes. Anatomical deformation (blebs) that occurred on some older (8-day-old) teratocytes probably resulted from enlargement or expansion of microvilli.

Index descriptors (in addition to those in title): Parasitoid, tobacco budworm, Hefiothis virescens, Braconidae, Noctuidae.

I N T R O D U C T I O N W h e n eggs of cer ta in h y m e n o p t e r a n pa ras i to ids ha tch , the e x t r a e m b r y o n i c m e m b r a n e (serosal m e m b r a n e ) tha t enve lops the deve lop ing pa ra s i t o id e m b r y o d issoc ia tes in to ind iv idua l o r smal l c lus ters of cells which, shor t ly a f te r re lease , acqu i re i n d e p e n d e n t ex i s tence and usually b e c o m e d i s t r ibu ted t h r o u g h o u t the hos t b o d y cavi ty via h e m o l y m p h flow (Tawfik, 1961; Sal t , 1968). T h e s e cells a re c o m m o n l y f o u n d in the h e m o l y m p h of hosts a t t a c k e d by B r a c o n i d a e but have also been found in hosts a t t a c k e d by P l a tygas t e r i dae (Hil l and E m e r y , 1937) and Sce l ion idae (Ger l i ng and O r i o n , 1973; S t r and et al., 1985; Vo lko f f and Colazza , 1992). T e r a t o c y t e s have b e e n d e s c r i b e d f rom at least 30 paras i t i c species in these 3 famil ies that a t t ack e i the r the egg, larval o r adu l t s tages of m o r e than 30 k n o w n host species in 5 d i f fe ren t insect o rders . T h e s e cells have been desc r ibed with severa l d i f fe ren t t e rms inc luding " t r o p h o s e r o s a ce l l s" ( Jackson , 1928), "g i an t ce l ls" ( Jackson , 1935; Huff , 1940; K i t a no , 1965; G e r l i n g and O r i o n , 1973), " t r o p h i c cel ls" (Sluss, 1968; Sluss and L e u t e n e g g e r , 1968), " t r o p h a m n i o n ce l l s"

173

174 D. ZHANG et al.

(Tremblay, 1966) and "teratocytes" (Vinson, 1970; Vinson and Lewis, 1973; Strand et al., 1985). It is the latter term that has become accepted and is frequently'used in the current literature.

Teratocytes do not divide after being liberated into the host hemolymph but both the ploidy number and size increase (Strand and Wong, 1991). The reported number of teratocytes per parasitoid larva appears to be species-specific (Vinson and Iwantsch, 1980) and ranges from 8 (Tremblay and Calvert, 1972) to more than 900 (this paper).

While the general role of teratocytes across groups is to alter host development so that the parasitoid's survival and reproduction are assured, it also seems that the major observed roles of teratocytes in different parasitoid-host systems may be different. Mechanistically, the primary roles of teratocytes have been suggested to be trophic, inhibitory, secretory and immunosuppressive (Vinson and Iwantsch, 1980; Dahlman, 1990, 1991; Dahlman and Vinson, 1993).

Recent evidence, particularly from studies with Microplitis croceipes and Heliothis virescens (Zhang and Dahlman, 1989; Zhang et al., 1992), shows that teratocytes produce some unknown substances that alter host development by affecting the host endocrine system. This study was conducted to characterize the development and fine structure of M. croceipes teratocytes within the host hemolymph for the purpose of correlating their cellular integrity with the potential roles played by these cells in the parasitoid-host relationship.

M A T E R I A L S A N D M E T H O D S

Insect culture Procedures for maintaining host larvae, H. virescens, and parasitoids, M. croceipes, under laboratory

conditions have been described (Zhang and Dahlman, 1989). Parasitism for in vivo teratocyte collection was achieved by exposing mated parasitoid females to pharate 4th-instar host larvae at a ratio of 1:8 for approximately 1 hr, while parasitism for parasitoid embryo collection was made by superparasitizing pharate 5th instar host larvae with female parasitoid wasps at a ratio of 1:2 (wasp/host). Parasitized larvae were individually maintained on artificial diet under 16 hr light/8 hr dark photoperiod conditions at 25 +_ 2°C. The parasitoid hatches about 40 hr after oviposition and teratocytes are released (Zhang and Dahlman, 1989). The age of parasitoid embryos was measured from the time of oviposition, while the age of teratocytes was measured from the time the embryo hatched.

