J Arthropod-Borne Dis, December 2016, 10(4): 454–461 S Bazrafkan et al.: Discrimination of … 454 http://jad.tums.ac.ir Published Online: October 04, 2016 Original Article Discrimination of Paederus fuscipes and Paederus littoralis by mtDNA-COI PCR-RFLP Sahar Bazrafkan 1, Hassan Vatandoost 1, Abbas Heydari 1, Hassan Bakhshi 1, Somayeh Panahi-Moghadam 1, Saedeh Hashemi-Aghdam 1, Fatemeh Mohtarami 1, Abbas Rahimi- foroushan 2, Sinan Anlaş 3, *Mansoreh Shayeghi 1, *Mohammad Ali Oshaghi 1, Seyed Mo- hammad Abtahi 4 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Isfahan University of Medical Sciences, Isfahan, Iran 3Celal Bayar University Alaşehir, Vocational School, Department of Entomology, Alaşehir, Manisa, Tur- key 4Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran (Received 26 Oct 2014; accepted 11 Jan 2015) Abstract Background: Linear dermatitis is endemic in Iran where most cases occur in the Caspian Sea coast and Fars prov- ince. The disease is caused by beetles of the genus Paederus which are active from early spring to beginning of au- tumn although its incidence rises from May to August. The classic taxonomy of Paederus spp. is based on the male genitalia that is very complex and needs expertise. In this study, we report a DNA-based method to discriminate Paederus fuscipes and Paederus littoralis (=syn: P. lenkoranus, P. ilsae). Methods: Type specimens were collected from north and south of Iran. Molecular typing of the species was per- formed using restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-am- plified fragments of mtDNA-COI. Results: Sequence analyses of the data obtained in this study showed significant DNA polymorphisms. There were 89 substitutions between COI sequences of the two species. The mtDNA-COI fragment comprises several useful species-specific restriction sites comprising HaeIII that could result in distinctively different species-specific PCR– RFLP profiles. The HaeIII enzyme cuts the 872 bp PCR amplicon of P. littoralis into 737 and 100 bp and two small nonvisible bands whereas it does not cut P. fuscipes amplicon into fragments. Conclusion: This study demonstrates that molecular typing is useful method and allows one to differentiate between two species and is recommended for discrimination of other Paederus species, which morphologically are indistin- guishable or very difficult to be distinguished. Keywords: Paederus, Linear dermatitis, mtDNA- COI, PCR-RFLP, Molecular typing Introduction The genus Paederus Fabricius (Staph- ylinidae: Coleoptera) is represented by 622 species worldwide and 85 species and sub- species in the Palaearctic region. The he- molymph of some species within the genus Paederus is nuisance as, once released, it causes linear dermatitis and conjunctivitis in humans. The symptoms are due to a toxic amide substance, which has been named Pederin (Pavan and Bo 1953) and makes up approximately 0.025% of an insect's weight (for P. fuscipes). Most cases of linear derma- titis in Iran occur in the Caspian Sea shore- line and Fars in south of Iran (Nikbakhtza- deh and Tirgari 2008). Systematics of Paederus beetles at species *Corresponding author: Dr Mansoureh Shayeghi, Email: mansorehshayeghi@yahoo.com, Dr Moham- mad Ali Oshaghi, Email: moshaghi@sina.tums.ac.ir J Arthropod-Borne Dis, December 2016, 10(4): 454–461 S Bazrafkan et al.: Discrimination of … 455 http://jad.tums.ac.ir Published Online: October 04, 2016 level is rather difficult and is based on the morphology of male primary and secondary sexual characters (Coiffait 1982). This makes them difficult to identify and limits their study and management. Consequently, this has made a very complicated history for Pae- derus taxonomy and has changed it dramat- ically and some species are treated as syno- nyms of each other and or downgraded to a single subspecies/species (Nikbakhtzadeh et al. 2012 and references herein). For example P. lenkoranus Scheerpeltz (Scheerpeltz 1957) P. littoralis ilsae (Bernhauer 1932) and P. ilsae (Coiffait 