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Virus-like Particles in the Mycetocytes of the Sweetpotato Whitefly,Bemisia tabaci(Homoptera, Aleyrodidae)
JOBNAME: 67#2 96 PAGE: 1 SESS: 4 OUTPUT: Mon Jun 10 13:29:39 1996 /xypage/worksmart/tsp000/67596b/6 JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.
67, 183–186 (1996)
0028
NOTE Virus-like Particles in the Mycetocytes of the Sweetpotato Whitefly, Bemisia tabaci (Homoptera, Aleyrodidae) In the past decade, the sweetpotato whitefly (SPW), Bemisia tabaci Gennadius, has increased in importance as a direct pest and as a vector of plant viruses (Faust, 1992; Brown, 1994). Whiteflies in the B. tabaci complex have been characterized as numerous distinct biotypes based on host range and biological, biochemical and/or genetic differences (Costa et al., 1993; Brown et al., 1995a,b). Whiteflies have intracellular organisms that are considered symbiotic in nature, and that are housed in specialized cells called mycetocytes (Buchner, 1965; Clark et al., 1992; Costa et al., 1993). During comparative morphological studies of the mycetocytes and associated bacteria in six populations of Bemisia spp., large crystalline aggregates of virus-like particles (VLPs) were observed in individuals of a Jatropha biotype population. A total of six populations of Bemisia were examined. These populations were identified by distinct esterase electromorphs (Costa and Brown, 1991; Costa et al., 1993a; Brown et al., 1995a) and have been characterized with respect to inability or ability to induce phytotoxic symptoms in plants (Costa and Brown, 1991, 1993a; Bedford et al., 1994) and differences in host preference and geminivirus transmission capabilities (Bedford et al., 1994; Brown et al., 1995b) and as having distinct mitochondrial 16S ribosomal DNA sequences (Brown et al., 1995b). Three populations of the silverleaf whitefly, Bemisia argentifolii Bellows and Perring, formerly B. tabaci B biotype, were examined: (1) a population originating from Hawaii, originally collected from squash (Cucurbita sp.) at the University of Hawaii Agricultural Experiment Station, at Poamoho, on the Hawaiian island of Oahu, in 1991, and maintained on cotton, Gossypium hirsutum L., in outdoor cages at the University of Hawaii at Manoa; (2) a population collected from Hibiscus sp. in greenhouses at the University of Florida campus (Gainesville, FL) in 1994; (3) a population originally collected from poinsettia plants (Euphorbia pulcherrima) in greenhouses at the University of Arizona (Tucson, AZ) in 1988 and maintained since that time on either cotton or pumpkin (Cucurbita maxima Duchesne) in greenhouses or growth rooms at the University of Arizona.
Three other populations of whitefly presently described as B. tabaci were examined: (1) a population characterized as the A biotype (or the A1 electromorph), originally collected from cotton in Phoenix, Arizona, in 1983, and maintained since that time on cotton or pumpkin in greenhouses or growth rooms at the University of Arizona; (2) a population characterized as the A2 biotype (or the A2 electromorph), originally collected from squash in a field in Culiacan, Mexico, in 1993, and subsequently maintained on cotton in a growth room at the University of Arizona; (3) a population of B. tabaci, Jatropha race (Bird 1957), originally collected from the field and maintained on J. gossypifolia in screen cages by J. Bird at the University of Puerto Rico (Rio Piedras, PR) for approximately 2 years. The Jatropha population has previously been referred to as the 0 biotype (Costa et al., 1993) and more recently as the N electromorph (Brown et al., 1995a) of B. tabaci. Whitefly mycetomes were dissected from third or fourth instar immatures in 4% glutaraldehyde and set in 2% agar blocks as previously described (Costa et al., 1993). Agar blocks were fixed in 4% glutaraldehyde overnight at 4°C. Samples were postfixed for 2 hr in 2% osmium tetroxide in distilled water, washed three times in 0.05 M sodium cacodylate buffer and three times in distilled water, and en bloc stained for 15 min in 2% uranyl acetate in distilled water. Samples were serially dehydrated, embedded in Spurr’s epoxy (Spurr, 1969), and tissues were sectioned and stained as previously described (Ullman et al., 1989). In each whitefly population examined, the mycetome was made up of a variable number of large specialized insect cells, the mycetocytes, that were of similar shape and structure and contained many bacterial-like organisms (Fig. 1). Among the six Bemisia populations examined, virus-like particles (VLPs) were observed only in samples of the Jatropha population. In this case, VLPs were found in mycetocytes of all six Jatropha individuals examined. VLPs were not observed in any of the 36 individuals examined from other Bemisia populations. The VLPs observed in Jatropha individuals were icosahedra of approximately 30 nm in diameter and appeared to be scattered throughout the mycetocytes among the endosymbiotic bacteria. Crys-
183 0022-2011/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
JOBNAME: 67#2 96 PAGE: 2 SESS: 4 OUTPUT: Mon Jun 10 13:29:39 1996 /xypage/worksmart/tsp000/67596b/6 184
NOTE
FIG. 1. Virus-like particles in crystalline aggregates (Cr) within a mycetocyte cell of Bemisia tabaci Jatropha biotype. P, pleomorphic organism; C, coccoid organism; m, mitochondria (×12,600).