Number, growth and viability o f teratocytes The following semi-in vitro cultural procedure was used to obtain the number of teratocytes released from

each parasitoid embryo. About 30 hr after superparasitization, hosts were dissected and parasitoid embryos collected, washed and individually maintained in a 100 ~1 drop of ex-cell 400 medium (JRH Biosciences, Lenexa, KS 66215) held in a covered 50 × 9 mm Petri dish. A few hours before the embryos hatched, dishes were placed on a rocker platform shaker and moved through a 50 ° arc at 25 rpm to facilitate the separation of teratocytes from the embryo. Actual counts of teratocytes from 15 embryos were made under a Wild Heerbrugg dissecting microscope at 25 ×.

Beginning from the 2nd day after parasitization of pharate 4th-instar hosts, hemolymph was drawn from each of 5 parasitized larvae randomly selected for each day. Each larva was sampled only once. Diameters of teratocytes were determined with a Laborlux 12 compound microscope at 400 × with an ocular scale calibrated to a stage micrometer. Measurements on 10 teratocytes, randomly selected from each of 5 larvae, were made at 24 hr intervals through the 9-day development period of the parasitoid. The means of these data were regressed on a cumulative Gaussian distribution to obtain a growth curve.

Viability of 1- through 9-day-old teratocytes derived from hosts parasitized as pharate 4th instars was tested by staining with carboxyfluorescein diacetate succinimidyl ester (CFSE) or propidium iodide (PI) (Molecular Probes Inc., Junction City, OR 97402), as described by Strand and Wong (1991). Staining is based on the principle that living cells readily take up CFSE and exhibit a green color under epifluorescent light, while PI binds to the DNA only in dead cells and exhibits an orange-red color under the same conditions.

Ultrastructure of Microplitis croceipes 175

Simultaneous use of both dyes permitted the determination of the number of living cells in a single preparation.

Preparation of M. croceipes embryos and teratocytes for light and transmission electron microscopy (TEM) studies

Groups of approximately ten 36- and 40-hr-old parasitoid embryos were submerged and washed in 0.13 M sodium cacodylate buffered primary fixative (pH7.4) containing 2% gluteraldehyde, 2% acrolein, 2% formaldehyde and 1.5% dimethyl sulfoxide in 1 mM CaCI2. One-, 3-, 6-, and 8-day-old in vivo teratocytes were placed into 1:he primary fixative held in a dichlorodimethylsilane-treated glass Petri dish. The dichlorodimethylsilane greatly reduced adherence of cells to the glass surface. Immediately after collection, teratocytes were washed in the primary fixative 5-8 times and then fixed for 1 hr at 4°C. After primary fixation, teratocytes were washed in 0.26 M sodium cacodylate, then further fixed in 1% OsO4 buffered with 0.13 M sodium cacodylate with 2% sucrose for 30 min. Fixed teratocytes were dehydrated with an ethanol series, embedded in Spurr's resin, and cured for 24 hr at 65°C.

Thick sections (c 1/zm) were stained with 1% toluidine blue in 1% sodium borate using a procedure similar to that described by Burns and Bretschneider (1981) and viewed and photographed with a Zeiss Axioskop light microscope with photographic attachments. Thin sections (c. 90 nm) were stained with uranyl acetate and lead citrate and examined under a Philips 400 transmission electron microscope. Sections from at least 30 different larvae were examined in this part of the study.

