J -Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … Archive of SID

Original Article Utility of mtDNA-COI Barcode Region for Phylogenetic Relationship and Diagnosis of Five Common Pest

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 species: germanica, orientalis, americana, Shelfordella lateralis, and 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 populations, but no genetic variation within other species. Accordingly, phylogenetic analysis demonstrates common monophyly for cockroach families in agreement with conventional . However S. longipalpa () 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 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 . 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 www.SID.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 … Archive of SID

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 (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 (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 www.SID.ir Published Online: May 27, 2017 J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … Archive of SID

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 in 70% alcohol. Details of the specimens extension 72 °C for 5 min in a Peqlab ther- used in this study are described in Table 1. mocycler machine. The PCR products were The specimens were morphologically identi- electrophoresed on 1% agarose gel with TAE fied according to the available identification buffer (40mM Tris-acetate and 1mM EDTA, keys (Pratt and Stojanovich 1966, Cochran pH 8.0), and stained with Ethidium bromide 1999). Fifty-seven different specimens be- (EtBr) at a concentration of 0.5µg/ml, along long to the five species (comprising 14 Amer- with a 100-bp ladder (SinaClon, Iran). ican cockroaches, 17 German cockroaches, ten Brown-banded cockroaches, eight Orien- Nucleotide sequencing and phylogenetic tal cockroaches, and eight Turkistan cock- analysing roaches) were used. Genomic DNA was ex- The amplified products of mtDNA COI tracted from the hind leg (femur) for larger gene for individuals of the five cockroach cockroaches whereas for small ones such species were sequenced bi-directionally and German and Brown-banded cockroaches the consensus nucleotide sequences were obtained. entire body was used. For DNA extraction, Multiple alignments of the nucleotide sequenc- the cockroach femur or entire body was iso- es were performed using ClustalW2 program lated and dried at 37 °C in an incubator. (http://www.ebi.ac.uk/Tools/msa/clustalw2). Samples were subsequently placed in liquid BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) nitrogen for 2–5 min and then homogenized software was used in order to confirm the ac- with autoclaved glass pestle. DNA was ex- curacy of sequences, discovering their identity tracted from the resultant homogenate using and rate of similarity with available data in the method described by Collins et al. (1987) GenBank. The obtained sequences in this and stored at -20 °C until used. study plus a subset of mtDNA-COI sequenc- 184 http://jad.tums.ac.ir www.SID.ir Published Online: May 27, 2017 J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … Archive of SID

