J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … Original Article Utility of mtDNA-COI Barcode Region for Phylogenetic Relationship and Diagnosis of Five Common Pest Cockroaches Saedeh Sadat Hashemi-Aghdam 1, Golnaz Rafie 1, Sanaz Akbari 1, *Mohammad Ali Oshaghi 2 1Deptartment of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran 2Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (Received 16 Mar 2015; accepted 17 Feb 2016) Abstract Background: Cockroaches are of vital importance medically and hygienically as they can disperse human patho- genic agents and are especially responsible for food contamination and spreading of food borne pathogens. In this study, part of mtDNA-COI gene of five common pest cockroaches was tested for diagnostic and phylogenetic pur- poses. Methods: We have described barcode region of mtDNA-COI gene of five cockroach species: Blattella germanica, Blatta orientalis, Periplaneta americana, Shelfordella lateralis, and Supella longipalpa, along with the development of a PCR-RFLP method for rapid detection and differentiation of these health pest species. Results: The PCR generates a single 710 bp-sized amplicon in all cockroach specimens, followed by direct se- quencing. AluI predicted from the sequencing data provided different RFLP profiles among five species. There was a significant intra-species variation within the American cockroach populations, but no genetic variation within other species. Accordingly, phylogenetic analysis demonstrates common monophyly for cockroach families in agreement with conventional taxonomy. However S. longipalpa (Ectobiidae) diverged as an early ancestor of other cockroaches and was not associated with other Ectobiidae. Conclusion: The PCR-RFLP protocol might be useful when the conventional taxonomic methods are not able to identify specimens, particularly when only small body parts of specimens are available or they are in a decaying condition. mtDNA-COI gene shows potentially useful for studying phylogenetic relationships of Blattodea order. Keywords: Cockroach, mtDNA-COI, PCR-RFLP, Molecular marker, Phylogeny Introduction Cockroaches are considered one of the most biological characters and their ecological as- successful groups of animals. Because cock- sociation with humans positioned them in roaches are so adaptable, they are almost found the list of dirty species by FDA as the great- around the world with most species living in est health hazard risk for transmitting human tropical and equatorial regions (Schal and Ham- diseases through food products (Olsen et al. ilton 1990). Cockroaches are synanthropic and 2001, Sulaiman et al. 2011). Cockroaches may endophilic, exhibit communicative behavior, transmit four strains of poliomyelitis virus, vi- attracted to dirt and filth that could contain ruses of encephalitis, yellow fever, coxsakie, human pathogens and human food products, and the eggs of seven species of pathogenic and harbors these pathogens. Cockroaches ringed worms. Moreover, twelve species of are present in homes, groceries, food ware- adult cockroaches are known as intermediate houses, restaurants, hotels, hospitals as well host for invertebrates, some species of bacteria as in sewer systems and rubbish bins. These including: Salmonella, Staphylococcus, Strep- 182 *Corresponding author: Dr Mohammad Ali Oshaghi, http://jad.tums.ac.ir E-mail: [email protected] Published Online: May 27, 2017 J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … tococcus, Escherichia coli, Proteus, Klebsiella, been gradually replacing the common Ger- Serratia and some protozoa such as: Giardia, man cockroach and to form breeding colo- Balantidium, Entamoba histolytica, Trichomo- nies in many parts of the world including nas and some of the pathogenic fungi such as Iran (Laddoni, personnel observation). Aspergillus. Cockroach species identification is tradi- Cockroaches coexist with approximately tionally based on morphological characteris- 150 species of bacteria, 60 species of fungi, six tics, however, morphology is not always a species of yeast, 90 species of protozoa, 45 suitable method for these types of studies species of pathogenic ringed worms and some and sometimes it suffers from deficiencies par- of the other worms (Pai et al. 2003, Mlso et ticularly when only small body parts of the al. 2005, Salehzadeh et al. 2007, Sookrung et specimen are available or they are in a decay- al. 2008, Karimizarchi and Vatani 2009, Fakoo- ing condition. Consequently, today, in addi- rziba et al. 2010, Akbari et al 2014). They tion to morphological, molecular markers, spe- also are a principal contributor to allergies cifically DNA-based molecular markers