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Determination of mode of transmission of the citrus red mite virus
JOURNAL
OF INVERTEBRATE
PATHOLOGY
Determination
D. K. REED,
26, 239-246
(1975)
of Mode of Transmission Citrus Red Mite Virus H. TASHIRO,’
AND
Boyden Entomological Laboratory, U.S. Department of Agriculture, Received
of the
J. B. BEAVERS2
Agricultural Research Service, Riverside, California 92502
December
12.1974
Successful inoculations of citrus red mites, Panonychus citri. with the nonoccluded virus were made by feeding healthy mites through membranes on contaminated lemon fruit. Manipulation of the membranes showed that healthy mites pick up the virus from contaminated surface substrates rather than from within plant cells. Electron microscopical studies substantiated that feces of infected mites should be highly infectious since the hindgut contains numerous virus rods.
INTRODUCTION None of the numerous studies made to elucidate the relationship between the normal feeding activities of the citrus red mite, Panonychus citri, and transmissions of a nonoccluded virus (Gilmore and Munger, 1963; Gilmore and Tashiro, 1966; Tashiro et al., 1970) have established the exact mode of transmission; however, each has contributed toward such knowledge. The present study involved the feeding of mites through inert membranes and electron microscopical observations of host-pathogen interactions. The results help to explain the mechanism of virus transmission from diseased to healthy mites.
the mites had been exposed to the inoculum, they were confined for disease development in Munger cells attached to green lemons where they fed on 15-cm2 areas (Munger and Gilmore, 1963). Incidence of infection was determined by mounting and clearing the mites in Hoyer’s medium for examination under polarized light for the characteristic birefringent crystals (Smith and Cressman, 1962). For electron microscopy, mites were prepared as reported previously by Reed and Hall (1972). Sections were examined in a Hitachi Hu 12 electron microscope operated at an accelerating voltage of 80 kV.
MATERIALS AND METHODS The mites used in the study were reared on green lemons at 27°C and 60% RH according to methods described by Munger and Gilmore (1963). All tests were made in the laboratory under similar conditions. Virus inoculum consisted of mites killed by the disease that were collected and stored at -27°C (Gilmore and Munger, 1963). After
In one test, a 90-mm disc of DuPont polyethylene 100 (DuPont De Nemours Co., Wilmington, Delaware)3 was floated on 25 ml of suspension in a 100 mm diam Petri dish, and seven healthy adult female mites were transferred to the membrane with a fine brush. A ring of lanolin at the edge of the disc prevented the mites from crawling into the suspension. They were allowed to feed for 2, 6, or 24 hr on one of the three treatments before they were transferred to the Munger cages. Treatments included 1 mg of triturated diseased mites/ml of 10%
1Department of Entomology, Agricultural Experiment Station, Cornell University, Geneva, New York 14456. 2USDA Horticultural Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Orlando, Florida 32803.
Transmission of Virus from Aqueous Suspensions Through an Inert Membrane
3Mention dorsement
239 Copyright All rights
6 1975 by Academic Press, Inc. of reproduction in any form reserved.
of a proprietary by the USDA.
