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Ultrastructure of teratocytes of Cardiochiles Nigriceps Viereck (Hymenoptera : Braconidae)
Int. J. Insect Mo,,phol. & Embryol. 3 (2): 293-304. 1974. Pergamon Press. Printed in Great Britain,
U L T R A S T R U C T U R E OF T E R A T O C Y T E S OF CARDIOCHILES NIGRICEPS VIERECK ( H Y M E N O P T E R A : BRACONIDAE)* S. BRADLEIGH VINSONt and J. R. SCOTT~ Texas A&M University, College Station, Texas 77843, U.S.A. (Accepted 14 January 1974)
Abstract--Teratocytes, cells from the embryonic membrane of the eggs of braconid parasitoids, increase in size following their liberation into the hemocoele of their host. Ultrastructural examination of these cells revealed a ramified nucleus, extensive vesicular rough endoplasmic reticulum, microvilli, and vesicles at the cell periphery. The extensive vesicular endoplasmic reticulum suggests a secretory function. The presence of cellular outpockets containing vesicles supports this view. All cells up to 4 days, following their liberation, appear very uniform. Two cell types develop in 5-6 ,:lays. Index descriptors (in addition to those in the title): Parasitoids, giant cells, trophic cells. Heliothis virescens.
INTRODUCTION TERATOCVTL:S (Salt, 1968) have been described from the hosts o f a n u m b e r o f parasitic braconids, ~tlthough their function in the hosts with respect to the d e v e l o p i n g p a r a s i t o i d is u n k n o w n . These cells have also been referred to as giant cells (Jackson, 1935; Huff, 1940), or t r o p h i c cells (Sluss, 1968). J a c k s o n (]935) r e p o r t e d that teratocytes arise from the e m b r y o n i c m e m b r a n e s o f b r a c o n i d parasitoids. W h e n the p a r a s i t o i d hatches, the cells (teratocytes) t h a t m a k e up the e m b r y o n i c m e m b r a n e s are liberated into the hemocoele o f their host, and begin to dissociate. The cells are c o n s u m e d 10 days following their liberation, at which time the entire host is c o n s u m e d b y the p a r a s i t o i d (Vinson, 1970). The fine structure o f these cells d u r i n g their developmen]: was investigated to explore their possible role in the p a r a s i t o i d - h o s t relationship. MATERIALS AND
METHODS
Host larwie, Heliothis virescens, were reared in the l a b o r a t o r y a c c o r d i n g to tile m e t h o d o f V a n d e r z a n t et al. (1962). P a r a s i t o i d s were m a i n t a i n e d in the l a b o r a t o r y a c c o r d i n g to the m e t h o d of Vinson et al. (1973). Third instar larvae were exposed to 3-day-old female * Approved for publication as TA-10810 by the Director, Texas Agricultural Experiment Station and conducted in cooperation with the USDA. Supported in part by NSF Grant GB-24282. i" To whom all correspondence should be addressed. Departments of Entomology and Biology-Electron Microscopy Center, respectively. 293
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parasitoids (C. nigriceps) in a petri dish. Following parasitism, the larvae were replaced on the artificial medium and held at 37C, 17 hr photophase and approximately 70'}~] RH. At various intervals, several parasitized larvae were dissected under Pringle's saline (Pringle, 1938), buffered at pH 7.4 with KOH, to liberate the teratocytes. These were rapidly collected according to the method described by Vinson (1970), and fixed in 2"51'.,i gluteraledhyde in a 0"066M caccodylate buffer at pH 7.4 for 2 hr at 4°C (Sabatini et al., 1964). The cells were washed in 6 changes of the above named buffer for 2 hr. The cells were post fixed in 1-33 of, osmium tetroxide buffered with 0"066M caccodylate at pH 7.4 for 1 hr at 4°C, and dehydrated in a graded ethanol series with 6 changes of 100~, ethanol at room temperature. Infiltration was accomplished in 3 stages; two 10 min changes of propylene oxide (Luft, 1961), 2 hr in a I : 1 mixture of propylene oxide and araldite-epon (Mollenhauer, 1964), and vacuum infiltrated with araldite-epon at 4 5 for I hr. The samples were cured overnight in a 60~C oven. Thick sections were stained with 1 ~!iitoluidine blue in 1 ~',i sodium borate (Pearse, 1960). Thin sections were stained in 2",~, uranyl acetate (Watson, 1958) followed by lead citrate (Reynolds, 1963). OBSERVATIONS The teratocytes of the braconid, Cardiochiles nigriceps Viereck are about 14 /zm in diameter following their liberation into the hemocoele of its host, Heliothis virescens (F.) (Vinson, 1970). They increase in diameter as the parasitoid larva develops, and reach about 350/zm in dia in 10 days. Approximately 200 cells are liberated by C. nigriceps at the time of egg hatch into the hemocoele of the host, and the cells do not increase in number. Teratocytes are approximately 18/zm in diameter 2 days after oviposition (approximately 12 hr following their liberation from the egg). The nucleus (N) is oval and appears to contain both chromatin and euchromatin. Nuclear pores (NP) are evident along the nuclear envelope (Fig. IA). The mitochondrial profiles (M) (Fig. I A) appear round to oval with an indistinctive matrix, poorly developed cristae, and are scarce in these young cells. A few granular inclusion bodies are seen at this stage. The endoplasmic reticulum consists of widely separated rough-surfaced cisternae of variable diameter, which show some continuity with the perinuclear cisterna. There are many unattached ribosomes, polysomes and microtubules. Scattered microvilli (MV) are present (Fig. IA). Sixty hours following the liberation from the egg the teratocytes grow to about 25/zm in diameter, and are uniform in appearance. The nucleus has begun to ramify and increase in size. Free ribosomes remain abundant between the cisternae of the rough endoplasmic reticulum (Fig. IC), which has become extensive and vesicular (VER) (Fig. I B). The intracisternal spaces of the endoplasmic reticulum contain a material of moderate electron
FIG. 1. (A) 12-hr old teratocyte from Heliothis vireseens, parasitized by Cardioehiles nigriceps showing rounded nucleus (N) with many nuclear pores (NP). A few microvilli (MV) are seen along with abundant polysomes between swollen endoplasmic reticulum. (B) 60-hr old teratocyte showing development of extensive vesiculated endoplasmic reticulum (VER). Note ramified nucleus (N) and nuclear pores (NP). (C) Same age as "B" showing ribosomes (R) surrounding vesicles containing a fine granular material. (D) Same age cell as "B'" showing granular bodies (GB) prevalent at this stage.
