Serosal cells of Biosteres longicaudatus HymenopteraBraconidaeUltrastructure and release of polypeptides.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 13:199-216 (1 990) Serosal Cells of Biosteres longicaudatus (Hymenoptera: Braconidae): Ultrastructure and Release of Polypeptides Pauline 0. Lawrence Department of Zoology, University of Florida, Gainesville, Florida As a prelude to a study i n vitro of the function of the embryonic serosa of the parasitic wasp Biosteres longicaudatus (Braconidae), the ultrastructure of serosas of different ages reared i n vivo and i n vitro were compared. The evidence suggests that the serosal capsule consists of one to three cell layers. The innermost (internal cells) which line the serosal capsule and the outermost (external) cells which are bathed by the host's hernolymph are secretory. Large, coated vesicles in the internal cells increase in number and size with age and, likely, take up and transport molecules into the serosa. Multivesicular bodies, known for their enzyme-degradative function, occur in external cells and are eventually extruded into the surrounding environment. Distinctive electrondense, rod-shaped particles appear in external cells within 8 h after larvae hatch, increase in number with larval age, and occur at the bases of microvilli. The latter appear electron dense with age and eventually they and the lobulated microvilli in internal cells fragment into the surrounding environment. To determine whether parasites and/or their serosas release substances into the host Anastrepha suspensa (Diptera: Tephritidae), hemolymph from unparasitized and superparasitized (> 1parasiteihost) pharate pupaewas analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PACE). Serosas and/or parasite larvae were incubated in artificial media and each of these was subjected to SDS-PAGE. A polypeptide, approximately 24 kilodaltons (Kd) occurred in the hemolymph of 24-h-old superparasitized pharate pupal hosts but not in the control. A similar polypeptide was observed in medium cultured with parasite larva and serosa as well as serosa alone, but was not i n their respective control media. This approximately 24-Kd band in SDS-PACE gels corresponds to a band i n the serosa homogenate and may be identical to it. Serosas and parasite larvae i n vivo and in vitro have similar protein profiles. Based upon these ultrastructural and electrophoretic studies, i t appears that the serosa of B. longicaudatus has a synthetic function, as has been reported Acknowledgments: I thank Mark Beveridge and Debbie Akin for assistance with the SDSPAGE and electron microscopy, respectively. Financial support from the National Science Foundation, DCB-8502235, and from the U.S. Department of Agriculture, CRGO-8700433, is gratef uIly acknowledged. Received September 11,1989; accepted December 19,1989. Address reprint requests to Pauline 0. Lawrence, Department of Zoology, 223 Bartram Hall, University of Florida, Gainesville, FL 32611. 0 1990 Wiley-Liss, Inc. 200 Lawrence for the extra-embryonic membrane of other parasitic Hymenoptera. It may sequester and degrade molecules from the host hemolymph and likely release newly synthesized as well as degraded products into the host. Key words: teratocytes, extra-embryonic membrane, polyacrylamide gel electrophoresis, parasitism-specific protein, parasite proteins, fruit fly proteins, hemolymph proteins, Diptera proteins, Hymenoptera proteins INTRODUCTION The extraembryonicmembranes or serosa of parasitic Hymenoptera perform nutritive and gaseous exchange functions [l].In some species an endocrine role is also suspected [Z]. Recent studies of parasitic Hymenoptera serosas suggest that they disrupt the host's cellular defence capabilities [3,4] and endocrine status . Thus, they may have major significance in influencing host-parasite interactions. In several parasitic wasp species the serosa disintegrates into enlarged, single cells or teratocytes [6-91. The results of the present study corroborate other reports [9,10] of secretory organelles in serosal cells (teratocytes)and support the hypothesis of their secretory function. However they indicate that serosal disintegrationinto giant, single-celled teratocytes is not a universal phenomenon among the parasitic Hymenoptera. Superparasitism(> 1parasitehost) of the tephntid fruit fly Amstrepha suspensa by the parasitic wasp Biosteres longicaudatus elevates the host's ecdysteroid titer [ll]. It also induces an increase in juvenile hormone levels and a concomitant decrease