J Arthropod-Borne Dis, September 2020, 14(3): 239–249 A Samiei et al.: The Phylogenetic Analysis of … 239 http://jad.tums.ac.ir Published Online: September 30, 2020 Original Article The Phylogenetic Analysis of Cimex hemipterus (Hemiptera: Cimicidae) Isolated from Different Regions of Iran Using Cytochrome Oxidase Subunit I Gene Awat Samiei1; *Mousa Tavassoli1; Karim Mardani2 1Department of Pathobiology, Faculty of Veterinary Medicine, Urmia University, Urmia, West Azerbaijan, Iran 2Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, Urmia, West Azerbaijan, Iran *Corresponding author: Dr Mousa Tavassoli, E-mail: m.tavassoli@urmia.ac.ir (Received 05 Sep 2018; accepted 24 Aug 2020) Abstract Background: Bedbugs are blood feeding ectoparasites of humans and several domesticated animals. There are scar- city of information about the bed bugs population throughout Iran and only very limited and local studies are availa - ble. The aim of this study is to assess the phylogenetic relationships and nucleotide diversity using partial sequences of cytochrome oxidase I gene (COI) among the populations of tropical bed bugs inhabiting Iran. Methods: The bedbugs were collected from cities located in different geographical regions of Iran. After DNA ex- traction PCR was performed for COI gene using specific primers. Then DNA sequencing was performed on PCR products for the all 15 examined samples. Results: DNA sequencing analysis showed that the all C. hemipterus samples were similar, despite the minor nu- cleotide variations (within the range of 576 to 697bp) on average between 5 and 10 Single nucleotide polymorphisms (SNPs). Subsequently, the results were compared with the database in gene bank which revealed close similarity and sequence homology with other C. hemipterus from other parts of the world. Conclusion: In conclusion, this study has demonstrated the ability of the COI gene to differentiate between the C. hemipterus populations from a few different locations in Iran. The current research is the first report of phylogenetic and genetic species diversity analysis conducted on C. hemipterus in Iran. These results provided basic information for further studies of molecular epidemiology, public health and pest control operators in Iran. Keywords: Bed bug; Cimex hemipterus; COI; Phylogenetic analysis; Iran Introduction “Bed bugs” is a term often applied to the approximately 90 species within the Cimicidae; of these, only Cimex lectularius, the common bed bug, and C. hemipterus, the tropical bed bug, show a strong host preference for hu- mans (1). Cimex hemipterus, is considered as the common tropical bedbug whereas C. lec- tularius is common in temperate climates (2). It has been well documented that bed bugs harbor at least 40 human pathogens, but there is no tangible evidence regarding the ability of routine mechanical transmission any of them (1, 3-6). All the daytime, bed bugs disappear and hide in inaccessible places such as: cracks and crevices in beds, wooden furniture, floors, and walls and reappear at night to feed from their preferred host, humans (7). Apart from the discomfort caused by the bite, bedbugs have been known to cause sec- ondary infections and psychological disorders (4). Chronic infestation can cause nervousness, lethargy, pallor, diarrhea, and even iron defi- ciency (8-10). Bedbugs infest any kind of tem- porary accommodations, especially hotels, ser- viced apartments, and cause severe problems. A resurgence of bed bugs has been reported Copyright © 2020 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, September 2020, 14(3): 239–249 A Samiei et al.: The Phylogenetic Analysis of … 240 http://jad.tums.ac.ir Published Online: September 30, 2020 from the United States, Canada, Australia, Europe, and some Asian countries during the past 15 years (11-16). The reasons for the ex- plosion have not yet been clarified; however, several factors such as increased rate of in- ternational travel, reduced use of residual in- secticides indoors, and insecticide resistance may play a role (17). Few studies have been undertaken on these bugs in Iran. Dehghani et al. examined an out- break of these bugs in 1998 in villages west of Kashan. Out of 495 houses in 10 villages there was 6.7% contamination1 (18). In that year, the National Association of Managing World Pests announced the contamination in New York at 6.7%. Shahraki et al. in 2000, examined an outbreak of bugs in university dormitories among 180 boys and 145 girls in Yasuj, western Iran. They reported 28.9% con- tamination