Técnica Molecular para a Identificação de Espécies Próximas de Trichogramma (Hymenoptera: Trichogrammatidae): T. rojasi Nagarkatti & Nagaraja e T. lasallei Pinto

ne Neotropical Entomology Neotrop. entomol. 1519-566X 1678-8052 Sociedade Entomológica do Brasil Londrina, PR, Brazil O sequenciamento da região ITS2 (espaço interno transcrito) do rDNA foi utilizado para identificar duas espécies próximas de Trichogramma: T. rojasi e T. lasallei, esta última recentemente constatada no Brasil. O DNA das amostras foi extraído utilizando-se Chelex 100. O produto de PCR foi ligado ao vetor pGEM-T ® e ambos foram então acondicionados em placas de Petri para o crescimento de colônias brancas, o que constata a ligação correta entre o vetor e o DNA. A partir dessas colônias, uma pequena quantidade (10µl) foi purificada utilizando material específico e seqüenciada. As seqüências foram alinhadas pelo programa ESEE 3.0. Os primers específicos utilizados para a amplificação do rDNA da região ITS2 das espécies de Trichogramma estudadas foram eficientes. O sequenciamento das duas espécies diferiu em comprimento e posição dos nucleotídeos, mostrando que são distintas. Os resultados mostraram que esta é uma boa técnica para identificação de espécies crípticas de Trichogramma, difíceis de identificar quando são utilizados apenas caracteres morfológicos. a10v30n4 SYSTEMATICS, MORPHOLOGY AND PHYSIOLOGY Molecular Tool for Identification of Closely Related Species of Trichogramma (Hymenoptera: Trichogrammatidae): T. rojasi Nagaraja & Nagarkatti and T. lasallei Pinto AMÉRICO I. CIOCIOLA JR.1, RANYSE B. QUERINO1, ROBERTO A. ZUCCHI1 AND RICHARD STOUTHAMER2 1 Depto. de Entomologia, Fitopatologia e Zoologia Agrícola, ESALQ/USP, Av. Pádua Dias, 11, CEP 13.418-900 Piracicaba, SP 2 Dept. of Plant Sciences, Laboratory of Entomology, Wageningen University, P.O. Box 8031, 6700 EH, Wageningen, The Netherlands Técnica Molecular para a Identificação de Espécies Próximas de Trichogramma (Hymenoptera: Trichogrammatidae): T. rojasi Nagarkatti & Nagaraja e T. lasallei Pinto RESUMO – O sequenciamento da região ITS2 (espaço interno transcrito) do rDNA foi utilizado para identificar duas espécies próximas de Trichogramma: T. rojasi e T. lasallei, esta última recentemente constatada no Brasil. O DNA das amostras foi extraído utilizando-se Chelex 100. O produto de PCR foi ligado ao vetor pGEM-T ® e ambos foram então acondicionados em placas de Petri para o crescimento de colônias brancas, o que constata a ligação correta entre o vetor e o DNA. A partir dessas colônias, uma pequena quantidade (10µl) foi purificada utilizando material específico e seqüenciada. As seqüências foram alinhadas pelo programa ESEE 3.0. Os primers específicos utilizados para a amplificação do rDNA da região ITS2 das espécies de Trichogramma estudadas foram eficientes. O sequenciamento das duas espécies diferiu em comprimento e posição dos nucleotídeos, mostrando que são distintas. Os resultados mostraram que esta é uma boa técnica para identificação de espécies crípticas de Trichogramma, difíceis de identificar quando são utilizados apenas caracteres morfológicos. PALAVRAS-CHAVE: Insecta, região ITS2, controle biológico, rDNA, parasitóide. ABSTRACT – The sequence of the ITS2 (internally transcribed spacer 2) region of the rDNA was used to identify two closely related Trichogramma species: T. rojasi and T. lasallei, the second species recently reported in Brazil. The DNA of the samples was extracted using Chelex 100. The PCR product was linked to a pGEM-T® vector and both were then placed into Petri dishes to grow white colonies, which demonstrate the correct ligation between the vector and the DNA. From these colonies, 10µl were purified and sequenced using a special kit, and the sequences aligned using the ESEE 3.0 software. Specific primers were efficient to amplify the ITS2 rDNA region of the Brazilian Trichogramma. The sequence of each species differed in length and in nucleotide position, showing that they are distinct. Thus, the results show that this technique is a good tool to identify Trichogramma cryptic species, otherwise difficult to identify when using only morphological characters. KEY WORDS: Insecta, ITS2 region, biological control, rDNA, parasitoids. Parasitoids of the genus Trichogramma are minute wasps (about 0,25mm in length) and are the most important egg parasitoids used in biological control programs