Department of Virology, Wageningen Agricultural University, Binnenhaven 11, 6709 PD, Wageningen, The Netherlands1
EMBRAPA/Hortaliças, 70359-970, PO Box 218, Brasília, DF, Brazil2
Author for correspondence: Dick Peters. Fax +31 317 484820. e-mail Dick.Peters{at}viro.dpw.wau.nl
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Abstract |
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Introduction |
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TSWV has a unique vector relationship with thrips in that the virus is acquired by the larvae but not by adults, while the ability to acquire virus rapidly declines during larval development (van de Wetering et al., 1996 ; Nagata et al., 1999
). After a temperature-dependent latent period, larvae at the end of their second stage and adults are able to transmit the virus (Wijkamp et al., 1993
). Until now, only eight thrips species of the genera Thrips and Frankliniella have been reported as vectors of tospoviruses (Mound, 1996
; Webb et al., 1998
).
Interspecific as well as intraspecific differences were found in vector competence in a study using four tospoviruses and four thrips species of six distinct populations (Wijkamp et al., 1995 ). Of the four viruses studied, TSWV was transmitted by all four thrips species, while Impatiens necrotic spot virus was transmitted solely by F. occidentalis with a high efficiency. Distinct differences were found in the transmission of TSWV by four T. tabaci populations studied. Three thelytokous populations, which propagate parthenogenetically and produce a progeny of only females (Moritz, 1997
), did not transmit TSWV. An arrhenotokous population transmitted the virus with low efficiency (Wijkamp et al., 1995
). Thrips of these populations lay two different types of eggs. Fertilized eggs, which are diploid, produce females; unfertilized eggs give only males (Moritz, 1997
). These results suggested the existence of several factors regulating the vector competence to transmit TSWV. Analysis of these factors may provide keys to explain the differences in transmission competence of the various vector species and the differential transmission of the various TSWV isolates by its vectors.
The amount of TSWV present in the adult thrips body is probably one of the main factors that affects vector competence and efficiency (Wijkamp et al., 1995 ; van de Wetering et al., 1996
). Virus accumulation, most likely, depends on the invasion and rate of replication in thrips tissues, presumably the midgut and salivary glands. Besides the accumulation of virus in these tissues, barriers, which permit or prevent virus translocation, may also play an important role (Nagata et al., 1999
). The existence of such barriers may explain the failure of thelytokous T. tabaci populations to transmit TSWV, as the primary cell cultures of these populations support replication of this virus (Nagata et al., 1997
). This observation suggests that thelytokous T. tabaci are susceptible to TSWV, but lack the ability to become transmitters.
Here, we report a comparative study performed to elucidate the stepwise development of the infection in the midgut and salivary glands of an efficiently transmitting F. occidentalis population and a non-transmitting thelytokous T. tabaci population.
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Methods |
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The transmission capacity of each newly emerged adult was tested using the local lesion assay on petunia leaf discs (Wijkamp & Peters, 1993 ). They were individually placed on a leaf disc in an Eppendorf tube for 2 days at 25 °C. The discs were then transferred to a 24-well plate and incubated for 3 days on water for the development of local lesions.
Accumulation of virus in thrips was measured by ELISA targeting the nucleocapsid (N) protein. The reaction was amplified as previously described (Wijkamp et al., 1995 ). Groups of five larvae, sampled at 0, 2, 4, 12, 24, 48, 72 h post-acquisition (p.a.), of prepupae, and of 2-day-old adults were triturated with 100 µl sample extraction buffer (Wijkamp et al., 1995
). Half of these extracts were used in ELISA and the other half stored to repeat the ELISA when necessary.
Midgut preparation and whole mount immunofluorescent staining (WMIS).
Dissected midguts and/or salivary glands from larvae and adults were fixed with cold acetone on object glasses at -20 °C (Nagata et al., 1999 ) and incubated for 1 h in PBS pH 7·2. This buffer contained 10% BSA to block non-specific reactions. The organs were incubated with polyclonal antibodies to the N protein (2 µg/ml) raised in a rabbit. To eliminate aspecific reactions the antisera were pre-absorbed with extracts from uninfected thrips for 2 h in 10% BSAPBS (Nagata et al., 1999
). Following this incubation the preparations were overlaid with 10 µg/ml pig anti-rabbit FITC conjugate (Nordic) in 10% BSAPBS for 1 h. After washing, the preparations were mounted in CitiFluor (Agar Scientific) and studied by fluorescence microscopy (Leica, Laborlux 5).
Immunohistochemical studies of thrips.
