J Arthropod-Borne Dis, March 2023, 17(1): 94–104 A Heydari et al.: Molecular Characterization of … 94 http://jad.tums.ac.ir Published Online: March 31, 2023 Original Article Molecular Characterization of Paederus Spp (Coleoptera: Staphylinidae, Paederinae) the Agent of Human Linear Dermatitis in the Caspian Sea Coast, North of Iran Abbas Heydari1, Sinan Anlaş2, Hasan Bakhshi3, Mona Koosha3, Nayyereh Choubdar3, Somayeh Panahi-Moghadam3, *Mohammad Ali Oshaghi3 1Department of Entomology, Garmsar Branch, Islamic Azad University, Garmsar, Iran 2Celal Bayar University, Alaşehir Vocational School, Department of Entomology, Alaşehir, Manisa, Turkey 3Department of Vector Biology and Control of Diseases, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran *Corresponding author: Dr Mohammad Ali Oshaghi, E-mail: moshaghi@sina.tums.ac.ir, oshaghima@yahoo.com (Received 06 Jan 2023; accepted 29 Mar 2023) Abstract Background: A combined morphological and molecular survey was performed to determine the agent of human linear dermatitis Paederus Fabricius, 1775 (Coleoptera: Staphylinidae, Paederinae) species composition in Mazandaran Province in the Caspian Sea coast in northern Iran, where most of linear dermatitis cases of the country occurred. Methods: Altogether, 397 Paederus specimens were collected from May to August 2021 and classified using morpho- logical characters and ITS2-rDNA sequence analysis. Results: Morphological investigation revealed that all the specimens were Paederus fuscipes. ITS2 polymerase chain reaction (PCR) direct-sequences and the profiles of restriction fragment length polymorphism (RFLP) derived from digestion of PCR products by HinfI, HpaII, and SalI enzymes were identical confirming the morphological results, im- plying that all specimens belonged to a single taxon. Conclusion: Paederus fuscipes (Fabricius, 1775) is considered the dominant taxon and responsible for linear dermatitis in Mazandaran Province. To our knowledge, we have provided the first molecular typing of Paederus beetles at the spe- cies level, suggesting that ITS2-rDNA characterization is an alternative tool for species discrimination of Paederus spp. Keywords: Paederus fuscipes; Rove beetle; Linear dermatitis; ITS2-rDNA; Iran Introduction Rove beetle (Staphylinidae) is the prime family of Coleoptera order with more than 6.000 species (1, 2). Paederus Fabricius, 1775, is cat- egorized into the tribe Paederini and subfam- ily Paederinae, presently encompasses around 490 species (3). According to Herman (2001), Paederinae subfamily is the third largest sub- family within the Staphylinidae family, and it is represented about 6.000 species (4). In the Palaearctic region, the genus Paederus is in- cluded 85 species and subspecies. Some Rove beetle are scavenger (5) or predator and act as biological agents in control of pests (6). Paederus beetles are nocturnal and attract- ed by incandescent and fluorescent lights. As Paederus species turn towards particularly flu- orescent light, they encounter with people. These insects that can enter homes at nights from an open window or underneath of door, excrete toxic substance in their hemolymph when they are disturbed or rushed. The hemolymph of some species within the Paederus has long been recognized to be an ir- ritation as, once released, it leads to human lin- ear dermatitis and conjunctivitis (7). The signs are as a result of a toxic amide substance, named Copyright © 2023 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/ mailto:moshaghi@sina.tums.ac.ir mailto:oshaghima@yahoo.com https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, March 2023, 17(1): 94–104 A Heydari et al.: Molecular Characterization of … 95 http://jad.tums.ac.ir Published Online: March 31, 2023 Pederin (C25 H45 O9 N, LD50: 0.14mg/kg rat i.p.) (8) and makes up roughly 0.025% of an adult P. fuscipes weight. After pederin touch- es the skin, this substance to be proportional to its amount may cause important results from a simple rash to large wounds. At least 20 spe- cies of Paederus beetles have been associated with linear dermatitis (9). Along with causing rigorous skin lesions, their ordinary substruc- ture accounts for similar antitumor and antiviral activities, cytotoxicity and interruption of DNA metabolism which are primarily based on ob- struction of eukaryotic protein biosynthesis (10– 13). At the same time owing to have the abil- ity of inhibiting protein and DNA synthesis and