in juvenile hormone esterase activity , resulting in delayed host larval-pupal metamorphosis . Similar effects were observed after injection of Heliothis virescens with teratocytes from the braconid parasite Microplitis uoceipes . In other studies, B . longicaudatus larvae cultured in artificial medium preconditioned with conspecifics and their serosas [ 141 or serosas alone [unpublished] suffered retarded growth and/or mortality. These observations led to the hypothesis that the parasite larva and/or its serosa may synthesize and release a substance(s) into the host's hemolymph that affects both the host and parasite conspecifics. As a prelude to in vitro studies, of serosal synthesis and function, the present study was initiated to 1)evaluate the ultrastructure and cellular integrity of serosas reared in vitro vs. those in vivo and 2) determine whether a substance(s) is released from the serosa. MATERIALS AND METHODS Rearing of Insects Fruit flies and parasites were reared at 27 k 2°C and 70-80% R.H. as previously described . First-day third-instar fruit fly larvae were parasitized for 3 h and 2 days later larvae that had pupariated within 4 h of each other were utilized for all experiments. Newly pupariated hosts contained parasite eggs, and 24-h-old pharate pupal hosts contained parasite first instars with and without serosas [12,14,15]. Subsamples of these parasitized hosts served Ultrastructure and Polypeptides of Serosal Cells 201 as a source of parasite larvae for in vivo studies and parasite eggs for in vitro culture of the resulting larvae. In Vitro Studies Parasitized fruit fly puparia (O-4-h-old)were dissected under sterile conditions in Goodwin’s IPL 52B medium (Hazleton, Lenexa, KS) as previously described [14,16]to remove fully embryonated (40-44-h-old) parasite eggs. The eggs were then placed in Goodwin‘s IPL 52B medium (340 mg/kg osmolarity) containing glutamine and gentamycin (1mg/ml, each) (Sigma, St. Louis, MO). The medium containing specimens for ultrastructural studies was supplemented with 20% FCS* (Hazleton) while that for electrophoresis was FCS-free. In the latter group larvae and/or serosas were each maintained in 150 ~1 of medium for 7 or 13 days. Culture medium maintained under similar conditions and for the same period without larvae and/or serosas served as the control. Light and Electron Microscopy Serosas from 0-, 4-, 8-, and 24-h-old larvae in vivo and from 4-, 8-, 24-, and 72-h-old parasite larvae in vitro were fixed in 2% gluteraldehyde, embedded in Spurr’s resin, and post-fixed in 2% uranyl acetate. Thick (> 1 Fm) longitudinal sections of O-h-old parasite larvae in vivo were viewed under the light microscope to determine the relationship between the intact serosa and the larval body. Thin sections (600 A) of serosa from larvae in vivo and in vitro were viewed under a JEOL transmission electron microscope at 80 KV. Gel Electrophoresis SDS-PAGE  of hemolymph from superparasitized and unparasitized 24-h-old fruit fly puparia and medium incubated for 7 or 13days (as described above) were performed. Serosas and parasite first instars dissected from the host (in vivo) and those reared in vitro were homogenized separately in sample buffer and centrifuged at 8,OOOg for 5 min at 4°C. Supernatants from each homogenate of first-instar parasites and their serosas in vivo and in vitro were electrophoresed to evaluate their constituent polypeptides. The electrophoresis parameters varied. Consequently, specific conditions are described for each figure (see figure legends). The polypeptide bands and molecular weight markers (Sigma, St. Louis, MO, and Bio-Rad, Richmond, CA)were visualized with Coomassie brilliant blue R-250 and/or silver stain (Bio-Rad). Prior to electrophoresis, a micro Bio-Rad protein assay was performed on each sample. RESULTS Upon hatching from the egg, the first-instar larva of B. Zongicaudatusis encased in an embryonic serosa (Fig. 1). Along the dorsal, cephalic, and anterio-ventral regions the serosa is one cell thick but in the ventro-abdominal area it is three cells thick. The innermost (internal) serosal cells line the serosal capsule and *Abbreviations used: FCS = fetal calf serum; JH= juvenile hormone; )HE = JHesterase; Kd = kilodaltons; Mvb = multivesicular bodies; PhP = pharate pupa or pharate pupal development; SDS = sodium dodecyl sulfate; VT = in vitro; W = in vivo. Figs. 1-3. Micrographs of Biosteres longicaudatus first instar and serosa. Fig. 1. Light micrograph of the longitudinal section of a newly hatched (0-h-old) parasite