Molecular, Cellular and Developmental Biology Program, Division of Biology, 232 Ackert Hall, Kansas State University, Manhattan, KS 66506, USA1
Author for correspondence: Rollie J. Clem. Fax +1 785 532 6653. e-mail rclem{at}ksu.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The progress of AcMNPV infection within the host has been best described for the cabbage looper caterpillar Trichoplusia ni. These T. ni caterpillars are extremely susceptible to AcMNPV infection, with the injection LD50 (dose required for 50% lethality) being reported as between 1 and 10 p.f.u. per larva (Clem & Miller, 1993 ) or less (Washburn et al., 1998
) and a feeding LD95 of 15 occlusion bodies per larva (Washburn et al., 1998
). Virus can be observed in the haemolymph of the infected insect within 10 h following ingestion of occluded virus and blood titres rise rapidly over the first 24 h of infection before reaching a plateau (Granados & Lawler, 1981
). Infection of haemocytes and trachea can be detected 1224 h after oral infection, followed by the spread of the virus to the epithelial and fat body tissues (Barrett et al., 1998
; Keddie et al., 1989
). Death of the caterpillar occurs four days after the initial infection and the time to death is only slightly shortened if the virus is delivered by injection (Washburn et al., 1995
). Studies of developmental and selected resistance in a number of susceptible caterpillar species suggest that the sloughing of infected midgut cells is the primary defence against baculovirus infection and that few insects are capable of resisting the virus once it crosses the midgut basal lamina (Volkman, 1997
; Washburn et al., 1998
).
The AcMNPV infection process has been examined in detail in only a handful of other lepidopteran species, including the tobacco budworm (Heliothis virescens), corn earworm (Helicoverpa zea) and armyworm (Pseudaletia unipuncta). The infection process of AcMNPV in H. virescens follows a pattern similar to that in T. ni, with a longer period between infection of midgut cells and movement of the virus across the basal lamina and a slightly slower progression of the virus through the insect tissues (Washburn et al., 1995 ). Injection of AcMNPV into P. unipuncta also produces an infection pattern resembling that of T. ni; however, this species is highly resistant to oral infection (Volkman, 1997
). Unlike the aforementioned species, H. zea is able to limit the progression of the virus after it has crossed the midgut by encapsulating the infected tracheal cells with haemocytes and by using the haemocytes, which appear to be resistant to infection, to clear virions from the blood (Washburn et al., 1996
; Trudeau et al., 2001
).
In the course of examining the role of apoptosis in insect defence, we noted that fall armyworm (Spodoptera frugiperda) caterpillars undergo a somewhat different response than T. ni to injection of AcMNPV budded virus. In this study, we used a recombinant strain of AcMNPV expressing enhanced green fluorescent protein (eGFP) to document the course of infection in the haemolymph, epidermal and fat body tissues of S. frugiperda and to examine the role of haemocytes in the dissemination of baculovirus infection following intrahaemocoelic injection.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Injections.
T. ni and S. frugiperda caterpillars were injected with measured doses of budded virus diluted in complete TC-100 media within 12 h of molting to the penultimate (S. frugiperda) or final (T. ni) instar. T. ni caterpillars were injected in the final instar to reduce injection-related mortality, which was less than 1% in each species. Intrahaemocoelic injections were performed with a Hamilton 26 gauge syringe by inserting the needle between the first and second abdominal segment of the caterpillar and depositing the virus at the rear of the abdomen.
Haemolymph extraction and examination.
For each experiment, cohorts of 10 caterpillars were examined every 24 h for external eGFP fluorescence and then sacrificed. Caterpillars were chilled for 30 min at 4 °C before haemolymph extraction. Haemolymph was collected by cutting an anal proleg and allowing the caterpillar to bleed on to a section of parafilm. Of the haemolymph, 5 µl was then transferred into 200 µl of ice-cold anti-coagulant buffer (4 mM NaCl, 40 mM KCl, 1·7 mM PIPES, 146 mM sucrose, 0·1% polyvinyl pyrrolidine, 8 mM EDTA, 9·5 mM citric acid and 27 mM sodium citrate). Samples were centrifuged at 320 r.p.m. and 4 °C to separate the cells from the haemolymph. Cells were fixed in 2% paraformaldehyde and examined on a Nikon Eclipse TE200 inverted microscope for the presence of eGFP fluorescence. A minimum of 100 cells was scored from each sample. Cell-free haemolymph samples were stored at -80 °C for determination of virus levels. Inspection of infected caterpillars was performed using a Nikon SMZ-10A binocular microscope equipped for eGFP fluorescence. Internal examinations were performed on a subset of each cohort of caterpillars to confirm infection status. Fat body was removed and viewed using the inverted microscope described above.
Haemolymph virus assay.