Ultra~,tructure of Teratocytes of Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae)
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opacity, and of fine granular appearance (Fig. I C). There appear to be more mitochondria with conventional cristae (Hackenbrock et al., 1971) and an increase in granular inclusion bodies ('GB) is observed in the cytoplasm (Fig. 1D). Poorly organized Golgi elements, like those sometimes observed in insect tissue (Smith, 1968), are observed. The microvilli are better developed. After 4 days following liberation, the teratocytes attain a diameter of approximately 50 /zm, and the nucleus becomes extensively ramified (Fig. 2). Two types can now be distinguished both by light and electron microscopy (Fig. 2A-C). In cell type I, the mitochondria remain unchanged. The rough endoplasmic reticulum is dilated, vesicular and abundant. Multivesicular bodies and few granular inclusion bodies are observed. Microvilli are very abundant (Figs. 2C, 3A) and appear to have small microfilaments or fibrils (F) extending a short distance into the cell cortex (Fig. 3B). A few multivesicular bodies (MVB) are seen among the mircovilli (Fig. 2C). No evidence of pinocytosis or pinocytic vesicles was observed. In cell type II (Fig. 3D) the cisternae of the vesicualted rough endoplasmic reticulum have enlarged, rounded and appear as vacuoles or inclusion bodies, although the vesicles remain surrounded by ribosomes ('R) (Fig. 3E, F), and are often connected (Fig. 3C). Some of these vesicles are seen at the cell surface (see arrow Fig. 3C), and many smaller vesicles appear among the microvilli (Fig. 3D). These vesicles are the dominant feature of cell type Ii, and closely packed ribosomes (R) and microtubules (MT) are abundant between the vesicles. A few myelin figures (MF) and lipid bodies (LB) are observed, but there appears to be an absence of multivesiculate, granular or dense bodies. The mitochondria (DM) have well-developed condensed cristae, indicating their active state (Hackenbrock, et al., 1971) (Fig. 4E). The number of microvilli has decreased with respect to the earlier cell or when compared to cell type I (Fig. 2C). In 5 to 6 days the teratocytes are 125-200 /zm in dia. The rough endoplasmic reticulum (LER) is the dominant feature of cell type I, and occurs as parallel profiles of cristae, reminiscent of finger prints particularly near the cell periphery ('Fig. 4B). The inside diameter of the cisternae is about 50 nm, and the cisternae are about 300 nm apart. In some areas of the teratocyte, the rough endoplasmic reticulum appears to be more vesicular. The vesicular endoplasmic reticulum is more extensive in some type I cells than others. Poorly organized Golgi (GA) is seen nearer the extensively ramified nucleus (Fig. 4E), and appears to give rise to vacuolar structures and multivesicular bodies. A few lipid inclusions are seen. Numerous dense, granular, and multivesicular inclusion bodies are observed throughout the cytoplasm in younger cells (Fig. 4A), and tend to be observed near the cell periphery in 7-day and older cells (Fig. 6A). Some of the inclusion bodies are observed at or just beyond the cell boundary. Numerous microvilli are present, and small cellular outpockets containing multivesiculate bodies or vesicles are observed (Fig. 4C). In addition, autophagic vacuoles (A), myelin figures ('MF) and points of cellular destruction (FCD) are seen indicating cellular recycling ('Fig. 4B, D). The nucleus is extensively ramified with much of the chromatin clumped next to the nuclear envelope. The mitochondria have well developed conventional cristae (Fig. 4B, E). FI(;. 2. (A) Thick sections of 4-day old teratocytes showing extensively ramified nuclei (N) and 2 cell types; darker staining cell type I and lighter staining cell type 11. (B) Higher magnification showing vacuolated cell type I!. (C) Electron micrograph showing swollen endoplasmic reticulum of cell type i, and vacuolated endoplasmic reticulum of cell type 11. Note reduced number of microvilli (MV) on cell type II and multivesiculate body (MVB).
Ultrastructure of Teratoctyes of Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae)
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Ultra:;tructure of Teratocytes of Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae)
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Flea. 5.
