Bulletin of Entomological Research (2005) 95, 309–312
DOI: 10.1079/BER2005361
Nuclear DNA content of the whitefly Bemisia tabaci (Aleyrodidae: Hemiptera) estimated by flow cytometry J.K. Brown1*, G.M. Lambert1, M. Ghanim3, H. Czosnek2 and D.W. Galbraith1 1
Department of Plant Sciences, The University of Arizona, Tucson, AZ 85721, USA: 2The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel: 3Department of Entomology, Agricultural Research Organization, Bet Dagan, Israel Abstract The nuclear DNA content of the whitefly Bemisia tabaci (Gennnadius) was estimated using flow cytometry. Male and female nuclei were stained with propidium iodide and their DNA content was estimated using chicken red blood cells and Arabidopsis thaliana L. (Brassicaceae) as external standards. The estimated nuclear DNA content of male and female B. tabaci was 1.04 and 2.06 pg, respectively. These results corroborated previous reports based on chromosome counting, which showed that B. tabaci males are haploid and females are diploid. Conversion between DNA content and genome size (1 pg DNA = 980 Mbp) indicate that the haploid genome size of B. tabaci is 1020 Mbp, which is approximately five times the size of the genome of the fruitfly Drosophila melanogaster Meigen. These results provide an important baseline that will facilitate genomics-based research for the B. tabaci complex. Keywords: Bemisia tabaci, nuclear DNA, genome, flow cytometry, Hemiptera Introduction The accurate estimation of the nuclear genome size for arthropods, some of which are important agricultural pests and/or insect vectors of plant viruses, is essential for undertaking a variety of genomics and proteomics efforts, and for evolutionary studies. Estimates of genome size are particularly useful when developing bacterial artificial chromosome (BAC)/sequence tagged connector (STC) based scaffold sequences, from which genes can be positionally cloned. In the absence of whole-genome sequence data, the genome sizes of a number of organisms, including those for higher animals and plants, have been estimated indirectly by determining the nuclear DNA content (haploid or diploid C-value), using Feulgen microspectrophotometry and flow cytometry (compiled by Gregory, 2001).
*Fax: ++1-520-621-8839 E-mail:
[email protected]
Among selected members of the class Insecta, Gregory & Hebert (2003) have determined the nuclear DNA content of approximately 60 species of lepidopterans in 15 families. This was accomplished using Feulgen image analysis of sperm compared to a sperm standard for Drosophila melanogaster Meigen (Diptera: Drosophilidae) with a haploid C-value of 0.18 pg. Results from these studies indicated haploid C-values of insects examined ranged from a low of 0.29 pg for the monarch butterfly Danaus plexippus (Linnaeus) (Lepidoptera: Danaidae) to 1.9 pg for the geometrid moth Euchlaena irraria (Barnes & McDunnough) (Lepidoptera: Geometridae). The nuclear DNA content also has been estimated for several insect species of economic importance. For example, the haploid C-value for the honeybee Apis mellifera Linnaeus (Hymenoptera: Apidae) was estimated to be 0.17 pg, while an estimate of 0.52 pg was made for the silkworm moth Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae), and 1.00 pg for the Chinese oak silkmoth Antheraea pernyi Gue´rin-Me´neville (Lepidoptera: Saturniidae). Further, the C-value has been estimated for a number of agriculturally important insect
310
J.K. Brown et al.
pests, including the gypsy moth Lymantria dispar (Linnaeus) (Lepidoptera: Lymantriidae) at 1.03 pg, the wax moth Galleria melonella (Linnaeus) (Lepidoptera: Pyralidae) at 0.50 pg, the tobacco budworm moth Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae) at 0.41 pg, and the flour beetle Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) at 0.21 pg. The C-value also has been estimated for a number of aphids, including the pea aphid Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae) and the green peach aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae) for which a range of 0.31–0.32 pg was obtained. (Compiled by Gregory, 2001.) In contrast, genome size estimates are entirely unavailable for certain groups of insects, and among others, this holds true for the order Hemiptera. The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is of great interest as an agricultural pest and as a vector of several important groups of plant viruses (Brown & Bird, 1992; Brown et al., 1995; Brown & Czosnek, 2002). The size of the B. tabaci genome is of fundamental interest owing to the status of this whitefly as a cryptic species, its haplo-diploid reproductive mode, and an interest on the part of many to elucidate the genetic basis for interactions between it and the prokaryotic endosymbionts that inhabit it (Costa et al., 1995; Zchori-Fein & Brown, 2002). Finally, there is a growing interest among those that study B. tabaci to determine the complete genome sequence for this whitefly, both as a prototype aleyrodid, and as a widely distributed hemipteran that feeds on cultivated and non-cultivated plants in subtropical/tropical habitats, nearly worldwide.