Thrips bodies were fixed, after amputating the legs and antenna, in Bouins Hollande sublime (Smid, 1998 ) and incubated under vacuum (Nagata et al., 1999
). After fixation, the specimens were embedded in Paraplast (Oxford Labware) and cut in 5 µm thick sections, which were mounted on object glasses. The sections were deparaffinized with xylene, rehydrated and incubated in PBS. After this treatment, the preparations were incubated with pre-absorbed N protein antibodies (1 µg/ml in PBS) for 2 h, washed with PBS and incubated with pig anti-rabbit antibodies conjugated with horseradish peroxidase (5 µg/ml in PBS containing 10% normal serum) for 1 h (Dako). After washing, the sections were incubated for 5 min with a substrate solution of 0·05% (w/v) diaminobenzidine (DAB) and 0·01% (v/v) hydrogen peroxide in 50 mM TrisHCl pH 7·6. Some sections were stained with Mayers haematoxylin (Sigma) to visualize the organs in detail and localize them correctly in the immunostained tissue. The samples were then mounted with DPX (Fluka) after dehydration and studied by light microscopy.
Electron microscopy.
Whole thrips specimens or dissected midguts were fixed in a 3% paraformaldehyde2% glutaraldehyde solution for 30 min in a microwave oven with water flow, dehydrated in a series of 50100% ethanol solutions, and embedded in LR-Gold (London Resin). The resin was polymerized by UV light irradiation at -20 °C. The 6070 nm thick slices were incubated for 2 h with 0·8 or 2·0 µg/ml pre-absorbed N or viral glycoprotein (G) antibodies, respectively, washed with 30 droplets of PBS, and incubated with gold-conjugated protein A for 1 h. After washing with PBS, the sections were post-fixed in 1% glutaraldehyde, contrasted with uranyl acetate and lead citrate, and studied with an electron microscope (Philips CM 12).
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Results |
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Just after acquisition (0 h p.a.), a lower virus titre was found in T. tabaci than in F. occidentalis. This difference can be explained by the ingestion of smaller amounts of infected plant material, and thus virus, by T. tabaci, although its larvae produce larger feeding damage spots than those of F. occidentalis. A difference in the rate at which the virus is digested cannot be excluded.
Adults of the F. occidentalis population transmitted TSWV at a rate of 67·3% (206/306 individuals) and those of T. tabaci at 0% (0/456 individuals) in three independent experiments using the petunia leaf disc assay (Wijkamp & Peters, 1993 ).
Different levels of midgut infections in F. occidentalis and T. tabaci as determined by WMIS
Infection of the intestinal tract is the first discernible sign, which can lead to transmission of the virus by the vector. The midguts of larvae at 72 and 96 h p.a. (second stage larvae and prepupae) and of adults were analysed by WMIS (see Methods) for infection by TSWV since a dramatic difference in the accumulation of virus was detected by ELISA between late second instars and adults (Fig. 1). The midguts of these larvae and adults of F. occidentalis were found to be infected (Table 1
). The infection was restricted to the midgut epithelial (Mg1) and midgut muscle cells of the anterior part of the midgut (Mg1 and Mg2) in larvae 72 and 96 h p.a. No infection could be discerned in the midgut epithelium of all adults, but was observed in the muscle cells of, almost, the entire midgut (Mg1, -2 and -3). These observations confirmed previous results that the virus is eliminated from epithelial cells, but not from muscle cells during the metamorphogenic pupation processes (Nagata et al., 1999
; front cover of this issue).
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No sign of infection was found by ELISA in any of the T. tabaci adults tested, while weak but positive reactions in midgut muscle cells were found in 57 out of 89 adults using WMIS. This technique appeared to be more sensitive than ELISA to detect infection of TSWV in thrips.
Development of TSWV infection in thrips of both populations
To monitor the stepwise infection in the midgut as well as in the salivary glands, thrips of both populations were collected at 24 (1st instar), 48, 72 (2nd instar), 96 (2nd instar or prepupae), 144 h (pupae) p.a. and in the adult stage, and cut in thin longitudinal sections. These sections were serologically analysed by targeting the N protein using the DAB substrate reaction, visualizing positive reactions as dark brown precipitates. These reactions were not observed in thrips of either of the populations fed on healthy plants and studied at the same intervals.
Distinct infections were observed in the midgut epithelial cells of F. occidentalis 24 h p.a. (Fig. 2a). These infections were only noticed in the anterior midgut (Mg1), and remained in most cases restricted in this region during the whole larval development of this thrips species (Fig. 2a
, b
). After pupation, no infections could be discerned in the midgut epithelium of adults, showing that multiplication was not resumed in this renewed tissue as reported earlier. Infections in the midgut of adults were only noticed in the visceral muscle cells, mostly in the whole midgut region (Fig. 2c
). Infections of the foregut were often clearly observed in adults of F. occidentalis.