are (Arruda et al. 2001, Arlian 2002). In most re- being widely used in animals systematic. Mo- gions of the world, residual insecticide sprays lecular data contributes to resolve the system- are applied into cracks and crevices of filthy atic issues in different organisms including places to control cockroaches (Limoee et al. cockroaches (Lazebnaya et al. 2005, Mukha 2006). et al. 2007, Pechal et al. 2008, Hashemi-Agh- Based on the current classification, cock- dam and Oshaghi 2014). However, the rela- roaches belong to the order Blattodea that tionships within Blattodea order are still un- comprises three superfamilies of Corydioi- certain in the literature. dea, Blaberoidea, and Blattoidea (Beccaloni Studying of the barcode region of cyto- and Eggleton 2013). Overall, 7570 living spe- chrome oxidase subunit I gene of mitochon- cies of Blattodea are currently recognized, of drial DNA (mtDNA-COI) has been proposed which 4641 are cockroaches (Beccaloni 2007) as a modern systematic tool applied in evo- and 2929 are termites (Krishna et al. 2013). lutionary and population study on species About 30 out of 4641 cockroach species are delimitation and taxonomy. This gene is one more associated with human habitations where of the largest genes in the metazoan mito- some species such as Periplaneta americana chondrial genome and its adaptability is (American), B. germanica (German), B. ori- more than the other mitochondria genes. It is entalis (Oriental) are well known as pests also one of the most important molecular (Valles et al. 1999, Schal and Hamilton 1990). markers used for molecular taxonomy and In addition, Shelfordella lateralis (Turkestan) systematic of living things and microorgan- and Supella longipalpa (Brown-banded) are isms (Hebert et al. 2003, Karimian et al. among the rapidly replacing known pest spe- 2014). Of different segments of the COI cies in some places of the world. Shelfordella gene, DNA barcode is selected and used as a lateralis is well distributed in central Asia, the standardized region in molecular systematic Caucasus Mountains, and northeastern Af- in literature. In fact, barcoding region pro- rica. Recently it has been rapidly replacing vides a common source of DNA sequence for the common oriental cockroach in urban areas identification and taxonomy of organisms (Hol- of the southwestern USA (Kim and Rust 2013) lingsworth et al. 2011), whereby scientists can and north of Iran (Oshaghi, personnel ob- compare living organisms. As of February servation). Supella longipalpa (Brown-band- 2013, the Barcode of Life Data systems data- ed) is a worldwide pest (Charlton et al. 1993) base (http://www.boldsystems.org) included and one of the most recent cockroaches has almost 2,000,000 barcode sequences from over 183 http://jad.tums.ac.ir Published Online: May 27, 2017 J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … 160,000 species of animals, plants and fungi. PCR-direct sequencing In this study, we designed a PCR-RFLP A fragment of 710bp of the COI mito- assay from the barcode region of mtDNA chondrial region designated as the barcoding COI sequences generated from five cockroach region was amplified by PCR using primers species, and attempted to infer the phyloge- 5΄-TTAAACTTCAGGGTGAC- netic position of those species in the Blat- CAAAAAATCA-3' (HCO2198) and 5΄-G todea more reliably. This diagnostic and in- GTCAACAAATCATAAAGATATTGG-3΄ ferring phylogeny might be a valuable tool for (LCO1490) (Folmer et al. 1994). PCRs were cockroach species identification for entomo- carried out in 20μL reaction using 2μL ge- logical studies and control measures strategies. nomic DNA. The PCR reaction consisted of 2µL PreMix (iNtRON®, South Korea), and Materials and Methods 18µL of a solution containing 200 nM each primer, and 2μL (50 ng) genomic DNA. The Sample collection and DNA extraction PreMix is a premixed solution containing Periplaneta americana, B. orientalis, B. ger- HotStarTaq DNA polymerase, PCR buffer, manica, Sh. lateralis and Su. longipalpa were and deoxynucleoside triphosphates (dNTPs), trapped between March and August 2012– with a final concentration of 1.5mM MgCl2 2013 from different locations such as dwell- and 200 mM each dNTP. The reactions were ing, hospital, confectionary, park and insec- run with an initial denaturation of 2 min at tarium in towns located in center, north and 94 °C and then followed first by 5 cycles at northwest of Iran using live-catch trap, box 94 °C for 40 sec, 45 °C for 40 sec, 72 °C for matches and hand catch. The specimens 1 min and then 35 cycles at 94 °C for 40 sec, were anesthetized in cold and then preserved 51 °C for 40 sec, 72 °C for 1 min, with a final