Ultras~:ructure of Teratocytes of Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae)
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S. BRADLEIGHVINSONand J. R. SCOTT Fic;. 3. (A) 4-day old cell type 1 teratocyte showing dilated vesicular endoplasmic reticulum (VER), indistinct mitochondria (M) and multivesiculate body (MVB). (B) Same cell type as '~A" showing microfilaments extending into cell cortex from microvilli. (C) Same cell type as ~'A" at a later stage showing vesicular endoplasmic reticulum. Note connections between vesicles (V-bar) and presence of vesicles at cell membrane (arrow), (D) 4-day old cell type 11 teratocyte showing extensive vacuolated endoplasmic reticulum, a few large vacuoles (LV), a myelin figure (MF), lipid body (LB), and the dense mitochondria (DM). Note vesicles at and beyond cell surface (V). (E) Same cell type as "~D" showing a mitochondrion with well-developed, condensed cristae. (F) High magnification of same cell type as '~D" showing vesicles surrounded by ribosomes and extensive microtubules (MT). FI(;. 4. (A) 5-day old cell type I teratocyte showing branched microvillii (BMV) and the multivesicular body (MVB). (B) Same type cell as "'A" showing extensive parallel profiles of endoplasmic reticulum (LER), lipid bodies (LB), orthodox mitochondria (M) and points of cellular destruction (FCD). Note small protrusions (P) or buds on microvilli (MV). (C) Same age cell as "'A" showing vesiculated body protruding from the cell. (D) Same age cell as "'A" showing myelin figures (MF), autophagic vacuole (A) and a point of cellular destruction (FCD) observed in local areas of cell. Note granular body (GB). (E) Same age cell as "A'" showing a Golgi area (GA) and a multivesicular body (MVB). Note well-developed microtubule (MT). FiG. 5. (A) An 8-day old cell type II teratocyte showing condensed mitochondria, numerous vesicles containing fibers (VF) or smaller vesicles (VCV) and lipid bodies. Note reduction in ribosomes between vesicles. (B) An 8-day old cell type I teratocyte showing ramified nucleus, mitochondria, vesicular and parallel profiles of endoplasmic reticulum (VER and LER, respectively). Note small blebs on the cell membrane (B). FiG. 6. (A) An 8-day old cell type I teratocyte showing dense bodies (DB) and a vesicle being exuded by cell. (B) A 10-day old cell type I teratocyte. Note extensively ramified nucleus and alternating vesicular and parallel profiles of rough endoplasmic reticulum (VER and LER, respectivelyl.
In cell type I1, the only observable changes in tile 5-6-day old cell are an increase in the size of the vesicles, a change in their content, and a decrease in the polysomes. These same changes are observed in older type lI cells (Fig. 5A). Some vesicles appear to c o n t a i n a fibrous material (VF) or smaller vesicles (VCV). M a n y vesicles are observed at the cell periphery, and some are observed next to the cell m e m b r a n e and a m o n g the microvilli. Autophagic and lipid vacuoles are infrequently observed. The m i t o c h o n d r i a remain condensed (Fig. 5A). Seven to 8 days after liberation, the teratocytes attain a diameter of 225 p,m. A few type II cells are observed with no obvious changes. In the type l cell (Fig. 5B) the nucleus continues to be extensively ramified, with nuclear pyknosis occurring in local areas, indicating cellular aging. In addition, there is an increase in the n u m b e r of autophagic vacuoles observed, further indicating cellular recycling or aging. Some areas of the cell, primarily near the cell periphery, contain parallel profiles of endoplasmic reticulum, while in other areas of the cell the endoplasmic reticulum is more vesicular, reminiscent of cells 30 hr following their liberation. Dense bodies (DB) are found t h r o u g h o u t the cytoplasm in 6- and 7-day old cells, while being more often located near the periphery in older cells (Fig. 6A). N u m e r o u s cytoplasmic whorls, Golgi vacuoles and mictotubules are observed t h r o u g h o u t the cytoplasm. A few lipid vacuoles are observed in older cells but are not extensive. The m i t o c h o n d r i a appear more n u m e r o u s but stratified in the cell a m o n g the more vesicular rough endoplasmic reticulum, a n d have well-developed cristae. As described, cellular outpockets c o n t a i n i n g multivesiculate and other inclusion bodies are observed a m o n g the extensive microvilli.
Ultrastructure of Teratocytes of Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae)
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Nine to ten days following liberation from the parasitoid egg, teratocytes average 300/xm in diameter. Both cell types continue to be observed, and appear little changed over those observed at 8 days. The nucleus remains extensively ramified with the cytoplasm containing extensive amounts of vesicular and parallel profiles of rough endoplasmic reticulum (Fig. 6B). Autophagic vacuoles, dense bodies, and lipid vacuoles are observed along with a few cellular outpockets. Between the 9th and 10th day, the teratocytes are consumed as the developing parasitoid larvae consume the host. DISCUSSION Most investigators of teratocytes have suggested that these cells function in the absorption of nutrient:~ from the host, thus weakening the host and supplying the parasitoid larvae with a ready food supply prior to the parasitoids emergence (Salt, 1968; Sluss and Leutenegger, 1968). Salt (1971) suggested that attrition of the host by the developing teratocytes weakened and rendered ineffective the ability of the host to encapsulate and destroy the parasitoid. However, Vinson (1972) reported that some foreign objects were encapsulated in the presence of teratocytes and that teratocytes were encapsulated when placed in alien hosts. Sluss and Leutenegger (1968) suggested that teratocytes are secretory in the early phase, but become primarily absorptive later in their development and serve as a food source for the developing parasitoid. In the present study, the young teratocytes appear to be relatively inactive as suggested by the simplicity of the mitochondria, absence of many inclusion bodies, Golgi, and the presence of a modreate amount of rough endoplasmic reticulum. Their rapid growth over the next 