NOTAS CIENTÍFICAS MOLECULAR CHARACTERIZATION OF WHITEFLY ( BEMISIA SPP . ) IN BRAZIL

RAPD analysis was used to evaluate the genetic variability of 12 Brazilian populations of Bemisia spp. (Hemiptera: Aleyrodidae). Ten primers were analysed in order to detect the polymorphism between the samples tested. The results showed that individuals from a colony maintained since 1983 had similar patterns of Bemisia tabaci originated from California, USA. The other samples analysed showed similar patterns to B. tabaci biotype B (=B. argentifolii), also from California, USA, indicating a great dissemination of this biotype in Brazil. Phenetic analysis of populations revealed high homogeneity between individuals of the B biotype of B. tabaci. CARACTERIZAÇÃO MOLECULAR DA MOSCA-BRANCA (BEMISIA SPP.) NO BRASIL RESUMO A análise de RAPD foi utilizada para avaliar a variabilidade genética de 12 populações brasileiras de Bemisia spp. (Hemiptera: Aleyrodidae). Foram analisados dez primers que permitiram a detecção de polimorfismo entre as amostras testadas. Os resultados obtidos mostraram que os indivíduos analisados provenientes de uma colônia mantida desde 1983 apresentaram perfis de RAPD próximos do padrão de B. tabaci oriunda da Califórnia, EUA. As outras populações analisadas apresentaram padrões semelhantes ao de B. tabaci raça B (=B. argentifolii), também oriunda da Califórnia, EUA, indicando a grande disseminação deste último biótipo no Brasil. A análise fenética dos dados dessas populações revelou uma alta homogeneidade entre os indivíduos do biótipo B de B. tabaci. The pest status of whiteflies of the genus Bemisia increased greatly in Brazil in the last few years. The type species of the genus, Bemisia tabaci (Gennadius), used to be considered important as a vector of virus disease, rather than as an insect pest. 1 Accepted for publication on June 11, 1999. 2 Biologist, Dr., Embrapa-Centro Nacional de Pesquisa de Recursos Genéticos e Biotecnologia (Cenargen), Caixa Postal 02372, CEP 70849-970 Brasília, DF. E-mail: vilarin@cenargen.embrapa.br 3 Agronomist, Dr., Embrapa-Cenargen. E-mail: myrian@cenargen.embrapa.br 4 Agronomist, Ph.D., Biology Dept., MSIRI, Reduit-Mauritius. E-mail: sjanabi@hotmail.com

A population of B. tabaci that was initially associated with unprecedent whitefly infestations in greenhouse-grown ornamentals in the USA, Caribbean Basin, and Europe, in late 1985 was designated as the B-biotype of B. tabaci (Costa & Brown, 1991;Brown et al., 1995) or B. argentifolii Bellows & Perring (Perring et al., 1993;Bellows et al., 1994).The existence of biotypes or host races of the whitefly, B. tabaci was proposed in the 1950s after the discovery that morphologically indistinguishable populations of B. tabaci exhibited measurably different biological traits with respect to host range, host-plant adaptability, and plant virus-transmission capabilities (Costa & Russell, 1975;Bird & Maramarosch, 1978;Brown et al., 1995).Recently recognized differences among populations of B. tabaci, the sweetpotato whitefly, and B. argentifolii, the silverleaf whitefly, represent either different biotypes of B. tabaci or are members of a species complex (Brown et al., 1995).The B type of B. tabaci (=B.argentifolii) has spread to many areas world wide due, probably, to the international trade of plants.
The introduction of B. tabaci biotype B in Brazil was first reported in 1991/92, in the State of São Paulo (Melo, 1992;Lourenção & Nagai, 1994).Investigations made after the first detection suggested that the insect was introduced into the country on ornamental plants commonly sold in the international market; a very heavy infestation of B. tabaci was observed in a greenhouse planted with Crysanthemum sp.(Lima et al., 1992;Oliveira & Lima, 1997).Since then, Villas Bôas et al. (1997) and Pedrosa et al. (1997), reported individuals of this species associated to symptoms of geminivirus in tomatoes and cabbage crops of central area of Brazil, in 1993.The favourable climate conditions and a great number of host plants may have played an important role on dissemination of this biotype.
The identification of a new species has been mainly based on crossing experiments, mating behavior, and isozymes analysis (Perring et al., 1993).The morphological characters used for whiteflies identification are not clearly and easily able to differentiate B. tabaci from B. tabaci biotype B (Bellows et al., 1994).