es of other cockroach species available in COI gene fragment of the species. However, GenBank were used for phylogenetic analy- the A-T contents did not show significant sis. The sequence data contained 16 species variations at the intra- or inter-species levels. belonged to four families of Cryptocercidae, A significant inter-species genetic diver- , Ectobiidae (Blattellidae) and Coryd- sity was observed among the five cockroach iidae (Polyphagidae) (Table 1). The sequence species characterized at the mtDNA-COI gene. of Myzus persicae (Accession No. JX844381) The most divergence rate (22.26%) was ob- was used as an out-group. Phylogenetic tree served between S. longipalpa and P. ameri- inferred using the neighbour-joining method cana and the least rate (11.57%) was seen embedded in ClustalW2 program and plotted between American and Oriental cockroach- using Software program Tree view (http:// es. However, the most genetic difference taxonomy.zoology.gla.ac uk/rod/rod.html). among the species was lowered to 0.0–9.1% at amino acid level indicating that most of PCR-RFLP the substitutions were silent or synonymous Based on sequence variation among the (Table 2). Generally, the brown-banded cock- five species, AluI restriction enzyme was se- roaches with the lowest similarity rate at DNA lected by manufacturer’s NEBcutter V2.0 (80%) and amino acid (92.86%) levels were program (http://tools.neb.com/nebcutter) and the most diverged species among the species a physical map provided (Fig. 1B). For re- (Table 2). Additionally, based on mtDNA striction fragment assays, 15µL of the PCR molecular clock (2% per MY), S. longipalpa products was digested in a 25µL reaction is suggested to have diverged from other spe- mixture containing 10 U of AluI (Vivantis) cies about 10 MY. At the intra-species level, and 2.5µL of the appropriate restriction buff- a considerable genetic polymorphism (2.62%) er at 37 °C overnight, following the instruc- was detected among the American cockroach tions of manufacturer. The digested products populations whereas no genetic variation has were fractionated on a 2.5% agarose gel and been noticed within other species. visualized by ethidium bromide staining un- Phylogenetic relationships among 16 species der ultraviolet light. belonged to four cockroach families Cryp- tocercidae, Blattidae, Ectobiidae (Blattellidae) Results and (Polyphagidae) were esti- mated based on the 648bp DNA sequences mtDNA-COI gene and phylogenetic analysis of the mitochondrial cytochrome oxidase sub- Result of PCR amplification revealed that unit I (COI) gene. A cladogram inferred using the primers could amplify a unique 710bp frag- the neighbor-joining method indicated mon- ment of the COI gene for all of the five cock- ophyly of each of the four families (Fig. 1). roach species. Of the 57 PCR amplified prod- However, S. longipalpa of Ectobiidae (Blat- ucts of the specimens, the mtDNA-COI nu- tellidae) was diverged early and formed a sep- cleotide sequences were generated for 26 spec- arate branch distinct from other members of imens. Of these, 14 sequences were generat- Ectobiidae. The phylogenetic tree showed that ed for P. americana, three sequences each for all species of that Blattidae grouped together B. germanica, S. lateralis, S. longipalpa, and B. and formed a main clad. In addition, they orientalis specimens. The nucleotide sequences were sister group of Ectobiidae (Blattellidae). have been deposited in the GenBank database The cladogram suggested that S. longipalpa under accession nos. JQ267476 to JQ267498 was the basal species and the early ancestor and KJ787108. On average, a high A-T content of other cockroaches. Following S. longipal- (65.7%) was evident in the amplified mtDNA- pa, Cryptocercidae was the basal families of 185 http://jad.tums.ac.ir www.SID.ir Published Online: May 27, 2017 J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … Archive of SID

other cockroaches. 275 to produce three bands of 251, 24, and 435bp for B. orientalis, at seven restriction PCR-RFLP sites to produce eight bands of 296, 15, 24, Based on nucleotide sequence analysis of 33, 24, 220, 50 and 48bp for B. germanica, the mtDNA-COI gene, a PCR-RFLP assay was at seven cut sites and therefore produces eight developed for the diagnosis and differentia- bands of 65, 54, 39, 138, 39, 132, 87 and 156 tion of the five cockroach species. It was ac- bp for S. longipalpa (Fig. 2). Although some complished by digesting the PCR product bands had approximate molecular weights (710bp) with AluI restriction enzymes. AluI (such as 255 and 251bp for S. lateralis and 132 cuts the PCR–amplified mtDNA-COI fragment and 138bp for S. longipalpa) or the bands of the five species at unique positions: at 251 with less than 100bp length were not easily and 455 to produce three bands of 251, 204 visible in agarose gel, still a distinctive and and 255bp for S. lateralis, at 167, 251, 392, and visible RFLP profile with AluI restriction 612 to produce five bands of 167, 84, 141, enzyme was obvious after restriction diges- 220 and 98bp for P. americana, at 251 and tion for each of these five species (Fig. 2A).

Fig. 1. Phylogenetic tree obtained by comparing 630 bp sequences of mtDNA-COI gene of cockroaches. G.B: GenBank. Myzus persicae with Accession No. JX844381 is used as outgroup

186 http://jad.tums.ac.ir www.SID.ir Published Online: May 27, 2017 J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … Archive of SID

Table 1. Details of sequence data used for cockroach phylogenetic analysis in this study. NS: Not stated

