rDNA-ITS2 characterization of Trichogramma species (Hymenoptera: Trichogrammatidae) in Turkey

Abstract

Background: ITS2 sequences can be used in systematic studies and proved to give reliable results in the distinguishing of Trichogramma species (Hymenoptera: Trichogrammatidae). Correct identifcation of natural enemies forms the
basis of a biological control program. The present study aimed to compare sequences of rDNA-ITS2 of Trichogramma
samples with those deposited in GenBank by using ITS2, as a barcode for reliable species identifcation and assessment of genetic diversity.
Results: Molecular identifcation methods were used to diferentiate 2 Trichogramma species collected from Adana
province of Turkey; Trichogramma brassicae (Bezdenko) and Trichogramma turkestanica Meyer (Hymenoptera: Trichogrammatidae). ITS2 sequences of samples ranged in size from 378 to 406 bp. The ITS2 sequences were aligned using
Clustal W, genetic distances and phylogenetic tree were calculated using MEGA V7.0. rDNA-ITS2 sequences of 37
specimens of Trichogramma confrmed in GenBank in the study. Also, secondary structures of ITS2 sequences were
predicted with the help of Mfold web server. All secondary structure constructions were performed at 37 °C using
RNA version 2.3 default parameters.
Conclusions: A molecular marker can be used successfully to distinguish closely related groups if it is a rapidly evolving and highly conserved gene region. In the study, it was shown that ITS2 was a reliable molecular marker in distinguishing species. Therefore, with rDNA-ITS2 sequence analysis, Trichogramma spp., which is a very important natural
enemy in biological control, has been identifed.

Full text

SümerErcanetal.
Egyptian Journal of Biological Pest Control (2022) 32:50
https://doi.org/10.1186/s41938-022-00549-z
RESEARCH
rDNA-ITS2 characterization ofTrichogramma
species (Hymenoptera: Trichogrammatidae)
inTurkey
Fahriye Sümer Ercan1*, Mevlüde Alev Ateş2 and Sevcan Öztemiz3
Abstract
Background: ITS2 sequences can be used in systematic studies and proved to give reliable results in the distinguish-
ing of Trichogramma species (Hymenoptera: Trichogrammatidae). Correct identification of natural enemies forms the
basis of a biological control program. The present study aimed to compare sequences of rDNA-ITS2 of Trichogramma
samples with those deposited in GenBank by using ITS2, as a barcode for reliable species identification and assess-
ment of genetic diversity.
Results: Molecular identification methods were used to differentiate 2 Trichogramma species collected from Adana
province of Turkey; Trichogramma brassicae (Bezdenko) and Trichogramma turkestanica Meyer (Hymenoptera: Tricho-
grammatidae). ITS2 sequences of samples ranged in size from 378 to 406 bp. The ITS2 sequences were aligned using
Clustal W, genetic distances and phylogenetic tree were calculated using MEGA V7.0. rDNA-ITS2 sequences of 37
specimens of Trichogramma confirmed in GenBank in the study. Also, secondary structures of ITS2 sequences were
predicted with the help of Mfold web server. All secondary structure constructions were performed at 37 °C using
RNA version 2.3 default parameters.
Conclusions: A molecular marker can be used successfully to distinguish closely related groups if it is a rapidly evolv-
ing and highly conserved gene region. In the study, it was shown that ITS2 was a reliable molecular marker in distin-
guishing species. Therefore, with rDNA-ITS2 sequence analysis, Trichogramma spp., which is a very important natural
enemy in biological control, has been identified.
Keywords: Internal transcribed spacer 2, Trichogramma, Molecular systematic, GenBank, Biological control
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Background
e internal transcribed spacer 2 (ITS2) region of nuclear
ribosomal DNA is one of the important DNA barcodes
because of the availability of conserved regions for
designing universal primers. is region has been used
for many years for species identification and phylogenetic
examination of Trichogramma. Since this region is pre-
served, a wide variety of taxa can be studied with specific
primers (Sumer etal. 2009). Stouthamer (2006) empha-
sized that the identification of protein and DNA regions
would be much more useful in the detection and diagno-
sis of parasitoids.
Trichogramma species are one of the most commonly
used groups of natural enemies of major crop pests.
Although the diagnosis at the species level is based on
the male genitalia, the presence of only female individu-
als under natural conditions is an important problem
for many species. In addition to being quite small and
morphologically indistinguishable, its morphology and
physiology are significantly influenced by environmental
factors. erefore, identification of these wasps is prob-
lematic and systematic clarification is needed. A wide
Open Access
Egyptian Journal of
Biological Pest Control
*Correspondence: fahriyesumer@gmail.com; fahriye.ercan@ahievran.edu.tr
1 Department of Plant Protection, Faculty of Agriculture, Kırşehir Ahi Evran
University, 40100 Kırşehir, Turkey
Full list of author information is available at the end of the article