Samples of haemolymphanti-coagulant mixture were serially diluted 10-fold in TC-100 media containing 10% FBS and added to 96-well plates seeded with TN-368 cells. For each set of wells, the highest dilution displaying eGFP expression from infected cells was recorded. Three replicates were scored for each sample and the mean highest dilution at which virus could be detected in the blood of the 10 insects in each cohort was calculated.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
T. ni caterpillars injected with 4x103 p.f.u. of vHSGFP displayed strong overall external eGFP fluorescence and strong internal fluorescence in the epithelial, fat body and tracheal tissue at 24 h after injection (data not shown). T. ni caterpillars injected with 40 p.f.u. of vHSGFP showed a reduced level of infection 24 h after injection, with most insects displaying scattered spots of infection internally and externally throughout the body but with a minority (6 of 20 caterpillars) having no external eGFP expression and no obvious internal sign of infection. However, by 48 h post-injection, all caterpillars receiving 40 p.f.u. of virus exhibited strong eGFP expression both internally and externally (data not shown).
T. ni injected with either 40 or 4x103 p.f.u. of vHSGFP showed nearly simultaneous expression of eGFP in epithelia, tracheal epithelia, haemocytes and fat body by 18 h post-injection. The majority of initial foci of infection in the fat body occurred in close proximity to the trachea but without extensive infection of the tracheal epithelium. Externally, foci initially appeared to be randomly distributed throughout the epithelia but, by 24 h, infection was strongest in the thoracic legs, prolegs and the mid-region of each abdominal segment.
At 24 h after injection, 30% of the S. frugiperda caterpillars receiving 2x105 p.f.u. of virus showed significant external infections, with large numbers of scattered foci consisting of one or a few cells (Table 1 and Fig. 3
). The remaining caterpillars either had no obvious external sign of infection or had only one or a few isolated foci of infection. The majority of the initial foci of infection were concentrated in the thoracic legs and ventral abdomen during the first 48 h after injection, with little eGFP expression occurring dorsally until the infection had become well established. Once the infection had become established, eGFP expression increased rapidly throughout the insect until by 72 h, 90% of the caterpillars displayed solid eGFP expression throughout the epithelium and fat body (Table 1
). Caterpillars injected with 2x104 p.f.u. showed little or no infection 24 h after injection but, by 48 h, 50% of the caterpillars displayed multiple foci of infection and, by 96 h, 85% of the caterpillars were showing solid eGFP expression throughout their tissues (Table 1
).
|
|
In contrast to infection in T. ni where eGFP expression appeared almost simultaneously in a number of different tissues, eGFP expression in S. frugiperda was not detected outside of the fat body between 12 and 18 h after injection. However, by 1824 h, eGFP expression could be observed externally within the body wall of infected S. frugiperda and internally in the tissues surrounding the spiracle entrances. By this time, infection within the fat body had spread from the initial foci to form web-like patterns of eGFP expression. eGFP-positive tracheal epithelial cells could also be observed at 1824 h after injection.
Haemolymph virus levels and haemocyte infection
T. ni caterpillars injected with 4x103 p.f.u. of vHSGFP exhibited a rapid build-up of virus in the haemolymph by 24 h after injection, with virus levels remaining high throughout the course of the infection (Fig. 4A). Similarly, by 24 h, the proportion of eGFP-positive haemocytes in these caterpillars had reached 57% and, by 48 h, eGFP could be detected in 90% of haemocytes (Fig. 5A
). T. ni caterpillars injected with 40 p.f.u. of vHSGFP displayed a slower initial build-up of infection. At the 24 h time-point, the proportion of eGFP-positive haemocytes in T. ni caterpillars injected with 40 p.f.u. was significantly lower than in caterpillars injected with 4x103 p.f.u. (Fig. 5A
). The levels of virus among these caterpillars were high 24 h after injection but displayed a lower mean and a greater degree of variation than the levels of virus in caterpillars injected with 4x103 p.f.u. (Fig. 4A
). However, by 48 h, these caterpillars had similarly high levels of eGFP-positive haemocytes and haemolymph virus levels.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
S. frugiperda, in contrast, is less susceptible to AcMNPV. The dose of virus required to kill 50 and 95% of the S. frugiperda caterpillars was in each case 1000 times greater than the dose required to kill an equal proportion of T. ni caterpillars. S. frugiperda caterpillars injected with vHSGFP showed a slower increase in the amount of budded virus in the haemolymph than T. ni caterpillars. Expression of eGFP was also slow to appear in the haemocytes of infected S. frugiperda caterpillars and significant infection of haemocytes was not observed until fairly late in the infection process. Although the proportion of infected haemocytes increased steadily throughout the course of the infection, by 120 h post-injection, at which time the S. frugiperda larvae were beginning to die, the proportion of eGFP-positive haemocytes was still significantly less than the proportion of infected haemocytes observed in T. ni at 48 h post-injection. Despite the slow build-up of virus in the haemolymph of S. frugiperda, the infection managed to spread into the epidermal and fat body tissues of S. frugiperda caterpillars relatively rapidly, although the rate at which these tissues were infected was dependent upon the dose of the initial inoculum.
