J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … 182 http://jad.tums.ac.ir Published Online: May 27, 2017 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 successful groups of animals. Because cock- roaches are so adaptable, they are almost found around the world with most species living in tropical and equatorial regions (Schal and Ham- ilton 1990). Cockroaches are synanthropic and endophilic, exhibit communicative behavior, attracted to dirt and filth that could contain human pathogens and human food products, and harbors these pathogens. Cockroaches are present in homes, groceries, food ware- houses, restaurants, hotels, hospitals as well as in sewer systems and rubbish bins. These biological characters and their ecological as- sociation with humans positioned them in the list of dirty species by FDA as the great- est health hazard risk for transmitting human diseases through food products (Olsen et al. 2001, Sulaiman et al. 2011). Cockroaches may transmit four strains of poliomyelitis virus, vi- ruses of encephalitis, yellow fever, coxsakie, and the eggs of seven species of pathogenic ringed worms. Moreover, twelve species of adult cockroaches are known as intermediate host for invertebrates, some species of bacteria including: Salmonella, Staphylococcus, Strep- *Corresponding author: Dr Mohammad Ali Oshaghi, E-mail: moshaghi@tums.ac.ir J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … 183 http://jad.tums.ac.ir Published Online: May 27, 2017 tococcus, Escherichia coli, Proteus, Klebsiella, Serratia and some protozoa such as: Giardia, Balantidium, Entamoba histolytica, Trichomo- nas and some of the pathogenic fungi such as Aspergillus. Cockroaches coexist with approximately 150 species of bacteria, 60 species of fungi, six species of yeast, 90 species of protozoa, 45 species of pathogenic ringed worms and some of the other worms (Pai et al. 2003, Mlso et al. 2005, Salehzadeh et al. 2007, Sookrung et al. 2008, Karimizarchi and Vatani 2009, Fakoo- rziba et al. 2010, Akbari et al 2014). They also are a principal contributor to allergies (Arruda et al. 2001, Arlian 2002). In most re- gions of the world, residual insecticide sprays are applied into cracks and crevices of filthy places to control cockroaches (Limoee et al. 2006). Based on the current classification, cock- roaches belong to the order Blattodea that comprises three superfamilies of Corydioi- dea, Blaberoidea, and Blattoidea (Beccaloni and Eggleton 2013). Overall, 7570 living spe- cies of Blattodea are currently recognized, of which 4641 are cockroaches (Beccaloni 2007) and 2929 are termites (Krishna et al. 2013). About 30 out of 4641 cockroach species are more associated with human habitations where some species such as Periplaneta americana (American), B. germanica (German), B. ori- entalis (Oriental) are well known as pests (Valles et al. 1999, Schal and Hamilton 1990). In addition, Shelfordella lateralis (Turkestan) and Supella longipalpa (Brown-banded) are among the rapidly replacing known pest spe- cies in some places of the world. Shelfordella lateralis is well distributed in central Asia, the Caucasus Mountains, and northeastern Af- rica. Recently it has been rapidly replacing the common oriental cockroach in urban areas of the southwestern USA (Kim and Rust 2013) and north of Iran (Oshaghi, personnel ob- servation). Supella longipalpa (Brown-band- ed) is a worldwide pest (Charlton et al. 1993) and one of the most recent cockroaches has been gradually replacing the common Ger- man cockroach and to form breeding colo- nies in many parts of the world including Iran (Laddoni, personnel observation). Cockroach species identification is tradi- tionally based on morphological characteris- tics, however, morphology is not always a suitable method for these types of studies and sometimes it suffers from deficiencies par- ticularly when only small body parts of the specimen are available or they are in a decay- ing condition. Consequently, today, in addi- tion to morphological, molecular markers, spe- cifically DNA-based molecular markers are being widely used in animals systematic. Mo- lecular data contributes to resolve the system- atic issues in different organisms including cockroaches (Lazebnaya et al. 2005, Mukha et al. 2007, Pechal et al. 2008, Hashemi-Agh- dam and Oshaghi 2014). However, the rela- tionships within Blattodea order are still un- certain in the literature. Studying of the barcode region of cyto- chrome oxidase subunit I gene of mitochon- drial DNA (mtDNA-COI) has been proposed as a modern systematic tool applied in evo- lutionary and population study on species delimitation and taxonomy. This gene is one of the largest genes in the metazoan mito- chondrial genome and its adaptability is more than the other mitochondria genes. It is also one of the most important molecular markers used for molecular taxonomy and systematic of living things and microorgan- isms (Hebert et al. 2003, Karimian et al. 2014). Of different segments of the COI gene, DNA barcode is selected and used as a standardized region in molecular systematic