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SümerErcanetal. Egyptian Journal of Biological Pest Control (2022) 32:50
variety of methods have been used to elucidate the clas-
sification of Trichogramma species. ese include mor-
phological features such as antennae structure and wing
veining, followed by morphological comparison of the
male reproductive system, and then a series of methods
including enzymatic analysis (Owen and Pinto 2004).
Esterase electrophoresis provided favorable results
for the differentiation of some Trichogramma species
(Sumer et al. 2008). However, limited variation in the
esterase locus allows for differentiation between a lim-
ited numbers of species (Silva et al. 1999). At the same
time, the samples must be fresh in order to be used in
alloenzyme analysis or they must be stored at -70ºC to
prevent enzyme degradation. erefore, researchers tried
alternative methods and used various molecular markers,
including RAPD (Ercan etal. 2012), RFLP (Vanlerberghe-
Masutti 1994), COI (Ercan et al. 2013), microsatellite
markers (Pizzol etal. 2005) and ITS regions of rDNA, for
diagnostic purposes (Ercan etal. 2011).
With studies based on DNA sequence analysis, identifi-
cation of cryptic species of Trichogramma, use of correct
species in production and release studies can be ensured.
e success of a biological control program is closely
related to the correct identification of the natural enemy
species (Silva 1999). Use of the most successful species in
terms of host search, parasitism capacity and tolerance to
environmental conditions will undoubtedly ensure suc-
cess. In the present study, secondary structures of ITS2
sequences of 2 Trichogramma species were predicted and
compared. Generally, secondary structure form of ITS2
was seen in RNA activities of cells. Even if it has different
combinations of nucleotide sequences, eukaryotic ITS2
region is common with 4 helices and motifs (Coleman
2007). With all these conserved motifs and helices ITS2
secondary structure supports more reliable perspective
of relationships at higher taxonomic levels (Zhang etal.
2015). Evaluation of base pair interactions in the second-
ary structure helps us to explain the phylogenetic infor-
mation to advance phylogenetic estimations (Telford etal
2005).
Generally, the aim of molecular systematic studies is to
elucidate the structure of the target population, to deter-
mine its phylogenetic boundaries, and to elucidate intra-
species and inter-species relationships. For this purpose,
in the present study, rDNA ITS2 gene region with nucle-
otide sequences and secondary structure form of these
sequences was used to identify Trichogramma species
collected from Adana province of Turkey.
Methods
Trichogramma samples
All samples were collected from Adana province of
Turkey in August 2018 & 2021 (A1-A25 coded samples
belong to the field study carried out in 2018, while A26-
A37 coded samples that carried out in 2021). Host insect
was Ostrinia nubilalis Hbn. (Lepidoptera: Crambidae)
and host plant was corn. A total of 37 Trichogramma
samples, hatched from the collected parasitized host
eggs, was transferred into 70% alcohol for DNA isolation.
DNA isolation
To extract DNA from Trichogramma samples, one wasp
from each sample was used. ey were ground in 60μl
5% Chelex-100 and 2 μl Proteinase K (20mg/ml) and
incubated at 1h at 55°C, followed by 10min at 96°C
(Stouthamer etal. 1999).
rDNA‑ITS2 amplication
e following primers were used for ITS2 amplifica-
tion: ITS2 forward, 5′-TGT GAA CTG CAG GAC ACA
TG-3′, and ITS2 reverse, 5′-GTC TTG CCT GCT CTGAG-
3′ (Stouthamer et al. 1999). e PCR was performed
in a total volume of 25μl. It contained 2μl DNA tem-
plate, 2.5μl PCR buffer (10X buffer with (NH4)2SO4),
5μl dNTPs (10mM stock solution), 0.5μl forward and
reverse primers, 0.2 μl Taq Polymerase (5 u/μl) and
14.3μl of sterile distilled water. e cycling program was
also the same as used by Stouthamer etal. (1999). e
size of PCR product was determined with 1% agarose gel
electrophoresis with a size standard.
After electrophoresis, PCR products were purified by
the Wizard® PCR Preps DNA Purification System. Fol-
lowing the purification, the PCR products were ligated
into a Pgem-T® Vector (Promega). 2 μl of the ligation
mix was transformed in the heat-shock cells of DH5-α
Escherichia coli and plated in a LB agar medium contain-
ing Ampicillin, X-GAL and IPTG. e plates were stored
overnight at 37°C. e next day, white colonies on each
plate were removed with sterile toothpicks, and the bac-
teria attached to the toothpicks were dispersed in Eppen-
dorf tubes containing 50μl sterile distilled water. Two μl
of this solution was used for PCR reaction using the ITS2
primers in the PCR reaction described above to deter-
mine the correct size of insert in the Pgem plasmid in
each sample (Ercan etal. 2013). e PCR products were
loaded on a standard TAE buffered agarose gel. en,
the gel was stained with ethidium bromide and viewed
and photographed under UV light. PCR products of 40
samples were then sent for automatic sequencing in a
sequencing facility (Atlas Biotechnology).
Secondary structure ofITS2 sequences
Secondary structures of ITS2 sequences and ΔG (Gibbs)
free energy calculations were predicted and calculated
with the help of Mfold web server (http:// unafo ld. rna.
albany. edu/?q= mfold/ RNA- Foldi ng- Form2.3) which