being a very strong toxic substance, peder- in has been habited in tumor studies in current years. The secretion or production of pederin depends on the activities of a Paederus bacte- rial endosymbiont (Pseudomonas sp.) (14–18). The production of pederin is mainly limited to adult female beetles which protects the beetles against predators (14, 15, 19). Males and lar- vae only accumulate pederin gained maternal- ly (i.e., through eggs) or by ingestion (13). Systematics of Paederus species is rather problematic and is relied on the male main and secondary sexual morphological characters (20). This has ended a very complex history for Pae- derus taxonomy and has reformed it intensely (21) and some species are regarded as syno- nyms of each other and or lowered to a single subspecies/species (22 and references herein). Recent advances in molecular methods have provided an extensive range of molecular mark- ers for taxonomy and systematics of insects (23–30). The ribosomal DNA (rDNA) of insect genome has many advantages comprising known PCR primers, easy to use, a high interspecies mutation rate, and low intraspecies mutation rate due to homogenization has acknowledged par- ticular attention for taxonomic and systematic studies of insects (24, 31–39). Earlier studies on the ecology, geographical distribution, medical importance, and fauna of Paederus beetles in Iran showed presence of fourteen Paederus subspecies or species in the country, in which six species P. littoralis ilsae, P. fuscipes, P. balachowskyi, P. balcanicus, P. riparius, and P. duplex are reported in Caspi- an Sea coast (Mazandaran, Guilan, and Goles- tan Provinces) (22). However, due to prob- lems in Paederus species identification, vari- ous lists of species have been reported for a same region. In north of country, for example, three species of P. pietschmanni (synonym of P. mesopotamicus), P. fuscipes, and P. specta- bilis have been reported by Janbaksh and Ar- dalan (1977) in Mazandaran Province at the Caspian Sea shore (40). Later, Majidi-Shad et al. (1989) registered three species of P. ripar- ius, P. littoralis and P. fuscipes, from the same region (41). Afterwards, P. balcanicus, P. fusci- pes, and P. kalalovae were reported from the same region by Nikbakhtzadeh and Tirgari (2008) (42). Ultimately, three species of P. bala- chowskyi (synonym of P. mesopotamicus), P. balcanicus, and P. fuscipes, was reported from the same region by Nikbakhtzadeh et al. (2012) (22). This discrepancy and inconsistency stimu- lated us to test genetic variation of ITS2-rDNA, as a powerful molecular marker, of Paederus specimens collected from the province to un- ravel systematic status of the Paederus beetles in the area. Materials and Methods Study area and Paederus collection Mazandaran Province (MP) is 23,833km2, situated on the southern coast of the Caspian Sea; clockwise it is bounded by the, Tehran, Al- borz, Golestan Semnan, Qazvin, and Guilan Ps. City of Sari is the capital and the largest city of the province. The province is geographically par- titioned into two parts: the mountainous areas, and the coastal plains. The Alborz Mountain Range borders the coastal belt and plains of the Caspian Sea. There is often snowstorm through- out most of the seasons in the Alborz regions, which run correspondence to the Caspian Sea's southern coast, partitioning the province into http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 94–104 A Heydari et al.: Molecular Characterization of … 96 http://jad.tums.ac.ir Published Online: March 31, 2023 many remote valleys. The province includes 15 counties including Sari, Babol, Amol, Behshahr, Babolsar, Chaloos, Tonekabon, Ramsar, Juybar, Qaem Shahr, Savad Kooh, Neka,Mahmood Abad, Noshahr, and Noor (Fig. 1). Adult Paederus samples were collected by mouth aspirator on various vegetation (partic- ularly rice) in early morning or afternoon and under soils at warm hours of daytime. In some areas, an ultraviolet black light was used for specimen collection from dusk to midnight at night as previously described by Nikbakhtza- deh and Tirgari (2008) (42). The collections were done mostly in rice farms in 33 sites belong to the 15 MP’s counties (Fig. 1, Table 1). The rice fields were selected randomly, and sampling was conducted throughout the growth time from May to August 2011. The captured specimens were kept in 70% ethanol till morphological and mo- lecular identification. Morphological identification The