jr. Ciociola, A.I.

Stouthamer, R.

Zucchi, R.A.

English

Wageningen Yield 

    Neotropical Entomology 30(2) 259June, 2001
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.
Neotropical Entomology 30(2): 259-262 (2001)
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
260
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.
of some nucleotides to be specific to Trichogramma. The
termocycler program used was: 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 (Qiagenâ).  After the purification the PCR
product was tied up to a pGEM-Tâ 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 37°C. 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 37°C. 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 60°C followed
by 5 min. at 95°C. 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 –80°C. The rest of the culture was used in a QIAprep
Miniprep kit (Qiagenâ) 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).
Table 1. Trichogramma spp. lines used on the sequence of the ITS2  region of  the rDNA.
Line Number Source/Collector Host
T. pretiosum TP-02 EPAGRI-SC/ Dirceu Pratissoli         -
T. pretiosum TP-03 Quixadá-CE/ Raul P. Almeida Alabama argillacea
T. pretiosum TP-04 Alegre-ES/ Dirceu Pratissoli Tuta absoluta
T. pretiosum TP-05 V. N. Imigrante-ES/ Dirceu Pratissoli T.  absoluta
T. pretiosum TP-06 Lavras-MG/ Lusinério Prezotti Helicoverpa zea
T. pretiosum TP-07 Piracicaba-SP/Lusinério Prezotti H. zea
T. pretiosum TP-10 Campo Novo dos Parecis-MT/Raul Almeida A. argillacea
T. pretiosum TP-11 Campina Grande-PB/Raul P. Almeida  Neuroptera eggs
T. pretiosum TP-12 Campina Grande-PB/Raul P. Almeida Trichoplusia ni
T. pretiosum TP-13 Barreias-BA/Ivan Cruz Spodoptera frugiperda
T. pretiosum TP-15 CNPMS/EMBRAPA(área n. 1)/Ivan Cruz H. zea
T. pretiosum TP-16 CNPMS/EMBRAPA(área n. 2)/Ivan Cruz H. zea
T. pretiosum TP-17 Dourados-MS/Paulo DeGrande A. argillacea
T. acacioi TAC-18 Botucatu-SP/ Elisabete Bernardi Euselasia sp.
T. galloi TG-19 Piracicaba-SP/Copersucar Diatraea saccharalis
T. lasallei TL-20 Rio Grande Farm, Curitiba-PR/L.A. Foerster Anticarsia gemmatalis
T. atopovirilia 1 TA-21 Venezuela/Francisco Ferrer         -
T. bruni TB-22 ESALQ/USP/João Cerignoni Heliconius phyllis
T. rojasi TR-23 Rio Grande Farm, Curitiba-PR/L.A. Foerster A. gemmatalis
1Population provided by the Department of Entomology, Wageningen University, The Netherlands.
Ciociola Jr. et al.
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 56°C followed by 10
minutes at 95°C.
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
    Neotropical Entomology 30(2) 261June, 2001
Figure 1. Agarose gel showing PCR products plus flanking
regions of 89 bp of the 5.8S and 24 bp of the 28S rDNA
genes, of five Brazilian Trichogramma species: Lines 1 and
8: molecular markers, Line 2: T. pretiosum (TP-07), Line 3:
T. galloi, Line 4: T. lasallei, Line 5: T. rojasi, Line 6: T.
acacioi, Line 7: negative control.
Table 2. Size (number of nucleotides) of the ITS2 region, plus flanking regions of 89 bp of the 5.8S and 24 bp of the 28S
rDNA genes, and the position of restriction sites generated by the enzymes Eco RI, Mse I, Pvu I and Nru I, belonging to some
species of Trichogramma.
Size of the
Strains ITS2 region Eco RI Mse I Pvu I Nru I
of  rDNA
T. acacioi 668 - 106 217 -
T. atopovirilia 669 - 699 - -
T. pretiosum 522-528 - - - -
T. galloi 556 192 - - -
T. lasallei 566 566 - - 161
T. rojasi 639 639 - - 639
T. bruni 662 - 98 204,  363 -
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 37°C 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).  Tricho-
gramma 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
262
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
Amornsak, W., G. Gordh & G. Graham. 1998. Detecting
parasitised eggs with polymerase chain reaction and DNA
sequence of Trichogramma australicum Girault
(Hymenoptera: Trichogrammatidae). Austr. J. Entomol.
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Cabot, E.L. 1995. ESEE V3.0s. Computer program
distributed by the University of Rochester, Rochester,
NY.
Campbell, B.C., J.D. Steffen-Campbell & J.H. Werren.
1993. Phylogeny of the Nasonia species complex
(Hymenoptera: Pteromalidae) inferred from an internal
transcribed spacer (ITS2) and 28S rDNA sequences.
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Hoy, M. 1994. Insect molecular genetics. An introduction to
principles and applications. Academic Press, Inc. San
Diego, California, 546p.
Kan, F.J.P.M. van, I. M.M.S. Silva, M. Schilthuizen, J.D.
Pinto & R. Stouthamer. 1996. Use of DNA-based
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genus Trichogramma. Proc. Exp. Appl. Entomol., N.E.V.
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Nagarkatti, S. & H. Nagaraja. 1971. Redescription of some
known species of Trichogramma,  showing the
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Bul. Entomol. Res. 61: 13-31.
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& J. D. Pinto. 1999. The utility of internally transcribed
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Table 3. Molecular key to seven Brazilian species of Trichogramma based on the PCR product plus flanking regions and
the enzymes Eco RI,  Mse l, Pvu I and Nru I.
1. PCR product bigger than 660 bp* ............................................................................................................................ 2
1' PCR  product smaller than 660 bp ............................................................................................................................ 3
2. PCR product cut by the enzyme  Mse I ..................................................................................................................... 4
2' PCR not cut by the enzyme  Mse I ........................................................................................................ T. atopovirilia
3. PCR product bigger than 550 bp .............................................................................................................................. 5
3' PCR product between 522-528 bp ........................................................................................................... T. pretiosum
4. PCR product cut once by the enzyme Pvu I ................................................................................................. T. acacioi
4' PCR product cut twice by the enzyme Pvu I .................................................................................................... T. bruni
5. PCR product cut by the enzyme Eco RI ......................................................................................................... T. galloi
5' PCR product not cut by the enzyme Eco RI .............................................................................................................. 6
6. PCR product cut once by the enzyme Nru I ................................................................................................. T. lasallei
6' PCR not cut by the enzyme Nru I .................................................................................................................. T. rojasi
* bp = base pairs
Received 25/VII/2000. Accepted 08/V/2001.
Ciociola Jr. et al.


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

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