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SümerErcanetal. Egyptian Journal of Biological Pest Control (2022) 32:50
were predicted at 37°C using RNA version 2.3 default
parameters by the program parameters (Zuker 2003).
Phylogenetic analysis
Aligned sequences were analyzed using the Kimura
2-Parameter (K2P) distance model (Kimura 1980) on
the Mega 7 program to determine the genetic diversity
index and inter- and intra-specific nucleotide differences
(Kumar etal. 2016). e probability of the best phyloge-
netic tree according to the polymorphism in the nucleo-
tide sequences was determined by Maximum Likelihood
(ML) analysis. e model with the lowest AIC value
(Akaike Information Criteria) was used to determine the
phylogenetic tree through the jModelTest v.0.1.1 pro-
gram to determine the most suitable model for sequence
change in Maximum Likelihood analyses (Posada 2008).
In addition, the reliability of the model phylogenetic trees
was checked with the 1000 replication Bootstrap test.
Results
Trichogramma samples were collected from O. nubila-
lis eggs in Adana province of Turkey. Twenty-five adults
emerged from the collected host eggs in 2018, 12 adults
were obtained from 2021 field collection. rDNA-ITS2
sequences of 37 specimens were blasted in GenBank
database of National Center for Biotechnology Informa-
tion to control the amplified region. e specimens were
compared them with all obtained homologous sequences
of other Trichogramma species in GenBank. Five of
the 37 specimens were identified as Trichogramma
turkestanica Meyer and 32 as Trichogramma brassicae
(Bezdenko) (Fig.1). e ITS2 sequences of T. turkestan-
ica samples varied between 378 and 380 bp in length,
while the ITS2 sequence length of all samples from T.
brassicae was determined as 406 bp. e phylogenetic
tree was constructed using the obtained sequence results.
A dendrogram was also constructed with sequences of
different Trichogramma species obtained from GenBank
(Fig.2).
In addition, secondary structures of both T. turkestan-
ica and T. brassicae were predicted by Mfold web server
(Fig.3). Branched structures in T. brassicae were remark-
able compared to T. turkestanica. Moreover, the similari-
ties and differences in the constructed phylogenetic tree
were also reflected in the secondary structure form of
ITS2 with helices and angels among species. erefore,
calculation of ΔG (Gibbs) free energy values with Mfold
program parameters for T. turkestanica and T. brassicae
based on the helices and angles in the secondary struc-
ture with thermodynamic calculations (Santa Lucia 1998)
were different based on species, as −136.50kcal/mol and
−146.60kcal/mol, respectively. So, secondary structure
form of ITS2 region was also used like a morphological
characteristic of species due to its clear visually of nucle-
otide sequences.
Discussion
Trichogramma spp. are known to be distributed world-
wide, represented by approximately 210 species (Pinto
2006). Up till now, 11 different Trichogramma species
have been identified in different cultivated plants and for-
est areas in Turkey. Considering their synonyms, 8 spe-
cies are known to exist in Turkey (Öztemiz etal. 2013).
However, the existence of only T. brassicae (Koca etal.
2018), T. euproctidis Girault (Ercan et al. 2011) and T.
evanescens (unpublished data) has been demonstrated in
Turkey using molecular methods.
Among the genetic markers, rDNA is found in all
organisms and contains various regions that evolve at
different rates. erefore, its use as a molecular marker
is preferred in studies comparing closely related species
and populations. It also plays an important role in the
molecular diagnosis of Trichogramma spp. In addition,
it is known that DNA-based methods are not affected
by the life stage or sex of Trichogramma species (Vanler-
berghe-Masutti 1994).
In the present study, 37 Trichogramma samples were
identified based on ITS2 sequences and T. brassicae and
T. turkestanica species were determined. T. evanescens, T.
brassicae and T. euproctidis were included in the Tricho-
gramma species evanescens group in Europe and T. turke-
stanica and T. euproctidis were used synonymously (Rohi
and Pintureau 2003). T. brassicae was first identified by
Bezdenko (Bezdenko 1968). Later, it was determined as
a valid species by morphometric (Pintureau 1993), bio-
chemical (Pintureau and Keita 1989) and DNA-based
studies (Laurent etal. 1996).
Silva et al. (1999) used ITS2 sequence analysis, as in
this study, to identify the 5 Trichogramma species in Por-
tugal. Honda et al. (2006) re-evaluated Trichogramma
species from Japan and identified 3 new species based on
the ITS2 sequence. In another study, new Trichogramma
species, T. itsybitsi, was found based on ITS2 sequences
analysis (Pinto etal. 2002). It has been reported that clas-
sical methods are not sufficient to diagnose micro-hyme-
noptera species at the racial level (Landry etal. 1993).
omson et al. (2003) used ITS2 sequence analysis to
identify Trichogramma species from South-eastern Aus-
tralia and found that the ITS2 size was different for each
species. Similarly, the size of ITS2 differed between spe-
cies in the present study.
In the study, while 25 of 37 Trichogramma specimens
were collected in 2018, the remaining 12 specimens
were collected in 2021. ese Trichogramma samples
were divided into 2 main groups. Five specimens were
included in a group named: T. turkestanica according to