larva from a pharate pupa of Anastrepha suspensa. The larva is surrounded by the serosal capsule ( S ) , which is about one cell thick along the dorsal (D) and ventro-cephalic regions and three cells thick in the ventro-abdominal area. The innermost (internal, I) cell layer lines the serosal capsule and the outermost (external, E) i s bathed by the host’s hemolymph. Ab = abdomen; HC = head capsule; Vn = venter. Fig. 2. Light micrograph showing parasite first instar and the ruptured serosa ( S ) still attached to the base of the parasite’s head capsule (HC) The serosa is ruptured within 2-3 h of hatching and shed from the parasite’s body24-36 h after hatching. Fig. 3. Transmission electron micrograph of an 8-h-old serosa from the ventral abdominal region of the larva to show the differences between external (E)and internal (I)cells. The middle cell (small arrows trace the outline of the cell) with a large nucleus (N) that occupies the majority of the cell lies between inner and outer cell layers. The outermost cells are characterized by long microvilli and accumulations of elongate, electron-dense particles (Ed) imrnediately beneath the cell membrane adjacent to the host‘s hemolymph. The innermost cells characteristically lack these dense accumulations and have short, irregular cytoplasmic projections. M = mitochondria. Ultrastructure and Polypeptides of Serosal Cells 203 are closest to the larval body while the outermost (external) ones are bathed by the host's hemolymph (Fig. 1). The single-layered cells of the serosa are ultrastructurally similar to these external cells. Two to 3 h after the larva hatches the serosa ruptures but remains attached at the ventral base of the head capsule (Fig. 2) and by 24-36 h the serosa is shed. The serosal cells each have a large nucleus (Fig. 3) and loose intercellular connections. The external cells contain electron-dense, rod-shaped particles beneath the plasma membrane but internal cells lack these particles (Fig. 3). Age-Related Ultrastructural Changes in External and Internal Serosal Cells External serosal cells. These cells from O-h-old parasite larvae in vivo have numerous rough endoplasmic reticula, mitochondria, and small clusters of vesicles that are characteristic of protein synthetic properties (Fig. 4). In O-h-old cells a diffuse electron-opaque material accumulatesbeneath the plasma membrane, and as the cells age, this material is replaced by elongate, electron-dense bodies which also increase in number (Figs. 5-7). Characteristicelongate microvilli with bulbous apices extend into the surrounding host hemolymph (Fig. 4).Their swollen apices enlarge with age (Figs. 4-6) and by 8 h seem to fragment into the surrounding host hemolymph (Fig. 6). While the microvilli of O-8-h-old cells are electron-lucent those in 24-h-old cells are electron-opaque, presumably as a result of incorporating some material from the underlying electron-dense bodies (Fig. 7). Indeed, electron-densebodies lie directly beneath some microvilli (Fig. 7). An elaborate array of large extracellular vesicles occurs along the plasma membrane of 24-h-old cells (Fig. 7). These vesicles are possibly the swollen apices of the microvilli and contain material at their periphery visually similar to that in the microvilli (Fig. 7). Large coated vesicles absent in O-h-old cells are numerous in 4-h-old cells (Fig. 5) but become fewer in 8-h-old cells (Fig. 6 ) . At 24 h the vesicles are smaller than at the two younger ages (Figs. 5-7). Lamellar or cytolysome bodies known to be associated with enzymatic activity  are evident in 24-h-old cells (Fig. 7). The external serosal cells of parasite larvae in vitro (Figs. 8-11), exhibit ultrastructural characteristics similar to those in vivo (Figs. 4-7). The diffuse material observed in O-h-old cells in vivo (Fig. 4) occurs in 4-h-old cells in vitro (Fig. 8) but by 8 and 24 h old in vitro, it is replaced by electron-dense bodies more numerous than their counterparts in vivo (Figs. 9,10, respectively).These particles also extend into the bases of the microvilli (Figs. 9,lO) and may account for the electron-opaque contents of the microvilli in 24-h-old cells in vivo and in vitro (Figs. 7, 11, respectively). As with their counterparts in vivo, 24-h-old external serosal cells in vitro have large, extracellular vesicles (Fig. 11). Internal serosal cells. These cells of O-h-old parasite larvae in vivo contain rough endoplasmic reticula, ribosomes, and aggregations of vesicles, and like the external cells, likely synthesize proteins (Fig. 12). The cells have sparsely distributed, short lobulate microvilli that extend into the serosal cavity and the wide intercellular spaces (Figs. 12, 16). By 4 h old the cells contain numerous large, coated vesicles which contain particulate material (Fig. 13). Eighthour-old internal cells contain similar coated vesicles (Fig. 14), some of which are directly attached to the plasma membrane (Fig. 15).By 8 h of age, internal cells contain multivesicular bodies (characterized by several small vesicles within 204 Lawrence Figs. 4-7 Ultrastructure and Polypeptides of Serosal Cells 205 a membrane) (Fig. 14). Lobulate microvilli in 8- and 24-h-old cells appear to fragment into the surrounding environment (Figs. 14,14). The internal serosal cells from larvae in vitro (Figs. 17-19) are ultrastructurally similar to their counterparts in vivo (Figs. 12-14). Coated vesicles seen in 4- and 8-h-old cells in vivo (Figs. 13-15) were also evident in vitro cells (not shown) and in the latter case, they persisted through 72 h of age (Fig. 19). Multivesicular bodies (Fig. 18) which also occur in 8-h-old cells in vivo (Fig. 14) are extruded from the cells in vitro (Figs. 19, 20). Presumably, a similar process of exocytosis occurs in vivo. Presence of a Parasitism-Specific Protein In Vivo and In Vitro To facilitate comparison among host and control hemolymph and parasite larva and serosa protein profiles, the bands in the A. suspensa hemolymph were arbitrarilygrouped into six categories (A-F) after SDS-PAGE analysis (Fig. 21). Hemolymph from 24-h-old PhP hosts contained a faint protein band, (Category E) that was not seen in the controls (Fig. 21). However, Category A-D and F bands were common to both groups (Fig. 21). To further confirm the presence of the Category E band and amplify it, a second gel with > 10 x the protein concentration used in Figure 21 was prepared (Fig. 22). Even at the higher protein concentration, the band is not detectable in the controls and aligns well with the 24-Kd molecular weight standard (Fig. 22). Thus, this -24-Kd protein is parasitism-specific. A protein with similar molecular mass was also detected in FCS-free medium in which parasite larvae and serosas were incubated for 13 days but it was absent from medium incubated for the same period without biological material (Fig. 23). Furthermore, FCS-free medium incubated for 7 days with serosas alone contained the same protein band (Fig. 24). Both culture media contained about five additional bands of moderate to high molecular mass (approximately 45-205 Kd) that were absent from their respective controls (Figs. 23, 24). Interestingly, these bands and the approxi- Figs. 4-7. Electron micrographsof external serosal cells from B. longicaudatuslarvae dissected from host puparia (in vivo) at different ages. Fig. 4. External serosal cell from a 0-h-old parasite in vivo. The cell has numerous rough endoplasmic reticula (rer), mitochondria (M), vesicles (V), and microtubules (mt). Traces of an electron-dense material (arrowheads), presumably protein, begin to accumulate beneath the plasma membrane. Elongate microvilli (Mv) with enlarged apices (double arrows) extend from the cells into the host’s hemolymph. Fig. 5. External serosal cell from a4-h-old parasite in vivo. The diffuse material (Fig. 4) now consists of distinct elongate bodies (arrowheads) along the periphery of the outer cell membrane (towards the host’s hemolymph). Coated vesicles (V), absent from younger cells, are now evident along with microtubules (mt) and aggregates of small particles (*), Microvilli (Mv) with swollen apices (double arrow) fragment into the surrounding host hemolymph. Fig. 6. External serosal cell from an 8-h-old parasite in vivo. More electron-dense material (Ed, arrowheads) accumulate beneath the plasma membrane and microvilli (Mv) fragmentation persists. V = coated vesicles; mt = microtubules. Fig. 7. External serosal cell from a 24-h-old parasite in vivo. Lamellar bodies (Lb, inset) are seen in some sections along with coated vesicles (V) which enlarge, apparently by small ones coalescing. Microvilli (Mv) containing electron-dense material (double arrowheads) seem to arise directly above the areas with electron-dense particles (Ed). An array of very large extracellularvesicles (Ev) is now evident on the outer cell membrane. M = mitochondrion. Figs. 8-11. Electron micrographs of external serosal cells from B. longicaudatus larvae of different ages reared in vitro. Fig. 8. External cell from 4-h-old serosa in vitro. An accumulation