1982) have been recently con- sidered synonymous (Nikbakhtzade 2012). Paederus fuscipes fuscipes Curtis (1826) was formerly known by the synonymous names of P. iliensis Coiffait (1970) and P. kalalovae Roubal (1932). These older spe- cies are downgraded to a single subspecies of P. fuscipes in the current systematics of Staphylinidae. Previous studies on the fauna, geograph- ical distribution, ecology and medical im- portance of Paederus beetles in Iran re- vealed presence of 14 Paederus species or subspecies in the country (Nikbakhtzadeh et al. 2012). However, due to difficulties in Paederus species discrimination, different lists of species have been reported for an identical region. In north of Iran, for exam- ple, Janbaksh and Ardalan (1977) reported three species of P. fuscipes, P. pietschmanni (synonym of P. mesopotamicus), and P. spectabilis in Mazandaran province at the Caspian Sea coast. Majidi-Shad et al. (1989) reported three species of P. fuscipes, P. ri- parius, and P. littoralis from the same re- gion. Later on, P. fuscipes, P. kalalovae and P. balcanicus were reported from the prov- ince by Nikbakhtzadeh and Tirgari (2008). Finally, Nikbakhtzadeh et al. (2012) reported three species of P. fuscipes, P. balachowskyi (synonym of P. mesopotamicus), and P. bal- canicus from the same area. In southern part, Nikbakhtzadeh (2002, 2008) reported P. il- sae and P. iliensis Coiffait from Fars Province. In 2012, P. littoralis ilsae Bernhauer (=syn: P. lenkoranus Scheerpeltz, P. ilsae) and P. fuscipes fuscipes Curtis (=syn: P. iliensis Coiffait, P. kalalovae Roubal) were reported from that area. Recent advances in DNA-based technol- ogy have made a wide range of molecular characteristics and markers available for tax- onomic and systematic studies of insects. One region of the insect genome including beetles that has received particular attention is the mitochondrial DNA (mtDNA). Mito- chondrial DNA with a fast mutation rate has significant variation in sequences between species. A 658 bp region (the Folmer region) of the mitochondrial cytochrome c oxidase subunit I (COI) gene was proposed as a po- tential barcode (Hebert et al. 2003). The mtDNA genes have many advantages including a relatively fast mutation rate, easy to use and known PCR primers. The dis- crepancy and inconsistency in the number of species in Iran plus difficulties in systemat- ics of Paederus species encouraged us to test sequence variation of mtDNA-COI, to intro- duce a molecular marker to discriminate P. fuscipes and P. littoralis, the two sympatric and common species in the Mediterranean basin including southern part of Iran. Materials and Methods Paederus specimens and morphological identification Adult specimens were collected by aspi- rator on rice plants and weeds in early morn- ing or afternoon and under clays at hot hours of day time. Collection of P. fuscipes and P. littoralis were performed mostly in rice fields in various locations of Mazanderan and Fars Province during the growing season from May to August 2011. The specimens were preserved in 70% ethanol and were sent to the insect molecular biology laboratory of the School of Public Health, Tehran Univer- J Arthropod-Borne Dis, December 2016, 10(4): 454–461 S Bazrafkan et al.: Discrimination of … 456 http://jad.tums.ac.ir Published Online: October 04, 2016 sity of Medical Sciences, Iran for molecular characterization. The pictorial key of Coiffait (1982) was used to identify the specimens to genus level. The specimens were then identi- fied to species level based on the habits and morphology of male primary and secondary sexual characters by the Turkish expert, S. Anlaş. DNA extraction Paederus specimen representative differ- ent populations of both species were selected for DNA analysis. Total genomic DNA was extracted from total body of individual sam- ples using DNeasy® Blood and Tissue Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The specimens were frozen prior to DNA extraction and then grinded in the