talline arrays of particles were as large as 2–3 mm in diameter (Fig. 2). The VLPs and crystalline aggregates were observed only in the cytoplasm of insect cells, but not within the nuclei of the insect mycetocyte cells or within the bacteria. In one sample in which ovarian tissue was inadvertently fixed along with mycetocytes, aggregates of VLPs were aligned along the exterior surface of ovarian nuclear membranes (Fig. 3), suggesting that they may be associated with insect rather than with bacterial cells. The sweetpotato whitefly is a well-known vector of several families of plant viruses worldwide, with the most common being the Geminiviridae (Brown, 1994). The morphology of the VLPs observed in this study resemble that known for several groups of plant viruses including the Tymoviruses, the Luteoviruses, and the Tombusviruses (Francki et al., 1991). To our knowledge, however, none of these groups contain viruses known to be transmitted by any whitefly species, nor are they reported to infect the plant genus, Jatropha. One plant virus known to infect Jatropha, Jatropha mosaic virus (JMV) is a geminivirus transmitted by B. tabaci (Bird, 1957; Francki et al., 1991); however, the VLPs observed in the immature Jatropha whiteflies are not consistent with the distinct paired or gemi-
nate morphology characteristic of the Geminivirus family. The size and shape of the VLPs observed in the Jatropha whiteflies, however, are consistent with those of several groups of invertebrate viruses including the Nodaviruses and the Picorna-like viruses known to infect aphids and other insect species (Adams and Bonami 1991; Francki et al., 1991; Moore and Eley, 1991). To our knowledge, VLPs belonging to any extant virus family have not previously been reported in whiteflies, or in the mycetocytes of other insects. Because whitefly mycetocytes are maternally transmitted to each developing egg (Buchner, 1965), the presence of virus-like particles in the mycetocytes raises the possibility that these VLPs are transmitted transovarially. In this study, examination of these insects was restricted to the mycetome area, thus, the distribution of virus particles in other areas of the whitefly body remains to be determined. KEY WORDS: Bemisia tabaci; insect pathogen; Jatropha; sweetpotato whitefly; virus; whitefly virus. We thank H. Markus Gailitis (University of Hawaii, Manoa) for technical assistance; and the Pacific Biomedical Research Center’s Biological Electron Microscope Facility (University of Hawaii, Manoa); the University of Arizona Electron Microscopy Center for
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NOTE
FIG. 2. Crystalline aggregate (Cr) of virus-like particles within a mycetocyte cell of Bemisia tabaci Jatropha biotype (×100,600).
use of their facilities; and Julio Bird for providing the Jatropha biotype and culture and for valuable insights on this unique whitefly biotype. This research was funded by the Governor’s Agricultural Coordinating Committee, State of Hawaii, Contract 91-27; a University of Hawaii Research and Training Revolving Fund Award; and a Research Centers in Minority Institutions Grant, RR-03061, National, Institutes of Health.
REFERENCES Adams, J. R., and Bonami, J. R.. 1991. “Atlas of Invertebrate Viruses” CRC Press, Boca Raton. Bird, J. 1957. A whitefly-transmitted mosaic of Jatropha gossypifolia. Tech. Paper Agric. Exp. Station P.R. 22, 1–35. Brown, J. K. 1994. A global position paper: The status of Bemisia tabaci Genn. as a pest and vector in world agroecosystems. FAO Plant Protein Bull. 42, 3–32. Brown, J. K., Coats, S., Bedford, I. D., Markham, P. G., Bird, J., and Frohlich, D. R. 1995a. Characterization and distribution of esterase electropmorphs in the whitefly, Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) Biochem Genetics 33, 205–314. Brown, J. K., Frohlich, D. R., and Rosell, R. C. 1995b. The sweetpotato or silverleaf whiteflies: Biotypes of Bemisia tabaci or a species complex? Annu. Rev. Entomol. 40, 511–534.