Preparation of M. croceipes embryos and teratocytes for scanning electron microscopy (SEM) studies Microplitis croceipes embryos and teratocytes for SEM observations were placed into Pringle's saline (pH,

7.4) (Pringle, 1938), washed 8-10 times with fresh saline, then washed 3 or 4 times, during 1 hr, in 0.1% gluteraldehyde in 0 1 M cacodylate buffer (pH, 7.0). The samples were then prepared using a procedure described by Nation (1983), which we modified in the following manner for continuous flow. The samples, suspended in the 0.1% gluteraldehyde, were placed into a 5 ml glass syringe attached to a stainless filter holder containing a 13 mm diameter, 0.4nm polycarbonate membrane filter. The filter holder inlet was attached to the barrel of the glass syringe and the outlet was connected via flexible tubing to a controlled-vacuum aspirator. Samples were dehydrated in 5 ml aliquots of an ethanol series at a flow rate of 1 ml/min. Following the dehydration, the samples were treated twice with 10ml of hexamethyldisilazane (Sigma Chemical Co., St. Louis, MO 63178). The filter was air-dried at room temperature, mounted on a specimen holder, coated with gold-palladium, and examined in a Hitachi S-800 Field Emission scanning electron microscope.

R E S U L T S

Number, growtk and viability of teratocytes By 36 hr, the serosal m e m b r a n e is well defined and the cells, which will become

tera tocytes , are clearly ou t l ined by the egg chor ion in S EM prepara t ions (Fig. 1A). The m e m b r a n e is orle cell- layer thick in all locat ions (Fig. 2A). A t egg hatch the serosal m e m b r a n e , which is still one cell- layer thick, rup tures (Figs 1B, 2B) with the aid of s t rong repea ted a b d o m i n a l cont rac t ions of the parasi toid larva. Some of the tera tocytes r ema in a t tached to the young paras i to id larva for several hours before release ei ther indiv idual ly or in clusters into the host hemocoel .

The m e a n n u m b e r of tera tocytes / larva (n = 15) de t e rmined from the in vitro cul tures of ind iv idua l embryos was 914 + 43 ( range 858-974). The growth pa t t e rn of in vivo tera tocytes in host h e m o l y m p h is shown in Fig. 3. Newly re leased cells have an average d i ame te r of 14.1 _+ 2 .4 /xm and are relat ively un i fo rm in size. Ini t ial ly, they appear spherical and pale g reen -ye l low in color. Over t ime, they increase in size unt i l they reach abou t 68/,Lm, after which, their size did not change. We regressed these data on a cumula t ive Gauss i an d is t r ibut ion and the regression line is shown in Fig. 3 (r e = 0.996). The fitted regression is Y = 9.2 + 29.4* {1 + erC[O.43*(X- 2.4)] }, where Y is the te ra tocyte d iamete r , X is t ime in days, and erf the s t andard Gauss i an er ror funct ion . Accord ing to this equa t ion , teratocyte growth rate increases for the first 2.4

176 D. ZHANG et al.

FIG. 1. (A) Scanning electron micrograph (SEM) of a 36 hr-old M. croceipes embryo. The egg chorion (C) follows the contours of the serosal membrane underneath it, outlining the individual cells. (B) SEM of a 40-hr-old hatching M. croceipes embryo. The serosal membrane dissociated into individual teratocytes (T). The egg chorion (C) broke up with the aid of strong, repeated

abdominal contractions of the hatching parasitoid.

Ultrastructure of Microplitis croceipes 177

C

2A

FIG. 2. (A) Longitudinal section through the anterior portion of a 36-hr-old M. croceipes embryo showing early stage of teratocytes (T) forming one single-cell layer serosal membrane; C = chorion. (B) After dissociation, teratoeytes (T) appeared to remain attached to the young

parasitoid larva (L) for several hours before release into host hemocoel; C = chorion.

178 D. ZHANG et al.

80

6O

E

40 Ip

E o

i:5

20

___e Measured / " - Predicted

Age (days)

FIG. 3. Actual and predicted growth curve of teratocytes collected from parasitized larvae at 24 hr intervals through the 9 day developmental period. Each point represents the average of 50 measurements . The vertical bars represent standard errors of means; where not shown, they are within the area of the symbol. The predicted curve was obtained by regressing the data on a cumulative Gaussian distribution with r 2 = 0.996. The fitted regression is Y = 9.2 + 29.4"(1 + erf[O.43*(X -2 .4) ] ) where Y is the teratocyte diameter , X is t ime in days and

err is the s tandard Gaussian error function.

days after which it begins to decrease. At that time, the teratocytes are about 40/zm in diameter and are growing at the rate of approximately 14~m/day (the maximum growth rate occurs at 2.4 days). As the growth rate decreases, the teratocyte diameter asymptotically approaches its mean maximum size of 68/xm and the regression estimate of the maximum size is 68.1 _+ 4.6/zm.

