Molecular key to seven Brazilian species of Trichogramma (Hymenoptera: Trichogrammatidae) using sequences of the ITS2 region and restriction analysis

Chave molecular para sete espécies brasileiras de Trichogramma (Hymenoptera: Trichogrammatidae) utilizando o sequenciamento da região ITS2 e análise de restrição

SYSTEMATICS, MORPHOLOGY AND PHYSIOLOGY

Molecular Key to Seven Brazilian Species of Trichogramma (Hymenoptera: Trichogrammatidae) Using Sequences of the ITS2 Region and Restriction Analysis

AMÉRICO I. CIOCIOLA JR.¹, ROBERTO A. ZUCCHI¹ AND RICHARD STOUTHAMER²

¹Departamento de Entomologia, Fitopatologia e Zoologia Agrícola, ESALQ/USP, Caixa postal 9, 13418-900, Piracicaba, SP, Brasil.

²Department of Plant Sciences, Laboratory of Entomology, Wageningen University, P.O. Box 8031, 6700 EH, Wageningen, The Netherlands.

Chave Molecular para Sete Espécies Brasileiras de Trichogramma (Hymenoptera: Trichogrammatidae) Utilizando o Sequenciamento da Região ITS2 e Análise de Restrição

RESUMO – Utilizando-se o sequenciamento da região ITS2 do DNA ribossomal juntamente com algumas enzimas de restrição, pôde-se construir uma chave molecular simples e precisa de algumas espécies brasileiras de Trichogramma. Esta chave é fácil de ser elaborada e resultados rápidos são obtidos na identificação desse pequeno parasitóide (0,25 mm). Usando-se essa metodologia, pode-se também verificar possíveis contaminações em criações de laboratório.

PALAVRAS-CHAVE: Insecta, DNA, enzimas, controle biológico.

ABSTRACT – Using the ITS2 sequences of the ribosomal DNA together with some restriction enzymes, a simple and precise molecular key to some Brazilian species of Trichogramma was created. This key is very easy to make and quick results can be obtained on the identification of this minute parasitoid (0.25 mm). The methodology presented is easily implemented and can be used to detect possible contaminations under laboratory rearings.

KEY WORDS: Insecta, DNA, enzymes, biological control.

In biological control programs using parasitoids of the genus Trichogramma (Hymenoptera: Trichogrammatidae), a very important step is the identification and the use of correct species to be released in the field. The identification of these wasps is difficult due to their size (0.25 mm in length) and only specialists can reliably identify specimens, using characteristics of the male genitalia. In the past, Trichogramma identification was based on body color and setation as morphological parameters. Later, Nagarkatti & Nagaraja (1971) found out the taxonomic use of male genitalic characteristics. This lead to an improvement of the identification of these minute wasps. Using this technique, around 200 species have been described (Pinto 1998). Due to the small size and few characters available, specific identification is still difficult, particularly when sibling species need to be distinguished. In addition, the specimen preparation for morphological identification is time consuming and requires much trainning. In this paper, the identification of this minute wasp was based on DNA sequences of the internally transcribed spacer (ITS-2). This technique can be applied by any lab with access to a PCR equipment and it allows a quick and precise Trichogramma species identification. At the species and intraspecific levels, the ITS1 and ITS2 regions have been often used as a taxonomic tool for insect identification (Campbell et al. 1993, Hoy 1994). The sequence and restriction analysis of the ITS2 rDNA has been described in recent studies as a tool for Trichogramma identification as well (Kan et al. 1996, Pinto et al. 1997, Stouthamer et al. 1999). Using this technique, it was possible to identify seven Trichogramma species and to elaborate a molecular key to these species recorded in Brazil.

Material and Methods

Trichogramma Cultures. Populations of seven species (Table 1): T. acacioi, from Botucatu-SP, T. galloi Zucchi from Copersucar Company, Piracicaba-SP, T. pretiosum Riley from several locations, T. rojasi Nagaraja & Nagarkatti and T. lasallei Pinto from Curitiba-PR, T. bruni from Piracicaba-SP, and one population of T. atopovirilia Oatman & Platner from Venezuela, were used to elaborate the molecular key. The population from Venezuela was used because no success on DNA extraction of one population of T. atopovirilia from Brazil was obtained.

DNA Extraction by Chelex-100. Five individuals from each isofemale population, preserved in 100% ethanol, were shaken in 100 ml of TE before grinding. The wasps were ground with 100 ml of 5% Chelex-100 and 4ml of proteinase K (20mg/ml), incubated at least 6h at 56ºC followed by 10 minutes at 95ºC.