FIG. 3. Virus-like particles (v) abutting the nuclear membrane in whitefly ovarian tissue. N, nucleus; m, mitrochondria (×18,450).
Buchner, P. 1965. “Endosymbiosis of Animals with Plant Microorganisms.” Interscience, New York. Clark, M. A., Baumann, L., Munson, M. A., Baumann, P., Campbell,
JOBNAME: 67#2 96 PAGE: 4 SESS: 4 OUTPUT: Mon Jun 10 13:29:39 1996 /xypage/worksmart/tsp000/67596b/6 186
NOTE
B. C., Duffus, J. E., Osborne, L. S., and Moran, N. A. 1992. The eubacterial endosymbionts of whiteflies (Homoptera: Aleyrodoidea) constitute a lineage distinct from the endosymbionts of aphids and mealybugs. Curr. Microbiol. 25, 119–123. Costa, H. S., Brown, J. K., Sivasupramaniam, S., and Bird, J. 1993. Regional distribution, insecticide resistance, and reciprocal crosses between the A and B biotypes of Bemisia tabaci. Insect Sci. Applic. 14, 255–266. Costa, H. S., Westcot, D. M., Ullman, D. E., and Johnson, M. W. 1993. Ultrastructure of the endosymbionts of the whitefly, Bemisia tabaci and Trialeurodes vaporariorum. Protoplasma 176, 106–115. Faust, R. M. 1992. Conference report and 5-year national research and action plan for development of management and control methodology for the sweetpotato whitefly, Houston, Texas, Feb. 18–21, 1992. U.S. Department of Agriculture, ARS, 107. Francki, R. I. B., Fauquet, C. M., Knudson, D. L., and Brown, F. (Eds.) 1991. “Classification and Nomenclature of viruses.” Fifth report of the International Committee on Taxonomy of Viruses. Arch. Virol. Suppl. 2, Springer-Verlag, New York.
H. S. COSTA* D. M. WESTCOT† D. E. ULLMAN† R. C. ROSELL‡ J. K. BROWN‡ M. W. JOHNSON† *Department of Entomology University of California Riverside, California 92521 †Department of Entomology University of Hawaii at Manoa Honolulu, Hawaii 96822 ‡Department of Plant Sciences University of Arizona Tucson, Arizona 85721 Received January 25, 1995; accepted November 3, 1995
67, 183–186 (1996)
0028
NOTE Virus-like Particles in the Mycetocytes of the Sweetpotato Whitefly, Bemisia tabaci (Homoptera, Aleyrodidae) In the past decade, the sweetpotato whitefly (SPW), Bemisia tabaci Gennadius, has increased in importance as a direct pest and as a vector of plant viruses (Faust, 1992; Brown, 1994). Whiteflies in the B. tabaci complex have been characterized as numerous distinct biotypes based on host range and biological, biochemical and/or genetic differences (Costa et al., 1993; Brown et al., 1995a,b). Whiteflies have intracellular organisms that are considered symbiotic in nature, and that are housed in specialized cells called mycetocytes (Buchner, 1965; Clark et al., 1992; Costa et al., 1993). During comparative morphological studies of the mycetocytes and associated bacteria in six populations of Bemisia spp., large crystalline aggregates of virus-like particles (VLPs) were observed in individuals of a Jatropha biotype population. A total of six populations of Bemisia were examined. These populations were identified by distinct esterase electromorphs (Costa and Brown, 1991; Costa et al., 1993a; Brown et al., 1995a) and have been characterized with respect to inability or ability to induce phytotoxic symptoms in plants (Costa and Brown, 1991, 1993a; Bedford et al., 1994) and differences in host preference and geminivirus transmission capabilities (Bedford et al., 1994; Brown et al., 1995b) and as having distinct mitochondrial 16S ribosomal DNA sequences (Brown et al., 1995b). Three populations of the silverleaf whitefly, Bemisia argentifolii Bellows and Perring, formerly B. tabaci B biotype, were examined: (1) a population originating from Hawaii, originally collected from squash (Cucurbita sp.) at the University of Hawaii Agricultural Experiment Station, at Poamoho, on the Hawaiian island of Oahu, in 1991, and maintained on cotton, Gossypium hirsutum L., in outdoor cages at the University of Hawaii at Manoa; (2) a population collected from Hibiscus sp. in greenhouses at the University of Florida campus (Gainesville, FL) in 1994; (3) a population originally collected from poinsettia plants (Euphorbia pulcherrima) in greenhouses at the University of Arizona (Tucson, AZ) in 1988 and maintained since that time on either cotton or pumpkin (Cucurbita maxima Duchesne) in greenhouses or growth rooms at the University of Arizona.