Regardless of age, almost all cells fluoresced bright green when stained with CFSE, indicating that they were viable. However, teratocytes which are 8 days old stained less intensely and a few cells (less than 3%) turned orange-red when stained with PI. Presumably, these cells were damaged during manipulation.

Microscopic observations The surface of newly released teratocytes (within 5 hr after liberation) appears

wrinkled (Fig. 4A). These cells are probably in the process of reconfiguring from the more angular shape required in the serosal membrane to the spherical shape acquired as free floating individual cells. Short and sparsely distributed microvilli are visible.

The microvilli covering 1-day-old teratocytes (Fig. 4B) are longer and appear to be more numerous than those on newly released teratocytes. The nucleus is large and ovoid, and the mitochondria appear round to oval with few cristae. Long, narrow cisternae of the endoplasmic reticulum (ER) as well as many unattached ribosomes are scattered in the cytoplasm (Fig. 5A).

Compared with 1-day-old teratocytes, the mean diameter of 3-day-old teratocytes is 2.4 times greater but the surface area is 5.8 times greater. However, the density of microvilli is approximately the same, so it is obvious that additional microvilli have formed. The nucleus has begun to ramify and increase in size. There appear to be more

Ultrastructure of Microplitis croceipes 179

FIG. 4. (A) SEM of a newly hatched teratocyte showing a wrinkled surface sparsely covered with short microvil]i. (B) SEM of a 1-day-old spherical teratocyte. Short and relatively sparse microvilli

are visible.

180 D. ZHAN~ et al.

FIG. 5. (A) Transmission electron micrograph (TEM) of a 1-day-old teratocyte showing a portion of its large nucleus (N), mitochondrial profiles (m) with sparse cristae, long ER cisternae and abundant unattached ribosomes scattered in the cytoplasm; mv = microvilli. (B) TEM of a 3-day-old teratocyte. The nucleus is ramified and abundant mitochondria (m) and glycogen (g) are present throughout the cytoplasm. (C) Three-day-old teratocyte at higher magnification. A lipid

droplet (Ld) is present; m = mitochondrion.

Ultrastructure of Microplitis croceipes 181

mitochondria scattered throughout the cytoplasm and some stored glycogen is evident (Fig. 5B). Free ribosomes are abundant and some lipid droplets are observed (Fig. 5C).

The microvilli have developed to a maximum coverage of the cell surface in 6-day-old teratocytes. The nucleus has increased in size and further ramified. Mitochondria are more abundant than in younger teratocytes (Fig. 6A). The ER profiles are distended and filled with flocculent material (Fig. 6B,C). Large clear areas with abundant glycogen rosettes are often observed (Fig. 6A,C).

Some 8-day-old teratocytes have blebs on their surface (Fig. 7A,B). It appears that the blebs develop by enlargement and coalescence of the microvilli and may become as large as the cell itself (Fig. 7B). TEM shows that the nuclei become further ramified throughout the interior of the cell. The ER cisternae are long and narrow and are often observed in parallel stacks (Fig. 8A). Mitochondrial profiles, glycogen granules and lipid droplets are abundant (Fig. 8B). Numerous profiles of the ER are present and "myelin" figures are gathered within large clear areas.

DISCUSSION Ogloblin (1925) first described the dissociation of the extraembryonic membrane, the

serosa, of some parasitic hymenopterans into unusually large cells, the teratocytes, when the parasitoid larva hatched. Since then, many studies on teratocyte origins, functions, and morphology have been conducted (reviewed by Dahlman and Vinson, 1993).