Accession Species Common name Family Origin Reference number relictus Relict Cryptocercidae JX144941 NS Direct Submission Periplaneta japonica Japanese Blattidae JQ350708 NS Direct Submission Periplaneta fuliginosa Smoky brown Blattidae AB126004 NS Direct Submission Periplaneta americana American Blattidae JQ267476 Iran: Kashan This study Periplaneta americana American Blattidae JQ267480 Iran: Tabriz This study Periplaneta americana American Blattidae JQ350707 NS Direct Submission Pelmatosilpha guyanae N.S. Blattidae EU253833 NS Direct Submission JQ267490- Blatta orientalis Oriental Blattidae Iran: Tabriz This study JQ267492 Blatta orientalis Oriental Blattidae EU253827 NS Direct Submission JQ267493- Shelfordella lateralis Turkestan Blattidae Iran: Assalem This study JQ267494 Shelfordella lateralis Turkestan Blattidae JQ267495 Iran: Tehran This study Phyllodromica iberica N.S. Ectobiidae AM600690 Spain: Zaragoza Direct Submission Phyllodromica subaptera N.S. Ectobiidae AM600683 Spain: Granada Direct Submission pensylvan- Pennsylvania Ectobiidae GU013646 Canada:Ontario Direct Submission ica wood Blattella bisignata Double-striped Ectobiidae JX233805 NS Direct Submission JQ267496- Blattella germanica German Ectobiidae Iran: Tehran This study JQ267498 Blattella germanica German Ectobiidae EU854321 China Direct Submission Supella longipalpa Brown-banded Ectobiidae KJ787108 Iran: Tehran This study Supella longipalpa Brown-banded Ectobiidae EU253834 NS Direct Submission N.S. Corydiidae JF700164 NS Direct Submission Polyphaga sp N.S. Corydiidae JQ267475 Iran: Tehran Direct Submission petiveriana Desert Corydiidae EU253835 NS Direct Submission Peach-potato Myzus persicae Aphididae JX844381 China Direct Submission aphid

A B

Fig. 2. Profile of AluI PCR-RFLP (A) and physical map (B) of mtDNA-COI (barcode region) for five cockroach specis.1: Shelfordella lateralis, 2: Periplaneta americana, 3: Blatta orientalis, 4:Blattella germanica, 5: Supella longipalpa

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Table 2. Genetic similarity rates of mtDNA-COI gene among five cockroach species of P. americana (American), B. orientalis (Oriental), S. lateralis (Turkestan), B. germanica (German), and S. longipalpa (Brown-banded) at DNA (648 bp, left and below asterisks) and amino acid (210 bases, right and above asterisks) levels

American Oriental Turkestan German Brown-banded American *** 98.57 100 96.19 92.38 Oriental 88.43 *** 98.57 95.71 91.90 Turkestan 87.65 88.27 *** 96.19 92.38 German 82.10 83.49 82.56 *** 92.86 Brown-banded 77.74 80.53 80.53 79.44 ***

Discussion

In this study, we developed a PCR-RFLP of cockroach body parts in food samples. method and defined a unique restriction en- Analysis of the data revealed a high inter- zyme (AluI) for the first time that can rapidly species variation (11.56–22.26%) in the bar- detect and differentiate the five pest species coding region of mtDNA COI gene across of cockroaches: P. americana, B. orientalis, B. these five cockroach species, suggesting the germanica, Sh. lateralis, and S. longipalpa. gene fragment is a good molecular marker for Using a simple conventional PCR and just the development of a diagnostic tool for the one single restriction enzyme (AluI) for dif- detection of food pests in food samples. DNA ferentiating of five cockroach species is the barcoding has been used as a shared resource advantages of this method in comparison of DNA sequences for identification and tax- with the only previous study that used two onomic clarification of many organisms, in- restriction enzymes and a more complicated cluding birds (Hebert et al. 2004), sea turtles nested-PCR for discrimination of four cock- (Vargas et al. 2009), eutherian mammals (Luo roach species (Sulaiman et al. 2011). PCR- et al. 2011), fishes (Mabragana et al. 2011) RFLP technique is a very cheap and rapid and (Barrett and Hebert 2005, Smith method for species identification of many et al. 2006, Wilson 2010). In the light of DNA organisms. This molecular assay was used to barcoding, researchers can compare different identify many species of organisms such as kinds of organisms and have access to their rodents (Oshaghi et al. 2011a), parasites genetic information providing evidence of (Oshaghi et al. 2009, 2010, 2011b), Anophe- interpreting systematic situation. Hebert et les (Oshaghi et al. 2008, Mehravaran et al. al. (2003) proposed the use of COI gene as 2011) and to determine blood type within an original source for species identification in guts (Chavshin et al. 2006, Maleki- the kingdom. Additionally, they demon- Ravasan et al. 2009). This will provide a strated that species-level assignments could cost-effective solution that so many speci- be obtained by creating a comprehensive COI. mens can be studied with no need to provide In addition to COI, many researchers used DNA sequencing. In this method, using a set various genes including rRNA (Kambham- of conserved primers, a tiny amount of DNA pati et al. 1995, Mukha et al. 2000, Lazebna- can be amplified and then be identified by ya et al. 2005), COII (Maekawa and Matsu- one restriction enzyme that overcomes lack moto 2000), ETS (Mukha et al. 2002, Mukha of ample amount of cockroach body parts. In et al. 2005), NTS (Mukha et al. 2007), ITS1 addition, this method can be used to deter- (Pechal et al. 2008) and complete mitochon- mine identity of museum specimens, unknown drial genome (Yamauchi et al. 2004, Zhang samples and malformed specimens or residues et al. 2010, Cameron et al. 2012, Xiao et al. 188 http://jad.tums.ac.ir www.SID.ir Published Online: May 27, 2017 J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … Archive of SID

