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

Background: ITS2 sequences can be used in systematic studies and proved to give reliable results in the distinguishing 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 assessment 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 distinguishing species. Therefore, with rDNA-ITS2 sequence analysis, Trichogramma spp . , which is a very important natural enemy in biological control, has been identified.


Background
The 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. This region has been used for many years for species identification and phylogenetic examination of Trichogramma. Since this region is preserved, a wide variety of taxa can be studied with specific primers (Sumer et al. 2009). Stouthamer (2006) emphasized that the identification of protein and DNA regions would be much more useful in the detection and diagnosis 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 individuals 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. Therefore, identification of these wasps is problematic and systematic clarification is needed. A wide Page 2 of 7 Sümer Ercan et al. Egyptian Journal of Biological Pest Control (2022) 32:50 variety of methods have been used to elucidate the classification of Trichogramma species. These include morphological 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 limited numbers of species . 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. With studies based on DNA sequence analysis, identification of cryptic species of Trichogramma, use of correct species in production and release studies can be ensured. The 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 success. 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 secondary structure helps us to explain the phylogenetic information 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 determine its phylogenetic boundaries, and to elucidate intraspecies and inter-species relationships. For this purpose, in the present study, rDNA ITS2 gene region with nucleotide sequences and secondary structure form of these sequences was used to identify Trichogramma species collected from Adana province of Turkey.

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. They 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 .
After electrophoresis, PCR products were purified by the Wizard ® PCR Preps DNA Purification System. Following 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 containing Ampicillin, X-GAL and IPTG. The plates were stored overnight at 37 °C. The next day, white colonies on each plate were removed with sterile toothpicks, and the bacteria attached to the toothpicks were dispersed in Eppendorf 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 determine the correct size of insert in the Pgem plasmid in each sample (Ercan et al. 2013). The PCR products were loaded on a standard TAE buffered agarose gel. Then, 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 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). The probability of the best phylogenetic tree according to the polymorphism in the nucleotide sequences was determined by Maximum Likelihood (ML) analysis. The model with the lowest AIC value (Akaike Information Criteria) was used to determine the phylogenetic tree through the jModelTest v.0.1.1 program 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. nubilalis 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 Information to control the amplified region. The 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). The ITS2 sequences of T. turkestanica 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. The 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. turkestanica and T. brassicae were predicted by Mfold web server (Fig. 3). Branched structures in T. brassicae were remarkable compared to T. turkestanica. Moreover, the similarities and differences in the constructed phylogenetic tree were also reflected in the secondary structure form of ITS2 with helices and angels among species. Therefore, 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 structure 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 nucleotide sequences. Among the genetic markers, rDNA is found in all organisms and contains various regions that evolve at different rates. Therefore, 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 (Vanlerberghe-Masutti 1994).

Discussion
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 Trichogramma species evanescens group in Europe and T. turkestanica 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), biochemical (Pintureau andKeita 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 Portugal. 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 classical methods are not sufficient to diagnose micro-hymenoptera species at the racial level (Landry et al. 1993). Thomson et al. (2003) used ITS2 sequence analysis to identify Trichogramma species from South-eastern Australia and found that the ITS2 size was different for each species. Similarly, the size of ITS2 differed between species 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. These Trichogramma samples were divided into 2 main groups. Five specimens were included in a group named: T. turkestanica according to ITS2 sequence analysis. The other 32 samples were in the 2nd main group. These 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
The ITS2 region is one of the most important molecular markers that can be used to compare closely related species, subspecies and populations. The correct identification of the natural enemy in a biological control program plays a key role in the success of this control. The absence of adequate and appropriate techniques for diagnosis makes the biological control program unsuccessful. Therefore, there is a need to develop molecular diagnostic tools to identify natural enemies and understand population dynamics. The use of molecular techniques in Trichogramma systematics has been made by many researchers for many years (Karimi 2012). The 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.