8 days does indicate the absorption of nutrients from the host. Thirty hr after liberation from the egg, teratocytes show the development of extensive vesicular endoplasmic reticulum. In some cells (Designated cell type II), the swelling of the cisternae continues, and by 60 hr, most of the cisternae have developed into enlarged vacuoles surrounded by ribosomes. These vacuoles are found at the cell border, suggesting that their contents may be liberated from the cell. In other cells (cell type I) the granular material, which is present in the cisternae, disappears and the rough endoplasmi,z reticulum develops extensive, evenly space, parallel profiles of cisternae. Although rough endoplasmic reticulum is present in all cells, its greatest development is most often seen in secretory cells (Fawcett, 1966). The presence of parallel profiles of extensive endoplasmic reticulum, and the presence of vesicular cisternae in teratocytes throughout their short life span suggets they are secretory. Further, the loss of material from the vesicular endoplasmic reticulum by most of the cells by 60 hr after their liberation from the egg supports this view. The reason for the retention of material in the cisternae of the endoplasmic reticulum and the subsequent enlargement of these cisternae in approximately 5-10 ~ of the cells is unknown. It may represent cycles of secretory activity, cells with different functions, or cells in a different stage of development. Vacuoles are seen near the surface of type I cells, and in secretory outpockets in type II cells, suggesting that secretion is occurring in both cell types. No evidence of pinocytosis was observed. Golgi are observed in these cells, and appear to give rise to multivesicular bodies, comparable to those observed in the corpus allatum of Leucophaea (Scharrer, 1971). The migration of the multivescular and dense bodies to the cell periphery, along with the absence of any extensive buildup of inclusion bodies in teratocytes, suggests that these bodies may be released from the cell. K
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The absence of glycogen reported by Vinson (1970) is supported by the observed lack of glycogen granules. The lack of any extensive b u i l d u p of glycogen, lipid or protein in the teratocytes does n o t support a general view that these cells primarily f u n c t i o n as a food source for developing parasitoids, as is suggested by Jackson (1928) a n d Sluss (1968). Teratocytes m a y serve as an i m p o r t a n t food source in some parasitoid-host associations, a n d Sluss a n d Leutenegger (1968) did observe glycogen, lipid a n d protein bodies in teratocytes from adult coccinellid beetles infested with the braconid, P e r i l i t u s coccinellae. Vinson a n d Lewis (1974) reported that teratocytes in hosts parasitized by M i c r o p l i t i s croceipes were n o t c o n s u m e d by the developing parasitoid. The potential i m p o r t a n c e of teratocytes in the regulation of the host physiology, a n d the i m p o r t a n c e of this regulation to the developing parasitoid has been overlooked. Vinson (1970) has shown that injection of teratocytes into hosts results in the failure of the host to pupate normally. The rapid growth, extensive development of the endoplasmic reticulum, a n d general cellular activity observed in these cells, coupled with the general absence of stored cellular products, suggests functions other than nutritional. REFERENCES FAWCET'f, D. W. 1966. The Cell. An atlas o f fine structure. Sanders. Philadelphia, London. HACKENBROCK,C. R., T. G. REHN, E. C. WE~NBACHand J. J. LEMASTERS.1971. Oxidative phosphorylation and ultrastructural transformation in mitochondria in the intact ascites tumor cell. J. Cell Biol. 51: 123-37. HuFf, C. G. 1940. Immunity in Invertebrates. Physiol. Res. 20: 68-88. JACKSON, D. J. 1935. Giant cells in insects parasitized by hymenopterous larvae. Nature (Lond.) 135: 1040-41. LtJFT, J. H. 1961. Improvements in epoxy embedding methods. J. Biophys. Biochem. Cytol. 9: 409. MOLLENHAUER, H. H. 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39: 11-14. PEARSE, A. G. E. 1968. Histochemistry. Theoretical and Applied, Little Brown, Boston. PRINGLE, J. W. S. 1938. Proprioception in insects. J. Exp. Biol. 15: 101-13. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17: 209-12. SABATINI, D. D., F. MtLLER and R. J. BARRNETT. 1964. Aldehydre fixation for morphological and enzyme histochemical studies with the electron microscope. J. Histochem. Cytochem. 12: 57-71. SALT, G. 1968. The resistance of insect parasitoids to the defense reactions of their hosts. Biol. Rev. 43: 200-32. SALT, G. 1971. Teratocytes as a means of resistance to cellular defense reactions. Nature (Lond.) 232: 639. SCHARRER, B. 1971. Histophysiological studies on the corpus allatum of Leucophaea moderae. V Ultrastructure of sites of origin and release of a distinctive cellular product. A. Zellforsch. 120: 1-16. SLUSS, R. 1968. Behavioral and anatomical responses of the convergent lady beetle to parasitism by Perilitus coccinellae (Schronk) (Hymenoptera: Braconidae). J. Invert. Pathol. 10: 2-27. SLUSS, R. R. and R. LEUTENEGGER.1968. The fine structure of the trophic cells of Perilitus coccinellae (Hym.: Braconidae). J. Ultrastruct. Res. 25: 441-51. SMITH, D. S. 1968. Insect cells their structure and function. Oliver & Boyd Edinburgh, VA. VANDERZANT,E. S., C. D. RICHARDSONand S. W. FORT, SR. 1962. Rearing of the bollworm on artificial diet. J. Econ. Entomol. 55: 140. V1NSON,S. B. 1970. Development and possible function of teratocytes in the host-parasite association. J. Invert. Pathol. 16: 93-101. VINSON, S. B. 1972. Factors involved in successful attack on Heliothis virescens by the parasitoid Cardioehiles nigriceps. J. Invert. Pathol. 20: 118-23. VINSON, S. B., F. S. GUILLOTand D. B. HAYS. 1973. Rearing of Cardiochiles nigriceps in the laboratory with Heliothis virescens as hosts. Ann. Entomol. Soc. Amer. 66: 1170-72. VINSON, S. B. and W. J. LEWIS. 1974. Teratocytes: Growth and numbers in the hemocoel of Heliothis virescens attacked by Mieroplitis croceipes (Cresson). J. Invert. Pathol. 22: 351-5. WATSON, M. L. 1958. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. CytoL 4: 475-78.