Kit supplied buffer and extraction followed according to the manu- facturer's directions. DNA amplification The COI (Cytochrome Oxidase subunit 1) region of mtDNA gene (mitochondrial) was amplified with the primer pair c1-j-2183 (5′- caacatttattttgattttttgg-3′) and tl-2-n-3014 (5′- ccattgcatctgccatatta-3′). These primers have already been introduced by Simon et al. (1994) and used for some Colleopteran. PCR amplification reaction conditions were: 5 μl 10× PCR-Buffer, 120 μM of each dNTPs, 50 pmol of each primer, 2 μl (about 100 ng) of template DNA, and 2.5 U of Taq polymerase (Sinaclone, Iran) in a 25 μl reaction volume. PCR amplification was performed with an Eppendorf thermal cycler (Germany). The cycling parameters were: 2 min initial de- naturation at 94 °C followed by 5 cycles of 30 sec at 94 °C, annealing at 45 °C for 40 sec and extension at 72 °C for 1 min and 35 cycles of 94 °C at 30 sec, annealing at 51 °C for 40 sec and extension at 72 °C for 1 min. The final extension step was 72 °C for 10 min (www.dnabarcodes2011.org). Double dis- tilled water was used as negative and well- characterized DNA samples were used as positive controls. Sequencing and PCR-RFLP The PCR products of COI fragment were purified from gels by using a gel purification kit, and subjected to sequencing. Sequencing was performed using an ABI 3730 sequencer machine by Bioneer (South Korea). The re- sultant sequences were checked to correct ambiguities. Homologies with the available sequence data in GenBank was checked by using basic local alignment search tool (BLAST) analysis software (www.ncbi.nlm. nih.gov/BLAST). The COI sequences ob- tained in this study was checked to obtain its physical map and to select restriction en- zymes by using the Nebcutter program (Vin- cze et al. 2003). Restriction enzymes were selected based on their positions (beginning, middle, and last part of PCR product), pro- files, costs, and availabilities in the market. Digestion of PCR products was performed in 25 μL of a solution containing 15 μL of PCR product mixed with 2.5 μL of enzyme buff- ers and 5 units of HaeIII restriction enzyme overlaid with two drops of mineral oil. The mixture was incubated at the temperature recommended by the enzyme supplier. An aliquot (14 μL) of the digestion product was mixed with 6 μL of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol), loaded on to a 1% agarose gel, and subjected to electrophoresis. Gels were stained with ethidium bromide (2 mg/ mL) and the RFLP profiles were visualized under ultraviolet light. Results Species identification and PCR-RFLP In this study, a total of 154 adult Paede- rus specimens were collected from the two Iranian provinces. The P. littoralis specimens and a subset of the morphologically-identi- fied P. fuscipes were subjected to mtDNA- J Arthropod-Borne Dis, December 2016, 10(4): 454–461 S Bazrafkan et al.: Discrimination of … 457 http://jad.tums.ac.ir Published Online: October 04, 2016 COI PCR amplification followed by PCR- direct sequencing. Sequencing was performed for both strands and the consensus data were submitted to GenBank with accession num- bers (ANs): KF724713, KF724714, KM07 1620, KM071621, KM071622, KM071623, KM071624, KM071625, KM071626, KM07 1627, KM071628 and KM071629. There were minor intra-species sequence variations between individuals of both species; howev- er, there were considerable DNA polymor- phisms between the two species. Subsequent sequence analysis confirmed identical 872 bp PCR amplicons for both species. An alignment of the 805 bp of the COI region from these sequences is shown in Fig. 1. The amount of sequence similarity between the two species was 88% and 98% at DNA and amino acid level respectively. Subsequently, a BLAST search on the sequences revealed that there were corresponding COI sequenc- es for P. fuscipes and P. littoralis species in Genbank respectively with 96% and 98% maximum identity. Sequence analysis of the COI fragments of both species revealed a number restriction sites