which approximated the current study after taking into consideration that the numbers of individuals were not close to our study (19). Haghi et al. reported that in Bah- man Amir, Mazandaran, Iran most bugs were found in bedrooms (56.54%), living rooms (31.25%), and kitchens (8.59%) (17). Also, from the 182 examined containers in Polour, Mazandaran Province, 164 (approximately 90.1%) had evidence of contamination by bed bugs (20). Genetic analysis of medically important insect species is absolutely required, because it provides useful information about vector transmission, disease epidemiology and dis- ease control (21). There is scarcity of infor- mation about the bed bug population through- out Iran and only very limited and local stud- ies are available (17, 20). Taxonomic and mor- phologic identification of bed bugs require highly experienced person and appropriate samples. Nowadays, molecular techniques such as nucleotide sequence analysis and phylo- genetic tree have been developed for taxo- nomic identification (22). In recent years, the application of high- resolution molecular markers has provided im- portant new insight into the population genet- ic structure and infestation dynamics of many insect pest species of public health concern (23-25). New molecular tools now make it feasible to not only accurately identify the number of populations actively infesting a building (24, 26) but also to elucidate dynam- ics and characteristics essential for understand- ing infestation patterns and history, e.g., lev- els of genetic diversity (a measure often as- sociated with population health (27), tem- poral stability of populations after pest con- trol efforts (28), and the presence or absence of genetic mutations associated with insecti- cide resistance (29). Population genetics studies on bed bugs have been completed using nuclear rRNA, mt DNA genes, and microsatellite loci as markers (25, 30-34). The aim of the first of these studies (Szalanski et al. 2008) shed light on the dispersal patterns of bed bugs during their recent global resurgence. All of the stud- ies on bed bugs thus far have found genetic diversity within human associated population to be low, resulting from a great deal of in- breeding (25, 30-32). Despite the importance of bed bugs in Iran, genetic evaluation has not yet been studied. Therefore, we aimed at this shortcoming and analyzed DNA se- quences of C. hemipterus gathered from dif- ferent parts of Iran, using partial sequences of cytochrome oxidase I gene (COI) and evaluated the genetic relationship between them. Materials and Methods Sample collection All procedures in this study were carried out in accordance with the guidelines of the Animal Ethics Committee of Faculty of Vet- erinary Medicine, Urmia University (AECVU) and supervised by authority of Sample col- lection Urmia University Research Council (UURC). http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 239–249 A Samiei et al.: The Phylogenetic Analysis of … 241 http://jad.tums.ac.ir Published Online: September 30, 2020 Geographically, there are 4 different zones in Iran, known as region 1: Caspian Sea (tem- perature: 8–26 °C, annual rainfall: 400–1,500 mm), region 2: Mountainous area (tempera- ture: -5–29 °C, annual rainfall: 200–500mm), region 3: Persian Gulf (temperature: 12.6–35 °C, annual rainfall: 200–300mm), and region 4: the Central Desert (temperature: -4–44 °C, rainfall: less than 100mm). The pattern of bed bug species distribution for the 4 different areas was determined according to the meth- od of Skerman and Hillard (35). Considering 10% prevalence, 95% confidence level and 5% error rate, 138 bed bugs collected and were used in this study. That way, adult bed bugs were collected from various locations such as infested hotels, residential houses and industrial buildings during May 2016 to Au- gust 2017, with the help of pest control com- panies. The following cities were selected from each region: Region 1: Sari and Rasht, region 2: Tehran, Isfahan, Urmia, Tabriz, Shi- raz, Saghez, Sanandaj, Kermanshah and Ha- madan, region 3: Ahvaz and Bandar Abbas, and region 4: Semnan. These locations were mapped by collecting the locality data via Google Earth (Fig. 1). Individual samples were collected by us- ing forceps then stored in a sample collec- tion bottle and preserved in 95% alcohol and stored at -20 °C until analysis onset. The in- sects were transferred to the laboratory of parasitology division, faculty of veterinary medicine, Urmia University and identified un- der a stereo microscope (Olympus SZ61, Olympus Corporation, Tokyo, Japan), using morphological keys described by Usinger (1) and Walpole (36). The pronotum which is the most distinguishing feature used to iden- tify the two bedbugs species. The pronotum of C. lectularius is wider than that of C. he- mipterus because of an upturned lateral flange on the margin of the pronotum on the thorax of C. lectularius which is absent in C. hemipterus (1). The dorsal and ventral sides of the Bedbug pronotum were observed to get a distinct image of the pronotum because the projecting edge of some were not very clear dorsally. DNA extraction Genomic DNA from Individual tropical bed bug of each collection site was extracted using DNA isolation kit, MBST (Molecular and Biological system transfer, Tehran, Iran) following the manufacturer’s instructions. For this, samples were grinded by pestle and placed into 1.5ml micro centrifuge tube and total DNA was eluted in 100µl of elution buffer. DNA quality and concentration from each specimen was determined spectrophoto- metrically using the NanoDrop (Thermo Sci- entific 2000c, United States) and stored in - 20 °C for further procedures. We control the contaminations by check the 260/230 ratio be- cause a poor ratio may be the result of a con- taminant absorbing at 230nm or less. Also we check the wavelength of the trough in the spectra; this should be at 230nm. Amplification of the mitochondrial COI and sequencing For PCR amplification of the COI, the 658bp amplicon, the forward and the reverse primers LEP-F (5′-ATT CAA CCA ATC ATA AAG ATA TNG G- 3′) and LEP-R (5′- TAW ACT TCW GGR TGTCCR AAR AAT CA-3′) were used (32). The PCR was con- ducted in 25µl total volume, each containing 2.5µl 10x PCR buffer, 2μL 50mM MgCl2, 0.5µl dNTPs, 3µl DNA template, 10 pico- mole forward and reverse primers (0.5µl for each), 0.5µl Taq DNA polymerase (Sinaclon, Iran) and 15.5µl ddH2O. The PCR cycling conditions set in the program were as fol- lows: initial denaturation at 94 °C for 5min followed by 35 cycles of 94 °C for 30sec (denaturation), 42 °C for 30sec (annealing), 72 °C for 45sec (extension) and a final ex- tension step of 72 °C for 2min. PCR products were analyzed by electrophoresis in 1.5% agarose gel to confirm that the samples con- http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 239–249 A Samiei et al.: The Phylogenetic Analysis of … 242 http://jad.tums.ac.ir Published Online: September 30, 2020 tained a single band. Then, stained with safe DNA stain gel and visualized with UV-Tran- silluminator (BTS-20M, Japan). Finally, Pu- rified PCR products were sent to SinaClon Company (Tehran, Iran) for sequencing. Nucleotide Diversity and Phylogenetic Tree Construction The nucleotide sequences of each species from various regions were aligned for varia- tion positions. Sequences were uploaded on NCBI to search for the most similar reference sequences, and positions of the COI were determined with the help of BLAST, availa- ble at NCBI. A total of eleven COI sequenc- es belong to C. hemipterus available in the Gene Bank were used to phylogenetic analy- sis, including 3, 3, 2, 1, 1 and 1 sequence re- lated to Malaysia, Bangladesh, Thailand, Czech Republic, USA and Iran, respectively. The Triatoma dimidiata (Hemiptera: Reduviidae), accession number JQ575031, was used as an out group. The alignment was manually ed- ited to remove any alignment errors using the aligning tool Clustal W (37) and exported as MEGA and FASTA format files. All the ob- tained nucleotide sequences were deposited in the GenBank with the assigned accession numbers (Table 1). Subsequently, phyloge- netic relationship was examined and con- structed by Maximum-likelihood method (ML) using the Molecular Evolutionary Genetics Analysis (MEGA), version 6.0. The reliabil- ity of an inferred tree was tested by 1000 bootstrap. The DNA sequence polymorphism analyses for determining nucleotide diversity were estimated using BioEdit Version 7, 0, 1 and Blastn software (38). Results The average size of COI fragment ob- tained from the amplified C. hemipterus was found to be 655bp which was at the expected PCR product size (approximate length 658 bp) for the all 15 examined samples, within the range of 576 to 697bp. The accepted COI sequences found in NCBI GenBank database showed that the percentage identity ranged from 97 to 100. Nucleotide sequences of COI obtained from this study were submitted to NCBI GenBank and then accession num- bers of MG770888 to MG77089, MG739319 to MG 739326, MG737714 and MG696803 were assigned to them (Table 1). After some processing, for example, de- leting and aligning sequences using Mega 6 Molecular Software, 362bp of partial COI from 15 sequences of C. hemipterus were ob- tained successfully. Construction of phylo- genetic tree is done based on mitochondrial COI sequencing by maximum likelihood (ML) method. The sequences obtained from the present study were compared with sequences of C. hemipterus and C. lectularius from dif- ferent parts of the world. As the tree shows, the samples were classified in three major clusters which confirm the genetic variation among different species of Cimex. All our isolates and those of other parts of the world were placed in one clustered together (G1) showing no significant difference between various regions despite the minor nucleotide variations. Consistently, they were far from C. lec- tularis and T. dimidiata clusters (G2 and G3, respectively). Our isolates were further clus- tered into three subgroups (Fig. 2). The first one (SG1) contained 9 isolates which are shown in the phylogenetic tree in Fig. 2. Se- quences from this subgroup clustered togeth- er with the C. hemipterus reference sequenc- es containing three nucleotide sequences from Bangladesh (MG552132, MG572242, MG 587917), three nucleotide sequences from Malaysia (KT851503 to KT851505), two nucleotide sequences from Thailand (JX 826469, JX826470), two nucleotide sequenc- es from Czech Republic (KF018754, GU 985538), one nucleotide sequences from Iran (KY560443) and one nucleotide sequences http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 239–249 A Samiei et al.: The Phylogenetic Analysis of … 243 http://jad.tums.ac.ir Published Online: September 30, 2020 from USA (JQ782821). The second one (SG2) contained two isolates collected from Ker- manshah (MG770888) and Esfahan (MG 739326). These sequences showed a signifi- cant nucleotide similarity with each other and were distinct from the both subgroups SG1 and SG3, and there is no reference sequence in GenBank that corresponds to this subgroup. Our third subgroup (SG3) contained four iso- lates collected from Hamedan (MG739319), Bandar Abbas (MG739324), Tehran north (MG739321) and Semnan (MG739325). Anal- ysis of these sequences showed a significant nucleotide similarity between Hamedan and Bandar Abbas and also between Tehran north and Semnan. This phylogenetic tree is also supported by a mean pair-wise distance that is calculat- ed at 0.005, suggesting that all of the C. he- mipterus populations studied are clustered together, showing no significant variance be- tween different regions despite minor nucle- otide variations, on average between 5 and 10 Single nucleotide polymorphisms (SNPs). Fig. 1. Map of Iran showing study locations of Cimex hemipterus collected for the present study. Abbreviations are listed in Table 1 Table 1. Collection site, isolated code, collection date and accession number of each bed bug in this study Isolated code Isolation source State Collection date Length /bp Longitude and Latitude Genbank Accession number Hm Hamadan Hamedan 20 June 2016 585bp 34.7989° N, 48.5150° E MG739319 Az Ahvaz Khozestan 25 June 2016 577bp 31.3183° N, 48.6706° E MG739320 Tn Tehran north Tehran 15 May 2016 608bp 35.6892° N, 51.3890° E MG739321 Ra Rasht Gilan 28 June 2017 669bp 37.2682° N, 49.5891° E MG739322 Sr Sari Mazandaran 10 June 2017 706bp 36.5659° N, 53.0586° E MG739323 http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 239–249 A Samiei et al.: The Phylogenetic Analysis of … 244 http://jad.tums.ac.ir Published Online: September 30, 2020 Ba Bandar abbas Hormozgan 23 July 2017 683bp 27.1832° N, 56.2666° E MG739324 Sm Semnan Semnan 20 July 2016 683bp 35.2256° N, 54.4342° E MG739325 Is Isfahan Isfahan 15 July 2017 734bp 32.6546° N, 51.6680° E MG739326 Kr Kermanshah Kermanshah 14 August 2016 669bp 34.3277° N, 47.0778° E MG770888 Sn Sanandaj Kordistan 10 August 2017 721bp 35.3219° N, 46.9862° E MG770889 Shz Shiraz Fars 23 May 2016 717bp 29.5918° N, 52.5837° E MG770890 Ur Urmia West Azerbaijan 22 May 2016 684bp 37.5498° N, 45.0786° E MG770891 Tb Tabriz East Azerbaijan 19 August 2017 722bp 38.0962° N, 46.2738° E MG770892 Ts Tehran south Tehran 17 May 2017 587bp 35.6892° N, 51.3890° E MG737714 Sz Saghez Kordistan 16 May 2017 705bp 36.2389° N, 46.2780° E MG696803 Fig. 2. Maximum likelihood (ML) tree inferred from sequences of the mitochondrial COI gene for 15 Cimex hemip- terus populations collected in Iran (*sign) and one outgroup (Triatoma dimidiata accession no. JQ575031.), Numbers at nodes indicate bootstrap values (%) of ML replicates, obtained by 1,000 replications Table 1: Continued … http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 239–249 A Samiei et al.: The Phylogenetic Analysis of … 245 http://jad.tums.ac.ir Published Online: September 30, 2020 Discussion There is a paucity of data and analysis re- garding phylogenetic relationships below in- fraorder Cimicomorpha (39, 40). The proposed evolutionary relationships of the taxa within superfamily Cimicoidea and the family Cimi- cidae are based on morphological characters, chromosome numbers, and host associations (1, 32). Previous studies have associated the Cimicidae with other families within the su- perfamily Cimicoidea using both morpholog- ical and molecular characters (39, 41). A to- tal evidence analysis using 16S, 18S, 28S and COI DNA sequence data and 73 morpho- logical characters (39) has determined 13 in- fraorder Cimicomorpha and superfamily Ci- micoidea are both monophyletic. The same study reported that the families Cimicidae, Polyctenidae and Curaliidae form a mono- phyletic clade (39). In this work, we studied the phylogeny of C. hemipterus populations come from dif- ferent regions of Iran, using the mitochon- drial COI gene sequences. The COI gene is a part of the mitochondrial DNA genes and has been used as a potent marker for molecu- lar phylogenetic studies, because it is species specific and appropriate for analyses of intra specific variations. The rate of evolvement in the COI gene is also relatively rapid which allow distinction at the species level and the identification of obscure species (42, 43). De- spite the vast outbreak of bedbug in various rural and urban regions of Iran, there are very limited and unsatisfactory reports about the prevalence (17, 20). Probably due to re- surgence and propagation of bed bugs in other countries, the population also has in- creased in Iran (44, 45). Limited public awa- reness, increase in internal and international travels, increase in utilization of second-hand furniture and resistance to pesticides may contribute to this resurgence (46, 47). There- fore, the resurgence and subsequent problems inspired us to perform phylogenetic analysis, as well as study genetic diversity and popu- lation dynamics. The perceived near extirpation of bed bugs from many areas around the world suggests a genetic bottleneck would have occurred, which would be reflected in low genetic di- versity across current bed bug populations (25, 30-34). However, all of the studies com- pleted thus far have found a relatively high genetic diversity between populations in dif- ferent locations. Such high diversity across populations is atypical for species that have undergone a recent and single founder event (30). Szalanski et al. (30) focused on genetic variation among various bed bug populations in USA, Canada and Australia. They exam- ined a partial sequence of the mitochondrial (mt) 16S rRNA gene and nuclear rRNA ITS- 1 region of 136 adult bed bugs sampled from 22 populations and found a relatively high genetic diversity in the 16S gene, and low diversity in the ITS-1 gene. The current research is the first report of phylogenetic and genetic species diversity analysis conducted on C. hemipterus in Iran. Our analysis showed one main cluster in phy- logenetic tree. Therefore, from the present study it can be concluded that C. hemipterus in Iran is an arthropod with low genetic di- versity with a potentially high capability for raise the levels of inbreeding. The previous study, done by Booth et al. (25) regarding molecular markers of bed bug infestation dy- namics within apartment buildings supported the same pattern of genetic diversity in C. lectularius as they reveal restricted genetic diversity. Similar researches also have con- ducted in other countries. Seri-Masran and Majid (45) studied genetic diversity of bed bugs in Malaysia. They considered 22 se- lected infested structures and consistent to our findings, they observed one main mono- phyletic clade. Another study was performed in Thailand and one main cluster of C. he- http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 239–249 A Samiei et al.: The Phylogenetic Analysis of … 246 http://jad.tums.ac.ir Published Online: September 30, 2020 mipterus was obtained when it was com- pared with C. lectularis isolates. Both of the aforementioned studies support our results. Given that the current phylogenetic and taxonomic relationships within the family Cimicidae are based on host relationships and morphology, it is possible that the inclu- sion