name does not imply
its en-
240
REED, TASHIRO
sucrose, 10% sucrose solution alone, and 1 mg of diseased mite material/ml of distilled water. Each of the nine treatments was replicated four times. Subsequent tests were made using various dilutions of virus inoculum in 10% sucrose. Healthy mites were allowed to feed for 6 hr before transferring them to Munger cages where mortality and crystal formation was tabulated after 7 days. Transmission of Virus from Inoculated Lemons Through Parajilm
In a second test, lemons previously supporting high populations of diseased mites were wrapped in thinly stretched Parafilm “M”@ (American Can Co., Neenah, Wisconsin). Healthy mites were transferred to such lemons and permitted to feed through the film for various periods of time after which ten mites each were transferred to clean lemons mounted in Munger cages. In the reciprocal test, diseased mites fed through parafilm on a clean fruit for 3 days; then ten healthy mites were placed on each fruit which had been stripped of the parafilm and mounted in Munger cages. The control consisted of ten healthy mites each feeding on lemons that were not covered with film. Each treatment was replicated six times. RESULTS Virus Transmissionfrom Suspension Through Membrane
Mites were examined at 3, 7, and 10 days after exposure, and the percentages of accuand disease were mulative mortality recorded. As Table 1 shows the highest levels of infection were produced by feeding for 2 or 6 hr on virusssucrose substrates. However, in all four replicates, crystals were found in mites exposed to each treatment. We can only speculate that massive doses of virus imbibed in 24 hr somehow interfered with crystal formation; or perhaps the humidity within the Petri dishes was high enough to inhibit the formation of crystals when mites were confined for 24 hr (Reed et al., 1974). Mortality and incidence of disease
AND BEAVERS TABLE 1 Transmission of Virus Disease to Mites Feeding on Aqueous Virus Suspensions Through Inert Membrane Time on substrate Or) 2 6 24 2 6
24 2 6 24
Corrected % mortality in 10 days
No-~ b exposed ’
Sucrose + virus 28 28 28 Virus + water 14 14 14 10% Sucrose only 14 14 14
% With crystals
100 100 11.2
84 82 18
-63.2 49.3 91.1
0 29 I
0 0 0
0 0 0
al mg diseased mites/l ml water or 10% sucrose solution. bEach treatment with 7 mites/replicate. TABLE 2 Influence of Concentration of Diseased Mite Suspension on Transmission to Mites Feeding For 6 hr Through an Inert Membrane Dilution of diseased mite suspension (mg/mV
Corrected % mortality in I days
% Mites with crystals
Control (virus free) 1:lOOO l:soo 1:250 1:lOO 1:lO 1:l
0 5.26 29.8 24.6 41.4 100.0 100.0
0 11 10 13 30 41 50
‘Each treatment consisted of 6 replicates of 5 mites/ cage.
were lower when mites were fed virus-distilled water. These results were anticipated because Reed et al. (1973) determined that sucrose acts as a protectant and extender for this virus in aqueous suspension. However, our results were such that they raise the question of whether the sucrose also enhances the viability and infectivity of suspensions or whether it was simply acting as a feeding stimulus. Table 2 reports effect of virus concentration on infection. A direct relationship
TRANSMISSION
OF
CITRUS
existed between concentration and infections, but the higher concentrations did not produce the expected high infections. Control mortality was higher than normal, but no mites showed virus infection. Virus Transmission from Lemons Through Parajilm
Early limited and inconclusive evidence led to the genera1 hypothesis that infected citrus red mites puncture epidermal cells with their stylets and contaminate that cell with virus as they feed on the cell contents. Healthy mites, in subsequent feedings, would then contact these cells and therefore contract the disease. This theory appears untenable in view of the number of epidermal cells and the improbability that another mite would contact the same cell, particularly in short feeding periods. Since the preceding experiments established that virus could be acquired through membranes, we used parafilm-covered lemons in an attempt to ascertain the mode of transmission of virus. When healthy mites were allowed to feed through such a film for 24 hr on contaminated lemons, 75% acquired disease (Table 3). When healthy mites fed directly on contaminated lemons, 73% became infected. TABLE 3 influence of Inoculation and of Parafilm Cover on Virus Infections in Citrus Red Mites “/o Mites Exposure
Experiment 1
of lemon’
Diseased mites (no film), healthy mites (no film) Diseased mites (no film), healthy mites (through film) Diseased mites (through film), healthy mites (no film) Control: healthy mites (no film), healthy mites (no film) ‘Each treatment mites/cage.