for discrimination of the species. Among them, an appropriate restriction site for HaeIII (GG↓CC) gave two distinct profiles of 737/100/23/12 bp fragments for P. littoralis and an intact 872 bp for P. fuscipes (Fig. 2). Of course, for P. littoralis, only the two 737 and 100 bp bands were visible because the small ones (23 and 12 bp) were not visible in the gel. P.fuscipes TTACCTGGATTTGGAATAATTTCTCATATTATCTCTTACAGAAGTGGAAAACAAGAAACT 60 P.littoralis TTACCAGGATTTGGAATAATTTCTCATATCATTTCTTACAGAAGAGGGAAACAAGAAACT ***** *********************** ** *********** ** ************ 60 P.fuscipes TTTGGAGCAATTGGGATAATTTATGCTATGCTTGCAATTGGTTTATTAGGTTTTATTGTA 120 P.littoralis TTTGGGGCAATAGGAATAATTTATGCTATATTAGCAATTGGTTTATTAGGTTTTATTGTT ***** ***** ** ************** * ************************** 120 P.fuscipes TGAGCTCATCATATATTTACTGTCGGAATGGACATTGATACTCGAGCTTACTTTACATCA 180 P.littoralis TGAGCCCATCATATATTTACAGTAGGTATAGATATTGATACACGAGCTTATTTTACCTCA ***** ************** ** ** ** ** ******** ******** ***** *** 180 P.fuscipes GCCACAATAGTAATTGCTGTTCCAACTGGAATTAAGGTTTTTAGATGAATAGGAACAATT 240 P.littoralis GCAACTATAGTAATTGCTGTACCTACAGGAATTAAAGTATTTAGTTGAATAGCAACAATT ** ** ************** ** ** ******** ** ***** ******* ******* 240 P.fuscipes TATGGTGGAAATTTAAATTTTAGCCCACCAATAATCTGAAGTTTAGGGTTTGTATTTTTA 300 P.littoralis TATGGAGGAAATTTAAATTTTAGACCCCCAATAATTTGAAGATTAGGTTTTGTATTTTTA ***** ***************** ** ******** ***** ***** ************ 300 P.fuscipes TTTACTGTCGGAGGATTAACTGGAGTAATTTTAGCTAATTCATCAATTGATATTGTATTA 360 P.littoralis TTTACTGTAGGAGGATTAACAGGAGTGATTTTAGCTAATTCATCAATTGATATTGTTTTA ******** *********** ***** ***************************** *** 360 P.fuscipes CATGACACATATTATGTTGTAGCTCATTTTCATTATGTCTTATCAATAGGGGCAGTATTT 420 P.littoralis CATGATACTTACTATGTAGTAGCTCACTTTCACTATGTTTTATCAATAGGAGCTGTTTTT ***** ** ** ***** ******** ***** ***** *********** ** ** *** 420 P.fuscipes GCTATTATAGCAGGGTTAGTACAATGATACCCAATATTTATTGGGTTAATATTAAACGAA 480 P.littoralis GCTATTATAGCAGGATTAGTGCAATGATTTCCAATATTCATTGGATTAATATTAAATGAA ************** ***** ******* ******** ***** *********** *** 480 P.fuscipes AAATATTTAAAAATTCAATTTTTAATTATATTTATTGGGGTAAATTTAACTTTTTTCCCT 540 P.littoralis AAATACTTAAAAATCCAATTTTTAATTATATTTATTGGGGTAAATTTAACATTTTTTCCT ***** ******** *********************************** ***** *** 540 P.fuscipes CAACATTTTTTAGGTTTATCAGGAATACCACGTCGATATTCAGATTACCCAGATGCTTAC 600 P.littoralis CAACATTTTTTAGGATTATCAGGAATACCTCGTCGATACTCAGATTACCCTGATGCTTAT ************** ************** ******** *********** ******** 600 P.fuscipes ACAATATGAAATGTAATTTCATCTATTGGATCAATAATTTCATTTATTGGAATTATATTC 660 P.littoralis ACAATATGGAACGTAATTTCATCTATTGGATCAATAATTTCATTTATTGGAATTATATTC ******** ** ************************************************ 660 P.fuscipes TTTTTATGAATTATTTGAGAAAGATTTATTTCAATACGAAAAATTATTGGAGCTCCAATC 720 P.littoralis TTTTTATGAATTATTTGAGAAAGATTTATTTCTATACGAAAAATTATTGGGGCCCCAATT ******************************** ***************** ** ***** 720 J Arthropod-Borne Dis, December 2016, 10(4): 454–461 S Bazrafkan et al.: Discrimination of … 458 http://jad.tums.ac.ir Published Online: October 04, 2016 P.fuscipes CCACCAACAGCATTAGAATGAATGCATTCATACCCCCCATCAGAACACACCTATTCTGAG 780 P.littoralis CCCCCAACTGCTTTAGAATGGATACATTCTTATCCACCCTCAGAGCATACTTACTCAGAA ** ***** ** ******** ** ***** ** ** ** ***** ** ** ** ** ** 780 P.fuscipes TTGCCTTTTATAACAATTAAGTTCT 805 P.littoralis TTACCTTTTATAACAATTAAATTCT ** ***************** **** 805 Fig. 1. Alignment of 752 bp of mtDNA-COI sequences of Paederus fuscipes and Paederus littoralis. Stars show the conserved position and gaps or dots indicate substitutions. The HaeIII restriction site (GGCC) for P. littoralis is shown at position of 705–708. Fig. 2. Digestion profiles of mtDNA-COI PCR products (872 bp) with HaeIII in Paederus fuscipes and Paederus littoralis. Lane 1 (872 bp) for P. fuscipes and lane 2 (737 and 100 bp) for P. littoralis, M: 100 bp molecular weight marker (SinaClon, Iran). Discussion To our knowledge, this is the first molec- ular investigation aiming at discrimination