of undefined electrondense material (arrowhead) similar to that seen in 0-h-old serosa in vivo (Fig. 4) is evident. A few microvilli (Mv) project into the surrounding medium (double arrows). Rough endoplasmic reticula (rer), mitochondria (M),and microtubules (mt) also occur in these cells. Accumulations of many small particles (*) are also evident. Fig. 9. The external cell of 8-h-old serosa in vitro. The electron-dense particles (Ed) are distinct and more numerous than in younger cells (Fig. 8 ) or in those of corresponding age in vivo (Fig. 6 ) . Some particles (double arrowheads) have projected into the bases of the microvilli (Mv). Microtubules (rnt)and mitochondria (M)are also numerous. V and * = coated vesicles. Fig. 10. By 24 h old the external serosal cells in vitro have developed an array of very large vesicles (Ev) as observed in vivo (Fig. 7). Electron-dense particles (Ed) push against the plasma membrane (double arrowheads). M = mitochondria; mt = microtubules; M v = microvilli. Fig. 11. Twenty-four-hour-old external serosal cells in vitro have an array of enlarged extracellular vesicles (Ev), interspersed with microvilli (Mv) that contain electron-dense material. A tight junction (J) connecting two external serosal cells i s evident. Ultrastructureand Polypeptidesof Serosal Cells 207 mately 24-Kd parasitism-specific band corresponded to bands in homogenates of the serosa (Fig. 24) as well as to bands in Categories A-C of A. suspensa hemolymph (Fig. 21). Homogenates of serosas and larvae cultured in vivo and in vitro (Figs. 25, 26, respectively) had similar overall protein profiles, suggesting that in vitro culture did not adversely affect them. In addition, parasite larvae and serosas in vivo (Fig. 25) and in vitro (Fig. 26) had protein bands corresponding to Categories A-D and F of A . suspensa hemolymph (Fig. 21), and to the -24-Kd protein in serosa-incubated medium (Fig. 24). The 24-Kd band in larvae, serosas, and serosa-cultured medium is shared only with the host but not with the unparasitized A . suspensa hemolymph (Figs. 21-26). DISCUSSION In some species, the serosa disintegrates into a number of single cells or teratocytes [3,4,8,10]. However, the in vivo and in vitro evidence indicate that in B. longicaudatusthe serosa persists as an intact organ (Fig. 3) even after detachment from the parasite’s body (figure not shown). There are no other reports of serosas remaining intact after hatching of other parasitic wasp species but this may reflect the limited number of species studied in this regard. Young teratocytes are thought to play a nutritive role in parasite larval development, presumably by sequestering substances from the host’s hemolymph and/or synthesizing others [1,7]. The sequestration of host substances by teratocytes/serosashas been suspected but not demonstrated. Concentrations of severalhost hemolymph proteins [19,20]including arylphorins , (Thomas, personal communication) change after parasitism. Protein bands (67-94 Kd) corresponding to the hemolymph storage proteins of the host also occur in larval parasite hemolymph . In the present study, separate homogenates of parasite larvae and serosas also have some protein bands with relative mobilities near the 200-205-Kd, 66-97.4-Kd, and 43-66.2-Kd bands of the host (Figs. 21, 24-26). Whether these proteins are the same as the host’s and are sequestered from host hemolymph or are synthesized in parasite larva and/or serosa remains to be determined. The uptake of radiolabeled ecdysteroids by parasite larvae and of amino acids by parasite larvae  and eggs  has been demonstrated in vitro. However, evidence for the role of the teratocytes/serosasin these processes or in sequesteringproteins is still lacking. The ultrastructural data implicate the serosa in sequestering substances from the host and further suggest age-related functions and/or processing of different molecules. The presence of coated vesicles at the membranes of internal serosal cells (Fig. 15) and their increase in size (volume) from 0-8 h (Figs. 4-6) through 12 h (not shown) suggest that they may pick up and sequester substances from the host. Their apparent decrease in size and association with the innermost plasma membrane of the serosa (Fig. 16) could also indicate the release of substance(s). Coated vesicles are known to transport proteins into and out of cells [18,25] and their pinocytic vesicles coalesce to form Mvb’s . In 8-h-old internal serosas Mvb’s become prominent (Figs. 14, 18). They are thought to contain acid