U L T R A S T R U C T U R E OF T E R A T O C Y T E S OF CARDIOCHILES NIGRICEPS VIERECK ( H Y M E N O P T E R A : BRACONIDAE)* S. BRADLEIGH VINSONt and J. R. SCOTT~ Texas A&M University, College Station, Texas 77843, U.S.A. (Accepted 14 January 1974)
Abstract--Teratocytes, cells from the embryonic membrane of the eggs of braconid parasitoids, increase in size following their liberation into the hemocoele of their host. Ultrastructural examination of these cells revealed a ramified nucleus, extensive vesicular rough endoplasmic reticulum, microvilli, and vesicles at the cell periphery. The extensive vesicular endoplasmic reticulum suggests a secretory function. The presence of cellular outpockets containing vesicles supports this view. All cells up to 4 days, following their liberation, appear very uniform. Two cell types develop in 5-6 ,:lays. Index descriptors (in addition to those in the title): Parasitoids, giant cells, trophic cells. Heliothis virescens.
INTRODUCTION TERATOCVTL:S (Salt, 1968) have been described from the hosts o f a n u m b e r o f parasitic braconids, ~tlthough their function in the hosts with respect to the d e v e l o p i n g p a r a s i t o i d is u n k n o w n . These cells have also been referred to as giant cells (Jackson, 1935; Huff, 1940), or t r o p h i c cells (Sluss, 1968). J a c k s o n (]935) r e p o r t e d that teratocytes arise from the e m b r y o n i c m e m b r a n e s o f b r a c o n i d parasitoids. W h e n the p a r a s i t o i d hatches, the cells (teratocytes) t h a t m a k e up the e m b r y o n i c m e m b r a n e s are liberated into the hemocoele o f their host, and begin to dissociate. The cells are c o n s u m e d 10 days following their liberation, at which time the entire host is c o n s u m e d b y the p a r a s i t o i d (Vinson, 1970). The fine structure o f these cells d u r i n g their developmen]: was investigated to explore their possible role in the p a r a s i t o i d - h o s t relationship. MATERIALS AND
METHODS
Host larwie, Heliothis virescens, were reared in the l a b o r a t o r y a c c o r d i n g to tile m e t h o d o f V a n d e r z a n t et al. (1962). P a r a s i t o i d s were m a i n t a i n e d in the l a b o r a t o r y a c c o r d i n g to the m e t h o d of Vinson et al. (1973). Third instar larvae were exposed to 3-day-old female * Approved for publication as TA-10810 by the Director, Texas Agricultural Experiment Station and conducted in cooperation with the USDA. Supported in part by NSF Grant GB-24282. i" To whom all correspondence should be addressed. Departments of Entomology and Biology-Electron Microscopy Center, respectively. 293
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parasitoids (C. nigriceps) in a petri dish. Following parasitism, the larvae were replaced on the artificial medium and held at 37C, 17 hr photophase and approximately 70'}~] RH. At various intervals, several parasitized larvae were dissected under Pringle's saline (Pringle, 1938), buffered at pH 7.4 with KOH, to liberate the teratocytes. These were rapidly collected according to the method described by Vinson (1970), and fixed in 2"51'.,i gluteraledhyde in a 0"066M caccodylate buffer at pH 7.4 for 2 hr at 4°C (Sabatini et al., 1964). The cells were washed in 6 changes of the above named buffer for 2 hr. The cells were post fixed in 1-33 of, osmium tetroxide buffered with 0"066M caccodylate at pH 7.4 for 1 hr at 4°C, and dehydrated in a graded ethanol series with 6 changes of 100~, ethanol at room temperature. Infiltration was accomplished in 3 stages; two 10 min changes of propylene oxide (Luft, 1961), 2 hr in a I : 1 mixture of propylene oxide and araldite-epon (Mollenhauer, 1964), and vacuum infiltrated with araldite-epon at 4 5 for I hr. The samples were cured overnight in a 60~C oven. Thick sections were stained with 1 ~!iitoluidine blue in 1 ~',i sodium borate (Pearse, 1960). Thin sections were stained in 2",~, uranyl acetate (Watson, 1958) followed by lead citrate (Reynolds, 1963). OBSERVATIONS The teratocytes of the braconid, Cardiochiles nigriceps Viereck are about 14 /zm in diameter following their liberation into the hemocoele of its host, Heliothis virescens (F.) (Vinson, 1970). They increase in diameter as the parasitoid larva develops, and reach about 350/zm in dia in 10 days. Approximately 200 cells are liberated by C. nigriceps at the time of egg hatch into the hemocoele of the host, and the cells do not increase in number. Teratocytes are approximately 18/zm in diameter 2 days after oviposition (approximately 12 hr following their liberation from the egg). The nucleus (N) is oval and appears to contain both chromatin and euchromatin. Nuclear pores (NP) are evident along the nuclear envelope (Fig. IA). The mitochondrial profiles (M) (Fig. I A) appear round to oval with an indistinctive matrix, poorly developed cristae, and are scarce in these young cells. A few granular inclusion bodies are seen at this stage. The endoplasmic reticulum consists of widely separated rough-surfaced cisternae of variable diameter, which show some continuity with the perinuclear cisterna. There are many unattached ribosomes, polysomes and microtubules. Scattered microvilli (MV) are present (Fig. IA). Sixty hours following the liberation from the egg the teratocytes grow to about 25/zm in diameter, and are uniform in appearance. The nucleus has begun to ramify and increase in size. Free ribosomes remain abundant between the cisternae of the rough endoplasmic reticulum (Fig. IC), which has become extensive and vesicular (VER) (Fig. I B). The intracisternal spaces of the endoplasmic reticulum contain a material of moderate electron
FIG. 1. (A) 12-hr old teratocyte from Heliothis vireseens, parasitized by Cardioehiles nigriceps showing rounded nucleus (N) with many nuclear pores (NP). A few microvilli (MV) are seen along with abundant polysomes between swollen endoplasmic reticulum. (B) 60-hr old teratocyte showing development of extensive vesiculated endoplasmic reticulum (VER). Note ramified nucleus (N) and nuclear pores (NP). (C) Same age as "B" showing ribosomes (R) surrounding vesicles containing a fine granular material. (D) Same age cell as "B'" showing granular bodies (GB) prevalent at this stage.