of Paederus fuscipes and Paederus littoralis by mtDNA-COI in literature. According to the present results, P. fuscipes and P. littoralis specimens can be easily distinguished by mtDNA-COI PCR-Restriction fragment length polymorphism (RFLP) using HaeIII. This assay could end up the discrepancy, incon- sistency, and difficulties present in systemat- ics of Paederus species, allowing studies re- garding their distribution, biology, and be- havior to proceed, as well as better under- standing of their role in linear dermatitis and antiviral and antitumor activities of the ped- erin presents in their hemolymph. Correct species identification is crucial in entomological surveys and application of con- trol measures for the species that are mor- phologically indistinguishable or difficult to be distinguished. Targeting correct species is particularly important where more than one species live sympatric which is the case for P. littoralis and P. fucipes in south of Iran (Nikbakhtzadeh et al. 2012). Paederus litto- ralis has been collected from different parts of Fars province (Nikbakhtzadeh et al. 2012). On the other hand, P. fuscipes has a world- wide geographical distribution and has been reported from different countries in Africa, Asia and Europe (Coiffait 1982, Smetana 2004). This species is reported from central and southern Iran and is the most frequent species in north of Iran (Janbakhsh and Ar- dalan 1977, Tirgari and Nikbakhtzadeh 2002, Nikbakhtzadeh and Tirgari 2008, Anlas and Newton 2010, Nikbakhtzadeh et al. 2012). Nowadays many researchers have been focused on medicinal insects such as Paede- rus species as well as horseflies, blister bee- tles and American cockroaches that have been well known due to their effects against various pathogens such as viruses and bacte- ria as well as diseases such as thrombosis and cancer (Richter et al. 1997, Witczak et al. 2012, Mosey et al. 2012). Pederin of Paede- Fig. 1. Continued… J Arthropod-Borne Dis, December 2016, 10(4): 454–461 S Bazrafkan et al.: Discrimination of … 459 http://jad.tums.ac.ir Published Online: October 04, 2016 rus species has been used in cancer studies in recent years. Pederin and its common sub- structure accounts for similar antiviral and antitumor activities, cytotoxicity and disrup- tion of DNA metabolism that are mainly based on inhibition of eukaryotic protein biosyn- thesis (Piel 2002). The production of pederin relies on the activities of endosymbiont bac- teria (Pseudomonas species) within Paederus (Kellner 2002, Piel et al. 2004). The manu- facture of pederin is largely confined to adult female beetles which protects the beetles against predators (Kellner and Dettner 1996, Kellner 2002). Larvae and males only store pederin acquired maternally (i.e., through eggs) or by ingestion (Piel 2002). The species commonly causing linear der- matitis are beetles of P. fuscipes in Asia (An- laş and Çevik 2008). Also amount of pederin and its related compounds might be variable in different species (Kellner and Dettner 1996). Therefore correct species identifica- tion is essential to study the biological effect of the molecule. In this study we used the COI gene of mtDNA genome, which is a known powerful molecular marker for molecular identifica- tion of various organisms. However, in addi- tion to COI, other interest marker such as ITS2 (rDNA), wingless, Topoisomerase I, and 28S might be useful in order to develop a molecular key for discrimination of Paede- rus species. There are considerable reports on using COI gene in species diagnosis, population genetics, and systematics of bee- tles in the literature (e.g. Andreev et al. 1998, Gallego and Galián 2001, Becerra 2004, Chatzimanolis et al. 2010, Germain et al. 2013). Also there are a few available COI sequence entries of P. littoralis, P. ruficollis, P. riparius, P. moesopotamicus, and P. fusci- pes in Genbank database. Species discrimina- tion can be achieved by using RFLP profiles on PCR products or designing species specif- ic primers