phosphatases, and in Catpodes ethius (Lepidoptera) are presumed to regulate the turnover of proteins between the fat body 208 Lawrence Figs. 12-16 Ultrastructure and Polypeptides of Serosal Cells 209 and hemolymph . The relationship of the internal serosal cells to the uptake, transport, and degradation of molecules deserve further study. Teratocytes by implication release substances into the host to effect a variety of changes including, presumably, a decrease in phenoloxidase activity [3,4] and juvenile hormone esterase levels  of the parasitized host. The present study is the first to provide evidence from in vitro studies of B. longicaudatus serosa that may be applicable to teratocytes. Six protein bands occur in medium cultured with B. longicuudutus serosa alone (Fig. 24). Five of these are of moderate to high molecular mass (43-205 Kd) and correspond to bands in the serosa (Figs. 23, 24), parasite larva (Figs. 25, 26), and host hemolymph (Figs. 21, 22). These five proteins likely represent excess amounts of commonly occurring molecules and could be excretory. The sixth, approximately 24-Kd band, also occurs in the host but not in unparasitized hemolymph and is likely synthesized in the larva and/or serosa and released into the hemolymph via the serosa. The role(s) of these six proteins, and particularly the novel 24-Kd protein, in altering the levels of ecdysteroids [ll],JH and JHE  in superparasitized A . suspensa pharate pupae, is under investigation. Since five of the six proteins are common to hosts and controls, they are unlikely to induce the observed endocrine changes in the host. The ultrastructural data provide evidence that both the external and internal serosal cells may be points of release. The presence of Mvb’s in vivo (Fig. 14) and their extrusion in vitro (Figs. 19,20) suggest a similar phenomenon in vivo. This and the fragmentation of microvilli, especially in the external cells (Figs. 5-7, 9-11), represent plausible avenues through which the six proteins detected in vitro may be released, An apparent decrease in diameter (and presumably, volume) of coated vesicles in 24-h-old serosal cells (Figs. 7, 16) may also be an indication of release. Figs. 12-16. Electron micrographs of internal serosal cells from 5. longicaudatus larvae dissected from host puparia (in vivo) at different ages. Fig. 12. The internal cells from 0-h-old serosa in vivo. Cells contain accumulations of vesicles (V) that appear to be associated with Golgi apparati, and rough endoplasmic reticula (rer). The cells are loosely connected and short cytoplasmic extensions occur intercellularly (**) and extracellularly (Cp). These projections are shorter than the microvilli seen in the external cells of the serosa of similar age (Fig. 3). Fig. 13. By4 h old the internal serosal cells in vivo contain numerous, large coated vesicles (V) and bodies of small particles (*). C p = cytoplasmic projection; mt = microtubules; N = nucleus. Fig. 14. By 8 h old, the internal serosal cells in vivo also contain multivesicular bodies (Mvb). By this age, t h e cytoplasmic projections (Cp) have increased in number. V = vesicles; mt = microtubules. Fig. 15. Cross section of Figure 14 showing the point of attachment of t h e internal serosa to t h e larval cuticle. Large coated vesicles (V) occur along t h e cell membrane. The larval cuticle (Cu) of t h e parasite has numerous projections, presumably setae (St) that help t o hold the serosa in place. Note coated vesicle (arrow) attached to plasma membrane closest to parasite larva. Fig. 16. Twenty-four-hour-old internal serosal cells in vivo. Small coated vesicles arise near the plasma membrane (large arrow) and apparently increase (V) in size by coalescing. Desmosomes (triple arrowhead) form local intercellular connections. Cp = cytoplasmic projection; mt = microtubules. 210 Lawrence Figs. 17-20 Ultrastructureand Polypeptides of Serosal Cells 21 1 Fig. 21. Protein profiles of hernolymph from unparasitized (UPP) and superparasitized (PP) 24-h-old Anastrepha suspensa PhF! Each sample of kg proteinilane was applied to a 15-20% gradient SDS gel. The gel was run at 150 V for 4 h at 4°C and the bands were visualized with Coornassie brilliant blue R-250 followed by silver stain. Five arbitrary categories (A-D and F) of polypeptides co-occur in superparasitized and unparasitized PhP hemolymph and a sixth (E) occurs only in superparasitized individuals. CategoryA = 2-3 bands near200 Kd; B =*2 major and 2-3 minor bands, 66.2-97.4 Kd; C = 3-4 minor bands, 43-66.2 Kd; D = a