The analysis of RAPD markers has been showed to be useful to recognize inter and intraspecific differences among Bemisia spp.(Gawel & Bartlett, 1993;Perring et al., 1993;Barro & Driver, 1997).Compared to isozymes, this analysis has the advantages to allow the use of alcohol preserved material, and to extensively cover the genome.
The purpose of this study was to evaluate the genetic variability of the complex species of Bemisia occurring in Brazil, using RAPD analysis.
Adults of Bemisia spp.were collected in different crops and regions in the years 1996/1997 (Table 1).They were kept in 100% ethanol until use.Adults from colonies in California, USA (Perring et al., 1993) were used as standards for B. tabaci and B. tabaci biotype B. Species identification was based on morphological characteristic.Four female specimens from each sample were selected for molecular analysis.
A modified DNA extraction protocol of Kazmer et al. (1995) was used to extract DNA.Individual insect was ground in a 1.5 mL microcentrifuge tube with 59 mL of well-stirred, sterile 5% Chelex-100 using a disposable micropestle, after which 1 mL of proteinase K (20 mg/mL) was added and mixed.The samples were incubated at 65°C for one hour and then for ten minutes at 95 o C. The tubes containing the DNA solution were spun for five seconds and then stored at -20°C until needed.
The PCR reactions were performed in a 50 mL volume containing 2 mL of previously prepared template DNA solution, two units of Taq polymerase (Cenbiotec, Porto Alegre, RS, Brazil), 5 mL of 10x polymerases recommended buffer, 200 mm of each dNTP (Pharmacia), and 0.4 mm primer.Amplification was carried out in 96-well PCR plates using the PTC-100 programmable thermal controller (MJ Research).The temperature profile used was one step at 94°C for three minutes, followed by 45 cycles of 93°C for one minute, 35°C for one minute and 72°C for two minutes, with final extension step at 72°C for five minutes.The reactions were maintained at 4°C until electrophoresis.PCR products were electrophoresed in 2% LE agarose gel prepared in 0.5x Trisborato-EDTA (TBE) buffer, stained with ethidium bromide (Sambrook et al., 1989), and photographed under UV light.Primers were selected according to Gawel & Bartlett (1993).Ten 10-mer primers (Operon Technologies, Alameda, CA) were used: OPA-02, OPA-03, OPA-04, OPA-05, OPA-10, OPA-11, OPA-13, OPA-15, OPA-17 and OPA-20.
DNA fingerprints were scored directly from the photographs.Only well resolved products were scored.The presence or absence of each fragment was considered as an independent character.RAPD markers were analyzed using NTSYS-pc V1.8 (Rohlf, 1993).A similarity matrix was calculated using Jaccard similarity coefficient.Clustering was done using the unweighted mean pair group arithmetic mean method (UPGMA) (Sneath & Sokal, 1973).
The DNA extraction protocol adopted proved to be simple and produced consistent yield within the time frame, which is conducive to performing largescale population genetics analysis on these insects.The amount of crude DNA extracted of each specimens in this study was more than sufficient to prepare all the PCR reactions.
The analysis of RAPD markers proved to be an efficient method to distinguish species on Bemisia samples collected in Brazil.Examples of fingerprinting data obtained is shown in Figs. 1 and 2   Only individuals from sample 1 (Table 1) presented similar RAPD patterns to individuals of B. tabaci from the USA.All the other specimens from different populations analyzed showed to be related to B. tabaci biotype B (Figs. 1  and 2).Therefore, the spread of biotype B associated with phytotoxic symptoms was confirmed by the RAPD analysis.Individuals presented no common bands with B. tabaci.
RAPD patterns showed to be also efficient markers to detect genetic variability within biotypes.A total of 25 bands were scored with the five primers selected, OPA-02, OPA-03, OPA-04, OPA-15 and OPA-20, presenting ten polymorphic bands among B. tabaci biotype B samples.An example of fingerprinting among these samples is shown in Fig. 3. Although, different genotypes were detected within and among samples, cluster analysis of the RAPD characters did not show clear phenetic groups related to host plants or geographical region.
This study is the first of a series that will be analysing the complex population of B. tabaci in Brazil, in order to develop integrated pest management programs.The identification of the biotype B of B. tabaci in our agricultural systems may have severe implications as most of the crops produced are for subsistence, cultivated in small areas such as 1 ha to 10 ha.
These results showed that RAPD markers could be a useful tool to resolve identification questions and to analyze genetic variability of Bemisia spp. in Brazil.Recognition of species and population variation is important to develop strategies for pest management.1).The primer used was OPA-02.
. Specimens of B. tabaci and