consisted
infected Experiment 2
73
65
15
55
0
0
0 of
0 6 replicates
of
10
RED
MITE
241
VIRUS
In the reciprocal test, healthy mites allowed to feed on the same sites that had been fed on through film by ten infected mites for 3 days, (the film was removed and the lemon was mounted in a clean cell) failed to develop disease after 14 days. Subsequent identical experiments produced the same results. Also, when parafilm-covered lemons were infested with 10, 20, 30, 40, and 50 infected mites for 4 days, 10 healthy mites placed on the lemon after the film was removed did not show any evidence of disease. However, when the parafilm stripped from the lemons was washed in 10% sucrose, which was subsequently fed to healthy mites through a membrane, 20% of the mites in each of two tests became infected. The parafilm was therefore contaminated by the feeding activities of the infected mites. Electron Microscopic
Observations
The results of the present test indicate that the virus healthy mites encounter when they feed in an area previously infected with diseased mites is on the surface, probably in the form of fecal material. Then if the contamination arises from fecal material rather than from cells contaminated with saliva, virus particles should be present in the hindgut of diseased mites. Electron microscopic studies showed such particles present at that location. The hindgut cells of citrus red mite have a peculiar vesiculate appearance and the organ is surrounded for most of its length by the ventriculus (Fig. 1). The nonoccluded rod-shaped virus particle responsible for the disease is formed within the nucleii of the midgut epithelium cells (Reed and Hall, 1972). The intimate contact between ventriculus and hindgut (Fig. 2) afford ample opportunity for virus rods to move from one to another. The hindgut cells display irregular filamentous strands of cytoplasm interspersed with mitochondria; however, this condition is not virus induced because it is present in both healthy and diseased mites and is apparently responsible for the vesiculate appearance in the hght mi-
REED,
TASHIRO
AND
BEAVERS
FIG. 1. Posterior region of citrus red mite in transverse section. HG, hindgut; MC, midgut. x 1000.
croscope. The nucleus of a hindgut epithelial cell in which many virus rods (ca. 194 x 58 nm) are present can be seen in Figure 3. In the mid- and hindgut shown in Figure 4, virus rods are interspersed within the hindgut along with mitochondria. Most of the rods in the hindgut are apparently enclosed in a protein matrix, but no membrane seemsto enclose the matrix. Figures 5 and 6 show the nucleus breaking down and liberating virus rods into the vesiculate cyto-
plasm. Remnants of the nuclear membrane can be seen. The protein matrix may help to explain why natural deposits of infected mites remain infectious so much longer than virus applied as an aqueous suspension; most of the virus in fecal deposits would be protected by the matrices; while aqueous suspensions would contain more unprotected virus from midgut cells. The virus rods present within the hindgut cytoplasm of infected mites as it
TRANSMISSION
OF CITRUS
RED
MITE
VIRUS
243
244
REED,
TASHIRO
AND
BEAVERS
FIGS. 2-4. Ultrathin section through midgut and hindgut region of citrus red mite. Figures 2 and 3 shows the intricate and irregular cytoplasmic strands of the hindgut. In Figure 4 can be seen the basement membrane (BM) separating the organs and the numerous mitochondria (M) within the hindgut cells. N, nucleus; Cy, cytoplasm; VR, virus rods. Figure 2-x7800; Figure 3-x 6400; Figure 4- x 25,600.
sloughs off are liberated into the lumen and are voided. Subsequently they act as infectious agents to healthy mites feeding in the same area. Perhaps the mites in puncturing cell walls of the substrate release the cell contents, which flow onto the surface to mix with the fecal material. Jeppson et al. (1975) has postulated that the mite stylets penetrate the plant cells, which then exude
their contents because of turgor pressure. They have reported that the “flaps” at the tip of the rostrum apparently overlap against the plant surface to provide a plungerlike cup. The vacuum thus produced by the pharyngeal pump draws up the exuded cell contents from the plant surface. Our finding that mites feeding on the surface of fruit are exposed to fecal contamination seems to
5-
FIGS. 5.6. Ultrathin section through x 20,500; Figure 6- x 20,250.
hindgut
showing
virus
rods
in the nucleus
being liberated
into the cytoplasm
both
singly
and in mattrix
bound
bundles.