against lepidopterous pests (Parra 1997). The taxonomy of this group is difficult due to its small size. Before the work done by Nagarkatti & Nagaraja (1971), who used the male genitalia of Trichogramma species for identification and started a modern taxonomy for this genus, the identification was made only using color and setation (Stouthamer et al. 1999). The use of the male genitalia to identify the almost 200 species of Trichogramma (Pinto 1998) is still a problem because some of them multiply by parthenogenesis. In addition to that, several populations are similar when the male genitalia is analysed. The ribosomal DNA (rDNA) has been reported as an important tool for taxonomic work since these ribosomal genes have been highly preserved throughout evolution (Orrego & Agudelo-Silva 1993, Hoy 1994, Pinto et al. 1997). The purpose of this work was to use the technique of sequencing the ITS2 region of the rDNA recently developed by Stouthamer et al. (1999), for the first time used in Brazil, to identify two Brazilian Trichogramma cryptic species, T. rojasi and T. lasallei. Material and Methods Trichogramma Cultures. Cultures of T. rojasi and T. lasallei were collected on soybeans at Rio Grande Farm, Curitiba county, Paraná state. The parasitoids were reared on Anagasta kuehniella (Zeller) (Lep.: Pyralidae) eggs, under 70±10% relative humidity, 25±2°C temperature and 14h photophase (Parra 1997). Taxonomy of T. rojasi and T. lasallei. Samples of males of each species were mounted on microscope slides using Hoyer's medium (Rosen & DeBach 1979) and the identification of the two species were based on the morphology of the genitalia and on the posterior setal track of the hind wing (Pinto 1998). DNA Extraction by Chelex-100. Five individuals from isofemale populations of each species were shaken in 100µl of TE, ground in 100 µl of 5% Chelex-100 and 4 µl of proteinase K (20 mg/ml) and incubated overnight at 56°C followed by 10 min. at 95°C. PCR, Cloning and Sequencing the ITS2 Region of the rDNA. A PCR was performed in a total volume of 50 µl using a Techne thermocycler. For one reaction, 5 µl of DNA template (1,6 µg/ml) were used, with 45 µl of the PCR mix [5 µl of 10x PCR-buffer, 1 µl of dNTP's (each at 10mM), 1µl of the ITS2 forward primer (5'-TGTGAACTGGAGGACACATG-3') located in the 5.8s region of rDNA, 1 µl of the reverse primer (5'-GTCTTGCCTGCTGAG-3'), located in the 28s region of the rDNA, 0.14 µl of TAQ polymerase 5 units/µl and 36,86 µl of bidestiled and autoclaved water]. The primers used to amplify the ITS2 region were the same used by Campbell et al. (1993) with modifications on the position of some nucleotides to be more specific to Trichogramma. The thermocycler program used was as follows: 3 min. at 94°C, followed by 33 cycles of 40 seconds at 94°C, 45 seconds at 55°C and 45 seconds at 72°C, with 5 min. at 72°C after the last cycle. The PCR product was purified with a QIAquick PCR purification kit (Qiagen ®). After purification, the PCR product was linked to a pGEM-T ® vector (Promega). The ligation (2 µl) mix was transformed in the heat shock cells off DH5-a E. coli and plated in a LB agar medium containing 3,5 ml of LB liquid medium with Ampicillin, X-GAL and IPTG and let to grow overnight in a shaker set at 250 rpm and 37°C. The bacterial culture (10 µl) was added to 100 µl Chelex 5%, and incubated during 15 min. at 60°C followed by extra 5 min. at 95°C. A PCR, with the final volume of 25 µl, was done to confirm if the correct DNA inserted had been cloned. When the reaction was positive, 850 µl of bacterial culture was added to 150 µl of 87% Glycerol and stored at -80°C. The remaining culture was used in a QIAprep Miniprep kit by Qiagen ® and sequenced on an Applied Biosystems automatic sequencer. Alignment of Sequences. The T. rojasi and T. lasallei sequences were manually aligned using the ESEE 3.0s sequence editor (Cabot 1995), and sent to the Genbank with the acession numbers AF-282237 and AF-282239, respectively. Results and Discussion The use of five individuals from each Trichogramma population with Chelex-100 to grind the samples and extract the DNA gave enough PCR products to be cloned and sequenced. The PCR product was enough to clone and sequence the ITS2 region of the rDNA. After sequencing it was found the length of the ITS2 region to be 453 bp (base pairs) for T. lasallei (TL-20) and 526 bp for T. rojasi (TR-23). By