Three other populations of whitefly presently described as B. tabaci were examined: (1) a population characterized as the A biotype (or the A1 electromorph), originally collected from cotton in Phoenix, Arizona, in 1983, and maintained since that time on cotton or pumpkin in greenhouses or growth rooms at the University of Arizona; (2) a population characterized as the A2 biotype (or the A2 electromorph), originally collected from squash in a field in Culiacan, Mexico, in 1993, and subsequently maintained on cotton in a growth room at the University of Arizona; (3) a population of B. tabaci, Jatropha race (Bird 1957), originally collected from the field and maintained on J. gossypifolia in screen cages by J. Bird at the University of Puerto Rico (Rio Piedras, PR) for approximately 2 years. The Jatropha population has previously been referred to as the 0 biotype (Costa et al., 1993) and more recently as the N electromorph (Brown et al., 1995a) of B. tabaci. Whitefly mycetomes were dissected from third or fourth instar immatures in 4% glutaraldehyde and set in 2% agar blocks as previously described (Costa et al., 1993). Agar blocks were fixed in 4% glutaraldehyde overnight at 4°C. Samples were postfixed for 2 hr in 2% osmium tetroxide in distilled water, washed three times in 0.05 M sodium cacodylate buffer and three times in distilled water, and en bloc stained for 15 min in 2% uranyl acetate in distilled water. Samples were serially dehydrated, embedded in Spurr’s epoxy (Spurr, 1969), and tissues were sectioned and stained as previously described (Ullman et al., 1989). In each whitefly population examined, the mycetome was made up of a variable number of large specialized insect cells, the mycetocytes, that were of similar shape and structure and contained many bacterial-like organisms (Fig. 1). Among the six Bemisia populations examined, virus-like particles (VLPs) were observed only in samples of the Jatropha population. In this case, VLPs were found in mycetocytes of all six Jatropha individuals examined. VLPs were not observed in any of the 36 individuals examined from other Bemisia populations. The VLPs observed in Jatropha individuals were icosahedra of approximately 30 nm in diameter and appeared to be scattered throughout the mycetocytes among the endosymbiotic bacteria. Crys-
183 0022-2011/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
JOBNAME: 67#2 96 PAGE: 2 SESS: 4 OUTPUT: Mon Jun 10 13:29:39 1996 /xypage/worksmart/tsp000/67596b/6 184
NOTE
FIG. 1. Virus-like particles in crystalline aggregates (Cr) within a mycetocyte cell of Bemisia tabaci Jatropha biotype. P, pleomorphic organism; C, coccoid organism; m, mitochondria (×12,600).
talline arrays of particles were as large as 2–3 mm in diameter (Fig. 2). The VLPs and crystalline aggregates were observed only in the cytoplasm of insect cells, but not within the nuclei of the insect mycetocyte cells or within the bacteria. In one sample in which ovarian tissue was inadvertently fixed along with mycetocytes, aggregates of VLPs were aligned along the exterior surface of ovarian nuclear membranes (Fig. 3), suggesting that they may be associated with insect rather than with bacterial cells. The sweetpotato whitefly is a well-known vector of several families of plant viruses worldwide, with the most common being the Geminiviridae (Brown, 1994). The morphology of the VLPs observed in this study resemble that known for several groups of plant viruses including the Tymoviruses, the Luteoviruses, and the Tombusviruses (Francki et al., 1991). To our knowledge, however, none of these groups contain viruses known to be transmitted by any whitefly species, nor are they reported to infect the plant genus, Jatropha. One plant virus known to infect Jatropha, Jatropha mosaic virus (JMV) is a geminivirus transmitted by B. tabaci (Bird, 1957; Francki et al., 1991); however, the VLPs observed in the immature Jatropha whiteflies are not consistent with the distinct paired or gemi-
nate morphology characteristic of the Geminivirus family. The size and shape of the VLPs observed in the Jatropha whiteflies, however, are consistent with those of several groups of invertebrate viruses including the Nodaviruses and the Picorna-like viruses known to infect aphids and other insect species (Adams and Bonami 1991; Francki et al., 1991; Moore and Eley, 1991). To our knowledge, VLPs belonging to any extant virus family have not previously been reported in whiteflies, or in the mycetocytes of other insects. Because whitefly mycetocytes are maternally transmitted to each developing egg (Buchner, 1965), the presence of virus-like particles in the mycetocytes raises the possibility that these VLPs are transmitted transovarially. In this study, examination of these insects was restricted to the mycetome area, thus, the distribution of virus particles in other areas of the whitefly body remains to be determined. KEY WORDS: Bemisia tabaci; insect pathogen; Jatropha; sweetpotato whitefly; virus; whitefly virus. We thank H. Markus Gailitis (University of Hawaii, Manoa) for technical assistance; and the Pacific Biomedical Research Center’s Biological Electron Microscope Facility (University of Hawaii, Manoa); the University of Arizona Electron Microscopy Center for
JOBNAME: 67#2 96 PAGE: 3 SESS: 4 OUTPUT: Mon Jun 10 13:29:39 1996 /xypage/worksmart/tsp000/67596b/6 185
NOTE
FIG. 2. Crystalline aggregate (Cr) of virus-like particles within a mycetocyte cell of Bemisia tabaci Jatropha biotype (×100,600).