to produce species specific prod- uct. However, PCR-RFLP method is reason- ably cheap, fast, and user friendly and has been used frequently in many laboratories involving species identification including in- sects and the microbes they transmit (Clark et al. 2001, Mukabana et al. 2002, Armstrong and Ball 2005, Oshaghi et al 2006a and 2006b, Greenstone 2006, Oshaghi et al 2008, 2009, 2010, Kato et al. 2010, Oshaghi et al 2011). Conclusion Further molecular studies using type or morphologically well-known species are now required to verify the species composition of Paederus in Iran and other countries in Eu- rope, Asia, and Africa. By performing mo- lecular typing of Paederus species in the future, we expect that the Paederus fauna of Iran and other countries will be identified more accurately. Acknowledgements This work was supported financially by Tehran University of Medical Sciences, Iran. References Andreev D, Breilid H, Kirkendall L, Brun LO, ffrench-Constantm RH (1998) Lack of nucleotide variability in a bee- tle pest with extreme inbreeding. In- sect Molec Biol. 7: 197–200. Anlaş S, Çevik IE (2008) Faunistic studies on Pederinae (Coleoptera: Staphylini- dae) in Manisa province, Turkey. Mun Ent Zool. 3: 665–674. Anlaş S, Newton AF (2010) Distributional checklist of the Staphylinidae (Cole- optera) of Iran, with new and addi- tional records. Linzer Biol Beitr. 42: 335–388. Armstrong KF, Ball SL (2005) DNA bar- codes for biosecurity: invasive species J Arthropod-Borne Dis, December 2016, 10(4): 454–461 S Bazrafkan et al.: Discrimination of … 460 http://jad.tums.ac.ir Published Online: October 04, 2016 identification. Philos Trans R Soc Lond B Biol Sci. 360(1462): 1813– 1823. Review Erratum in: Philos Trans R Soc Lond B Biol Sci. 360(1464): 2373. Becerra JX (2004) Molecular systematics of Blepharida beetles (Chrysomelidae: Alticinae) and relatives. Mol Phyloge- net Evol. 30: 107–117. Chatzimanolis S, Cohen IM, Schomann A, Solodovnikov A (2010) Molecular phy- logeny of the mega-diverse rove beetle tribe Staphylinini (Insecta, Coleoptera, Staphylinidae). Zool Scr. 39: 436–449. Clark TL, Meinke LJ, Foster JE (2001) PCR-RFLP of the mitochondrial cyto- chrome oxidase (subunit I) gene pro- vides diagnostic markers for selected Diabrotica species (Coleoptera: Chrysomelidae). Bull Entomol Res. 91(6): 419–427. Coiffait H (1982) Coléoptères Staphylinidae de la région Paléarctique occidentale. IV. Sous-famille Pederinae. Tribus Pederini 1 (Paederi, Lathrobii). Nouv Rev Entomol. 12(4): 1–440. Gallego D, Galián J (2001) The internal transcribed spacers (ITS1 and ITS2) of the rDNA differentiates the bark beetle forest pests Tomicus destruens and T. piniperda. Insect Molec Biol. 10: 415– 420. Germain JF, Chatot C, Meusnier I, Artige E, Rasplus JY, Cruaud A (2013) Molecu- lar identification of Epitrix potato flea beetles (Coleoptera: Chrysomelidae) in Europe and North America. Bull Ento- mol Res. 1: 1–9. Greenstone MH (2006) Molecular methods for assessing insect parasitism. Bull Entomol Res. 96(1): 1–13. Hebert PD, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proc Biol Sci. 270 (1512): 313–321. Janbakhsh B, Ardalan A (1977) Rove beetles (Coleoptera: Staphylinidae) and their medical importance. Iran J Public Health. 6: 70–77. Kato H, Gomez EA, Cáceres AG, Uezato H, Mimori T, Hashiguchi Y (2010) Mo- lecular epidemiology for vector re- search on leishmaniasis. Int J Environ Res Public Health. 7(3): 814–826. Kellner RLL (2002) Molecular identification of an endosymbiotic bacterium associ- ated with pederin biosynthesis in Pae- derus sabaeus (Coleoptera: Staph- ylinidae). Insect Biochem Molec Biol. 32: 389–395. Kellner RLL, Dettner K (1996) Differential efficacy of toxic pederin in deterring potential arthropod predators of Pae- derus (Coleoptera: Staphylinidae) off- spring. Oecol. 