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Infection of the larvae and adults of the T. tabaci population was less pronounced (Fig. 2fj
) than in those of F. occidentalis, especially in larvae at 24 h p.a. and in adults. The infection became more prominent in the midgut of larvae at 48 (Fig. 2b
) and 72 h p.a. Since T. tabaci moulted to prepupae almost 1 day earlier (7296 h p.a.) than F. occidentalis (96144 h p.a.), elimination of epithelial cells with the virus by histolysis consequently occurred 1 day earlier. No infection could be found in the salivary glands before pupation, in the pupal stages or in adults in any of the T. tabaci thrips studied.
The development of the infection in thrips of both populations was evaluated by an infectivity index, in which 0 stands for no infection, 1 for an infection restricted to the midgut, 2 for infection of the midgut and foregut, and 3 for infection of the midgut, foregut and salivary glands. The infection rate was indexed at 24, 48, 72, 96 and 144 h p.a. and in adults using eight to 25 thrips per interval. The average values obtained (Fig. 3) were almost equal to the tendency found by ELISA for the accumulation of virus in thrips of both populations (Fig. 1
). These results show that virus accumulation and the extent to which the tissues are infected are positively correlated. The distinct vector specificity cannot, therefore, be explained by a susceptibility of the different tissues, but by an impeded migration of the virus to the salivary glands in T. tabaci.
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Although many thrips were dissected, only a small number of midgutligamentsalivary gland complexes were obtained. The first, but weak, infections in the salivary glands of F. occidentalis were observed at 72 h p.a. These infections often consisted of small patches, visible as a small row of positive signals at the connection site between the ligaments and salivary glands. Infections in the ligaments always preceded those in the salivary glands (Table 2). Infections restricted to the ligament always preceded those in the salivary gland. Infections in salivary glands were never observed when infections could not be discerned in the ligaments (Table 2
). These observations support the hypothesis that the salivary glands become infected by a process of virus migration in which the ligaments are involved.
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Electron microscopic observations on midgut cells
Infection of the midgut epithelium and muscle cells has immunohistologically been demonstrated by light microscopy in both thrips species. To elucidate the virus structures accumulating in the infected cells, ultra-thin sections of the midgut of second instar larvae 72 h p.a. and adults were studied by electron microscopy. Gold-labelled antibodies to the viral N protein and glycoproteins (G1/G2) were used to follow the virus infection process.
In F. occidentalis larvae 72 h p.a., many large aggregates of nucleocapsids were observed in the cytoplasm of the epithelial cells of the Mg1 (Fig. 4a). In two out of four thrips examined, virus particle-like structures were only found in the extracellular spaces of the basal labyrinth of the Mg1 epithelium (Fig. 4b
) and in the cytoplasm of visceral muscle cells in Mg1 (Fig. 4c
, d
). These structures were specifically labelled with antibodies to both the N protein (Fig. 4b
, c
) and the G1 and G2 proteins (Fig. 4d
), and, hence, could be identified as genuine TSWV particles. They were roughly spherical and their diameter was estimated to be approximately 100 nm (Fig. 4b
d
), similar to the size of TSWV particles seen in plants.
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No enveloped virus particles, however, were seen in the midgut muscle cells of adults, while nucleocapsid aggregates were found in these cells in two out of three F. occidentalis adults studied. The assembly of enveloped virus particles seems therefore to be a transient event in the midgut basal labyrinth and midgut visceral muscle tissues of larvae. The lack of nucleocapsid aggregates in the cytoplasm of adult midgut epithelial cells confirms the results obtained by light microscopy.
In T. tabaci, nucleocapsid aggregates, although less abundant than in F. occidentalis, were the only virus structures found in the Mg1 epithelium (Fig. 4e) and visceral muscle cells (Fig. 4f
) of larvae 72 h p.a. and in visceral muscle cells of adults. Enveloped virus particles were not observed in the midgut of these larvae and adults. From these observations, it may be concluded that the virus is less apt to escape to muscle cells or the haemocoel in T. tabaci than in F. occidentalis.
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Discussion |
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Virus could not be detected in the midgut epithelium of the adults of either population after pupation. The loss of the virus in the midgut epithelial cells may result from the elimination of the virus with these cells in the renewal processes, which take place during pupation (Müller, 1926 ). However, virus could readily be detected in the muscle cells of all three midgut regions in F. occidentalis adults, and much less in these cells of the non-transmitting T. tabaci. This large difference in muscle cell infection suggests that the virus is either translocated at a high rate from the epithelial cells to the muscle cells in F. occidentalis or that the muscle cells of this population are highly permissive for TSWV.
