Serosal cells of Biosteres longicaudatus HymenopteraBraconidaeUltrastructure and release of polypeptides.

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.
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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 [5]. 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 [12], resulting in delayed host
larval-pupal metamorphosis [13]. Similar effects were observed after injection
of Heliothis virescens with teratocytes from the braconid parasite Microplitis uoceipes
[5]. 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 [15]. 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 [17] 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 [18] 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
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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 [21], (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 [22]. 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 [23]and of amino acids by parasite larvae [22] and eggs [24]
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
[26]. 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
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Figs. 12-16
Ultrastructure and Polypeptides of Serosal Cells
209
and hemolymph [26]. 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 [5] 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 [12] 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.
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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.
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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 [7]. 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) [27], Pieris r a p e
parasitized by Cutesia glomerata (Hymenoptera: Ichneumonidae) [20], and
Manducu sexta parasitized by C. cungregata [28], 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 [22]). 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 [29] 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
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