Ultra~,tructure of Teratocytes of Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae)
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opacity, and of fine granular appearance (Fig. I C). There appear to be more mitochondria with conventional cristae (Hackenbrock et al., 1971) and an increase in granular inclusion bodies ('GB) is observed in the cytoplasm (Fig. 1D). Poorly organized Golgi elements, like those sometimes observed in insect tissue (Smith, 1968), are observed. The microvilli are better developed. After 4 days following liberation, the teratocytes attain a diameter of approximately 50 /zm, and the nucleus becomes extensively ramified (Fig. 2). Two types can now be distinguished both by light and electron microscopy (Fig. 2A-C). In cell type I, the mitochondria remain unchanged. The rough endoplasmic reticulum is dilated, vesicular and abundant. Multivesicular bodies and few granular inclusion bodies are observed. Microvilli are very abundant (Figs. 2C, 3A) and appear to have small microfilaments or fibrils (F) extending a short distance into the cell cortex (Fig. 3B). A few multivesicular bodies (MVB) are seen among the mircovilli (Fig. 2C). No evidence of pinocytosis or pinocytic vesicles was observed. In cell type II (Fig. 3D) the cisternae of the vesicualted rough endoplasmic reticulum have enlarged, rounded and appear as vacuoles or inclusion bodies, although the vesicles remain surrounded by ribosomes ('R) (Fig. 3E, F), and are often connected (Fig. 3C). Some of these vesicles are seen at the cell surface (see arrow Fig. 3C), and many smaller vesicles appear among the microvilli (Fig. 3D). These vesicles are the dominant feature of cell type Ii, and closely packed ribosomes (R) and microtubules (MT) are abundant between the vesicles. A few myelin figures (MF) and lipid bodies (LB) are observed, but there appears to be an absence of multivesiculate, granular or dense bodies. The mitochondria (DM) have well-developed condensed cristae, indicating their active state (Hackenbrock, et al., 1971) (Fig. 4E). The number of microvilli has decreased with respect to the earlier cell or when compared to cell type I (Fig. 2C). In 5 to 6 days the teratocytes are 125-200 /zm in dia. The rough endoplasmic reticulum (LER) is the dominant feature of cell type I, and occurs as parallel profiles of cristae, reminiscent of finger prints particularly near the cell periphery ('Fig. 4B). The inside diameter of the cisternae is about 50 nm, and the cisternae are about 300 nm apart. In some areas of the teratocyte, the rough endoplasmic reticulum appears to be more vesicular. The vesicular endoplasmic reticulum is more extensive in some type I cells than others. Poorly organized Golgi (GA) is seen nearer the extensively ramified nucleus (Fig. 4E), and appears to give rise to vacuolar structures and multivesicular bodies. A few lipid inclusions are seen. Numerous dense, granular, and multivesicular inclusion bodies are observed throughout the cytoplasm in younger cells (Fig. 4A), and tend to be observed near the cell periphery in 7-day and older cells (Fig. 6A). Some of the inclusion bodies are observed at or just beyond the cell boundary. Numerous microvilli are present, and small cellular outpockets containing multivesiculate bodies or vesicles are observed (Fig. 4C). In addition, autophagic vacuoles (A), myelin figures ('MF) and points of cellular destruction (FCD) are seen indicating cellular recycling ('Fig. 4B, D). The nucleus is extensively ramified with much of the chromatin clumped next to the nuclear envelope. The mitochondria have well developed conventional cristae (Fig. 4B, E). FI(;. 2. (A) Thick sections of 4-day old teratocytes showing extensively ramified nuclei (N) and 2 cell types; darker staining cell type I and lighter staining cell type 11. (B) Higher magnification showing vacuolated cell type I!. (C) Electron micrograph showing swollen endoplasmic reticulum of cell type i, and vacuolated endoplasmic reticulum of cell type 11. Note reduced number of microvilli (MV) on cell type II and multivesiculate body (MVB).
Ultrastructure of Teratoctyes of Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae)
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Ultra:;tructure of Teratocytes of Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae)
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FIG. ,4.
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Flea. 5.
