|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online June 26, 2009
Journal of Experimental Biology 212, 2261-2268 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.025718
A supracellular system of actin-lined canals controls biogenesis and release of virulence factors in parasitoid venom glands
1 Biology Department MR526, City College of the City University of New York,
138th street and Convent Avenue, New York, NY 10031, USA
2 Department of Entomology, University of Kentucky, Lexington, KY 40546,
USA
* Author for correspondence (e-mail: sgovind{at}ccny.cuny.edu)
Accepted 27 April 2009
 |
Summary |
|---|
 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Key words: host, pathogen, parasitoid, venom gland, virulence, brush border, canal
|
INTRODUCTION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
).
During oviposition, endoparasitic Hymenoptera produce and deliver venom,
mutualistic viruses, polydnaviruses (PDVs) or virus-like particles (VLPs) into
their hosts. These factors are produced in the female reproductive tract or
within organs associated with it. Based on ultrastructural studies, both PDVs
and VLPs are described as virus-like, but they have distinct morphology, and
are produced in different tissues. Both PDVs and VLPs effectively block the
activation of the host's immune defenses (notably encapsulation and
melanization), conferring evolutionary advantage to parasitoids
(Rizki and Rizki, 1990;
Chiu et al., 2001;
Whitfield and Asgari, 2003;
Labrosse et al., 2005;
Morales et al., 2005;
Pennacchio and Strand,
2006).
In contrast to PDVs, VLPs appear not to encapsidate nucleic acid.
Furthermore, whereas PDVs are synthesized and assembled in the nuclei of calyx
cells, VLPs are detected in the lumen of the venom gland and reservoir
(Webb and Strand, 2005;
Morales et al., 2005;
Chiu et al., 2006).
The biogenesis and origin of PDVs has been the subject of much
investigation. Recent characterization of polydnaviral genomes
(Webb and Strand, 2005) has
provided insight into their relationship with their respective wasps. Although
the relationship of PDVs with their wasps is somewhat controversial, there is
general agreement that PDVs are vectors that deliver the wasp's genetic
material into the host and are likely to have evolved from a mutualistic or
symbiotic ancestral insect virus (Federici
and Bigot, 2003; Whitfield and
Asgari, 2003). The biogenesis and composition of VLPs are only now
being unraveled (see below), and their origin is not well understood.
Campoletis sonorensis (Ichneumonidae), a parasitoid of
lepidopteran pests of corn, cotton and several vegetable crops, has a
well-studied PDV (Campoletis sonorensis ichnovirus; CsIV), which replicates
from an integrated provirus in the ovarian calyx cells
(Stoltz and Vinson, 1979;
Webb and Strand, 2005). The
contents of C. sonorensis venom gland are, however, less well
characterized, although available evidence
(Webb and Summers, 1990;
Luckhart and Webb, 1996)
suggests that, like the VLPs of Drosophila parasitoids
(Rizki and Rizki, 1990),
C. sonorensis venom proteins perform immune suppressive functions,
which are important for parasitoid survival.
Wasps of the genus Leptopilina spp. infect Drosophila
spp. and produce VLPs (Rizki and Rizki,
1990; Labrosse et al.,
2005; Morales et al.,
2005; Schlenke et al.,
2007). The venom glands of the highly-virulent sister species,
L. heterotoma and L. victoriae, produce polymorphic, 300
nm-wide, spiked VLPs (Rizki and Rizki,
1990; Morales et al.,
2005). VLP spikes interact with immune-active blood cells
(lamellocytes) of the host, leading to their lysis
(Rizki and Rizki, 1990;
Morales et al., 2005;
Chiu et al., 2006). In each
case, lamellocyte lysis is attributed to the presence of a protein of about 40
kDa [p40 in L. heterotoma and p47.5 in L. victoriae
(Chiu et al., 2006)]. The
activities of venom gland factors, including VLPs
(Chiu and Govind, 2002),
provide one possible explanation for the broad host range observed for L.
heterotoma (Schlenke et al.,
2007).
In addition to VLPs, a filamentous virus has been identified in the
ovipository apparatus of specific strains of L. boulardi females.
Their presence has been correlated to the development of superparasitic
behavior by the infected parasitoid wasps
(Varaldi et al., 2006). L.
boulardi-17, is also a highly virulent and pathogenic wasp strain that
produces a variety of products including VLPs in its venom gland. Unlike
L. heterotoma venom, L. boulardi-17 venom is unable to lyse
host lamellocytes. The host range of L. boulardi-17 is restricted,
relative to that of L. heterotoma. Significantly, these wasps with
different infection strategies yield remarkably distinct host responses, as
measured by genome-wide gene expression changes
(Schlenke et al., 2007).