of molecular data could cause a restruc- turing of the systematic relationships (1, 32). In addition to COI genes, the DNA sequenc- es of the entire mitochondrial genome of C. hemipterus may be useful for population ge- netics studies. There are several reports in the country using molecular methods for spe- cies identification of insects (48-58). Conclusion These results provided basic information for further studies of molecular epidemiolo- gy and control of C. hemipterus infestation to the public, medical association, entomol- ogists and pest control operators in Iran. Acknowledgements The authors gratefully thank Iranian Pest Control Companies from all states in collab- oration in the bed bugs sample collection. We also grateful to Dr A Nazarizadeh and Dr M Soltani Eini for their fruitful assistance in manuscript writing and helpful advice. References 1. Usinger RL (1966) Monograph of Cimici- dae (Hemiptera, Heteroptera). Ento- mological Society of America, College Park, Maryland, p. 582. 2. Karunaratne S, Damayanthi B, Fareena M, Fareena MHJ, Imbuldeniya V, Heming- way J (2007) Insecticide resistance in the tropical bedbug Cimex hemipterus. Pest Biochem Physiol. 88(1): 102–107. 3. Cooper R, Harlan H (2004) Ectoparasites, part 3: bed bugs and kissing bug. Mal- lis’ handbook of pest control, Cleve- land. 2004(1): 494–529. 4. Goddard J, de-Shazo R (2009) Bed bugs (Cimex lectularius) and clinical conse- quences of their bites. Jama. 301(13): 1358–1366. 5. Wang C, Tsai WT, Cooper R, White J (2011) Effectiveness of bed bug moni- tors for detecting and trapping bed bugs in apartments. J Econ Entomol. 104(1): 274–278. 6. Doggett SL, Dwyer DE, Peñas PF, Russell RC (2012) Bed bugs: clinical relevance and control options. Clin Microbiol Rev. 25(1): 164–192. 7. Elston D, Stockwell S (2000) What's eat- ing you? Bedbugs. Cutis. 65(5): 262– 264. 8. Paul J, Bates J (2000) Is infestation with the common bedbug increasing? BMJ. 320(7242): 1141. 9. Masetti M, Bruschi F (2007) Bedbug in- festations recorded in Central Italy. Parasitol Int. 56(1): 81–83. 10. Reinhardt K, Siva-Jothy MT (2007) Bi- ology of the bed bugs (Cimicidae). Annu Rev Entomol. 52(1): 351–374. 11. Hwang SW, Svoboda TJ, De-Jong IJ, Kabasele KJ, Gogosis E (2005) Bed bug infestations in an urban environ- ment. Emerg Infect Dis. 11(4): 533– 538. 12. Cooper R (2006) Bed bugs--still more questions than answers: A need for re- search and public awareness. Am En- tomol. 52(2): 111–112. 13. Romero A, Potter MF, Potter DA, Haynes KF (2007) Insecticide resistance in the bed bug: a factor in the pest’s sudden resurgence? J Med Entomol. 44(2): 175– 178. 14. Doggett SL, Russell RC (2008) The re- surgence of bed bugs, Cimex spp. (He- miptera: Cimicidae) in Australia. Int Conf http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 239–249 A Samiei et al.: The Phylogenetic Analysis of … 247 http://jad.tums.ac.ir Published Online: September 30, 2020 Urban Pests (ICUP). 6(1): 407–425. 15. Kilpinen O, Jensen KMV, Kristensen M (2008) Bed bug problems in Denmark, with a European perspective. Int Conf Urban Pests (ICUP). 1316(1): 395–399. 16. Wang L, Xu Y, Zeng L (2013) Resur- gence of bed bugs (Hemiptera: Cimici- dae) in mainland China. Fla Entomol. 96(1): 131–136. 17. Haghi SFM, Behbodi M, Hajati H, Shafaroudi MM (2014) Prevalence of bed bug (Cimex lectularius) in human settlement area of Bahnamir, Iran. Asian Pac J Trop Dis. 4(2): 786–789. 18. Dehghani R, Asadi MA, Ahmadi M (1998) prevalence survey of bed bug infestation in residential homes in the villages of western Kashan. Feyz Sci J. 14(1): 71–76 19. Shahraki GH, Rayegan A, Fararouei M (2000) Prevalence of bed bug and its biting in student dormitories. J Yasuj Univers Med Sci. 5(19): 25–33 20. Dehghani R, Hashemi A, Takhtfiroozeh S, Chimehi E (2015) Bed bug (Cimex lectularis) outbreak: A cross-sectional study in Polour, Iran. Iran J Dermatol. 19(1): 16–20. 21. Tabachnick WJt, Black IW (1995) Mak- ing a case for molecular population ge- netic studies of arthropod vectors. Par- asitol Today. 11(1): 27–30. 22. Kress WJ, Erickson DL (2008) DNA barcodes: genes, genomics, and bioin- formatics. Proc Natl Acad Sci USA. 105(8): 2761–2762. 23. Endersby NM, Hoffman AA, White VL, Lowenstein S, Ritchie S, Johnson EH, Rapley LP, Ryan PA, Nam VS, Yen NT, Kittiyapong P, Weeks AR (2009) Genetic structure of Aedes aegypti in Australia and Vietnam revealed by microsatellite and exon primed intron crossing markers suggests feasibility of local control options. J Med Entomol. 46(5): 1074–1083. 