Ultras~:ructure of Teratocytes of Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae)
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S. BRADLEIGHVINSONand J. R. SCOTT Fic;. 3. (A) 4-day old cell type 1 teratocyte showing dilated vesicular endoplasmic reticulum (VER), indistinct mitochondria (M) and multivesiculate body (MVB). (B) Same cell type as '~A" showing microfilaments extending into cell cortex from microvilli. (C) Same cell type as ~'A" at a later stage showing vesicular endoplasmic reticulum. Note connections between vesicles (V-bar) and presence of vesicles at cell membrane (arrow), (D) 4-day old cell type 11 teratocyte showing extensive vacuolated endoplasmic reticulum, a few large vacuoles (LV), a myelin figure (MF), lipid body (LB), and the dense mitochondria (DM). Note vesicles at and beyond cell surface (V). (E) Same cell type as "~D" showing a mitochondrion with well-developed, condensed cristae. (F) High magnification of same cell type as '~D" showing vesicles surrounded by ribosomes and extensive microtubules (MT). FI(;. 4. (A) 5-day old cell type I teratocyte showing branched microvillii (BMV) and the multivesicular body (MVB). (B) Same type cell as "'A" showing extensive parallel profiles of endoplasmic reticulum (LER), lipid bodies (LB), orthodox mitochondria (M) and points of cellular destruction (FCD). Note small protrusions (P) or buds on microvilli (MV). (C) Same age cell as "'A" showing vesiculated body protruding from the cell. (D) Same age cell as "'A" showing myelin figures (MF), autophagic vacuole (A) and a point of cellular destruction (FCD) observed in local areas of cell. Note granular body (GB). (E) Same age cell as "A'" showing a Golgi area (GA) and a multivesicular body (MVB). Note well-developed microtubule (MT). FiG. 5. (A) An 8-day old cell type II teratocyte showing condensed mitochondria, numerous vesicles containing fibers (VF) or smaller vesicles (VCV) and lipid bodies. Note reduction in ribosomes between vesicles. (B) An 8-day old cell type I teratocyte showing ramified nucleus, mitochondria, vesicular and parallel profiles of endoplasmic reticulum (VER and LER, respectively). Note small blebs on the cell membrane (B). FiG. 6. (A) An 8-day old cell type I teratocyte showing dense bodies (DB) and a vesicle being exuded by cell. (B) A 10-day old cell type I teratocyte. Note extensively ramified nucleus and alternating vesicular and parallel profiles of rough endoplasmic reticulum (VER and LER, respectivelyl.
In cell type I1, the only observable changes in tile 5-6-day old cell are an increase in the size of the vesicles, a change in their content, and a decrease in the polysomes. These same changes are observed in older type lI cells (Fig. 5A). Some vesicles appear to c o n t a i n a fibrous material (VF) or smaller vesicles (VCV). M a n y vesicles are observed at the cell periphery, and some are observed next to the cell m e m b r a n e and a m o n g the microvilli. Autophagic and lipid vacuoles are infrequently observed. The m i t o c h o n d r i a remain condensed (Fig. 5A). Seven to 8 days after liberation, the teratocytes attain a diameter of 225 p,m. A few type II cells are observed with no obvious changes. In the type l cell (Fig. 5B) the nucleus continues to be extensively ramified, with nuclear pyknosis occurring in local areas, indicating cellular aging. In addition, there is an increase in the n u m b e r of autophagic vacuoles observed, further indicating cellular recycling or aging. Some areas of the cell, primarily near the cell periphery, contain parallel profiles of endoplasmic reticulum, while in other areas of the cell the endoplasmic reticulum is more vesicular, reminiscent of cells 30 hr following their liberation. Dense bodies (DB) are found t h r o u g h o u t the cytoplasm in 6- and 7-day old cells, while being more often located near the periphery in older cells (Fig. 6A). N u m e r o u s cytoplasmic whorls, Golgi vacuoles and mictotubules are observed t h r o u g h o u t the cytoplasm. A few lipid vacuoles are observed in older cells but are not extensive. The m i t o c h o n d r i a appear more n u m e r o u s but stratified in the cell a m o n g the more vesicular rough endoplasmic reticulum, a n d have well-developed cristae. As described, cellular outpockets c o n t a i n i n g multivesiculate and other inclusion bodies are observed a m o n g the extensive microvilli.
Ultrastructure of Teratocytes of Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae)
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Nine to ten days following liberation from the parasitoid egg, teratocytes average 300/xm in diameter. Both cell types continue to be observed, and appear little changed over those observed at 8 days. The nucleus remains extensively ramified with the cytoplasm containing extensive amounts of vesicular and parallel profiles of rough endoplasmic reticulum (Fig. 6B). Autophagic vacuoles, dense bodies, and lipid vacuoles are observed along with a few cellular outpockets. Between the 9th and 10th day, the teratocytes are consumed as the developing parasitoid larvae consume the host. DISCUSSION Most investigators of teratocytes have suggested that these cells function in the absorption of nutrient:~ from the host, thus weakening the host and supplying the parasitoid larvae with a ready food supply prior to the parasitoids emergence (Salt, 1968; Sluss and Leutenegger, 1968). Salt (1971) suggested that attrition of the host by the developing teratocytes weakened and rendered ineffective the ability of the host to encapsulate and destroy the parasitoid. However, Vinson (1972) reported that some foreign objects were encapsulated in the presence of teratocytes and that teratocytes were encapsulated when placed in alien hosts. Sluss and Leutenegger (1968) suggested that teratocytes are secretory in the early phase, but become primarily absorptive later in their development and serve as a food source for the developing parasitoid. In the present study, the young teratocytes appear to be relatively inactive as suggested by the simplicity of the mitochondria, absence of many inclusion bodies, Golgi, and the presence of a modreate amount of rough endoplasmic reticulum. Their rapid growth over the next 