In their review, Dahlman and Vinson (1993) suggested that the egg volume of parasitic hymeaopterans increases several hundred-fold with concomitant growth of the serosal membrane and stretching of the egg chorion. Differentiation of the cells forming the serosal membrane probably occurs during embryogenesis. However, the structural arrangement of these cells has not been described in detail, except in the case of Cardiochiles nigriceps reported by Pennacchio et al. (1992). They found, at the time of egg hatch, that anterior and posterior to the embryo, the serosal membrane was several cell layers thick. Our observations of the M. croceipes embryo clearly indicate that the entire serosal membrane is composed of a single-cell layer at the time the egg hatches.

When first dissociated and released into host hemocoel, teratocytes are relatively uniform in size. As they develop, the volume of individual teratocytes usually increases manifold. The increased size and ramification of the nucleus suggests that they become highly polyploid, as was observed in the teratocytes from a related species, Microplitis demolitor (Strand and Wong, 1991). Our data show an approximate 125-fold increase in volume. However, not all teratocytes reached the maximum size. This phenomenon has been observed from both in vivo (Sluss and Leutenegger, 1968; Vinson, 1970; Vinson and Lewis, 1973; Gerling and Orion, 1973; Strand et al., 1985; Volkoff and Colazza, 1992) and in vitro (Strand et al., 1988; Strand and Wong, 1991) systems. Volkoff and Colazza (1992) described 3 different class sizes of teratocytes that seem to co-exist in the., egg parasitoid, Trissolcus basalis. Because the environmental and nutritional differences in either of these in vivo or in vitro systems is relatively uniform, the causes for ~Lhe variation of teratocyte size could result either from the sequence of dissociation of the cells from the serosal membrane at the time of larval hatch, the location of the cell on the serosal membrane and/or a genetically determined fate.

182 D. ZHANG et al.

FiG. 6. (A) TEM of a 6-day-old teratocyte. The nucleus (N) of this teratocyte is ramified and quite large. Mitochondria (m) appear to be abundant. Large areas of glycogen rosettes (g) are observed primarily near the nucleus. (B) TEM of a 6-day-old teratocyte. In some 6-day-old teratocytes, the ER cisternae are distended. Golgi (Go) complexes are present. (C) TEM of a 6-day-old teratocyte.

The cisternae are filled with flocculent material (*). Glycogen (g) rosettes are plentiful.

Ultrastructure of Microplitis croceipes 183

FIG. 7. (A) SEM of an 8-day-old teratocyte with developing blebs (b) on the surface of the cell. (B) TEM of an 8-day-old in vitro teratocyte. Some blebs (b) grew very large.

184 D. ZHANG et al.

F~G. 8. (A) TEM of an 8-day-old teratocyte. The nucleus (N) is extensively ramified. The ER profiles are long and arranged in parallel arches adjacent to the nucleus. (B) TEM of an 8-day-old teratocyte. Lipid droplets (Ld) and glycogen (g) are abundant. Numerous profiles of the ER are

present and "myelin" figures (MF) are gathered within large clear areas; m = mitochondria.

Ultrastructure of Microplitis croceipes 185

Vinson and Lewis (1973) reported a relatively constant 750 teratocytes/M, croceipes larva, based on data obtained from dissections of H. virescens larval hosts. It is reasonable to assume that not all of the teratocytes released from the M. croceipes parasitoid embryo were counted from a dissected host and the observed number of teratocytes could only be approximate. In contrast, we found 914 + 43 teratocytes/ embryo with our in vitro system. However, Strand and Wong (1991) found no significant difference between the number of teratocytes reported from in vivo dissection and in vitro culturing of M. demolitor embryos, and Pennacchio et al. (1992) reported no significant difference between in vivo and in vitro numbers of teratocytes from C. nigriceps.

The ultrastru,cture of newly released teratocytes suggests that they may be relatively inactive. They have ovoid nuclei, simple mitochondria, a limited number of ribosomes and a moderate: number of ER profiles. The microvilli are short and relatively sparse. Our observations of the lack of extensive glycogen, lipid or protein in the newly released and 1-day-old teratocytes are similar to those of Vinson and Scott (1974) and do not support a general view that younger teratocytes serve as a food source for the developing parasitoid.