using the ITS2 region of the rDNA it was possible to distinguish these two closely related species, and several differences were found among the sequences showing that the species are distinct (Table 1). Based on the taxonomic studies of T. lasallei and T. rojasi by Pinto (1998) and together with the sequence of each population studied T. lasallei was confirmed as a new record for Brazil. The report of T. lasallei for Brazil is recent (Ranyse Barbosa Querino, personal communication), and this species is morphologically similar to T. rojasi, already found in the country. The two species are sympatric and were collected in the state of Paraná. The morphological characteres to differentiate these two species were discussed by Pinto (1998), where T. lasallei differs from T. rojasi primarily by the broad genital capsule, the more anterior position of the ventral process, the narrower and slightly longer setae on the male flagellum, the shorter anterior mesoscutellar setae, and the shorter posterior setal track of the hind wing. On T. rojasi the genital capsule is only ca. 0.3 as wide as long, the ventral processes are positioned at less than 0.1 the basal distance, and the posterior track of the hind wing consists of ca. 12 elongate setae and reaches the wing apex. By contrast, in T. lasallei the genital capsule is ca. 0.35 as wide as long, the ventral process is positioned at 0.2 the basal distance, and the posterior track of the hind wing is short, consisting of only 5-8 setae and does not approach the wing apex. As seen, there are small differences between T. rojasi and T. lasallei and, in some cases, the identification of these parameters is difficult (Pinto 1998). So, to clarify the identification of these two species, the sequence of their ITS2 regions of the rDNA can easily be done to confirm the taxonomic data. Sequences of the ITS2 region of the rDNA are used as data bank to compare with sequences already done with new samples not yet sequenced (Ciociola Jr. 2000). The sequences obtained in this work were sent to the Genbank and it is available on internet. As for T. lasallei and T. rojasi, differences were found in the size of the ITS2 region of the rDNA and also on the position of some nucleotides, thereby showing that they are different species (Table 1). Alignment of Sequences. The sequences of the ITS2 region aligned showed, with the ESEE 3s program (Cabot 1995), that differences were found between the populations studied. It is possible to identify closely related species of Trichogramma from Brazil using the size of the ITS2 region plus the flanking region. This technique has several advantages over RAPD because only few individuals are needed to extract the DNA and the specific primers for Trichogramma are already known. In RAPD many insects are necessary and several primers must be tested (Landry et al. 1993, Vanlerberghe-Masutti 1994). Another difficulty is that problems with transportability may occur (Stouthamer et al.1999). The ITS2 method has also advantages over allozyme markers because samples can be better preserved by storage in 100% alcohol or in liquid nitrogen. Furthermore, the ITS2 of the rDNA contains more options for species-specific characters than a single allozyme with at least 2-5 distinguishable alleles (Stouthamer et al. 1999). An additional advantage of this technique is that it is relatively unexpensive and gives quick results. It is important to know that the sequence of the ITS2 of the rDNA cannot be used alone to identify Trichogramma species. Morphology is very important and the samples must be analyzed in this respect before the molecular work is done. With the ITS2 sequence, it is also possible to check for possible contamination during laboratory rearings. The use of PCR for Trichogramma can enable entomologists to determine levels of parasitism under field conditions without delay (Amornsak et al. 1998). When the sequences of the species present in one country is already available, it is possible to determine if a new species is present or not by simply comparing the sequences of the populations under study. This technique is easy to be used but requires basic laboratory facilities. The use of this molecular tool may solve many problems with Trichogramma identification in Brazil and improves the adoption of biological control using this minute wasp. 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