Another significant difference we observed between the two species was the order in which tissues became infected. In T. ni, eGFP expression appeared simultaneously in a number of different susceptible tissues, including fat body, tracheal and body wall epithelium and haemocytes at around 1218 h post-injection. However, in S. frugiperda, it was clear that the fat body was the tissue where infection first occurred. Infection appeared to spread from the fat body to tracheal and body wall epithelium by 1824 h post-injection but significant numbers of haemocytes did not become infected until much later.
Earlier studies of the pathology of AcMNPV in T. ni and H. virescens have involved either oral or tracheal inoculation and have reported a steady progression of infection from the midgut into the trachea and haemocoel and from there into the epidermis and fat body. In our examination of the pathology of AcMNPV in S. frugiperda, we observed that the initial foci of infection occurred well within the fat body of the caterpillars before infection could be detected in the trachea, haemocytes or body wall. The mechanism by which the virus penetrated the fat body is unclear; however, the close proximity of trachea to many of the foci of infection suggest that the virus may be able to utilize the tracheoblast cells as a conduit to travel into and infect susceptible tissues. The lack of eGFP expression within the tracheal system at this early time indicates that such a transportation mechanism would have to occur without infecting the tracheoblast cells or by using tracheoblast cells that are infected but in which viral DNA is not being transcribed, since the heat shock promoter used to drive eGFP expression in this study is expressed constitutively in most, if not all, cell types. The process of using infected tracheoblast cells to rapidly transport other virions deeper into the host tissues has been suggested as a mechanism by which MNPV baculoviruses are able to rapidly cross the midgut basal lamina before the midgut epithelium is sloughed off (Grandos, 1978 ; Granados & Lawler, 1981
; Washburn et al., 1999
) and enter other organs in T. ni (Engelhard et al., 1994
). A similar mechanism may be involved during the infection of S. frugiperda, allowing the virus to rapidly penetrate deep into the tissues of its host during the very early stages of the infection.
In general, the mechanisms by which a caterpillar could resist baculovirus injected into its body fall into two basic classes: those that operate at the level of the infected cell and those that operate at the level of the organism. The large difference in susceptibility to AcMNPV infection between T. ni caterpillars and S. frugiperda and the slower progression of the infection in the latter species strongly suggests that S. frugiperda possesses a partially effective mechanism of virus resistance, capable of preventing infection from low doses of virus but incapable of stopping infection once the virus has become established. In particular, the observation that injecting 2x105 p.f.u. of virus into S. frugiperda caterpillars is sufficient to initiate only 3050 initial foci of infection indicates that either large quantities of virus are inactivated and/or removed from the haemolymph following injection or that S. frugiperda cells are much more resistant to AcMNPV infection than are T. ni cells. Stocks of AcMNPV titrated on S. frugiperda cell lines produce a virus titre approximately 40 times lower than when titrated on T. ni cells (Danyluk & Maruniak, 1987 ), an observation that suggests that a cellular-level resistance mechanism may be at least partially responsible for the difference in susceptibility between these two caterpillar species. However, of the cellular mechanisms of resistance to baculovirus infection that have been described, including apoptosis (Clem & Miller, 1993
), protein synthesis shutdown (Du & Thiem, 1997
) and incompatibility between virus replication machinery and the host cell (Croizier et al., 1994
), none appear to be involved in limiting the ability of S. frugiperda caterpillars to be infected with AcMNPV.
Organism-level resistance mechanisms that would block infection by injected virus have been described for the species H. zea (Trudeau et al., 2001 ) but there is little indication that similar mechanisms are functioning within S. frugiperda. S. frugiperda haemocytes do not form melanotic capsules around infected tissue and, unlike H. zea haemocytes, a large proportion of the S. frugiperda haemocytes do eventually become infected. The more gradual increase in the proportion of infected haemocytes in S. frugiperda relative to T. ni suggests that these cells may have a role in resisting infection and removing virus from the blood, similar to the situation in H. zea (Trudeau et al., 2001
). However, alternative explanations, such as rapid replacement of infected haemocytes, removal of infected haemocytes by phagocytic tissue or slower up-take of virus by the haemocytes cannot be ruled out.