Figure
g WI
246
REED, TASHIRO AND BEAVERS
substantiate his theory. Piercing-sucking mouthparts would be unlikely to pick up enough virus to produce infections. REFERENCES J. E., AND MUNGER, F. 1963. Stability and transmissibility of a viruslike pathogen of the citrus red mite. J. Invertebr. Pathol., 5, 141-151. GILMORE, J. E., AND TASHIRO, H. 1966. Fecundity, longevity, and transinfectivity of citrus red mite (Panonychus citri) infected with a noninclusion virus. GILMORE,
J. Invertebr.
Pathol..
8,334-339.
JEPPSON,L. R., H. H. KIEFER,AND E. W. BAKER. 1975. Mites Injurous to Economic Plants,” 528 pp. Univ. Calif. Press. Berkeley and Los Angeles, California. MUNGER, F., AND GILMORE, J. E. 1963. Equipment and techniques used in rearing and testing the citrus red mite. In “Advances in Acarology” (J. Naegele, cd.)
Comstock Publ. Assoc. Cornell Univ. Press, Ithaca, New York. REED, D. K., AND HALL, I. M. 1972. Electron microscopy of a rod-shaped noninclusion virus infecting the citrus red mite. J. Invertebr. fathol.. 20,272 -278. REED, D. K., HENDRICKSON,R. M., JR., RICH, J. R., AND SHAW, J. G. 1973. Laboratory evaluation of extenders for the noninclusion cirus of the citrus red mite. J. Invertebr. Pathol.. 22, 182-185. REED, D. K., RICH, J. E., AND SHAW, J. G. 1974. Inhibition of formation of birefringent crystals by high humidity on citrus red mites infected with virus.J. Invertebr.
Pathol..
23,285-288.
CRESSMAN, A. W. 1962. Birefringent crystals in virus-diseased citrus red mites. J. Insect
SMITH,
K. M., AND
Pathol.,
4,229-236.
BEAVERS, J. B., GROZA, M., AND MOFC. 1970. Persistence of the citrus red mite noninclusion virus on lemon fruit and in intact dead mites. J. Invertebr. Pathol.. 16,63-68.
TASHIRO, FITT,
H.,
OF INVERTEBRATE
PATHOLOGY
Determination
D. K. REED,
26, 239-246
(1975)
of Mode of Transmission Citrus Red Mite Virus H. TASHIRO,’
AND
Boyden Entomological Laboratory, U.S. Department of Agriculture, Received
of the
J. B. BEAVERS2
Agricultural Research Service, Riverside, California 92502
December
12.1974
Successful inoculations of citrus red mites, Panonychus citri. with the nonoccluded virus were made by feeding healthy mites through membranes on contaminated lemon fruit. Manipulation of the membranes showed that healthy mites pick up the virus from contaminated surface substrates rather than from within plant cells. Electron microscopical studies substantiated that feces of infected mites should be highly infectious since the hindgut contains numerous virus rods.
INTRODUCTION None of the numerous studies made to elucidate the relationship between the normal feeding activities of the citrus red mite, Panonychus citri, and transmissions of a nonoccluded virus (Gilmore and Munger, 1963; Gilmore and Tashiro, 1966; Tashiro et al., 1970) have established the exact mode of transmission; however, each has contributed toward such knowledge. The present study involved the feeding of mites through inert membranes and electron microscopical observations of host-pathogen interactions. The results help to explain the mechanism of virus transmission from diseased to healthy mites.
the mites had been exposed to the inoculum, they were confined for disease development in Munger cells attached to green lemons where they fed on 15-cm2 areas (Munger and Gilmore, 1963). Incidence of infection was determined by mounting and clearing the mites in Hoyer’s medium for examination under polarized light for the characteristic birefringent crystals (Smith and Cressman, 1962). For electron microscopy, mites were prepared as reported previously by Reed and Hall (1972). Sections were examined in a Hitachi Hu 12 electron microscope operated at an accelerating voltage of 80 kV.