in literature. In fact, barcoding region pro- vides a common source of DNA sequence for identification and taxonomy of organisms (Hol- lingsworth et al. 2011), whereby scientists can compare living organisms. As of February 2013, the Barcode of Life Data systems data- base (http://www.boldsystems.org) included almost 2,000,000 barcode sequences from over J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … 184 http://jad.tums.ac.ir Published Online: May 27, 2017 160,000 species of animals, plants and fungi. In this study, we designed a PCR-RFLP assay from the barcode region of mtDNA COI sequences generated from five cockroach species, and attempted to infer the phyloge- netic position of those species in the Blat- todea more reliably. This diagnostic and in- ferring phylogeny might be a valuable tool for cockroach species identification for entomo- logical studies and control measures strategies. Materials and Methods Sample collection and DNA extraction Periplaneta americana, B. orientalis, B. ger- manica, Sh. lateralis and Su. longipalpa were trapped between March and August 2012– 2013 from different locations such as dwell- ing, hospital, confectionary, park and insec- tarium in towns located in center, north and northwest of Iran using live-catch trap, box matches and hand catch. The specimens were anesthetized in cold and then preserved in 70% alcohol. Details of the specimens used in this study are described in Table 1. The specimens were morphologically identi- fied according to the available identification keys (Pratt and Stojanovich 1966, Cochran 1999). Fifty-seven different specimens be- long to the five species (comprising 14 Amer- ican cockroaches, 17 German cockroaches, ten Brown-banded cockroaches, eight Orien- tal cockroaches, and eight Turkistan cock- roaches) were used. Genomic DNA was ex- tracted from the hind leg (femur) for larger cockroaches whereas for small ones such German and Brown-banded cockroaches the entire body was used. For DNA extraction, the cockroach femur or entire body was iso- lated and dried at 37 °C in an incubator. Samples were subsequently placed in liquid nitrogen for 2–5 min and then homogenized with autoclaved glass pestle. DNA was ex- tracted from the resultant homogenate using the method described by Collins et al. (1987) and stored at -20 °C until used. PCR-direct sequencing A fragment of 710bp of the COI mito- chondrial region designated as the barcoding region was amplified by PCR using primers 5΄-TTAAACTTCAGGGTGAC- CAAAAAATCA-3' (HCO2198) and 5΄-G GTCAACAAATCATAAAGATATTGG-3΄ (LCO1490) (Folmer et al. 1994). PCRs were carried out in 20μL reaction using 2μL ge- nomic DNA. The PCR reaction consisted of 2µ L PreMix (iNtRON®, South Korea), and 18µ L of a solution containing 200 nM each primer, and 2μL (50 ng) genomic DNA. The PreMix is a premixed solution containing HotStarTaq DNA polymerase, PCR buffer, and deoxynucleoside triphosphates (dNTPs), with a final concentration of 1.5mM MgCl2 and 200 mM each dNTP. The reactions were run with an initial denaturation of 2 min at 94 °C and then followed first by 5 cycles at 94 °C for 40 sec, 45 °C for 40 sec, 72 °C for 1 min and then 35 cycles at 94 °C for 40 sec, 51 °C for 40 sec, 72 °C for 1 min, with a final extension 72 °C for 5 min in a Peqlab ther- mocycler machine. The PCR products were electrophoresed on 1% agarose gel with TAE buffer (40mM Tris-acetate and 1mM EDTA, pH 8.0), and stained with Ethidium bromide (EtBr) at a concentration of 0.5µ g/ml, along with a 100-bp ladder (SinaClon, Iran). Nucleotide sequencing and phylogenetic analysing The amplified products of mtDNA COI gene for individuals of the five cockroach species were sequenced bi-directionally and consensus nucleotide sequences were obtained. Multiple alignments of the nucleotide sequenc- es were performed using ClustalW2 program (http://www.ebi.ac.uk/Tools/msa/clustalw2). BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) software was used in order to confirm the ac- curacy of sequences, discovering their identity and rate of similarity with available data in GenBank. The obtained sequences in this study plus a subset of mtDNA-COI sequenc- J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … 185 http://jad.tums.ac.ir Published Online: May 27, 2017 es of other cockroach species available in GenBank were used for phylogenetic analy- sis. The sequence data contained 16 species belonged to four families of Cryptocercidae, Blattidae, Ectobiidae (Blattellidae) and Coryd- iidae (Polyphagidae) (Table 1). The sequence of Myzus persicae (Accession No. JX844381) was used as an out-group. Phylogenetic tree inferred using the neighbour-joining method embedded in ClustalW2 program and plotted using Software program Tree view (http:// taxonomy.zoology.gla.ac uk/rod/rod.html). PCR-RFLP Based on sequence variation among the five species, AluI restriction enzyme was se- lected by manufacturer’s NEBcutter V2.0 program (http://tools.neb.com/nebcutter) and a physical map provided (Fig. 1B). For re- striction fragment assays, 15µ L of the PCR products was digested in