Thus, the female reproductive tracts of parasitoid wasps, and the organs associated with it, represent a privileged environment for the production of a variety of virulence factors. These organs also house microscopic entities that play a critical role in host–parasitoid interactions. The characterization of these organs is, therefore, important for understanding not only the parasitoid–host interaction in the context of the host's immune physiology, but also the role of VLPs, viruses and symbionts in the co-evolution of the host and its parasitoids.
To understand the biogenesis and nature of virulence factors produced in venom glands, we compared the structures of these organs from parasitoid species that produce either PDVs or VLPs. We show that, regardless of their infection strategy, venom glands from all the examined species have a supracellular system of canals. Within the secretory cell itself, the membrane surface of the proximal end of each canal has a brush border morphology, lined with bundles of polymerized actin. We infer the physiological function of this novel canal system from a study of the close and continuous association of p40, an abundant and functionally important VLP component of L. heterotoma.
|
MATERIALS AND METHODS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
);
L. victoriae by Morales et al.
(Morales et al., 2005); and
G. xanthopoda by Melk and Govind
(Melk and Govind, 1999). Wasp
species were maintained on the y w strain of Drosophila
melanogaster and have been previously described
(Morales et al., 2005;
Chiu et al., 2006;
Sorrentino et al., 2002).
C. sonorensis was reared on Heliothis virescens.
Venom gland dissection and immunolocalization
Venom glands from female wasps of the different species were dissected
under a stereomicroscope in PBS (pH 7.0), fixed in 3% paraformaldehyde (in 2%
sucrose PBS solution, pH 7.6), rinsed 3 times in PBS, stained with TRITC
(tetramethyl rhodamine isothiocyanate)-labeled phalloidin for 20 min to
highlight actin filaments (0.5 µg ml–1; Invitrogen
Molecular Probes, Eugene, OR, USA). Samples were rinsed three times in PBS,
and counterstained with the nuclear stain DAPI
(4',6-diamidino-2-phenylindole; 300 nmoll–1;
Invitrogen), and mounted in Vectashield (Vector Laboratories, Burlingame, CA,
USA).
L. heterotoma VLP protein p40 distribution was visualized with a
mouse polyclonal antiserum obtained after injection of purified VLP
preparations. It has been shown to be effective in a number of studies. (1)
This serum specifically reacts with the most abundant 40 kDa protein in
denatured protein preparations of purified L. heterotoma VLPs, and
cross reacts with 47.5 kDa protein from purified L. victoriae VLPs
(Chiu et al., 2006). (2)
Anti-p40 antigen is localized to the spikes of mature L. heterotoma
or L. victoriae VLPs, as well as on (or in the vicinity of) VLPs at
different stages of biogenesis from the venom gland [transmission electron
microscopy studies (Chiu et al.,
2006)]. (3) Anti-p40 antibody reacts with hemocytes from infected
animals, but not with hemocytes from control animals
(Chiu et al., 2006), consistent
with the presence of intact VLPs within these cells
(Rizki and Rizki, 1994). (4)
In in vitro experiments, this antiserum also inhibits lysis of host
hemocytes, an activity previously associated with VLPs
(Rizki and Rizki, 1990;
Chiu et al., 2006). Thus
anti-p40 is used to track VLPs from initial stages of protein synthesis in
secretory cells, their biogenesis in the venom gland, and after oviposition
within host hemocytes.
After fixation, samples for immunolocalization were permeabilized with 0.5%
Triton X-100 in PBS (pH 7.4) at 4°C, rinsed three times and incubated with
blocking solution (2% BSA, 0.01% Tween20 in PBS, pH 7.4) for 30 min at
37°C, and incubated with anti-p40 antibody (1:500 dilution in blocking
solution) (Chiu et al., 2006)
for 2 h at 37°C. Samples were rinsed five times with blocking solution and
incubated with a FITC (fluorescein isothiocyanate)-conjugated anti-mouse
secondary antibody (1:200 in blocking solution; Invitrogen) for 1 h at
37°C. Samples were then counterstained with TRITC-phalloidin and DAPI and
mounted in Vectashield. Samples were viewed with a Zeiss LSM510 confocal
microscope at room temperature.
