The
The
Balsam fir sawfly larvae were collected from balsam fir branches at the leading edges of the balsam fir sawfly population outbreak in the Corner Brook-Deer Lake region beginning in 1999 [
Evidence of NeabNPV infection was only observed in the epithelial cells of the midgut (Figures
Analysis of cytopathic effects of NeabNPV infection in
Cytopathic effects |
NeabNPV infected |
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Uninfected | 5 hpi | 8 hpi | 12 hpi | 24 hpi | 48 hpi | 72 hpi | ||
Virogenic stroma | % cells infected* | 0.00 | 55.88 | 60.98 | 76.71 | 81.82 | 91.66 | 94.59 |
Sample size | 84 | 68 | 81 | 73 | 66 | 120 | 138 | |
SD | 0.0000 | 0.5040 | 0.4939 | 0.4256 | 0.3887 | 0.2787 | 0.2292 | |
Uninfected‡ |
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5 hpi‡ |
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8 hpi‡ |
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12 hpi‡ |
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24 hpi‡ |
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48 hpi‡ |
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Nucleocapsid assembly | % cells* | 0.00 | 0.00 | 31.71 | 54.79 | 68.82 | 91.66 | 94.59 |
Sample size | 84 | 68 | 81 | 73 | 66 | 120 | 138 | |
SD | 0.0000 | 0.0000 | 0.4711 | 0.5011 | 0.4693 | 0.2787 | 0.2292 | |
Uninfected‡ |
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5 hpi‡ |
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8 hpi‡ |
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12 hpi‡ |
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24 hpi‡ |
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48 hpi‡ |
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72 hpi‡ | ||||||||
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Occlusion body formation | % cells* | 0.00 | 0.00 | 0.00 | 0.00 | 25.76 | 66.66 | 81.08 |
Sample size | 84 | 68 | 81 | 73 | 66 | 120 | 138 | |
SD | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.4497 | 0.4754 | 0.3971 | |
Uninfected‡ |
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5 hpi‡ |
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8 hpi‡ |
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12 hpi‡ |
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24 hpi‡ |
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48 hpi‡ |
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Occlusion body maturation | % cells* | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 8.69 | 48.65 |
Sample size | 84 | 68 | 81 | 73 | 66 | 120 | 138 | |
SD | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.0000 | 0.6642 | 0.5067 | |
Uninfected‡ |
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5 hpi‡ |
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8 hpi‡ |
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12 hpi‡ |
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24 hpi‡ |
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48 hpi‡ |
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72 hpi‡ |
‡Dunn’s multiple comparison tests.
Whole mount of a healthy balsam fir sawfly larva midgut. The central region is opaque due to the presence of a food bolus within the gut. Fat body (FB) is present at the lower left. Photomicrograph. Scale bar = 1 mm.
Whole mount of a balsam fir sawfly larva midgut with a patent NeabNPV infection. Nuclei in the epithelial cells throughout the midgut are opaque due to the presence of numerous NeabNPV occlusion bodies. Photomicrograph. Scale bar = 1 mm.
Cross-section of a healthy larval midgut. Epithelial cells are elongated and abutted against circular (CM) and longitudinal (LM) muscles; tracheole epithelial cells (TE) are also present. The nucleus (N) is elongated and centrally located in the cell. Heterochromatin (Hc) and invaginations of the nuclear envelope (arrowhead) are evident. Three successive stages of what appears to be a secretory process from these cells (a, b, and c) are apparent. Epoxy section, photomicrograph. Scale bar = 20
Detail of nuclear envelope invaginations in uninfected midgut epithelial cell. Visible are the nucleoplasm (NP), cytoplasm (CP), nuclear pores in cross (arrowhead) and longitudinal (arrow) sections, and rough endoplasmic reticulum (rER). Electron micrograph. Scale bar = 1
The basal end of an uninfected midgut epithelial cell (ME) abutting against the basal lamina (BL), a tracheole epithelial cell (TE), and a muscle cell (MC). Invaginations of the midgut epithelial cell plasma membrane (arrows) anchor the cell to the basal lamina. Mitochondria (M) were also seen in this region of the midgut cell. Electron micrograph. Scale bar = 0.5
Uninfected midgut (ME), and tracheole epithelial cells (TE) with basal lamina (BL) in between. Invaginations of the midgut epithelial cell plasma membrane (arrows) anchor the cell to the basal lamina. A tracheole (T), mitochondrion (M), and microtubules (arrowhead) can be seen in the tracheole epithelial cell. A muscle cell (MC) lies adjacent to the tracheole epithelial cell. Electron micrograph. Scale bar = 1
Apical end of an uninfected midgut epithelial cell. The cytoplasm contains mitochondria (M), rough endoplasmic reticulum (rER), and, near where microvilli (MV) extend out into the midgut lumen, a reticulate network (RN). Electron opaque material (arrows) was observed around mitochondria and the reticulate network. The zonula continua separating two epithelial cells can be seen on the left (arrowheads). Electron micrograph. Scale bar = 1
Electron opaque material, similar in appearance to that seen in Figure
Cross-section of midgut epithelial cells at 48 hpi with NeabNPV. The basal ends of the epithelial cells abut against circular (CM) and longitudinal (LM) muscles. A nidus of regenerative cells (Ni) lies between two epithelial cells. The nucleus (N) appears enlarged, and invaginations (arrow) of the nuclear envelope are present. Dark-staining bodies (arrowhead) (perhaps the same as those observed in Figure
Entry of NeabNPV virions into host epithelial cells and nuclei was not observed. Within 5 h of infection by NeabNPV, there was a proportional decrease in the size of microvillar borders and hypertrophy of nuclei in relation to the area of cells as a whole (Table
Cytopathic effects due to NeabNPV infection resulting in reduction in the microvillar border and nuclear hypertrophy based on mean values of the proportional surface area of each compared with the area of the affected cells from 0 to 72 hpi. Proportional mean values and standard deviations (SD) were determined for each time point. Kruskal-Wallis nonparametric and Dunn’s multiple comparison tests were performed for each cytopathic effect to obtain a measure of the statistical significance of each trait over the course of infection. All Kruskal-Wallis tests revealed significant differences between the time points with a KW value of 52.74 or greater.