24. Crissman JRW, Booth RG, Santangelo DM, Mukha EL, Vargo EL, Schal C (2010) Population genetic structure of the German cockroach (Blattodea: Blat- tellidae) in apartment buildings. J Med Entomol. 47(4): 553–564. 25. Booth W, Saenz VL, Santangelo RG, Wang C, Schal C, Vargo EL (2012) Mo- lecular markers reveal infestation dy- namics of the bed bug (Hemiptera: Cimi- cidae) within apartment buildings. J Med Entomol. 49(3): 535–546. 26. Pizarro JC, Gilligan LM, Stevens L (2008) Microsatellites reveal a high population structure in Triatoma infestans from Chu- quisaca, Bolivia. PLoS Negl Trop Dis. 2(3): e202. 27. Paup C, Brengues C, Kamgang B, Herve JP, Fontenille D, Simard F (2008) Gene ßow between domestic and sylvan pop- ulations of Aedes aegypti (Diptera: Cu- licidae) in North Cameroon. J Med En- tomol. 45(3): 391–400. 28. Perez de Rosas AR, Segura EL, Garcia BA (2007) Microsatellite analysis of ge- netic structure in natural Triatoma in- festans (Hemiptera: Reduviidae) pop- ulations from Argentina: its implication in assessing the effectiveness of Cha- gas disease vector control programmes. Mol Ecol. 16(7): 1401–1412. 29. Yoon KS, Kwon DH, Strycharz JP, Hol- lingsworth CS, Lee SH, Clark JM (2008) Biochemical and molecular analysis of deltamethrin resistance in the common bed bug (Hemiptera:Cimicidae). J Med Entomol. 45(6): 1092–1101. 30. Szalanski AL, Austin JW, McKern JA, Steelman CD, Gold RE (2008) Mito- chondrial and ribosomal internal tran- scribed spacer 1 diversity of Cimex lec- tularius (Hemiptera: Cimicidae). J Med Entomol. 45(2): 229–236. 31. Vargo EL, Booth W, Saenz V, Santange- lo RG, Schal C (2011) Genetic analysis of bed bug infestations and popula- http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 239–249 A Samiei et al.: The Phylogenetic Analysis of … 248 http://jad.tums.ac.ir Published Online: September 30, 2020 tions. Proceedings of the Seventh In- ternational Conference on Urban Pests. 32. Balvín O, Munclinger P, Kratochvíl L, Vilímová J (2012) Mitochondrial DNA and morphology show independent evolutionary histories of bedbug Cimex lectularius (Heteroptera: Cimicidae) on bats and humans. Parasitol Res. 111(1): 457–469. 33. Davies TGE, Field LM, Williamson MS (2012) The re-emergence of the bed bug as a nuisance pest: implications of resistance to the pyrethroid insecticides. Med Vet Entomol. 26(3): 241–254. 34. Booth W, Balvin O, Vargo EL, Vilímová J, Schal C (2015) Host association drives genetic divergence in the bed bug, Ci- mex lectularius. Mol Ecol. 24(5): 980– 992. 35. Skerman K, Hillard J (1967) A handbook for studies of helminth parasites of ru- minants. FAO, Near East Anim Health Inst. 2(1): 1–3. 36. Walpole D (1987) External morphology of the legs of two species of bed bugs (Hemiptera: Cimicidae). J Entomol Soc South Afr. 50(1): 193–201. 37. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flex- ible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25(24): 4876–4882. 38. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic acids symposium series, [London]: Information Retrieval Ltd., c1979-c2000. 41(1): 95–98. 39. Schuh RT, Weirauch C, Wheeler WC (2009) Phylogenetic relationships with- in the Cimicomorpha (Hemiptera: Het- eroptera): a total evidence analysis. Sys Entomol. 34(1): 15–48. 40. Kocher A, Guilbert E, Lhuillier E, Muri- enne J (2015) Sequencing of the mito- chondrial genome of the avocado lace bug Pseudacysta perseae (Heteroptera, Tingidae) using a genome skimming approach. C R Biol. 338(3): 149–160. 41. Cui Y, Xie Q, Hua J, Dang K, Zhou J, Liu X, Wang G, Yu X, Bu W (2013) Phylogenomics of Hemiptera (Insecta: Paraneoptera) based on mitochondrial genomes. Sys Entomol. 38(1): 233–245. 42. Hebert PD, Penton EH, Burns JM, Jan- zen DH, Hallwachs W (2004) Ten spe- cies in one: DNA barcoding reveals cryptic species in the neotropical skip- per butterfly Astraptes fulgerator. Proc Natl Acad Sci USA. 101(41): 14812– 14817. 43. Ballard JWO, Rand DM (2005) The pop- ulation biology of mitochondrial DNA and its phylogenetic implications. Annu Rev Ecol Evol Syst. 36(1): 621–642. 44. Tawatsin A, Lorlertthum K, Phumee A, Thavara U, Boon-Long J, Boonserm R, Siriyasatien P (2013) Discrimination be- tween tropical bed bug Cimex hemip- terus and common bed bug Cimex lec- tularius (Hemiptera: Cimicidae) by PCR- RFLP. Thai J Vet Med. 43(3): 421–427. 45. Seri Masran SNA, Ab-Majid AH (2017) Genetic diversity and phylogenetic re- lationships of Cytochrome C Oxidase Subunit I in Cimex hemipterus (Hemip- tera: Cimicidae) populations in Malay- sia. J Med Entomol. 54(4): 974–979. 