MATERIALS AND METHODS The mites used in the study were reared on green lemons at 27°C and 60% RH according to methods described by Munger and Gilmore (1963). All tests were made in the laboratory under similar conditions. Virus inoculum consisted of mites killed by the disease that were collected and stored at -27°C (Gilmore and Munger, 1963). After
In one test, a 90-mm disc of DuPont polyethylene 100 (DuPont De Nemours Co., Wilmington, Delaware)3 was floated on 25 ml of suspension in a 100 mm diam Petri dish, and seven healthy adult female mites were transferred to the membrane with a fine brush. A ring of lanolin at the edge of the disc prevented the mites from crawling into the suspension. They were allowed to feed for 2, 6, or 24 hr on one of the three treatments before they were transferred to the Munger cages. Treatments included 1 mg of triturated diseased mites/ml of 10%
1Department of Entomology, Agricultural Experiment Station, Cornell University, Geneva, New York 14456. 2USDA Horticultural Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Orlando, Florida 32803.
Transmission of Virus from Aqueous Suspensions Through an Inert Membrane
3Mention dorsement
239 Copyright All rights
6 1975 by Academic Press, Inc. of reproduction in any form reserved.
of a proprietary by the USDA.
name does not imply
its en-
240
REED, TASHIRO
sucrose, 10% sucrose solution alone, and 1 mg of diseased mite material/ml of distilled water. Each of the nine treatments was replicated four times. Subsequent tests were made using various dilutions of virus inoculum in 10% sucrose. Healthy mites were allowed to feed for 6 hr before transferring them to Munger cages where mortality and crystal formation was tabulated after 7 days. Transmission of Virus from Inoculated Lemons Through Parajilm
In a second test, lemons previously supporting high populations of diseased mites were wrapped in thinly stretched Parafilm “M”@ (American Can Co., Neenah, Wisconsin). Healthy mites were transferred to such lemons and permitted to feed through the film for various periods of time after which ten mites each were transferred to clean lemons mounted in Munger cages. In the reciprocal test, diseased mites fed through parafilm on a clean fruit for 3 days; then ten healthy mites were placed on each fruit which had been stripped of the parafilm and mounted in Munger cages. The control consisted of ten healthy mites each feeding on lemons that were not covered with film. Each treatment was replicated six times. RESULTS Virus Transmissionfrom Suspension Through Membrane
Mites were examined at 3, 7, and 10 days after exposure, and the percentages of accuand disease were mulative mortality recorded. As Table 1 shows the highest levels of infection were produced by feeding for 2 or 6 hr on virusssucrose substrates. However, in all four replicates, crystals were found in mites exposed to each treatment. We can only speculate that massive doses of virus imbibed in 24 hr somehow interfered with crystal formation; or perhaps the humidity within the Petri dishes was high enough to inhibit the formation of crystals when mites were confined for 24 hr (Reed et al., 1974). Mortality and incidence of disease
AND BEAVERS TABLE 1 Transmission of Virus Disease to Mites Feeding on Aqueous Virus Suspensions Through Inert Membrane Time on substrate Or) 2 6 24 2 6
24 2 6 24
Corrected % mortality in 10 days
No-~ b exposed ’
Sucrose + virus 28 28 28 Virus + water 14 14 14 10% Sucrose only 14 14 14
% With crystals
100 100 11.2
84 82 18
-63.2 49.3 91.1
0 29 I
0 0 0
0 0 0
al mg diseased mites/l ml water or 10% sucrose solution. bEach treatment with 7 mites/replicate. TABLE 2 Influence of Concentration of Diseased Mite Suspension on Transmission to Mites Feeding For 6 hr Through an Inert Membrane Dilution of diseased mite suspension (mg/mV
Corrected % mortality in I days
% Mites with crystals
Control (virus free) 1:lOOO l:soo 1:250 1:lOO 1:lO 1:l
0 5.26 29.8 24.6 41.4 100.0 100.0
0 11 10 13 30 41 50
‘Each treatment consisted of 6 replicates of 5 mites/ cage.