Cytopathic effects | NeabNPV infected | |||||||||
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Uninfected | 1 hpi | 2 hpi | 5 hpi | 8 hpi | 12 hpi | 24 hpi | 48 hpi | 72 hpi | ||
Microvillar reduction | Mean* | 25.45 | 25.49 | 25.27 | 14.94 | 15.83 | 18.26 | 18.15 | 10.20 | 15.14 |
Sample size | 84 | 79 | 65 | 68 | 81 | 73 | 66 | 120 | 138 | |
SD | 0.0717 | 0.0728 | 0.0763 | 0.0363 | 0.0407 | 0.0660 | 0.0532 | 0.0562 | 0.0790 | |
Uninfected‡ |
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1 hpi‡ |
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2 hpi‡ |
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5 hpi‡ |
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8 hpi‡ |
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12 hpi‡ |
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24 hpi‡ |
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Nuclear hypertrophy | Mean† | 29.35 | 28.92 | 29.54 | 42.35 | 42.35 | 38.15 | 37.36 | 43.87 | 48.70 |
Sample size | 84 | 79 | 65 | 68 | 81 | 73 | 66 | 120 | 138 | |
SD | 0.1160 | 0.1073 | 0.1127 | 0.1572 | 0.1572 | 0.1535 | 0.1127 | 0.6447 | 0.0871 | |
Uninfected‡ |
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1 hpi‡ |
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2 hpi‡ |
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5 hpi‡ |
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8 hpi‡ |
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12 hpi‡ |
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24 hpi‡ |
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48 hpi‡ |
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72 hpi‡ |
†Proportional surface area of the nucleus compared with the surface area of the entire cell (%).
‡Dunn’s multiple comparison tests.
Basal end of a midgut epithelial cell at 48 hpi. A layer of basal lamina (BL) lies between the midgut cell and a muscle cell (MC). Mitochondria (M) and a bacterium (B) can be seen in the cytoplasm of the midgut cell in between the invaginations of the plasma membrane (arrows). These plasma membrane invaginations appear irregular compared with those in Figures
Basal end of a midgut epithelial cell at 48 hpi similar to that seen in Figure
Early stage in the formation of vesicles (arrows) accumulating electron opaque material in the cytoplasm of a midgut epithelial cell at 48 hpi. Electron micrograph. Scale bar = 1
On either side of a mitochondrion (M) are vesicles at later stages of formation than those observed in Figure
Midgut epithelial cell at 72 hpi. Present in the cytoplasm are vesicles containing an electron opaque material (arrows) and bacteria (B). Structures (arrowhead) appearing to consist of lengths of a thread-like material were first observed at 72 hpi. Electron micrograph. Scale bar = 1
Cross-section of midgut epithelium at 72 hpi. Midgut epithelial cells abut circular (CM) and longitudinal (LM) muscle, and regenerative cells (Ni) are present. Virogenic stroma (VS) can be seen within the hypertrophied nucleus. Secretory activity appears to have been reduced or has ceased as no secretions, such as those seen in Figures
Virogenic stroma in the nucleus of a midgut epithelial cell at 72 hpi. Viral capsids (Cd) and nucleocapsids (Nd) can be seen nucleocapsids being occluded (arrows) into forming occlusion bodies (OBs). Electron micrograph. Scale bar = 1
Virogenic stroma in the nucleus of a midgut epithelial cell at 96 hpi. Viral capsids (Cd) and nucleocapsids (Nd) can be seen in longitudinal section and in cross-section (arrowhead). Electron micrograph. Scale bar = 1
Virogenic stroma in the nucleus of a midgut epithelial cell at 96 hpi showing a circular body (CB) and nucleocapsid (arrows) occlusion into occlusion bodies (OBs). Electron micrograph. Scale bar = 0.5
Occlusion of nucleocapsids into occlusion bodies (OBs) in the nucleoplasm of a midgut epithelial cell at 96 hpi. Two nucleocapsids (arrows) have been cut in clear cross-section. Microtubules (arrowheads) are present in the cell cytoplasm (CP). Electron micrograph. Scale bar = 0.5