In summary, we have provided evidence that the establishment of systemic infection by AcMNPV in S. frugiperda does not require significant infection of haemocytes, despite the injection of large amounts of budded virus directly into the haemolymph. Instead, the first tissue infected is the fat body, followed by epidermal cells lining the trachea and body wall. Around 50 % of the haemocytes eventually become infected but most do not become infected until very late in the infection process after the virus has already spread to other tissues. In this study, we infected caterpillars by intrahaemocoelic injection in order to examine virus pathogenesis and organism-level resistance without the complications of the midgut barrier. In a natural setting, caterpillars would be infected by feeding on occluded virus particles. It will thus be of interest to compare these results with the pattern of infection that is seen following the establishment of infection by feeding of occluded virus.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clem, R. J. & Miller, L. K. (1993). Apoptosis reduces both the in vitro replication and the in vivo infectivity of a baculovirus. Journal of Virology 67, 3730-3738.[Abstract]
Croizier, G., Croizier, L., Argaud, O. & Poudevigne, D. (1994). Extension of Autographa californica nuclear polyhedrosis virus host range by interspecific replacement of a short DNA sequence in the p143 helicase gene. Proceedings of the National Academy of Sciences, USA 91, 48-52.[Abstract]
Danyluk, G. M. & Maruniak, J. E. (1987). In vivo and in vitro host range of Autographa californica nuclear polyhedrosis virus and Spodoptera frugiperda nuclear polyhedrosis virus. Journal of Invertebrate Pathology 50, 207-212.[Medline]
Du, X. & Thiem, S. M. (1997). Responses of insect cells to baculovirus infection: protein synthesis shutdown and apoptosis. Journal of Virology 71, 7866-7872.[Abstract]
Engelhard, E. K., Kam-Morgan, L. N. W., Washburn, J. O. & Volkman, L. E. (1994). The insect tracheal system: a conduit for the systemic spread of Autographa californica M nuclear polyhedrosis virus. Proceedings of the National Academy of Sciences, USA 91, 3224-3227.[Abstract]
Flipsen, J. T. M., Martens, J. W. M., van Oers, M. M., Vlak, J. M. & van Lent, J. W. M. (1995). Passage of Autographa californica nuclear polyhedrosis virus through the midgut epithelium of Spodoptera exigua larvae. Virology 208, 328-335.[Medline]
Granados, R. R. (1978). Early events in the infection of Heliothis zea midgut cells by a baculovirus. Virology 90, 170-174.[Medline]
Granados, R. R. & Lawler, L. A. (1981). In vivo pathway of Autographa californica baculovirus invasion and infection. Virology 108, 297-308.
Granados, R. R. & Williams, K. A. (1986). In vivo infection and replication of baculoviruses. In The Biology of Baculoviruses , pp. 89-108. Edited by R. R. Granados & B. A. Federici. Boca Raton, FL:CRC Press.
Keddie, B. A., Aponte, G. W. & Volkman, L. E. (1989). The pathway of infection of Autographa californica nuclear polyhedrosis virus in an insect host. Science 243, 1728-1730.[Medline]
Kirkpatrick, B. A., Washburn, J. O., Engelhard, E. K. & Volkman, L. E. (1994). Primary infection of insect tracheae by Autographa californica M nuclear polyhedrosis virus. Virology 203, 184-186.[Medline]
O'Reilly, D. R., Miller, L. K. & Luckow, V. A. (1992). Baculovirus Expression Vectors: a Laboratory Manual. New York: W. H. Freeman.
Trudeau, D., Washburn, J. O. & Volkman, L. E. (2001). Central role of hemocytes in Autographa californica M nucleopolyhedrovirus pathogenesis in Heliothis virescens and Helicoverpa zea. Journal of Virology 75, 996-1003.
Volkman, L. E. (1997). Nucleopolyhedrovirus interactions with their insect hosts. Advances in Virus Research 48, 313-348.[Medline]
Wang, X., Ooi, B. G. & Miller, L. K. (1991). Baculovirus vectors for multiple gene expression and for occluded virus production. Gene 100, 131-137.[Medline]
Washburn, J. O., Kirkpatrick, B. A. & Volkman, L. E. (1995). Comparative pathogenesis of Autographa californica M nuclear polyhedrosis virus in larvae of Trichoplusia ni and Heliothis virescens. Virology 209, 561-568.[Medline]
Washburn, J. O., Kirkpatrick, B. A. & Volkman, L. E. (1996). Insect protection against viruses. Nature 383, 767.
Washburn, J. O., Kirkpatrick, B. A., Haas-Stapleton, E. & Volkman, L. E. (1998). Evidence that the stilbene-derived optical brightener M2R enhances Autographa californica M nucleopolyhedrovirus infection of Trichoplusia ni and Heliothis virescens by preventing sloughing of infected midgut epithelial cells. Biological Control 11, 58-69.
Washburn, J. O., Lyons, E. H., Haas-Stapleton, E. J. & Volkman, L. E. (1999). Multiple nucleocapsid packaging of Autographa californica nucleopolyhedrovirus accelerates the onset of systemic infection in Trichoplusia ni. Journal of Virology 73, 411-416.
Received 29 January 2002;
accepted 27 March 2002.