major band -26-28 Kd; E = a new, diffuse minor band,=24 Kd (double arrowheads); F = a band similar in intensity to that in D,just below 14.4 Kd. Figs. 17-20. Electron micrographs of internal serosal cells from B. longicaudatus larvae of different ages in vitro. Fig. 17. The internal serosal cellsfrom 4-h-old larvae in vitro are similar in ultrastructure to those in vivo (Figs. 13-16). They contain microtubules (mt) and bodies of many small particles (*I. Cp = cytoplasmic projections; M = mitochondria; N = nucleus; V = coated vesicle arising near plasma membrane; rer = rough endoplasmic reticula. Fig. 18. Eight-hour-old internal serosal cells in vitro. Multivesicular bodies (Mvb) and microtubules (mt) occur as they do in vivo (Fig. 14). Cp = cytoplasmic projections; M = mitochondria; mt = rnicrotubule. Fig. 19. Seventy-two-hour-old internal serosal cells in vitro. Multivesicular bodies (Mvb) bud into the intercellular space. Cp = cytoplasmic projection; rnt = microtubule; V, * = coated vesicles. The serosa is usually shed from the larva within 2-3 days after hatching. Hence, 72-h-old serosa are usually free in the medium or sometimes attached to the larval head capsule by a few cells. Fig. 20. Higher magnification of Figure 19 showing multivesicular bodies (Mvb) that have budded from the internal serosal cell into the medium. 212 Lawrence PP UP MW 205.OKd 1 16.OKd 97.4Kd 66.OKd 45.OKd 29.OKd 24.OKd 18.4Kd 14.3Kd Fig. 22. Amplification of the Category E protein in Anastrepha suspensa PhP host hernolymph. One hundred fifty micrograms and 156 kg protein (i.e., 1.2 PI hemolymph equivalents each) of hemolymph from 24-h-old superparasitized (PP)and unparasitized (UP) PhP, respectively, were loaded per lane. The 12% SDS gel was run at 180 V, 4"C, for 4 h. The bands were stained in Coomassie brilliant blue R-250 followed by silver stain. The Category E band (double arrowheads) i s a distinct, approximately 24-Kd band in the PP hernolymph but is absent from the UP lanes. Molecular weight standards (MW)were placed in outer lanes. The synthetic activity of teratocytes has yet to be demonstrated but the presence of rough endoplasmicreticula and other organellesin young teratocytes [3,6] provide circumstantialevidence for protein synthesis. Indeed, proteins and lipids have been detected in young teratocytes but proteins and glycogen were found in old cells . In the present study, young (O-8-h-old) serosas of B. Zungicaudutus also contain the appropriate apparatus for protein synthesis (e.g./ Figs. 4,12). The appearance of the novel 24-Kd protein in vitro and its presence in serosa and parasite larval body (Figs. 24-26) but absence from unparasitized fruit fly pharate pupae (Fig. 21) suggest that it is newly synthesized. Its site of synthesis could be the host as a response to parasitism and it then could be sequestered by the parasite body and/or serosa. Alternatively, it could be synthesized by parasite or serosa and released from the latter. This approximately 24-Kd protein was not detected in medium incubated with parasite eggs and larvae alone (data not shown). Furthermore, it did not appear in parasitized host hemolymph until about 60 h after parasitization (i.e./ in the hemolymph of 12-16-h-old pharate pupal hosts; data not shown). It is therefore unlikely that this protein was introduced by the avipositing parasite female. In Musca domestica parasitized by Nasonia vitripennis (Hymenoptera:Pteromalidae) , Pieris r a p e parasitized by Cutesia glomerata (Hymenoptera: Ichneumonidae) , and Manducu sexta parasitized by C. cungregata , parasitism-induced proteins appeared within a few hours (early) and after several days (late) of parasitism. Ultrastructure and Polypeptidesof Serosal Cells MW PCM CM 21 3 MW 205I.OKd 1168.OKd 97'.4Kd 66.OKd 66.OKd 45 .OKd 458.OKd 34.7Kd 29.OKd 24 .OKd Fig. 23. SDS-PAGE of FCS-free IPL52B medium after incubation of Biosteres longicaudatus first instars and serosas for 13 days. Parasite-conditioned (PCM) and control (CM) media at 2.4 pg and 2.6 pg . protein, respectively, were applied to each lane of a 10% SDS gel which was run at 180 V for 4 h at 4°C. The gel was stained in Coomassie brilliant blue R-250 and twice in silver. A major band (double arrowheads) occurs at1124 Kd in the PCM but is absent from the CM. This band has a migration pattern similar to that observed in superparasitized host hemolyrnph (Figs. 21,22). About five additional bands (arrows), each corresponding to at least one band in Group A, B, and C of unparasitized A. suspensa PhP (Fig. 