The CRBC standard was prepared both as an external standard, analysed separately from the whitefly sample and also as an internal standard in which case an aliquot (1–4 ml) of CRBC was added directly to the whitefly nuclear suspension before staining.
Materials and methods
Results and Discussion
Whitefly samples
The mean nuclear DNA content for male and female B. tabaci was determined to be 1.04 and 2.06 pg, respectively, using chicken red blood cells as standards and propidium iodide as the fluorochrome (Johnston et al., 1999). Based on the value of 1 pg DNA = 980 Mbp (Bennet et al., 2000), the whitefly male and female genomes were estimated to be 1019 and 2019 Mbp, respectively. These estimates were based on the mean of nine (56.55; SD = 6.68) and eight (111.9; SD = 4.47) measurements each for males and females, respectively, which were collected from the colony on two different days. These results corroborated previous reports based on chromosome counting, which showed that B. tabaci males are haploid and females are diploid (Blackman & Cahill, 1998). The experimental results were highly reproducible as indicated by the standard error (SE). The coefficients of variation (CVs) for the individual measurements ranged from 8.0 to 12.0, which are slightly higher than desirable, but the DNA peaks were discrete and unambiguous (fig. 1), adding credence to the validity of the measurements. When the estimated genome size for B. tabaci was further compared with that for A. thaliana (fig. 1), the 1C peak of male B. tabaci (at 125) was located between the plant 4n (90) and 8n (180) peaks. Taking the haploid C-value of A. thaliana as 0.16 pg (Bennett et al., 2003), the nuclear DNA content of the male B. tabaci was estimated at 1C = 0.89 pg, a value close to that obtained when chicken red blood cells was used as standard. The ability to obtain the complete genome sequence for animals, including insects (http://www.genomenewsnet work.org/resources/sequenced_genomes/genome_guide_p1.shtml) now permits a comparison of the haploid Cvalues obtained by indirect methods with actual sequencing data. For D. melanogaster, the genome size of 1C = 0.18 pg,
The Arizona ‘B biotype’ of B. tabaci (Costa & Brown, 1991) was used to estimate the genome size for the B. tabaci complex. Male and female whiteflies were reared on cotton and maintained in a laboratory colony at the University of Arizona since 1981. The sex of the adult male and female whiteflies was determined by viewing whiteflies under a light microscope. Sexed male and female whiteflies were placed in separate polypropylene tubes and held at 4 C until analysis was carried out, which was within approximately 2 h of collection. Approximately 50 individual males or females were pooled for each sample. Pooled samples were analysed for males (n = 9) and females (n = 8) on two separate days.
Calibration and size standards Calibration of the flow cytometer was performed according to standard protocols using fluorescent calibration beads (DNA Check, Beckman-Coulter, Florida or Align Flow, Molecular Probes, Oregon) to verify instrument performance. Samples of known DNA content (Arabidopsis thaliana (L.) (Brassicaceae) or Nicotiana tabacum (L.) (Solanaceae) were prepared and analysed prior to the analysis of the unknowns to verify that all systems, buffers and protocols were performing properly. The standard used for determination of DNA content was chicken red blood cells (CRBC) collected previously and held at x80 C. The DNA content and the means of determining the DNA content for the standard has been described by Johnston et al. (1999), and the value assigned to the standard is 3.0-pg (diploid C-value).
Sample preparation and flow cytometry Adult whiteflies (50 insects) and A. thaliana leaves (50– 100 mg) were chopped using a single-edged razor blade in an ice-cold buffer consisting of 45 mM magnesium chloride, 30 mM sodium citrate, 20 mM MOPS (3-N-morpholinopropane sulphonic acid), 0.01% v/v Triton X-100, pH 7.0 (Galbraith et al., 1983, 1998). Samples were purified by passage through a 30 mm mesh filter and the volume was adjusted to 2 ml. Samples were stained with propidium iodide (stock solution 1 mg mlx1 in water) and incubated with DNase-free ribonuclease A at a final concentration of 10 mg mlx1 for 5 min at room temperature to eliminate ribonucleic acids. Propidium iodide was added to a final concentration of 50 mg mlx1 and samples were incubated on ice in the dark for 15 min before analysis. Propidium iodide stained samples were analysed using a Cytomation MoFLo cytometer (Cytomation, Fort Collins, Colorado, USA) equipped with a 488 nanometer (nm) laser excitation source operated at an output of 300 mW. Fluorescence emission was collected using a 630/40 band-pass filter. Histograms were processed using Summit software (Cytomation, Fort Collins, Colorado).