Three-dimensional reconstructions
Z stacks of confocal images were loaded to Amira 3.1.1 (Mercury Computer
Systems,
www.tgs.com),
the appropriate voxel size was chosen and images were processed to obtain the
isosurfaces display. Image resampling was performed to obtain the desired
resolution in order to emphasize the most important features in the image.
Images were saved as tagged image file format files with Adobe Photoshop
(6.0).
|
). Images
were collected with a Zeiss transmission electron microscope (EM902) at room
temperature.
Cytochalasin treatment
Ten L. heterotoma-14 female wasps were fed cytochalasin D (1 mg
ml–1 diluted in a 1:1 honey:water solution) for 12 h. Venom
glands were dissected and p40 immunolocalization was performed as described
above.
|
RESULTS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
)].
These wasps and Ganaspis xanthopoda [Figitidae
(Melk and Govind, 1999;
Chiu et al., 2001)] regularly
infect larval stages of Drosophila species. The free-living adult
females (Fig. 1A–D) are
roughly half the size of the adult D. melanogaster, but significantly
smaller than adults of Campoletis sonorensis, an ichneumonid
parasitoid (Fig. 1E) that
infects some larval Lepidoptera species.
|
Confocal images of DAPI/TRITC-phalloidin-labeled venom glands show that
despite differences in size and shape, all glands share the presence of
prominent actin-rich structures inside their cells
(Fig. 2B). In venom glands of
all five wasps, two different cell types are distinguished: first, small and
narrow cells that line the lumen of the venom gland. We have previously
referred to this layer of cells as the intimal layer in venom glands of L.
heterotoma and L. victoriae
(Morales et al., 2005;
Chiu et al., 2006). This cell
layer is apposed to a second, peripheral layer of larger, evidently polyploid,
secretory cells, which contain the actin-lined canals
(Fig. 2B). A three-dimensional
reconstruction of L. heterotoma venom gland confirms that each
secretory cell only has one unbranched canal that originates roughly
perpendicular to the long axis of the venom gland
(Fig. 2C,D). The large nucleus
of the secretory cell is found peripherally, whereas nuclei of the smaller
cells of the intimal layer are located deeper within the organ
(Fig. 2D).
A brush border lines the proximal end of the canal
High magnification confocal images of the L. heterotoma gland
reveal that there are two distinct regions in the canals: the proximal portion
(proximal to site of origin), roughly 20 µm in length, positioned deep
within the secretory cell, which is strongly positive for TRITC-phalloidin
(rough canal, R), and a distal portion, roughly 10µm long, which is closer
to the lumen of the gland, where TRITC-phalloidin staining is faint (smooth
canal, S; Fig. 3A,B).
|
Detailed electron microscopy of L. victoriae
(Morales et al., 2005) and
L. heterotoma (Chiu et al.,
2006) venom glands shows a very similar organization of canal
structures in these species. The rough canal is lined by numerous microvillous
folds, each of which is 0.4 µm in length
(Fig. 3C,D, mv). This surface
organization is reminiscent of the brush borders found in intestinal and other
cells with secretory or absorptive functions. Cross sections of the rough
canal reveal that microvilli surround the lumen of the canal completely
(Fig. 3D, mv). The canal lumen
has electron-dense particles (Fig.
3D). These particles are strikingly similar to structures found in
rough canals of L. heterotoma, which, in immuno-EM studies, stain
positively with anti-p40 antibody (Fig.
4) (Chiu et al.,
2006) (Fig.
4A–C). The peripheral region of the canal lumen itself is
lined with a mesh-like membranous structure in which electron-dense particles
are also found (Fig. 3D). We
note that the `mesh' appearance may arise simply from a tangential section of
the very tips of tightly-packed microvilli, and may not be an additional
structure as interpreted here.
|
The distal, smooth portion of the canal is not lined with microvilli.
Instead, the smooth canal is lined with a chitinous layer. A closer
examination of the region where the rough region ends and the smooth region
begins, reveals an abrupt transition from the highly folded microvillar
membrane region to the tightly extended chitinous covering
(Fig. 3C,E, arrowhead). The
smooth canal opens directly into the lumen of the venom gland
(Fig. 3C,F). Thus, VLP contents
synthesized in secretory cells are released directly into the venom gland
lumen (L, Fig. 3F)
(Morales et al., 2005;
Chiu et al., 2006). The chitin
covering lining the smooth canal (Fig.
3F, arrowhead) appears to be continuous with the lining of the
venom gland lumen.