Teratocytes raarkedly increase in size during the first 4 days after liberation. By the 3rd day, the nuclei become ramified, extensive ER profiles arranged in parallel arches are formed, large polymorphic mitochondria appear and free ribosomes accumulate. The microvilli are well developed by the first day but continue to increase in total number as the cell enlarges. These features suggest increased metabolic activity within the teratocytes and support our physiological observations that 3-day-old teratocytes significantly influence host development (Zhang and Dahlman, 1989).

A prominent difference between the younger and older teratocytes is the accumula- tion of lipid droplets and glycogen in 6- and 8-day-old teratocytes. Perhaps the older cells are shifting to a primarily absorptive role. Teratocytes from this species are not ingested by parasitoid larvae (Vinson and Lewis, 1973) so the function of an accumulation of these compounds would not be to serve as a nutrient source for the parasitoid.

Presumably, ~:he contents of the teratocytes are released into the host hemocoel when the blebs rupture. The anatomical deformation (blebs) that occurs on some 8-day-old teratocytes clearly results from the enlargement or coalescence of microvilli. This may be comparable to the vesicular bodies among the microvilli observed by Vinson and Scott (1974) in C. nigriceps teratocytes. The bleb structure on the surface of some teratocytes alorkg with the deformation of other cells and numerous lipid droplets and myelin figures may indicate decreased viability of these cells. These structural changes correlate with the observation of decreased CFSE uptake by older teratocytes. If the teratocytes perform a beneficial role for the parasitoid, deterioration at this time is logical because the parasitoid larva has already emerged from its host. The teratocyte deterioration we observed is not unique because Volkoff and Colazza (1992) reported an increased proportion of irregularly shaped teratocytes with increasing age of T. basalis parasitoids.

The secretory functions of teratocytes cannot be ignored. Koscielski et al. (1978) suggested that teratocytes secrete or synthesize unique proteins and/or perhaps other substances that may satisfy specific physiological requirements essential for the growth and development of the parasitoid. It is likely that the suppression of phenoloxidase

186 D. ZnANG et al.

ac t i v i t y ( T a n a k a a n d W a g o , 1990) , t h e d e c r e a s e d j u v e n i l e h o r m o n e e s t e r a s e a c t i v i t y ,

e c d y s t e r o i d t i t e r s ( Z h a n g et al . , 1992) a n d o t h e r p h e n o m e n a r e l a t e d to t r e a t m e n t o f

h o s t l a r v a e w i t h t e r a t o c y t e s al l r e s u l t f r o m e f f ec t s o f t e r a t o c y t e s e c r e t o r y p r o d u c t s . I n

f ac t , D a h l m a n (1991) r e p o r t e d t h a t H. v i re scens l a r v a e i n j e c t e d w i t h in v i t ro M .

c r o c e i p e s t e r a t o c y t e s e c r e t o r y p r o d u c t s e x p e r i e n c e d d e l a y e d g r o w t h a n d l a r v a l m o r t a l i t y

w i t h s y m p t o m s s i m i l a r to t h o s e o b s e r v e d in i n s e c t s t r e a t e d w i t h e i t h e r in v i v o o r in v i t ro

t e r a t o c y t e s o r i n s e c t s t h a t w e r e p a r a s i t i z e d . T h u s , it is l i ke ly t h a t t h e p r i m a r y f u n c t i o n

o f t e r a t o c y t e s f r o m M . c r o c e i p e s is to s e c r e t e b i o l o g i c a l l y a c t i v e u n i q u e p r o t e i n

p r o d u c t s t h a t a l t e r p h y s i o l o g i c a l p r o c e s s e s in t h e h o s t .

Acknowledgements--We express our appreciation to T.J. Neary and Erik Siegel for their technical assistance, Becky Wilson, Traci Smith and Shelly Miller for help with insect rearing and Dr Grayson Brown for assistance with statistical analysis. Support of USDA Competitive Research Grant 89-37250, USDA Cooperative Agreement no. 58-6430-2-114, and a grant from R.J. Reynolds are gratefully acknowledged. This is paper 93-07-52 of the Kentucky Agricultural Experiment Station, Lexington, KY.

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