use of their facilities; and Julio Bird for providing the Jatropha biotype and culture and for valuable insights on this unique whitefly biotype. This research was funded by the Governor’s Agricultural Coordinating Committee, State of Hawaii, Contract 91-27; a University of Hawaii Research and Training Revolving Fund Award; and a Research Centers in Minority Institutions Grant, RR-03061, National, Institutes of Health.
REFERENCES Adams, J. R., and Bonami, J. R.. 1991. “Atlas of Invertebrate Viruses” CRC Press, Boca Raton. Bird, J. 1957. A whitefly-transmitted mosaic of Jatropha gossypifolia. Tech. Paper Agric. Exp. Station P.R. 22, 1–35. Brown, J. K. 1994. A global position paper: The status of Bemisia tabaci Genn. as a pest and vector in world agroecosystems. FAO Plant Protein Bull. 42, 3–32. Brown, J. K., Coats, S., Bedford, I. D., Markham, P. G., Bird, J., and Frohlich, D. R. 1995a. Characterization and distribution of esterase electropmorphs in the whitefly, Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) Biochem Genetics 33, 205–314. Brown, J. K., Frohlich, D. R., and Rosell, R. C. 1995b. The sweetpotato or silverleaf whiteflies: Biotypes of Bemisia tabaci or a species complex? Annu. Rev. Entomol. 40, 511–534.
FIG. 3. Virus-like particles (v) abutting the nuclear membrane in whitefly ovarian tissue. N, nucleus; m, mitrochondria (×18,450).
Buchner, P. 1965. “Endosymbiosis of Animals with Plant Microorganisms.” Interscience, New York. Clark, M. A., Baumann, L., Munson, M. A., Baumann, P., Campbell,
JOBNAME: 67#2 96 PAGE: 4 SESS: 4 OUTPUT: Mon Jun 10 13:29:39 1996 /xypage/worksmart/tsp000/67596b/6 186
NOTE
B. C., Duffus, J. E., Osborne, L. S., and Moran, N. A. 1992. The eubacterial endosymbionts of whiteflies (Homoptera: Aleyrodoidea) constitute a lineage distinct from the endosymbionts of aphids and mealybugs. Curr. Microbiol. 25, 119–123. Costa, H. S., Brown, J. K., Sivasupramaniam, S., and Bird, J. 1993. Regional distribution, insecticide resistance, and reciprocal crosses between the A and B biotypes of Bemisia tabaci. Insect Sci. Applic. 14, 255–266. Costa, H. S., Westcot, D. M., Ullman, D. E., and Johnson, M. W. 1993. Ultrastructure of the endosymbionts of the whitefly, Bemisia tabaci and Trialeurodes vaporariorum. Protoplasma 176, 106–115. Faust, R. M. 1992. Conference report and 5-year national research and action plan for development of management and control methodology for the sweetpotato whitefly, Houston, Texas, Feb. 18–21, 1992. U.S. Department of Agriculture, ARS, 107. Francki, R. I. B., Fauquet, C. M., Knudson, D. L., and Brown, F. (Eds.) 1991. “Classification and Nomenclature of viruses.” Fifth report of the International Committee on Taxonomy of Viruses. Arch. Virol. Suppl. 2, Springer-Verlag, New York.
H. S. COSTA* D. M. WESTCOT† D. E. ULLMAN† R. C. ROSELL‡ J. K. BROWN‡ M. W. JOHNSON† *Department of Entomology University of California Riverside, California 92521 †Department of Entomology University of Hawaii at Manoa Honolulu, Hawaii 96822 ‡Department of Plant Sciences University of Arizona Tucson, Arizona 85721 Received January 25, 1995; accepted November 3, 1995