pictorial keys of Coiffait (1982) were used to determine the specimens to genus lev- el (20) and then we send them to the specialist (Dr Sinan Anlaş laboratory) in Turkey for spe- cies identification. The specimens were iden- tified on the base of habitus and sound struc- ture of male basal and secondary sexual char- acters (Fig. 2). DNA extraction A subset (n=66) of Paederus specimens’ representative of the populations and counties, were chosen for DNA analysis. Total body of individual samples was used for genomic DNA extraction using DNeasy® Blood and Tissue Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Preceding to DNA extraction, the specimens were frozen and then pounded in the Kit supplied buffer and extraction followed based on the manufactur- er's directions. DNA amplification The primer pair ITS3f (5′-gcatcgatgaagaac- gcagc-3’) and ITS4r (5′- tcctccgcttattgatatgc-3’) was used to amplify ITS2 (Internal Transcribed Spacer number 2) region of rDNA gene of the specimens. The primer pair has already been proposed by White et al. (1990) and utilized for some Coleopteran (43). PCR amplification cock- tail included 5μl 10× PCR-Buffer, 120μM of each dNTPs, 50pmol of each primer, 2μl (around 100ng) of template DNA, and 2.5U of Taq polymerase (Sinaclon, Iran) in a 25μl reaction volume. PCR amplification was accomplished with an Eppendorf thermal cycler (Germany). The cycling program was: 3min denaturation at 94 °C followed by 35 cycles of 1min at 94 °C, 1min at 52 °C, and 2min at 72 °C, and a final 7min chain elongation. Well-characterized DNA samples and double distilled water (ddH2O) were used as positive and negative controls. Sequencing and PCR-RFLP A subset (n=17) of the specimen PCR prod- ucts were refined from gels by using a gel pu- rification kit and used for sequencing. An ABI 3730 sequencer machine by Bioneer (South Ko- rea) was used for sequencing the samples. The ambiguities of resultant sequences were veri- fied and their homologies with the accessible sequence data in GenBank was blasted by using basic local alignment search tool (BLAST) analy- sis software (www.ncbi.nlm.nih.gov/BLAST). To thrifty in time and cost of sequencing, the rest of specimens were tested by restriction enzyme fragment polymorphism (RFLP) assay. To do this, the ITS2 sequences attained in pre- sent study was tested to prepare its physical map and to choose restriction/digestion enzymes by using the Nebcutter program (44) (Fig. 3). Re- striction/digestion enzymes were selected ac- cording to their sites on the PCR product, costs, profiles, and obtainability in the marketplace. Di- gestion or cutting of PCR products was ac- complished in 25μL of a solution containing 15μL of PCR product associated with 2.5μL of enzyme buffers and 5 units of individual re- striction enzymes (HinfI, HpaII, and SalI) over- laid with two droplets of mineral oil. The blend was nursed at the temperature suggested by the http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 94–104 A Heydari et al.: Molecular Characterization of … 97 http://jad.tums.ac.ir Published Online: March 31, 2023 enzyme supplier. An aliquot part (14μL) of the restricted product was blended with 6μL of load- ing buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol), loaded on to a 2% agarose gel, and electrophoresed. Gels were contaminated with ethidium bromide (2mg/mL) and the RFLP outlines were envisioned under ultraviolet light. Results Species identification and PCR-RFLP During the study period, a total of 397 adult Paederus specimens were gathered by hands or black-light trap. Except one speci- men of Philonthous cf. sp which belongs to Staphylininae subfamily, (tribe Staphylinini, subtribe Philonthinina), all the specimens were classified morphologically as Paederus fusci- pes (Fig. 2). This species was the predominant taxon representing 99.75% (396) of the total number of collected rove beetles. A subsec- tion of the morphologically distinguished P. fuscipes were laid open to ITS2-rDNA PCR amplification. A 495bp fragment was amplified for all tested specimens. The ITS2 PCR prod- ucts of a total of 17 morphologically identi- fied P. fuscipes were selected based on their geographical origin for sequence characteriza- tion. The specimens were sequenced for both strands and the consensus data were submitted to GenBank database with GenBank identifi- cation numbers (IDs): KC414867- KC414883 (Table 1). The amplified fragment comprises 126bp of 