Cross-section of midgut epithelial cells at 96 hpi. Clear gaps (arrowheads) separate the basal ends of the epithelial cells from adjacent circular (CM) and longitudinal (LM) muscles. Virogenic stroma (VS) and occlusion bodies (OBs) occupy much of the enlarged nucleus on the left. Note that the level of NeabNPV development does not appear to be the same in the three visible nuclei. Large vacuoles (V) can be seen in the cytoplasm of two of the cells. Epoxy section, photomicrograph. Scale bar = 20
Nucleus of a midgut epithelial cell at 96 hpi. Heterochromatin (Hc), virogenic stroma (VS), and NeabNPV occlusion bodies (OB) lie within the nuclear envelope (NE). Electron micrograph. Scale bar = 2
Midgut epithelial cell at 96 hpi. A thread-like body (arrowhead), similar to that seen in Figure
Cross-section of midgut epithelium at 120 hpi. The enlarged nucleus of the cell, to the left of center, is full of occlusion bodies (OB). The basal end of this cell lies proximal to circular muscle but the cytoplasm in this region of the cell appears vacuolated (between arrowheads). Bacteria (arrows) are present in the hemocoel adjacent to longitudinal muscles (LM). An epithelial cell has detached (DC) from the midgut epithelium and is free within the gut lumen near the microvilli (MV) of the surrounding, still attached, midgut cells. Epoxy section, photomicrograph. Scale bar = 20
Cross-section of midgut epithelium at 120 hpi. One cell has detached (DC) from the midgut epithelium. This process results in gaps in the epithelium layer where there are no cells lying adjacent to circular (CM) and longitudinal (LM) muscles. Masses of bacteria (B) lie, in the gut lumen, adjacent to an epithelial cell with an enlarged nucleus containing virogenic stroma (VS) and occlusion bodies (OBs). There appears to be a disruption in the cytoplasm (between arrows) of the epithelial cell to the left of the gap in the epithelium. Microvilli (MV) are evident in this and the adjoining epithelial cell. Epoxy section, photomicrograph. Scale bar = 20
Area of cell cytoplasmic lysis (CL) adjacent to the nuclear envelope (NE) of a midgut epithelial cell at 120 hpi. Occlusion bodies (OBs) are present within the nucleus. Electron micrograph. Scale bar = 1
Occlusion bodies free in the midgut lumen at 120 hpi. Note the calyx-like covering (arrows) surrounding each occlusion body. Electron micrograph. Scale bar = 1
Cross-section of a balsam fir larva at a late stage of NeabNPV infection. Viral occlusion bodies (OBs) lie in the ectoperitrophic space between the peritrophic membrane (PM) and the midgut epithelium (ME). Tissues visible in the hemocoel include circular (CM) and longitudinal (LM) muscle, fat body (FB), and larger muscle masses (Mu) beneath the cuticle (C). Paraplast section, photomicrograph. Scale bar = 50
Whole mount of a midgut from a live, adult, female balsam fir sawfly. Masses of NeabNPV occlusion bodies can be seen within the nuclei (arrows) of midgut epithelial cells. Also visible are fat body (FB) and trachea (Tr) from which tracheoles (T) branch. Photomicrograph. Scale bar = 50
Within the
To date, neither
Insect midgut epithelial cells are generally short lived and are rapidly replaced by regenerative cells at the basal end and in between the epithelial cells [
With OB formation initiation in 81% and maturation in 49% of epithelial cells in NeabNPV-infected midgut within 72 hpi (Table
The epithelial cells of healthy
As is the case with HabrGV gut infections [
This paper on the cytology of NeabNPV infection in balsam fir sawflies is quite possibly the first detailed account of gammabaculovirus pathology since the earlier work of Bird and Whalen in 1954 [
This paper was supported through grants to C. J. Lucarotti and D. B. Levin from the Natural Sciences and Engineering Research Council (NSERC) Industrial Research Partnership with industrial partner, Forest Protection Limited (Lincoln, NB, Canada) and the NSERC BioControl Network, and through grants from Natural Resources Canada, Canadian Forest Service to C. J. Lucarotti. The authors gratefully acknowledge Nancy MacAfee for her assistance with the