46. Delaunay P, Blanc V, Del Giudice P, Levy-Bencheton A, Chosidow O, Marty P, Brouqui P (2011) Bedbugs and in- fectious diseases. Clin Infect Dis. 52(2): 200–210. 47. Ryan ET, Wilson ME, Kain KC (2002) Illness after international travel. New Engl J Med. 347: 505–516. 48. Naddaf SR, Oshaghi MA, Vatandoost H, Assmar M (2003) Molecular character- ization of Anopheles fluviatilis species complex in the Islamic Republic of Iran. East Mediterr Health J. 9 (3): 257–265. http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ J Arthropod-Borne Dis, September 2020, 14(3): 239–249 A Samiei et al.: The Phylogenetic Analysis of … 249 http://jad.tums.ac.ir Published Online: September 30, 2020 49. Sedaghat MM, Vonne-marie Y, Nicoles- cuz G, Koliopoulos G, Athanassios K, Zounos K, Oshaghi MA, Vatandoost H, Harbach R (2003) Morphological and molecular characterization of Anopheles (Anopheles) sacharovi Favre, a primary vector of malaria in the Middle East. Syst Entomol. 28: 241–256. 50. Barghamadi Z, Moosa-kazemi SH, Oshaghi MA, Vatandoost H, Shahraki G (2016) species identification of the Anopheles fluviatilis complex using phylogene-tic analysis PCR-sequencing in southwest- ern Iran. Acta Med Mediterr. 32: 2069. 51. Sedaghat MM, Linton YM, Oshaghi MA, Vatandoost H, Harbach RE (2003) The Anopheles maculipennis complex (Dip- tera: Culicidae) in Iran: molecular char- acterization and recognition of a new species. Bull Entomol Res. 93(6): 527– 535. 52. Khoshdel-Nezamiha F, Vatandoost H, Oshaghi MA, Azari-Hamidian S, Mi- anroodi RA, Dabiri F (2016) Molecular characterization of mosquitoes (Diptera: Culicidae) in Northwestern Iran by using rDNA-ITS2. Jpn J Infect Dis. 69(4): 319–322. 53. Oshaghi MA, Sedaghat MM, Vatandoost H (2003) Molecular characterization of the Anopheles maculipennis complex in the Islamic Republic of Iran. East Med- iterr Health J. 9(4): 659–566. 54. Karimian F, Ali Oshaghi MA, Sedaghat MM, Waterhouse ME, Vatandoost H, Hanafi-Bojd AA, Maleki Ravasan N, Chavshin AR (2014) Phylogenetic anal- ysis of the oriental-palearctic-afrotropi- cal members of Anopheles (Culicidae: Diptera) Based on Nuclear rDNA and Mitochondrial DNA Characteristics. Jpn J Infect Dis. 67: 361–367. 55. Oshaghi MA, Yaghoobi F, Vatandoost H, Abai MR, Akbarzadeh K (2006) Anopheles stephensi biological forms: Geographical distribution and malaria transmission in malarious regions of Iran. Pak Bio Sci. 9 (2): 294–298. 56. Chavshin AR, Oshaghi MA, Vatandoost H, Hanafi-Bojd AA, Raeisi A, Nikpoor F (2014) Molecular characterization, bi- ological forms and sporozoite rate of Anopheles stephensi in southern Iran. Asian Pac J Trop Biomed. 4(1): 47–51. 57. Mehravaran A, Oshaghi MA, Vatandoost H, Abai MR, Ebrahimzadeh A, Moazeni Roodib A, Gorouhi A (2011) First re- port on Anopheles fluviatilis U in south- eastern Iran. Acta Trop. 117(2): 76–81. 58. Oshaghi MA, Shemshad KH, Yaghobi- Ershadi MR, Pedram M, Vatandoost H, Abai MR, Akbarzadeh K, Mohtarami F (2007) Genetic structure of the malaria vector Anopheles superpictus in Iran using mitochondrial cytochrome oxi- dase (COI and COII) and morphologic markers: A new species complex? Ac- ta Trop. 101(3): 241–248. http://jad.tums.ac.ir/ http://jad.tums.ac.ir/ javascript:void(0) javascript:void(0) javascript:void(0) javascript:void(0) javascript:void(0) https://www.ncbi.nlm.nih.gov/pubmed/?term=Vatandoost%20H%5BAuthor%5D&cauthor=true&cauthor_uid=26743141 https://www.ncbi.nlm.nih.gov/pubmed/?term=Oshaghi%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=26743141 https://www.ncbi.nlm.nih.gov/pubmed/?term=Azari-Hamidian%20S%5BAuthor%5D&cauthor=true&cauthor_uid=26743141 https://www.ncbi.nlm.nih.gov/pubmed/?term=Mianroodi%20RA%5BAuthor%5D&cauthor=true&cauthor_uid=26743141 https://www.ncbi.nlm.nih.gov/pubmed/?term=Mianroodi%20RA%5BAuthor%5D&cauthor=true&cauthor_uid=26743141 https://www.ncbi.nlm.nih.gov/pubmed/?term=Dabiri%20F%5BAuthor%5D&cauthor=true&cauthor_uid=26743141 https://www.ncbi.nlm.nih.gov/pubmed/26743141 https://www.ncbi.nlm.nih.gov/pubmed/?term=Oshaghi%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=24144130 https://www.ncbi.nlm.nih.gov/pubmed/?term=Vatandoost%20H%5BAuthor%5D&cauthor=true&cauthor_uid=24144130 https://www.ncbi.nlm.nih.gov/pubmed/?term=Hanafi-Bojd%20AA%5BAuthor%5D&cauthor=true&cauthor_uid=24144130 https://www.ncbi.nlm.nih.gov/pubmed/?term=Raeisi%20A%5BAuthor%5D&cauthor=true&cauthor_uid=24144130 https://www.ncbi.nlm.nih.gov/pubmed/?term=Nikpoor%20F%5BAuthor%5D&cauthor=true&cauthor_uid=24144130 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3819495/