were lower when mites were fed virus-distilled water. These results were anticipated because Reed et al. (1973) determined that sucrose acts as a protectant and extender for this virus in aqueous suspension. However, our results were such that they raise the question of whether the sucrose also enhances the viability and infectivity of suspensions or whether it was simply acting as a feeding stimulus. Table 2 reports effect of virus concentration on infection. A direct relationship
TRANSMISSION
OF
CITRUS
existed between concentration and infections, but the higher concentrations did not produce the expected high infections. Control mortality was higher than normal, but no mites showed virus infection. Virus Transmission from Lemons Through Parajilm
Early limited and inconclusive evidence led to the genera1 hypothesis that infected citrus red mites puncture epidermal cells with their stylets and contaminate that cell with virus as they feed on the cell contents. Healthy mites, in subsequent feedings, would then contact these cells and therefore contract the disease. This theory appears untenable in view of the number of epidermal cells and the improbability that another mite would contact the same cell, particularly in short feeding periods. Since the preceding experiments established that virus could be acquired through membranes, we used parafilm-covered lemons in an attempt to ascertain the mode of transmission of virus. When healthy mites were allowed to feed through such a film for 24 hr on contaminated lemons, 75% acquired disease (Table 3). When healthy mites fed directly on contaminated lemons, 73% became infected. TABLE 3 influence of Inoculation and of Parafilm Cover on Virus Infections in Citrus Red Mites “/o Mites Exposure
Experiment 1
of lemon’
Diseased mites (no film), healthy mites (no film) Diseased mites (no film), healthy mites (through film) Diseased mites (through film), healthy mites (no film) Control: healthy mites (no film), healthy mites (no film) ‘Each treatment mites/cage.
consisted
infected Experiment 2
73
65
15
55
0
0
0 of
0 6 replicates
of
10
RED
MITE
241
VIRUS
In the reciprocal test, healthy mites allowed to feed on the same sites that had been fed on through film by ten infected mites for 3 days, (the film was removed and the lemon was mounted in a clean cell) failed to develop disease after 14 days. Subsequent identical experiments produced the same results. Also, when parafilm-covered lemons were infested with 10, 20, 30, 40, and 50 infected mites for 4 days, 10 healthy mites placed on the lemon after the film was removed did not show any evidence of disease. However, when the parafilm stripped from the lemons was washed in 10% sucrose, which was subsequently fed to healthy mites through a membrane, 20% of the mites in each of two tests became infected. The parafilm was therefore contaminated by the feeding activities of the infected mites. Electron Microscopic
Observations
The results of the present test indicate that the virus healthy mites encounter when they feed in an area previously infected with diseased mites is on the surface, probably in the form of fecal material. Then if the contamination arises from fecal material rather than from cells contaminated with saliva, virus particles should be present in the hindgut of diseased mites. Electron microscopic studies showed such particles present at that location. The hindgut cells of citrus red mite have a peculiar vesiculate appearance and the organ is surrounded for most of its length by the ventriculus (Fig. 1). The nonoccluded rod-shaped virus particle responsible for the disease is formed within the nucleii of the midgut epithelium cells (Reed and Hall, 1972). The intimate contact between ventriculus and hindgut (Fig. 2) afford ample opportunity for virus rods to move from one to another. The hindgut cells display irregular filamentous strands of cytoplasm interspersed with mitochondria; however, this condition is not virus induced because it is present in both healthy and diseased mites and is apparently responsible for the vesiculate appearance in the hght mi-
REED,
TASHIRO
AND
BEAVERS
FIG. 1. Posterior region of citrus red mite in transverse section. HG, hindgut; MC, midgut. x 1000.