a 25µ L reaction mixture containing 10 U of AluI (Vivantis) and 2.5µ L of the appropriate restriction buff- er at 37 °C overnight, following the instruc- tions of manufacturer. The digested products were fractionated on a 2.5% agarose gel and visualized by ethidium bromide staining un- der ultraviolet light. Results mtDNA-COI gene and phylogenetic analysis Result of PCR amplification revealed that the primers could amplify a unique 710bp frag- ment of the COI gene for all of the five cock- roach species. Of the 57 PCR amplified prod- ucts of the specimens, the mtDNA-COI nu- cleotide sequences were generated for 26 spec- imens. Of these, 14 sequences were generat- ed for P. americana, three sequences each for B. germanica, S. lateralis, S. longipalpa, and B. orientalis specimens. The nucleotide sequences have been deposited in the GenBank database under accession nos. JQ267476 to JQ267498 and KJ787108. On average, a high A-T content (65.7%) was evident in the amplified mtDNA- COI gene fragment of the species. However, the A-T contents did not show significant variations at the intra- or inter-species levels. A significant inter-species genetic diver- sity was observed among the five cockroach species characterized at the mtDNA-COI gene. The most divergence rate (22.26%) was ob- served between S. longipalpa and P. ameri- cana and the least rate (11.57%) was seen between American and Oriental cockroach- es. However, the most genetic difference among the species was lowered to 0.0–9.1% at amino acid level indicating that most of the substitutions were silent or synonymous (Table 2). Generally, the brown-banded cock- roaches with the lowest similarity rate at DNA (80%) and amino acid (92.86%) levels were the most diverged species among the species (Table 2). Additionally, based on mtDNA molecular clock (2% per MY), S. longipalpa is suggested to have diverged from other spe- cies about 10 MY. At the intra-species level, a considerable genetic polymorphism (2.62%) was detected among the American cockroach populations whereas no genetic variation has been noticed within other species. Phylogenetic relationships among 16 species belonged to four cockroach families Cryp- tocercidae, Blattidae, Ectobiidae (Blattellidae) and Corydiidae (Polyphagidae) were esti- mated based on the 648bp DNA sequences of the mitochondrial cytochrome oxidase sub- unit I (COI) gene. A cladogram inferred using the neighbor-joining method indicated mon- ophyly of each of the four families (Fig. 1). However, S. longipalpa of Ectobiidae (Blat- tellidae) was diverged early and formed a sep- arate branch distinct from other members of Ectobiidae. The phylogenetic tree showed that all species of that Blattidae grouped together and formed a main clad. In addition, they were sister group of Ectobiidae (Blattellidae). The cladogram suggested that S. longipalpa was the basal species and the early ancestor of other cockroaches. Following S. longipal- pa, Cryptocercidae was the basal families of J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … 186 http://jad.tums.ac.ir Published Online: May 27, 2017 other cockroaches. PCR-RFLP Based on nucleotide sequence analysis of the mtDNA-COI gene, a PCR-RFLP assay was developed for the diagnosis and differentia- tion of the five cockroach species. It was ac- complished by digesting the PCR product (710bp) with AluI restriction enzymes. AluI cuts the PCR–amplified mtDNA-COI fragment of the five species at unique positions: at 251 and 455 to produce three bands of 251, 204 and 255bp for S. lateralis, at 167, 251, 392, and 612 to produce five bands of 167, 84, 141, 220 and 98bp for P. americana, at 251 and 275 to produce three bands of 251, 24, and 435bp for B. orientalis, at seven restriction sites to produce eight bands of 296, 15, 24, 33, 24, 220, 50 and 48bp for B. germanica, at seven cut sites and therefore produces eight bands of 65, 54, 39, 138, 39, 132, 87 and 156 bp for S. longipalpa (Fig. 2). Although some bands had approximate molecular weights (such as 255 and 251bp for S. lateralis and 132 and 138bp for S. longipalpa) or the bands with less than 100bp length were not easily visible in agarose gel, still a distinctive and visible RFLP profile with AluI restriction enzyme was obvious after restriction diges- 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 J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … 187 http://jad.tums.ac.ir Published Online: May 27, 2017 Table 1. Details of sequence data used for cockroach phylogenetic analysis in this study. NS: Not stated Species Common name Family Accession Origin Reference number Cryptocercus 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 Blatta orientalis Oriental Blattidae JQ267490- JQ267492 Iran: Tabriz This study Blatta orientalis Oriental Blattidae EU253827 NS Direct Submission Shelfordella lateralis Turkestan Blattidae JQ267493- JQ267494 Iran: Assalem This study 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 Parcoblatta pensylvan- ica Pennsylvania wood Ectobiidae GU013646 Canada:Ontario Direct Submission Blattella bisignata Double-striped