The entire canal structure consists of a merger of two distinct structural
entities: (1) the rough canal containing a highly modified membrane adapted
for efficient trafficking of the molecular components, which will presumably
assemble into VLPs, and (2) the smooth canal that may simply be an
invagination of the extracellular space that is tailored for efficient
delivery. We speculate that VLP precursors and partially assembled particles
continue to undergo assembly and maturation once components of secretory cells
are delivered into the venom gland lumen
(Morales et al., 2005;
Chiu et al., 2006)
(Fig. 3F).
Secretion dynamics of the venom protein p40
To understand the function of canals in the secretion and delivery of VLP
components, we studied the association of p40, a previously identified
virulence factor from L. heterotoma VLPs (see Materials and methods)
(Chiu et al., 2006). Immunogold
staining of p40 in the venom gland of L. heterotoma demonstrates its
association with (1) the microvillar region of the rough canals
(Fig. 4A, mv), (2) along the
wall and within the lumen of the smooth canal
(Fig. 4B), and (3) with VLP
precursors in the lumen of the venom gland
(Fig. 4C). Thus, VLP precursors
produced in secretory cells are secreted into the rough canal and are
delivered into the venom gland lumen via the smooth canal.
Indirect immunofluorescence labeling of p40 throughout the organ (Fig. 4D–H) reveals differences in p40 localization within the gland. In cells of the anterior end of the venom gland, p40 is widely distributed through the cytoplasm of the secretory cells, whereas little, if any, p40 is detected in the immediate proximity of or within the canals (Fig. 4D). In cells at the posterior end of the same gland, p40 is strongly associated with canals (Fig. 4E). At higher magnification, these posterior secretory cells also show p40 localized in scattered spots in the cytoplasm, but the staining is concentrated mainly within the canals, either colocalizing with the actin-rich microvilli or inside the lumen (Fig. 4F,G). When the venom fluid was collected on a slide and stained with anti-p40 antibody, p40 signal was strong and spotty (Fig. 4H, arrowheads). This signal probably reflects the association of p40 with VLPs as observed in transmission electron microscopy (Fig. 4C). Variations in p40 localization in the venom gland most probably reflect the physiological and functional dynamics of the organ and the association of p40 with different stages of VLP biogenesis.
In order to assess the role of the actin scaffold in the structural
maintenance of the canal system, we tested the effect of systemically
administered cytochalasin D (see Materials and methods), an inhibitor of actin
polymerization (Blankson et al.,
1995). In general, the overall integrity of the wasp venom gland
is compromised and the size of the venom gland is significantly reduced
(Fig. 4I) in treated animals.
Secretory cells in the posterior portion of such venom glands are no longer
present; here the canals, devoid of their actin sheath, hang from the inner
core of the gland in which the venom gland lumen is still outlined by cells of
the intimal layer. This observation suggests that the cuticular chitinous
lining of the smooth canals helps support these canals. p40 staining
(Fig. 4I,J) is concentrated in
the lumen of the canals, whereas the lumen of the gland is only weakly
positive for the staining. These results are consistent with the idea that the
actin-rich microvillar region, which normally surrounds the canals, regulates
the rate of molecular traffic between the secretory cell and the canal lumen.
This proximal (rough) region of the canal can, in fact, be considered a
specialized zone of the venom gland lumen itself, where VLP components
accumulate to sufficiently high concentration and undergo early steps of
assembly.
A digital reconstruction of the isosurfaces (obtained from Z stacks of confocal images; Fig. 5A,B) supports our interpretation of results from EM and confocal studies. It also confirms that the distribution of p40 is different in the anterior and posterior parts of the venom gland, as seen in Fig. 4D,E above. In cells of the posterior-most part of the gland, p40 signal is found almost exclusively around (Fig. 5C) or inside (Fig. 5C,D) the canals.
|
|
DISCUSSION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
We propose a model (Fig. 5E) for the role of canals in the assembly and delivery of virulence factors. p40 is initially produced in the perinuclear region of secretory cell cytoplasm (Fig. 5A,B). It then moves towards the secretory cell membrane in the microvillar region of the canals (Fig. 5C,D). Here, p40 is secreted into the canal lumen, passing through a putative extracellular mesh. Once in the canal lumen, p40 associates with other components to assemble into VLP precursors, which undergo additional structural changes as they are released into the venom gland lumen. Aggregates, containing p40 travel along the gland lumen to reach the reservoir, where they eventually mature into VLPs.