The salivary glands of F. occidentalis thrips, which transmitted as second instars, became infected before pupation. No or only a limited infection could be detected in these glands of larvae and adults, which fail to transmit. The early infection in the larval salivary glands strongly indicates that the virus has to reach these glands before pupation to become transmitters. This view is supported by the observation that most viruliferous F. occidentalis adults were converted to transmitters before pupation when they acquire the virus early in the first stage of their larval development (Wijkamp & Peters, 1993 ). Those adults which acquired virus but did not transmit as larvae and also not as adults in the first days after emerging, did not become transmitters even after a long period of incubation. This observation implies that the virus is translocated at a low rate, if at all, to the salivary glands in F. occidentalis adults.
A complete virus particle seems to be essential to initiate an infection in the thrips cells, as shown in primary cell cultures (Nagata et al., 1999 ). No infection occurs when these cultures are inoculated with nucleocapsid preparations. This failure has to be explained by the absence of cellular receptor binding sites for the N proteins at the cell surface. The presence of proteins in the thrips which bind to one of the G proteins has been shown (Bandla et al., 1998
; Kikkert et al., 1998
). They found a 50 and a 92 kDa protein, respectively. Recently, Medeiros et al. (2000)
demonstrated that the 50 kDa protein is a candidate for TSWV entry in the midgut.
The efficient replication of TSWV in the midgut epithelial cells may result in a rather thorough infection of the muscle cells and a timely migration of the virus to the salivary glands. This successful replication seems to be one of the factors determining the vector competence. The lower rate of virus accumulation in the midguts of the non-competent T. tabaci population compared to F. occidentalis has to be explained by a lower rate of replication and a restricted translocation of the virus. This conclusion is supported by the limited replication of the virus in primary cell cultures derived from this T. tabaci population (Nagata et al., 1999 ).
The restricted virus spread in the visceral muscle tissue of T. tabaci (Fig. 3) implies that the virus escapes less efficiently from its midgut epithelium than in F. occidentalis. Assembly of virus particles at the plasmalemma of the midgut epithelial cells may be essential to enter midgut muscle cells as the acquisition of an envelope plays a fundamental role in virus release and re-entry into neighbouring cells. Since the midgut muscle cells of T. tabaci occasionally become infected, it is tempting to speculate that lower amounts of complete virus particles are assembled in the T. tabaci population than in F. occidentalis. To infect the muscle cells, the virus should pass the basal lamina, which is a thick extracellular matrix lying on the basal membrane of the midgut (Lerdthusnee et al., 1995
; Kaslow & Welburn, 1997
). Still a mechanism to explain how the virus particle passes this matrix is not fully elucidated; it is well known that the basal lamina acts as physical barrier for virus circulation. The thickness of this layer may play a role as a barrier regulating the transmission efficiency, as shown for La Crosse virus (LaCV; Grimstad & Walker, 1991
). Paulson et al. (1989)
demonstrated that the transmission of LaCV by Aedes triseriatus was primarily controlled by a midgut escape barrier, which was partially overcome by introducing the virus directly in the haemocoel. This technique could not successfully be used in our study.
Virus infection was readily detected in the ligaments and salivary glands in the larvae and adults of competent F. occidentalis. The observation that infection in the ligament preceded the infection of the salivary glands, and that the salivary gland infection was always accompanied by ligament infection (Table 2) strongly suggests that virus migration to the salivary glands occurs through this tissue. TSWV had been thought to migrate, like other arboviruses, from the midgut to the salivary glands through the haemocoel, although TSWV particles have not yet been encountered in the haemolymph in any study (Ullman et al., 1995
). The failure of thrips to become viruliferous after injecting adults with infectious virus particles (data not shown) supports the idea that the virus in the haemolymph does not serve as source for infection of the salivary glands.
The results obtained show that vector competence is determined by the degree to which the salivary glands become infected. Partial or weak infections in salivary glands rarely lead to virus transmission (Nagata et al., 1999 ). A heavy infection in the salivary glands and transmission (50% of male individuals) were observed for the males of an arrhenotokous T. tabaci population, while the salivary glands of its non-transmitting females were not infected (data not shown). This observation suggests that the absence of infection of the salivary glands of the poor transmitting population is due to the failure to deliver the virus to this organ before pupation.
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Footnotes |
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References |
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Received 6 July 2001;
accepted 13 November 2001.