PCR, Cloning by Escherichia coli and Sequencing the ITS2 Region of the rDNA. The PCR was done in a total volume of 50 ml using a Techne thermocycler. For one reaction, 5 ml of DNA template were used, plus 45 ml of the PCR mix (5 ml of PCR buffer, 1ml of dNTP's each in a 10mM concentration), 1 ml of the ITS2 forward primer (5'-TGTGAACTGCAGGACACATG-3') located in the 5.8s region of rDNA, 1 ml of the reverse primer (5'-GTCTTGCCTGCTCTGAG-3'), located in the 28s region of rDNA, 0.14 ml of superTAQ polymerase (Sphaero-Q 5 units/ml and 36.86 ml of bidestiled and autoclaved water). The primers used to amplify the ITS2 region were the same used by Campbell et al. (1993) with sequence modification of some nucleotides to be specific to Trichogramma. The termocycler program used was: 3 min. at 94ºC, followed by 33 cycles of 40 seconds at 94ºC, 45 seconds at 55ºC and 45 seconds at 72ºC, with 5 min. at 72ºC after the last cycle.

The PCR product was purified with a QIAquick PCR purification kit (QiagenÒ). After the purification the PCR product was tied up to a pGEM-TÒ vector (Promega), 2 ml of the Ligation mix was transformed in the heatshock cells of DH5-a E. coli and plated in a LB agar medium containing Ampicillin, X-GAL and IPTG. The plates were stored overnight at 37ºC. The next day, white colonies were picked up with a sterile toothpick from the plates and placed into tubes containing 3.5 ml of LB liquid medium and grown overnight in a shaker set to 250 rpm and 37ºC. To confirm that the correct piece of DNA had been cloned, a PCR reaction with a template extracted from the bacterial culture was performed. Ten microliters of bacteria culture was added to 100 ml 5% Chelex and incubated for 15 min. at 60ºC followed by 5 min. at 95ºC. The PCR was performed in a final volume of 25 ml. If indeed an ITS-2 had been cloned, 850 ml of the bacteria culture was added with 150 ml of 87% glycerol and stored at –80ºC. The rest of the culture was used in a QIAprep Miniprep kit (QiagenÒ) to purify the plasmid, which was used for the sequencing in a Applied Biosystems automatic sequencer.

Alignment of the Sequences and Restriction Analysis. The sequences of the six Trichogramma species were aligned manually using ESEE 3.0s sequence editor (Cabot 1995). The sequence sizes data and restriction analysis were used to elaborate a molecular key. The PCR products were digested with Eco RI, Mse I, Pvu I and Nru I in a total volume of 10 ml, and incubated at 37ºC for 1h.

Results and Discussion

The five individuals used for DNA extraction were enough to obtain good PCR products (Fig. 1) for cloning and for sequencing. All the sequences (Table 1) were sent to the Genbank and have the accession numbers: AF282222-AF282239. Using the sizes of the PCR products (Fig. 1) with the enzymes Eco Rl, Mse l , Pvu I and Nru I (Table 2), all the species were distinguished in this work. The key made (Table 3) shows that the sequence size of the ITS2 region of the rDNA of some species of Trichogramma is useful and can be used as a tool to quickly identify species. It is important to know that this key fits well for the populations studied in this work. More samples are needed in order to construct a complete key in the future with all other Trichogramma species from Brazil. With a molecular key of all Brazilian species as well as all the sequences, it can be easily known if new species are occurring in Brazil just comparing the recorded sequences of each species. Currently, 14 species are recorded in Brazil (Zucchi & Monteiro 1997).

Sequences of the ITS2 region of the rDNA presented in this paper show their importance for identification of Trichogramma species, which were previously identified only by morphological characteristics. Using the sequence of the ITS2 region, closely related species of Trichogramma can also be distinguished (Stouthamer et al. 1999). Trichogramma identification using RAPD-PCR (Vanlerberghe-Masutti 1994) has several problems when compared to what is presented in this work. In RAPD there are problems of transportability and at least 50 individuals are required to obtain good results. The technique used in this study is relatively cheap and gives quick results not only on Trichogramma identifications, but it also allows to monitorate laboratory rearings for possible contaminations. Another advantage of this technique is that in field parasitism surveys, a quick rate of parasitism on collected host eggs can be determined by PCR (Amornsak et al. 1998). Morphological data are extremely important when combined with molecular work. Both taxonomy data and sequences of the ITS2 region of the rDNA can give a precise identification of Trichogramma species, and it will be an improvement for the use of this parasitoid in biological control programs against several lepidopterous pests. To make a complete key from Brazilian Trichogramma species more samples must be collected, which will be done by comparing the sequences presented in this work with the taxonomic data of a new sample.

Acknowledgements

The authors are thankful to CAPES and CNPq by the financial support to this research, and to all those who have sent Trichogramma samples. They also thank Bertha Koopmanschap for her help in applying the molecular technique, and to Dr. José R. P. Parra (ESALQ/USP) for his comments on this paper.

Literature Cited

Received 25/VII/2000. Accepted 08/V/2001.

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Publication Dates

• Publication in this collection
  13 Aug 2004
• Date of issue
  June 2001

History

• Accepted
  08 May 2001
• Received
  25 July 2000