croscope. The nucleus of a hindgut epithelial cell in which many virus rods (ca. 194 x 58 nm) are present can be seen in Figure 3. In the mid- and hindgut shown in Figure 4, virus rods are interspersed within the hindgut along with mitochondria. Most of the rods in the hindgut are apparently enclosed in a protein matrix, but no membrane seemsto enclose the matrix. Figures 5 and 6 show the nucleus breaking down and liberating virus rods into the vesiculate cyto-
plasm. Remnants of the nuclear membrane can be seen. The protein matrix may help to explain why natural deposits of infected mites remain infectious so much longer than virus applied as an aqueous suspension; most of the virus in fecal deposits would be protected by the matrices; while aqueous suspensions would contain more unprotected virus from midgut cells. The virus rods present within the hindgut cytoplasm of infected mites as it
TRANSMISSION
OF CITRUS
RED
MITE
VIRUS
243
244
REED,
TASHIRO
AND
BEAVERS
FIGS. 2-4. Ultrathin section through midgut and hindgut region of citrus red mite. Figures 2 and 3 shows the intricate and irregular cytoplasmic strands of the hindgut. In Figure 4 can be seen the basement membrane (BM) separating the organs and the numerous mitochondria (M) within the hindgut cells. N, nucleus; Cy, cytoplasm; VR, virus rods. Figure 2-x7800; Figure 3-x 6400; Figure 4- x 25,600.
sloughs off are liberated into the lumen and are voided. Subsequently they act as infectious agents to healthy mites feeding in the same area. Perhaps the mites in puncturing cell walls of the substrate release the cell contents, which flow onto the surface to mix with the fecal material. Jeppson et al. (1975) has postulated that the mite stylets penetrate the plant cells, which then exude
their contents because of turgor pressure. They have reported that the “flaps” at the tip of the rostrum apparently overlap against the plant surface to provide a plungerlike cup. The vacuum thus produced by the pharyngeal pump draws up the exuded cell contents from the plant surface. Our finding that mites feeding on the surface of fruit are exposed to fecal contamination seems to
5-
FIGS. 5.6. Ultrathin section through x 20,500; Figure 6- x 20,250.
hindgut
showing
virus
rods
in the nucleus
being liberated
into the cytoplasm
both
singly
and in mattrix
bound
bundles.
Figure
g WI
246
REED, TASHIRO AND BEAVERS
substantiate his theory. Piercing-sucking mouthparts would be unlikely to pick up enough virus to produce infections. REFERENCES J. E., AND MUNGER, F. 1963. Stability and transmissibility of a viruslike pathogen of the citrus red mite. J. Invertebr. Pathol., 5, 141-151. GILMORE, J. E., AND TASHIRO, H. 1966. Fecundity, longevity, and transinfectivity of citrus red mite (Panonychus citri) infected with a noninclusion virus. GILMORE,
J. Invertebr.
Pathol..
8,334-339.
JEPPSON,L. R., H. H. KIEFER,AND E. W. BAKER. 1975. Mites Injurous to Economic Plants,” 528 pp. Univ. Calif. Press. Berkeley and Los Angeles, California. MUNGER, F., AND GILMORE, J. E. 1963. Equipment and techniques used in rearing and testing the citrus red mite. In “Advances in Acarology” (J. Naegele, cd.)
Comstock Publ. Assoc. Cornell Univ. Press, Ithaca, New York. REED, D. K., AND HALL, I. M. 1972. Electron microscopy of a rod-shaped noninclusion virus infecting the citrus red mite. J. Invertebr. fathol.. 20,272 -278. REED, D. K., HENDRICKSON,R. M., JR., RICH, J. R., AND SHAW, J. G. 1973. Laboratory evaluation of extenders for the noninclusion cirus of the citrus red mite. J. Invertebr. Pathol.. 22, 182-185. REED, D. K., RICH, J. E., AND SHAW, J. G. 1974. Inhibition of formation of birefringent crystals by high humidity on citrus red mites infected with virus.J. Invertebr.
Pathol..
23,285-288.
CRESSMAN, A. W. 1962. Birefringent crystals in virus-diseased citrus red mites. J. Insect
SMITH,
K. M., AND
Pathol.,
4,229-236.
BEAVERS, J. B., GROZA, M., AND MOFC. 1970. Persistence of the citrus red mite noninclusion virus on lemon fruit and in intact dead mites. J. Invertebr. Pathol.. 16,63-68.
TASHIRO, FITT,
H.,