Nuclear DNA content of Bemisia tabaci 300
a
250
2n
200 150 100 50
311
Determining the complete genome sequence for B. tabaci will yield a plethora of informative data and permit these latter hypotheses to be tested based on genomics and proteomics analyses. The genome sequence also can be mined to inquire about innumerable aspects relevant to whitefly taxnomy (Costa & Brown, 1991; Brown et al., 1995), toxicology, insect sciences, virus-vector relationships (Brown & Czosnek, 2002), and endosymbiont-host interactions (Costa et al., 1995; Zchori-Fein & Brown, 2002), among others.
0 0
50
100 150 200 250 300 350 400 450 500
300 250
b
n
200 150 100 50 0 0
50
100 150 200 250 300 350 400 450 500
300 c
250 200
2n
150
4n
8n
100 50 0 0
50
100 150 200 250 300 350 400 450 500
Fig. 1. Flow cytometric determination of the nuclear DNA content of diploid female and haploid male Bemisia tabaci showing the relative DNA staining of nuclei. The nuclear genome size of (a) female and (b) male B. tabaci is approximately 2.06 and 1.04 pg, respectively. (c) Arabidopsis. The x-axis = channel number and the y-axis = number of nuclei collected.
obtained by flow cytometry (Vieira et al., 2002; Bennett et al., 2003) and Feulgen densitometry (Rasch et al., 1971; Mulligan & Rasch, 1980) using chicken cells as standard at 1C = 1.25 pg, is in robust agreement with the *180 Mbp obtained by sequencing the complete insect genome (Adams et al., 2000). The DNA content of the male B. tabaci 1C = 1.04 pg suggests that the haploid genome of the whitefly comprises approximately 1000 Mbp. This value indicates that B. tabaci genome size is five to six times larger than that of Drosophila, 1000 Mbp compared to 180 Mbp. In further contrast, the B. tabaci genome is approximately six times larger than that of the honey bee A. mellifera, four times that of the mosquito Anopheles gambiae Giles (Diptera: Culididae) (Holt et al., 2002), three times that of the aphid M. persicae, and twice that of the silkworm moth B. mori. It is presumed that B. tabaci does not have considerably more genes than the Drosophila spp. genome at *13,500. Hence, it is likely that a large portion of the genome of the whitefly B. tabaci does not encode genes, and may have a relatively high proportion of highly repetitive non-coding DNA sequences.
References Adams, M.D., Celniker, S.E., Holt, R.A., Evans, C.A., Gocayne, J.D., Amanatides, P.G., Scherer, S.E., Li, P.W., Hoskins, R.A., Galle, R.F., George, R.A., Lewis, S.E., Richards, S., Ashburner, M., Henderson, S.N., Sutton, G.G., Wortman, J.R., Yandell, M.D., Zhang, Q., Chen, L.X., Brandon, R.C., Rogers, Y.H., Blazej, R.G., Champe, M., Pfeiffer, B.D., Wan, K.H., Doyle, C., Baxter, E.G., Helt, G., Nelson, C.R., Gabor, G.L., Abril, J.F., Agbayani, A., An, H.J., AndrewsPfannkoch, C., Baldwin, D., Ballew, R.M., Basu, A., Baxendale, J., Bayraktaroglu, L., Beasley, E.M., Beeson, K.Y., Benos, P.V., Berman, B.P., Bhandari, D., Bolshakov, S., Borkova, D., Botchan, M.R., Bouck, J., Brokstein, P., Brottier, P., Burtis, K.C., Busam, D.A., Butler, H., Cadieu, E., Center, A., Chandra, I., Cherry, J.M., Cawley, S., Dahlke, C., Davenport, L.B., Davies, P., de