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SümerErcanetal. Egyptian Journal of Biological Pest Control (2022) 32:50
Fig. 1 Phylogenetic tree based on ITS2 gene region of Trichogramma specimens of 37 different studied samples (AD1-AD37) was constructed by
using the Maximum Likelihood method based on the Kimura 2-parameter model with a uniform distribution to model evolutionary rate differences
among sites. Numbers on the branches indicate the bootstrap values of MP

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SümerErcanetal. Egyptian Journal of Biological Pest Control (2022) 32:50
Fig. 2 Phylogenetic tree based on ITS2 gene region of Trichogramma specimens was constructed by using the Maximum Likelihood method
based on the Kimura 2-parameter model with a discrete Gamma distribution to model evolutionary rate differences among sites. Numbers on the
branches indicate the bootstrap values of MP

Page 6 of 7
SümerErcanetal. Egyptian Journal of Biological Pest Control (2022) 32:50
ITS2 sequence analysis. e other 32 samples were in the
2nd main group. ese samples were named T. brassicae
according to ITS2 sequence analysis. According to the
genetic distance relationship, the samples in both groups
were completely similar in themselves.
Conclusions
e ITS2 region is one of the most important molecu-
lar markers that can be used to compare closely related
species, subspecies and populations. e correct identi-
fication of the natural enemy in a biological control pro-
gram plays a key role in the success of this control. e
absence of adequate and appropriate techniques for diag-
nosis makes the biological control program unsuccessful.
erefore, there is a need to develop molecular diagnos-
tic tools to identify natural enemies and understand pop-
ulation dynamics.
e use of molecular techniques in Trichogramma sys-
tematics has been made by many researchers for many
years (Karimi 2012). e molecular technique used in
this study contributed to the correct identification of
Trichogramma species distributed in Turkey. It is obvious
that there are species spread in the country and waiting
to be identified molecularly.
Abbreviations
ITS2: Internal transcribed spacer 2; rDNA-ITS2: Internal transcribed spacer
regions of ribosomal DNA.
Acknowledgements
Not applicable.
Author contributions
FSE was responsible for molecular data and prepared the manuscript. SÖ
collected insect samples. MAA prepared the data. All authors contributed to
writing and editing the manuscript. All authors read and approved the final
manuscript.
Funding
Not applicable.
Availability of data and materials
All data generated or analyzed during this study are included in this published
article.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Fig. 3 Secondary structures predictions and ΔG (Gibbs) free energy values (at the bottom) of T. turkestanica (a) T. brassicae (b) were calculated and
analyzed at mFOLD web server (http:// www. unafo ld. org/ mfold/ appli catio ns/ rna- foldi ng- form- v2. php)

Page 7 of 7
SümerErcanetal. Egyptian Journal of Biological Pest Control (2022) 32:50
Author details
1 Department of Plant Protection, Faculty of Agriculture, Kırşehir Ahi Evran
University, 40100 Kırşehir, Turkey. 2 Department of Agricultural Biotechnology,
Faculty of Agriculture, Kırşehir Ahi Evran University, 40100 Kırşehir, Turkey.
3 Department of Plant Protection, Faculty of Agriculture, Düzce University,
Düzce, Turkey.
Received: 24 December 2021 Accepted: 30 April 2022
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