A novel supracellular system of canals in the venom gland of parasitoid wasps
The many species of Hymenoptera (bees, ants and wasps) vary widely in
lifestyle, behavior and geographic distribution. Virtually, all Hymenoptera
have venom glands, but the nature and function of the venom components can
vary from pain-inducing defense molecules, hormone-like development
regulators, to immune suppressive factors
(Gnatzy and Volknandt, 2000).
Despite this functional diversity, the organization of the secretory unit
appears to be conserved among species with very different lifestyles.
Secretory units from venom glands of the free-living Apis mellifera
(Roat et al., 2006), the
gall-inducer cynipoid wasps (Vårdal,
2006), and other parasitoids
(Edson et al., 1982;
Quicke et al., 1992;
Uçkan, 1999;
Gnatzy and Volknandt, 2000;
Britto and Caetano, 2003;
Wan et al., 2006;
Zhu et al., 2007) have
previously been reported. Interestingly, Noirot and Quennedey
(Noirot and Quennedey, 1974)
have described similar canals in the class III epidermal gland cells from many
insects, using conventional microscopy techniques.
Our study reveals for the first time the scope of canal organization at the whole organ level, and at the same time, correlates the ultrastructural components of the secretory unit of the venom gland to the steps of release and sub-assembly of specific VLP components. The organization of the membrane into a brush border suggests the need for efficient exchange between intracellular and extracellular environments, consistent with the secretory function of the organ. The unique organization where rough canal (brush border) invaginates into the cytoplasm of the secretory cells appears to be an adaptation to increase the surface to volume ratio of the secretory cells. By confining the membrane area that is predominantly involved in the exchange process within the cell volume, the brush border organization helps keep the size of the venom gland small.
Venom glands are easy to isolate and secretory units possess a distinctive morphology that is manipulatable by systemic administration of drugs. These features offer an opportunity to study the unique structural aspects of the canal system, and the assembly and release of particles at a whole organ level.
|
Footnotes |
|---|
|
References |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Blankson, H., Holen, I. and Seglen, P. O. (1995). Disruption of the cytokeratin cytoskeleton and inhibition of hepatocytic autophagy by okadaic acid. Exp. Cell Res. 218,522 -530.[CrossRef][Medline]
Britto, F. B. and Caetano, F. H. (2003). Ultramorphology of the venom glands and its histochemical relationship with the convoluted glands of Polistes versicolor (Hymenoptera, Vespidae). Microsc. Acta 12,473 -474.
Chiu, H. and Govind, S. (2002). Natural infection of D. melanogaster by virulent parasitic wasps induces apoptotic depletion of hematopoietic precursors. Cell Death Differ. 9,1379 -1381.[CrossRef][Medline]
Chiu, H., Sorrentino, R. P. and Govind, S. (2001). Suppression of the Drosophila cellular immune response by Ganaspis xanthopoda. Adv. Exp. Med. Biol. 484,161 -167.[Medline]
Chiu, H., Morales, J. and Govind, S. (2006).
Identification and immuno-electron microscopy localization of p40, a protein
component of immunosuppressive virus-like particles from Leptopilina
heterotoma, a virulent parasitoid wasp of Drosophila. J. Gen.
Virol. 87,461
-470.