Pablos, B., Delcher, A., Deng, Z., Mays, A.D., Dew, I., Dietz, S.M., Dodson, K., Doup, L.E., Downes, M., Dugan-Rocha, S., Dunkov, B.C., Dunn, P., Durbin, K.J., Evangelista, C.C., Ferraz, C., Ferriera, S., Fleischmann, W., Fosler, C., Gabrielian, A.E., Garg, N.S., Gelbart, W.M., Glasser, K., Glodek, A., Gong, F., Gorrell, J.H., Gu, Z., Guan, P., Harris, M., Harris, N.L., Harvey, D., Heiman, T.J., Hernandez, J.R., Houck, J., Hostin, D., Houston, K.A., Howland, T.J., Wei, M.H., Ibegwam, C., Jalali, M., Kalush, F., Karpen, G.H., Ke, Z., Kennison, J.A., Ketchum, K.A., Kimmel, B.E., Kodira, C.D., Kraft, C., Kravitz, S., Kulp, D., Lai, Z., Lasko, P., Lei, Y., Levitsky, A.A., Li, J., Li, Z., Liang, Y., Lin, X., Liu, X., Mattei, B., McIntosh, T.C., McLeod, M.P., McPherson, D., Merkulov, G., Milshina, N.V., Mobarry, C., Morris, J., Moshrefi, A., Mount, S.M., Moy, M., Murphy, B., Murphy, L., Muzny, D.M., Nelson, D.L., Nelson, D.R., Nelson, K.A., Nixon, K., Nusskern, D.R., Pacleb, J.M., Palazzolo, M., Pittman, G.S., Pan, S., Pollard, J., Puri, V., Reese, M.G., Reinert, K., Remington, K., Saunders, R.D., Scheeler, F., Shen, H., Shue, B.C., Siden-Kiamos, I., Simpson, M., Skupski, M.P., Smith, T., Spier, E., Spradling, A.C., Stapleton, M., Strong, R., Sun, E., Svirskas, R., Tector, C., Turner, R., Venter, E., Wang, A.H., Wang, X., Wang, Z.Y., Wassarman, D.A., Weinstock, G.M., Weissenbach, J., Williams, S.M., Woodage, T., Worley, K.C., Wu, D., Yang, S., Yao, Q.A., Ye, J., Yeh, R.F., Zaveri, J.S., Zhan, M., Zhang, G., Zhao, Q., Zheng, L., Zheng, X.H., Zhong, F.N., Zhong, W., Zhou, X., Zhu, S., Zhu, X., Smith, H.O., Gibbs, R.A., Myers, E.W., Rubin, G.M. & Venter, J.C. (2000) The genome sequence of Drosophila melanogaster. Science 287, 2185–2195. Bennett, M.D., Bhandol, P. & Leitch, I.J. (2000) Nuclear DNA content amounts in angiosperms and their modern uses– 807 new estimates. Annals of Botany 86, 859–909. Bennett, M.D., Leitch, I.J., Price, H.J. & Johnston, J.S. (2003) Comparisons with Caenorhabditis (*100 Mb) and Drosophila (*175 Mb) using flow cytometry show genome size in
312
J.K. Brown et al.
Arabidopsis to be *157 Mb and thus *25% larger than the Arabidopsis Genome Initiative estimate of *125 Mb. Annals of Botany 91, 547–557. Blackman, R.L. & Cahill, M. (1998) The karyotype of Bemisia tabaci (Hemiptera: Aleyrodidae). Bulletin of Entomological Research 88, 213–215. Brown, J.K. & Bird, J. (1992) Whitefly-transmitted geminiviruses in the Americas and the Caribbean Basin: past and present. Plant Disease 76, 220–225. Brown, J.K. & Czosnek, H. (2002) Whitefly-transmitted viruses. pp. 65–100 in Plumb, R.T. & Callow, J.A. (Eds) Advances in botanical research. New York, Academic Press. Brown, J.K., Frohlich, D.R. & Rosell, R.C. (1995) The sweet potato or silverleaf whiteflies: biotypes of Bemisia tabaci or a species complex? Annual Review of Entomology 40, 511–534. Costa, H.S. & Brown, J.K. (1991) Variation in biological characteristics and esterase patterns among populations of Bemisia tabaci, and the association of one population with silver leaf symptom induction. Entomologia Experimentalis et Applicata 61, 211–219. Costa, H.S., Wescot, D.M., Ullman, D.E., Rosell, R., Brown, J.K. & Johnson, M.W. (1995) Morphological variation in Bemisia endosymbionts. Protoplasma 