107: 293–300. Majidi-Shad B, Janbakhsh B, Bagheri-Ze- nooz E (1989) The species of genus Paederus (Col. Staphylinidae) that caused human dermatitis in the south- ern region of Caspian Sea. The 9th Plant Protection Congress of Iran, 1989, Iranian Society of Plant Protec- tion, Mashhad, Iran, p. 40 Mosey RA, Floreancig PE (2012) Isolation, biological activity, synthesis, and me- dicinal chemistry of the pederin/ my- calamide family of natural products. Nat Prod Rep. 29(9): 980–995. Mukabana WR, Takken W, Knols BG (2002) Analysis of arthropod blood- meals using molecular genetic mark- ers. Trends Parasitol. 18(11): 505–509. Nikbakhtzadeh MR, Naderi M, Safa P (2012) Faunal diversity of Paederus fabricius 1775 (Coleoptera: Staphylin- idae) in Iran. Insecta Mundi. 267: 1–9. Nikbakhtzadeh MR, Tirgari S (2008) Medi- cally Important Beetles of Iran. J Ven- om Anim Toxins incl Trop Dis.14: 597–618. J Arthropod-Borne Dis, December 2016, 10(4): 454–461 S Bazrafkan et al.: Discrimination of … 461 http://jad.tums.ac.ir Published Online: October 04, 2016 Oshaghi MA, Chavshin AR, Vatandoost H (2006) Analysis of mosquito blood- meals using RFLP markers. Exp Para- sitol. 114(4): 259–264. Oshaghi MA, Rafinejad J, Choubdar N, Piazak N, Vatandoost H, Telmadarraiy Z, Mohtarami F, Ravasan NM (2001) Discrimination of relapsing fever Bor- relia persica and Borrelia microtti by diagnostic species-specific primers and polymerase chain reaction-restriction fragment length polymorphism. Vector Borne Zoonotic Dis. 11(3):201-207. Oshaghi MA, Rasolian M, Shirzadi MR, Mohtarami F, Doosti S (2010) First re- port on isolation of Leishmania tropica from sandflies of a classical urban Cu- taneous leishmaniasis focus in southern Iran. Exp Parasitol. 126(4):445-450. Oshaghi MA, Ravasan NM, Hide M, Ja- vadian EA, Rassi Y, Sedaghat MM, Mohebali M, Hajjaran H (2009) De- velopment of species-specific PCR and PCR-restriction fragment length poly- morphism assays for L.infantum/L. donovani discrimination. Exp Parasi- tol. 122(1):61-65. Oshaghi MA, Yaaghoobi F, Abaie MR (2006) Pattern of mitochondrial DNA variation between and within Anoph- eles stephensi (Diptera: Culicidae) bi- ological forms suggests extensive gene flow. Acta Trop. 99(2–3): 226–233. Oshaghi MA, Yaghobi-Ershadi MR, Shemshad K, Pedram M, Amani H (2008) The Anopheles superpictus complex: intro- duction of a new malaria vector com- plex in Iran. Bull Soc Pathol Exot. 101(5): 429–434. Pavan M, Bo G (1953) Pederin, toxic princi- ples obtained in the crystalline state from the beetle Paederus fuscipes Curt. Physiol comp Oecologia. 3: 307–312. Piel J (2002) Polyketide synthesis-peptide synthetase gene cluster from an un- cultured bacterial symbiont of Paede- rus beetles. Proceedings of the Na- tional Academy of Sciences. 99: 14002–14007. Piel J, Höfer I, Hui D (2004) Evidence for a symbiosis island involved in horizontal acquisition of Pederin biosynthetic ca- pabilities by the bacterial symbiont of Paederus fuscipes beetles. J Bacteriol. 186: 1280–1286. Richter A, Kocienski P, Raubo P, Davies DE (1997) The in vitro biological activities of synthetic 18-O-methyl mycalamide B, 10-epi-18-O-methyl mycalamide B and pederin. Anticancer drug des. 12 (3): 217–227. Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P (1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am. 87: 651–701. Smetana A (2004) Staphylinidae (except subfamilies Pselaphinae and Scaphidi- inae). Catalogue of Palaearctic Cole- optera II. Hydrophiloidea-Histeroidea- Staphylinoidea. In: Löbl I, Smetana A (Eds): Apollo Books, Stenstrup Den- mark, pp. 237–699. Tirgari S, Nikbakhtzadeh MR (2002) Paede- rus beetles (Coleoptera: Staphylini- dae): An urban problem in Iran. 4th In- ternational Conference on Urban Pests. In: Jones SC, Zhai, J, Robinson WH (Eds): Pocahontas Press, Charleston, South Carolina, pp. 401–407. Vincze T, Posfai J, Roberts RJ (2003) NEBcutter: A program to cleave DNA with restriction enzymes. Nucleic Ac- ids Res. 31: 3688–3691. Witczak ZJ, Rampulla RM, Bommareddy A (2012) Mycalamides, pederin and psymberin as natural carbohydrates and potential antitumor agents: past and future perspectives. Mini Rev Med Chem. 12(14): 1520–1532.