8 days does indicate the absorption of nutrients from the host. Thirty hr after liberation from the egg, teratocytes show the development of extensive vesicular endoplasmic reticulum. In some cells (Designated cell type II), the swelling of the cisternae continues, and by 60 hr, most of the cisternae have developed into enlarged vacuoles surrounded by ribosomes. These vacuoles are found at the cell border, suggesting that their contents may be liberated from the cell. In other cells (cell type I) the granular material, which is present in the cisternae, disappears and the rough endoplasmi,z reticulum develops extensive, evenly space, parallel profiles of cisternae. Although rough endoplasmic reticulum is present in all cells, its greatest development is most often seen in secretory cells (Fawcett, 1966). The presence of parallel profiles of extensive endoplasmic reticulum, and the presence of vesicular cisternae in teratocytes throughout their short life span suggets they are secretory. Further, the loss of material from the vesicular endoplasmic reticulum by most of the cells by 60 hr after their liberation from the egg supports this view. The reason for the retention of material in the cisternae of the endoplasmic reticulum and the subsequent enlargement of these cisternae in approximately 5-10 ~ of the cells is unknown. It may represent cycles of secretory activity, cells with different functions, or cells in a different stage of development. Vacuoles are seen near the surface of type I cells, and in secretory outpockets in type II cells, suggesting that secretion is occurring in both cell types. No evidence of pinocytosis was observed. Golgi are observed in these cells, and appear to give rise to multivesicular bodies, comparable to those observed in the corpus allatum of Leucophaea (Scharrer, 1971). The migration of the multivescular and dense bodies to the cell periphery, along with the absence of any extensive buildup of inclusion bodies in teratocytes, suggests that these bodies may be released from the cell. K
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The absence of glycogen reported by Vinson (1970) is supported by the observed lack of glycogen granules. The lack of any extensive b u i l d u p of glycogen, lipid or protein in the teratocytes does n o t support a general view that these cells primarily f u n c t i o n as a food source for developing parasitoids, as is suggested by Jackson (1928) a n d Sluss (1968). Teratocytes m a y serve as an i m p o r t a n t food source in some parasitoid-host associations, a n d Sluss a n d Leutenegger (1968) did observe glycogen, lipid a n d protein bodies in teratocytes from adult coccinellid beetles infested with the braconid, P e r i l i t u s coccinellae. Vinson a n d Lewis (1974) reported that teratocytes in hosts parasitized by M i c r o p l i t i s croceipes were n o t c o n s u m e d by the developing parasitoid. The potential i m p o r t a n c e of teratocytes in the regulation of the host physiology, a n d the i m p o r t a n c e of this regulation to the developing parasitoid has been overlooked. Vinson (1970) has shown that injection of teratocytes into hosts results in the failure of the host to pupate normally. The rapid growth, extensive development of the endoplasmic reticulum, a n d general cellular activity observed in these cells, coupled with the general absence of stored cellular products, suggests functions other than nutritional. REFERENCES FAWCET'f, D. W. 1966. The Cell. An atlas o f fine structure. Sanders. Philadelphia, London. HACKENBROCK,C. R., T. G. REHN, E. C. WE~NBACHand J. J. LEMASTERS.1971. Oxidative phosphorylation and ultrastructural transformation in mitochondria in the intact ascites tumor cell. J. Cell Biol. 51: 123-37. HuFf, C. G. 1940. Immunity in Invertebrates. Physiol. Res. 20: 68-88. JACKSON, D. J. 1935. Giant cells in insects parasitized by hymenopterous larvae. Nature (Lond.) 135: 1040-41. LtJFT, J. H. 1961. Improvements in epoxy embedding methods. J. Biophys. Biochem. Cytol. 9: 409. MOLLENHAUER, H. H. 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39: 11-14. PEARSE, A. G. E. 1968. Histochemistry. Theoretical and Applied, Little Brown, Boston. PRINGLE, J. W. S. 1938. Proprioception in insects. J. Exp. Biol. 15: 101-13. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17: 209-12. SABATINI, D. D., F. MtLLER and R. J. BARRNETT. 1964. Aldehydre fixation for morphological and enzyme histochemical studies with the electron microscope. J. Histochem. Cytochem. 12: 57-71. SALT, G. 1968. The resistance of insect parasitoids to the defense reactions of their hosts. Biol. Rev. 43: 200-32. SALT, G. 1971. Teratocytes as a means of resistance to cellular defense reactions. Nature (Lond.) 232: 639. SCHARRER, B. 1971. Histophysiological studies on the corpus allatum of Leucophaea moderae. V Ultrastructure of sites of origin and release of a distinctive cellular product. A. Zellforsch. 120: 1-16. SLUSS, R. 1968. Behavioral and anatomical responses of the convergent lady beetle to parasitism by Perilitus coccinellae (Schronk) (Hymenoptera: Braconidae). J. Invert. Pathol. 10: 2-27. SLUSS, R. R. and R. LEUTENEGGER.1968. The fine structure of the trophic cells of Perilitus coccinellae (Hym.: Braconidae). J. Ultrastruct. Res. 25: 441-51. SMITH, D. S. 1968. Insect cells their structure and function. Oliver & Boyd Edinburgh, VA. VANDERZANT,E. S., C. D. RICHARDSONand S. W. FORT, SR. 1962. Rearing of the bollworm on artificial diet. J. Econ. Entomol. 55: 140. V1NSON,S. B. 1970. Development and possible function of teratocytes in the host-parasite association. J. Invert. Pathol. 16: 93-101. VINSON, S. B. 1972. Factors involved in successful attack on Heliothis virescens by the parasitoid Cardioehiles nigriceps. J. Invert. Pathol. 20: 118-23. VINSON, S. B., F. S. GUILLOTand D. B. HAYS. 1973. Rearing of Cardiochiles nigriceps in the laboratory with Heliothis virescens as hosts. Ann. Entomol. Soc. Amer. 66: 1170-72. VINSON, S. B. and W. J. LEWIS. 1974. Teratocytes: Growth and numbers in the hemocoel of Heliothis virescens attacked by Mieroplitis croceipes (Cresson). J. Invert. Pathol. 22: 351-5. WATSON, M. L. 1958. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. CytoL 4: 475-78.