189, 194–202. Galbraith, D.W., Harkins, K.R., Maddox, J.M., Ayres, N.M., Sharma, D.P. & Firoozabady, E. (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220, 1049–1051. Galbraith, D.W., Dolezel, J., Lambert, G. & Macas, J. (1998) DNA and ploidy analyses in higher plants. pp. 7.6.1–22 in Robinson, J.P. et al. (Eds) Current protocols in cytometry. New York, Wiley. Gregory, T.R. (2001) Animal Genome Size Database. http:// www.genomesize.com Gregory, T.R. & Hebert, P.D.N. (2003) Genome size variation in lepidopteran insects. Canadian Journal of Zoology 81, 1399–1405. Holt, R.A., Mani Subramanian, G., Halpern, A., Sutton, G.G., Charlab, R., Nusskern, D.R., Wincker, P., Clark, A.G., Ribeiro, J.M.C., Wides, R., Salzberg, S.L., Loftus, B., Yandell, M., Majoros, W.H., Rusch, D.B., Lai, Z., Kraft, C.L., Abrill, J.F., Anthouard, V., Arensburger, P., Atkinson, P.W., Baden, H., de Berardinis, V., Baldwin, D., Benes, V., Biedler, J., Blass, C., Bolanos, R., Boscus, D., Barnstead, M., Cai, S., Center, A., Chatuverdi, K.,
Christophides, G.K., Chrystal, M.A., Clamp, M., Cravchik, A., Curwen, V., Dana, A., Delcher, A., Dew, I., Evans, C.A., Flanigan, M., Grundschober-Freimoser, A., Friedli, L., Gu, Z., Guan, P., Guigo, I.R., Hillenmeyer, M.E., Hladun, S.L., Hogan, J.R., Hong, Y.S., Hoover, J., Jaillon, O., Ke, Z., Kodira, C., Kokoza, E., Koutsos, A., Letunic, I., Levitsky, A., Liang, Y., Lin, J.-J., Lobo, N.F., Lopez, J.R., Malek, J.A., McIntosh, T.C., Meister, S., Miller, J., Mobarry, C., Mongin, E., Murphy, S.D., O’Brochta, D.A., Pfannkoch, C., Qi, R., Regier, M.A., Remington, K., Shao, H., Sharakhova, M.V., Sitter, C.D., Jyoti Shetty, Smith, T.J., Strong, R., Sun, J., Thomasova, D., Ton, L.Q., Topalis, P., Tu, Z., Unger, M.F., Walenz, B., Wang, A., Wang, J., Wang, M., Wang, X., Woodford, K.J., Wortman, J.R., Wu, M., Yao, A., Zdobnov, E.M., Zhang, H., Zhao, Q., Zhao, S., Zhu, S.C., Zhimulev, I., Coluzzi, M., della Torre, A., Roth, C.W., Louis, C., Kalush, F., Mural, R.J., Myers, E.W., Adams, M.D., Smith, H.O., Broder, S., Gardner, M.J., Fraser, C.M., Birney, E., Bork, P., Brey, P.T., Venter, J.C., Weissenbach, J., Kafatos, F.C., Collins, F.H. & Hoffman, S.L. (2002) The genome sequence of the malaria mosquito Anopheles gambiae. Science 298, 279. Johnston, S.J., Bennett, M.D., Rayburn, A.L., Galbraith, D.W. & Price, H.J. (1999) Reference standards for the determination of DNA content of plant nuclei. American Journal of Botany 86, 609–613. Mulligan, P.K. & Rasch, E.M. (1980) The determination of genome size in male and female germ cells of Drosophila melanogaster by DNA-Feulgen cytophotometry. Histochemistry 66, 11–18. Rasch, E.M., Barr, H.J. & Rasch, R.W. (1971) The DNA content of sperm of Drosophila melanogaster. Chromosoma 33, 1–18. Vieira, C., Nardon, C., Arpin, C., Lepetit, D. & Bie´mont, C. (2002) Evolution of genome size in Drosophila. Is the invader’s genome being invaded by transposable elements? Molecular Biology and Evolution 19, 1154–1161. Zchori-Fein, E. & Brown, J.K. (2002) Diversity of prokaryotes associated with Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae). Annals of the Entomological Society of America 95, 711–718.
(Accepted 25 January 2005) Ó CAB International, 2005