Ectobiidae JX233805 NS Direct Submission Blattella germanica German Ectobiidae JQ267496- JQ267498 Iran: Tehran This study 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 Eupolyphaga sinensis N.S. Corydiidae JF700164 NS Direct Submission Polyphaga sp N.S. Corydiidae JQ267475 Iran: Tehran Direct Submission Therea petiveriana Desert Corydiidae EU253835 NS Direct Submission Myzus persicae Peach-potato aphid Aphididae JX844381 China Direct Submission 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 A B J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … 188 http://jad.tums.ac.ir Published Online: May 27, 2017 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 method and defined a unique restriction en- zyme (AluI) for the first time that can rapidly detect and differentiate the five pest species of cockroaches: P. americana, B. orientalis, B. germanica, Sh. lateralis, and S. longipalpa. Using a simple conventional PCR and just one single restriction enzyme (AluI) for dif- ferentiating of five cockroach species is the advantages of this method in comparison with the only previous study that used two restriction enzymes and a more complicated nested-PCR for discrimination of four cock- roach species (Sulaiman et al. 2011). PCR- RFLP technique is a very cheap and rapid method for species identification of many organisms. This molecular assay was used to identify many species of organisms such as rodents (Oshaghi et al. 2011a), parasites (Oshaghi et al. 2009, 2010, 2011b), Anophe- les (Oshaghi et al. 2008, Mehravaran et al. 2011) and to determine blood type within insect guts (Chavshin et al. 2006, Maleki- Ravasan et al. 2009). This will provide a cost-effective solution that so many speci- mens can be studied with no need to provide DNA sequencing. In this method, using a set of conserved primers, a tiny amount of DNA can be amplified and then be identified by one restriction enzyme that overcomes lack of ample amount of cockroach body parts. In addition, this method can be used to deter- mine identity of museum specimens, unknown samples and malformed specimens or residues of cockroach body parts in food samples. Analysis of the data revealed a high inter- species variation (11.56–22.26%) in the bar- coding region of mtDNA COI gene across these five cockroach species, suggesting the gene fragment is a good molecular marker for the development of a diagnostic tool for the detection of food pests in food samples. DNA barcoding has been used as a shared resource of DNA sequences for identification and tax- onomic clarification of many organisms, in- cluding birds (Hebert et al. 2004), sea turtles (Vargas et al. 2009), eutherian mammals (Luo et al. 2011), fishes (Mabragana et al. 2011) and insects (Barrett and Hebert 2005, Smith et al. 2006, Wilson 2010). In the light of DNA barcoding, researchers can compare different kinds of organisms and have access to their genetic information providing evidence of interpreting systematic situation. Hebert et al. (2003) proposed the use of COI gene as an original source for species identification in the animal kingdom. Additionally, they demon- strated that species-level assignments could be obtained by creating a comprehensive COI. In addition to COI, many researchers used various genes including rRNA (Kambham- pati et al. 1995, Mukha et al. 2000, Lazebna- ya et al. 2005), COII (Maekawa and Matsu- moto 2000), ETS (Mukha et al. 2002, Mukha et al. 2005), NTS (Mukha et al. 2007), ITS1 (Pechal et al. 2008) and complete mitochon- drial genome (Yamauchi et al. 2004, Zhang et al. 2010, Cameron et al. 2012, Xiao et al. J Arthropod-Borne Dis, June 2017, 11(2): 182–193 SS Hashemi-Aghdam et al.: Utility of mtDNA … 189 http://jad.tums.ac.ir Published Online: May 27, 2017 2012, Chen 2013) for systematic classifica- tion of different species of cockroaches. The inferred relationships among cock- roach families (S. longipalpa + (S. longipalpa + (Blattidae + (Cryptocercidae + (Corydiidae + Ectobiidae)))) is partly in agreement with some previously published analyses (Kambhampati 1995, Maekawa and Matsumoto 2000, Lo et al. 2007). Furthermore, S. longipalpa has been introduced as the early ancestor of other cockroaches by the phylogenetic trees in- ferred from both amino acid and DNA se- quence data where it did not group with Ec- tobiidae or indeed nest within cockroaches. It is in controversy with earlier morphology- based phylogenetic hypotheses as well as molecular study using a combination of four mitochondrial (16S and COI+COII) and nu- clear (18S and 28S ribosomal subunits) loci indicating monophyletic topology of Blabe- roidea superfamily (Djernæ et al. 2002). A wider sampling of Blaberoidea including Blaberidae is highly required in order to test the topological situation of S. longipalpa. Conclusion The results of this study demonstrate the utility of the mtDNA-COI gene as a valuable and powerful molecular marker in unravel- ing medically important cockroach species, particularly when morphological characters are subtle. 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