2012, Chen 2013) for systematic classifica- References tion of different species of cockroaches. The inferred relationships among cock- Akbari S, Oshaghi MA, Hashemi-Aghdam roach families (S. longipalpa + (S. longipalpa + SS, Hajikhani S, Oshaghi G, Shirazi (Blattidae + (Cryptocercidae + (Corydiidae + MH (2015) Aerobic bacterial commu- Ectobiidae)))) is partly in agreement with some nity of American cockroach Peripla- previously published analyses (Kambhampati neta americana, a step toward finding 1995, Maekawa and Matsumoto 2000, Lo et suitable paratransgenesis candidates. J al. 2007). Furthermore, S. longipalpa has been Arthropod Borne Dis. 9(1): 35–48. introduced as the early ancestor of other Arlian LG (2002) Arthropod allergens and cockroaches by the phylogenetic trees in- human health. Annu Rev Entomol. 47: ferred from both amino acid and DNA se- 395–433. quence data where it did not group with Ec- Arruda LK, Vailes LD, Ferriani VPL, Santos tobiidae or indeed nest within cockroaches. ABR, Pomés A, Chapman MD (2001) It is in controversy with earlier morphology- Cockroach allergens and asthma. J Al- based phylogenetic hypotheses as well as lergy Clin Immunol. 107(3): 419–428. molecular study using a combination of four Barrett RD, Hebert PD (2005) Identifying mitochondrial (16S and COI+COII) and nu- spiders through DNA barcodes. Can J clear (18S and 28S ribosomal subunits) loci Zool. 83(3): 481–491. indicating monophyletic topology of Blabe- Beccaloni G, Eggleton P (2013) Order Blat- roidea superfamily (Djernæ et al. 2002). A todea. Zootaxa. 3703(1): 046-048. wider sampling of Blaberoidea including Beccaloni GW (2007) Pecies File Online. is highly required in order to test Version 1.2/4.0. World Wide Web elec- the topological situation of S. longipalpa. tronic publication (A regularly updated world catalogue of extant Blattodea excluding the Termitoidae (termites)). Conclusion Available at: http://www.Blattodea. SpeciesFile.org The results of this study demonstrate the Cameron SL, Lo N, Bourguignon T, Sven- utility of the mtDNA-COI gene as a valuable son GJ, Evans TA (2012) A mitochon- and powerful molecular marker in unravel- drial genome phylogeny of termites ing medically important cockroach species, (Blattodea: Termitoidae): robust sup- particularly when morphological characters port for interfamilial relationships and are subtle. Moreover, the mtDNA-COI gene molecular synapomorphies define ma- shows potentially advantageous for under- jor clades. Mol Phylogenet Evol. 65 standing phylogenetic relationships in this (1): 163–173. order. Charlton RE, Webster FX, Zhang A, Schal C, Liang D, Sreng I, Roelofs WL (1993) Acknowledgements Sex pheromone for the brownbanded cockroach is an unusual dialkyl-subs- Tehran University of Medical Sciences tituted alpha-pyrone. Proc Natl Acad (TUMS) financially supported this study. Sci USA. 90(21): 10202–10205. The authors declare that there is no conflict Chavshin AR, Oshaghi MA, Vatandoost H, of interests. Pourmand MR, Raeisi A, Enayati AA, Mardani N, Ghoorchian S (2012) Iden- tification of bacterial microflora in the 189 http://jad.tums.ac.ir www.SID.ir Published Online: May 27, 2017 J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … Archive of SID

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