Ribosomal DNA internal transcribed spacer 2 sequence analysis and phylogenetic comparison of seven cockroach species in northwestern Iran

(2019) 12:53 Farmani et al. BMC Res Notes https://doi.org/10.1186/s13104-019-4089-3 BMC Research Notes Open Access RESEARCH NOTE Ribosomal DNA internal transcribed spacer 2 sequence analysis and phylogenetic comparison of seven cockroach species in northwestern Iran Mostafa Farmani1,2, Hamidreza Basseri3, Behzad Norouzi4 and Saber Gholizadeh1,2* Abstract Objectives: The current study was conducted to identify cockroach species (Blattodea) of northwestern Iran in public places using morphological characteristics and ribosomal DNA internal transcribed spacer 2 (rDNA-ITS2). Sequences were analyzed with Basic Local Alignment Search Tool (BLAST) searches, Neighbor-Joining methods based on and Tamura-Nei phylogenetic analyses. In addition, eight cockroach rDNA-ITS2 sequences from China, India, Iran and the United States obtained from GenBank were compared to those obtained in this study. Results: Specimens collected in Iran were identified as Periplaneta americana (L.), Shelfordella lateralis (Walker), Blatta orientalis (L.) (Blattodea: Blattidae), Blattella germanica (L.), Supella longipalpa (F.) (Blattodea: Ectobiidae), Polyphaga aegyptiaca (L.), and Polyphaga saussurei (Dohrn) (Blattodea: Corydiidae). rDNA-ITS2 nucleotide sequence analysis showed 100% similarity between P. aegyptiaca and P. saussurei species collected from Iran despite morphological differences. However, ITS2 sequence of P. americana submitted from China showed 30.49–31.71% difference to P. americana sequences from Iran and the United States. The results highlight the importance of morphological identification of cockroach species before conducting molecular techniques. Keywords: rDNA-ITS2, Phylogeny, Blattidae, Ectobiidae, Corydiidae Introduction Cockroach is one of the most important urban pests in the world. They are mostly known for their role in allergies and can transmit some diseases to human [1–4]. Mirzayans reported 24 species of cockroaches in Blattidae, Ectobiidae, and Corydiidae families in Iran [5]. More recent surveys by Hanafi-Bojd, Sadaghiyani and Hashemi-Aghdam, Oshaghi reported 3 families, 14 genera, and 26 species in Iran [6, 7]. Two more species are Parcoblatta sp. [8], and Polyphaga sp. [7]. Mitochondrial and nuclear molecular markers are used for the precise identification of cockroach species and *Correspondence: 1 Cellular and Molecular Research Center, Urmia University of Medical Sciences, Urmia, Iran Full list of author information is available at the end of the article their phylogenetic relationship [9]. rDNA-ITS2 might be appropriate for mushrooms and Diptera, but it does not mean that it is appropriate for (some) cockroaches [10–12]. Ribosomal DNA has not been used frequently for identification of cockroach species [13]. ITS2 length varies among different cockroach species [13], and ITS2 sequences are often more polymorphic between species than within species [14]; therefore, it could be useful for molecular identification of sibling species [15]. A better understanding of the cockroach ecology and taxonomy is essential for the successful pest control program. Thus, a routine survey of the cockroach population in northwestern Iran will greatly contribute to the success of the control program. This study was designed to identify morphologically and molecularly cockroach species in northwestern Iran and to determine the phylogenetic relationships (using rDNA-ITS2 sequences) © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Farmani et al. BMC Res Notes (2019) 12:53 among these species. New set of primers were designed as universal primers to amplification of rDNA-ITS2 fragment in cockroach species. Main text Methods Cockroach collection and morphological identification The majority of cockroaches used in this study were collected manually by searching their shelters using flashlight at night from 30 locations in Urmia, Iran (37°33′19″N 45°04′21″E) in 2013–2015. Plastic bottle traps and sticky cards (n = 10 in each location) baited with sugar, biscuits and dried breads were used to collect cockroaches in houses, hospitals, dormitories and landfill. Trappings were conducted from 6:00 p.m. to 6:00 a.m. in 2015. Cockroaches were captured from the plastic bottle traps and transferred to containers individually. The inner surface of the plastic bottle traps was coated with butter to prevent escape. Adult cockroaches were killed at − 20 °C and identified using morphological keys [5, 6]. Voucher specimens were deposited in the Entomology Laboratory at the School of Public Health (SPH), Urmia Medical Sciences University (UMSU), Urmia, Iran. DNA extraction Genomic DNA was extracted from the thorax of individual cockroach species, stored in 70% ethanol, using YTA Genomic DNA Extraction Mini Kit (Yekta Tajhiz Azma, Tehran, Iran). Based on manufacturer’s instruction, a 25-mg tissue sample was removed from the thorax of each cockroach with a surgical blade and homogenized in 200 µL TG1 buffer by grinding with a micropestle containing liquid nitrogen. After adding 20 µL proteinase K, the mixture was incubated at 60 °C for 1–2 h. 200 μL of TG2 buffer was added and re-incubated for 10 min at 70 °C, after which 200 μL cold ethanol was added. The mixture was transferred to TG Mini Column and centrifuged for 1 min at 8000 rpm. The flow-through was discarded and TG Mini Column was transferred to a new Collection Tube. DNA was washed with 500 μL of W1 and 750 μL of wash buffers by centrifuging for 1 min at 14,000 rpm. Total DNA was eluted to the elution tube by adding 100 μL elution buffer or ddH2O (pH 7.5–9.0) and stored at 4 °C or − 20 °C until use [16]. Primer designing and PCR amplification Cockroach-specific primers (5.8S TGGGTCGATGAA GAACGC and 28S ATTCAGCGGGTAGTCTCG) were designed based on cockroach rDNA sequences available in the GenBank (GenBank ID: AF005243, KF899831, and EU306665) using the softwares Gene Runner (Hastings Page 2 of 5 Software Inc. 1994) and Standard Nucleotide BLAST [17]. PCR reactions of ITS2 fragment were performed in a total volume of 25 µL master mix. Each reaction contained 2 µL genomic DNA, 12.5 μL PCR Master Mix (Yekta Tajhiz Azma, Tehran, Iran), 0.2 μL Taq polymerase, 1 μL each primer (forward and reverse), and 8.3 μL ddH2O. The PCR amplification profile was set as follows: initial template denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 54 °C for 1 min, (...truncated)