Edson, K. M., Barlin, M. R. and Vinson, S. B. (1982). Venom apparatus of braconid wasps: comparative ultrastructure of reservoirs and gland filaments. Toxicon 20,553 -562.[Medline]
Federici, B. A. and Bigot, Y. (2003). Origin and evolution of polydnaviruses by symbiogenesis of insect DNA viruses in endoparasitic wasps. J. Insect Physiol. 49,419 -432.[CrossRef][Medline]
Gnatzy, W. and Volknandt, W. (2000). Venom gland of the digger wasp Liris niger: morphology, ultrastructure, age-related changes and biochemical aspects. Cell Tissue Res. 302,271 -284.[CrossRef][Medline]
Labrosse, C., Stasiak, K., Lesobre, J., Grangeia, A., Huguet, E., Drezen, J. M. and Poirie, M. (2005). A RhoGAP protein as a main immune suppressive factor in the Leptopilina boulardi (Hymenoptera, Figitidae)-Drosophila melanogaster interaction. Insect Biochem. Mol. Biol. 35, 93-103.[CrossRef][Medline]
Luckhart, S. and Webb, B. A. (1996). Interaction of a wasp ovarian protein and polydnavirus in host immune suppression. Dev. Comp. Immunol. 20, 1-21.[CrossRef][Medline]
Melk, J. P. and Govind, S. (1999). Developmental analysis of Ganaspis xanthopoda, a larval parasitoid of Drosophila melanogaster. J. Exp. Biol. 202,1885 -1896.[Abstract]
Morales, J., Chiu, H., Oo, T., Plaza, R., Hoskins, S. and Govind, S. (2005). Biogenesis, structure, and immune-suppressive effects of virus-like particles of a Drosophila parasitoid, Leptopilina victoriae. J. Insect Physiol. 51,181 -195.[CrossRef][Medline]
Noirot, C. and Quennedey, A. (1974). Fine structure of insect epidermal glands. Annu. Rev. Entomol. 19,61 -80.[CrossRef]
Pennacchio, F. and Strand, M. R. (2006). Evolution of developmental strategies in parasitic Hymenoptera. Annu. Rev. Entomol. 51,233 -258.[CrossRef][Medline]
Quicke, D. L. J., Tunstead, J., Vincente Falco, J. and Marsh, P. M. (1992). Venom gland and reservoir morphology in the Doryctinae and related braconid wasps (Insecta, Hymenoptera, Braconidae). Zool. Scr. 21,403 -416.[CrossRef]
Rizki, R. M. and Rizki, T. M. (1990).
Parasitoid virus-like particles destroy Drosophila cellular immunity.
Proc. Natl. Acad. Sci. USA
87,8388
-8392.
Rizki, T. M. and Rizki, R. M. (1994). Parasitoid-induced cellular immune deficiency in Drosophila. Ann. NY Acad. Sci. 712,178 -194.[CrossRef][Medline]
Roat, T. C., Cornelio Ferreira Nocelli, R. and da Cruz Landim, C. (2006). The venom gland of queens of Apis mellifera (Hymenoptera, Apidae): morphology and secretory cycle. Micron 37,717 -723.[CrossRef][Medline]
Schlenke, T., Morales, J., Govind, S. and Clark, A. G. (2007). Contrasting infection strategies in generalist and specialist wasp parasitoids of Drosophila melanogaster. PLoS Pathog. 3,1486 -1501.[Medline]
Sorrentino, R. P., Carton, Y. and Govind, S. (2002). Cellular immune response to parasite infection in the Drosophila lymph gland is developmentally regulated. Dev. Biol. 243,65 -80.[CrossRef][Medline]
Stoltz, D. B. and Vinson, S. B. (1979). Viruses and parasitism in insects. Adv. Virus Res. 24,125 -171.[Medline]
Uçkan, F. (1999). The morphology of the venom apparatus and histology of venom gland of Pimpla turionellae (L.) (Hym: Ichenumonidae) females. Tr. J. Zool. 23,461 -466.
Varaldi, J., Petit, S., Boulétreau, M. and Fleury, F. (2006). The virus infecting the parasitoid Leptopilina boulardi exerts a specific action on superparasitism behaviour. Parasitology 132,747 -756.[CrossRef][Medline]
Vårdal, H. (2006). Venom gland and reservoir morphology in cynipoid wasps. Arthropod. Struct. Dev. 35,127 -136.[CrossRef][Medline]
Wan, Z. W., Wang, H. Y. and Chen, X. X. (2006). Venom apparatus of the endoparasitoid wasp Opius caricivorae Fischer (Hymenoptera: Braconidae): morphology and ultrastructure. Microsc. Res. Tech. 69,820 -825.[CrossRef][Medline]
Webb, B. A. and Strand, M. R. (2005). The biology and genomics of polydnaviruses. In Comprehensive Molecular Insect Science (ed. L. I. Gilbert, K. Iatrou and S. S. Gill), pp.323 -360. San Diego, CA: Elsevier.
Webb, B. A. and Summers, M. D. (1990). Venom
and viral expression products of the endoparasitic wasp Campoletis
sonorensis share epitopes and related sequences. Proc. Natl.
Acad. Sci. USA 87,4961
-4965.
Whitfield, J. B. and Asgari, S. (2003). Virus or not? Phylogenesis of polydnaviruses and their wasp carriers. J. Insect Physiol. 49,397 -405.[CrossRef][Medline]
Zhu, J. Y., Ye, G. Y. and Hu, C. (2007). Morphology and ultrastructure of the venom apparatus in the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae). Micron 39,926 -933.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