21), occur in the PCM but not in the CM. Molecular weight markers (MW) were applied to the outer lanes. Some early proteins are presumed to be introduced by the ovipositing wasp [19, 271). However, an early appearing protein is induced presumably by polydnaviruses from the female wasp ). The 24 Kd reported here appears to be derived from the parasite's serosa. Whether it is synthesized by the serosa or by wasp-associatedviruses  that could be in the serosa or larval body, and then transported to the serosa remains to be determined. Thus, the ultrastructure of the serosa of B. Zongicuudatus indicates its ability to effect a dynamic interchange of molecules between host and parasite milieu. Some of these molecules are likely sequestered from the host and degraded, while others like the approximately 24-Kd protein may be synthesized de nwo and released into the host for functions yet undetermined. 214 Lawrence MWkM-iPCMVVT1 MW 205 97 66 66 .OKd 45 45 .OKd 29 24..OKd 18.4Kd 14.3 K d Fig. 24. Relationship between the 24-ZKd protein in incubation medium and proteins in the serosa. Forty microliters of Goodwin’s IPL 528 medium held without parasites (control medium, CM) for 7 days were applied to a 12% SDS gel at approximately 2.6 pg protein/lane. The same volume and approximate protein concentration of medium conditioned (PCM) for 7 days with parasite serosa was also applied to the gel. Twenty microliters (approximately 12 pg) of the supernatant from about 13 one-day-old serosas (VVTI) were applied to each lane of the gel. Molecular weight standards (MW) were placed in outer lanes. The gel was run at 50 V overnight at room temperature; it was stained in Coomassie brilliant blue R-250 and twice in silver. As in Figure 23, a 24-Kd band (double arrowheads) occurs in the PCM but not in the CM. This band also corresponds to a band in the serosa (WTI). Five additional bands (arrows) in the PCM do not occur in the CM but each corresponds to at least one band in VVTI. These bands also fall into Categories A-C of host and control hernolymph in Figure 21. The three groups are: (A) just below 205 Kd-one band; (B) between 66 and 97.4 Kd-two major bands; (C) above 45 Kd-one maior and one minor band. Figs. 25, 26. Comparisons of polypeptides in Biosteres longicaudatus first instars (LI) and their serosas (TI) from newly pupariated hosts (in vivo, W) (Fig. 25) and in first instars (L7) and their serosas (T7) reared in vitro (VT) for 7 days (Fig. 26). Ten micrograms of protein for each sample were loaded on each lane of a 12% SDS gel. Molecular weight markers (MW) were applied to the outer lanes. The gels were run at 120 V and 4°C for 5 h. Bands were visualized with silver stain. Serosa and larvae share several bands or group of bands some of which are similar to those in Categories A-D and F of A. suspensa hemolymph (Fig. 21). The approximately 24-Kd band occurs in both serosa and larval samples in vivo (Fig. 25) and in vitro (Fig. 26) (double arrowheads) and also corresponds to the 24-Kd band in host hemolymph (Figs. 21, 22). Serosas and larvae in vivo (Fig. 25) also share several bands with their counterparts in vitro (Fig. 26) Ultrastructureand Polypeptides of Serosal Cells MW VVT1 VVL1 215 MW 2OOKd 97.4Kd 97.4Kd 66.2Kd 66.2Kd 43.OKd 43.OKd 3 1.OKd 2 1.5Kd 14.4Kd 31.OKd 2 1.5Kd 14.4Kd 200.OKd + 116.3Kd + 97.4Kd + 200.OKd 66.2Kd + 66.2Kd 43.OKd -+ 43.OKd 116.3Kd 97.4Kd 31 .OKd 2 1.5Kd 14.4Kd LITERATURE CITED 1. Ivanova-Kasas OM: Polyembryony in insects. In: Developmental Systems: Insects. Counce SJ, Waddington CH, eds. Academic Press, Nay York, pp 243-271 (1972). 2. Joiner RL, Vinson SB, Benskin JB: Teratocytes as a source of juvenile hormone activity in a parasitoid-host relationship. Nature New Biol245, 120 (1973). 3. Wag0 H, Tanaka T: Ultrastructural and functional maturation of teratocytes of Apunfeles kariyui. Arch Insect Biochem Physiol23,187-197 (1990). 4. Kitano H, Wag0 H, Arakawa T: Possible role of teratocytes of the gregarious parasitoid, ApunfeIes glomerutu in the suppression of phenoloxidase activity in the larval host, Pieris rupae crucivoru. Arch Insect Biochem Physiol13, 177-185 (1990). 5. Zang D, Dahlman DL: Microplitis croceipes teratocytes cause developmental arrest of Heliofhis virescens larvae. Arch Insect Biochem Physiol12,51-61 (1989). 216 Lawrence 6. Strand MR, Vinson SB, Nettles WC, Jr, Xie ZN: In vitro culture of the egg parasitoid Telonornus heliothidis: The role of teratocytes and medium composition in development. 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