3’ end of 5.8S ribosomal DNA gene, 312bp internal transcribed spacer number 2 (ITS2), and 57bp of 5’ end of 28S ribosomal DNA gene. The sequences were relatively high (58.4%) GC content. Among the sequences, there was no substitution or indel throughout the 495bp of the fragment indicating identical sequences. Afterward, a BLAST search on the sequences showed that there were some match- ing ITS2 sequences for P. fuscipes species in GenBank including a specimen from south of Iran (GenBank ID: KM086334) with 98.75% identity), and a specimen from China (Gen- Bank ID: MZ172410) with 96.34% identity. Also, there were some homologous sequences with 89.45% maximum identity belonged to P. mesopotamicus from Turkey (GenBank ID: KC414866) and with 85.18% maximum identity belonged to P. littoralis from south of Iran (GenBank ID: KM098054). Sequence analysis of P. fuscipes ITS2 frag- ment revealed appropriate restriction sites for HpaII (CC↓GG) at 146, SalI (G↓TCGAC) at 221, and HinfI (G↓ANTC) at 310 nucleotide positions (Fig. 3). Restriction digestion of the ITS2 PCR products gave two fragments of 310/185, 146/349, and 221/274bp for HinfI, HpaII, and SalI respectively for all the 396 morphologically identified P. fuscipes. Fig. 1. Map of the area study (Mazandaran Province) and the collection sites (stars) of Paederus specimens http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 94–104 A Heydari et al.: Molecular Characterization of … 98 http://jad.tums.ac.ir Published Online: March 31, 2023 Fig. 2. Details of Paederus fuscipes (A)- habitus; (B)- aedeagus in ventral view (C)- aedeagus in dorsal view (D)- aedeagus in lateral view. Scale bars: 1.0mm (A); 0.2 mm (Figs. B-D) Fig. 3. Physical map of 495bp fragment of ITS2-rDNA region of Paederus fuscipes. The HpaII, SalI, and HinfI re- striction sites at 146, 221, and 310 nucleotid positions are shown by arrows http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 94–104 A Heydari et al.: Molecular Characterization of … 99 http://jad.tums.ac.ir Published Online: March 31, 2023 Table 1. Details of morphologically identified Paederus fuscipes specimens collected in this study Location (township) Date 2011 Site No. Further Identifi- cation Method GenBank Ac- cession Number Mahmood-Abad May Zardab, Hezarpey-Shomali Abolhassan-Abad 11 12 Sequencing PCR-RFLP KC414874 June May Ahlmarestaghe-Shomali Ahlmarestaghe- Jonobi 11 12 PCR-RFLP PCR-RFLP KC414875 July Mirdeh-Olya, Daboye-Shomali 27 PCR-RFLP NA Amol May Bisheh-Mahaleh 13 Sequencing KC414873 June Roodbar 12 PCR-RFLP NA June Aghozin, Linkooh 10 PCR-RFLP NA July Osko-Mahaleh, Mirza KochakKhan Jungle, Linkooh 13 PCR-RFLP NA April Marzango 21 Sequencing KC414878 Ghaem-Shahr June Haji-Kola, Balanjan 9 PCR-RFLP NA June Ahangar-Kola, Noukandeh 14 Sequencing KC414872 Noor June Banafsheh, Mianrood 12 Sequencing KC414877 Babol June Markazi Kati Tayebi, Babolkenar 11 Sequencing KC414871 July Mansour-Kandeh, Feyziyeh 8 PCR-RFLP NA Babolsar June Sorkh-Dasht, Bahmanmir 17 PCR-RFLP NA Location (township) Date 2011 Site No. Further Identifi- cation Method GenBank Ac- cession Number Sari June Jafar-Abad, Banaft 15 PCR-RFLP NA July Taher-Abad, Roodpeye-Shomali 16 Sequencing KC414879 Fereydoon-Kenar June Shahrae-Daryasar, Barikrood 10 Sequencing KC414880 Juybar June Seraj-Mahaleh, Siahrood 11 PCR-RFLP NA Neka June Ghaleh-Sare-Olya, Peyrajeh 12 PCR-RFLP NA Behshahr June Abbas-Abad, Kouhestan 11 Sequencing KC414876 Gelogah June Mosayeb Mahaleh, Tooska, Spring 16 Sequencing KC414870 Shirgah June Kati-Lateh 14 PCR-RFLP NA Pool-Sefid July Pool-Sefid 10 Sequencing KC414881 Zirab July Zirab 12 PCR-RFLP NA Alasht July Larzaneh 11 Sequencing KC414882 Noushahr July Kajour, Khachak 9 PCR-RFLP NA Chalous July Namak Abrood, Kalarestake-Gharbi 8 Sequencing KC414869 Tonekaboun July Kelarabad, Yalbandan, 10 Sequencing KC414868 Ramsar July Janate-Roodbar, Grasma-Sar 8 Sequencing KC414883 Abbas-Abad July Kelar-Abad 11 PCR-RFLP NA Royan Aug. Noor 10 Sequencing KC414867 Discussion To our knowledge, this is the pioneer mo- lecular analysis at population level aiming at Identifying Paederus beetles in literature. Based on the results of present study, the Paederus spp population in Mazandaran Province in the Caspian Sea shore almost exclusively consists of P. fuscipes. In the other hand, previous studies (22, 41, 42) reported the presence of five more species including P. kalalovae, P. littoralis, P. balcanicus, P. riparius, and P. balachowskyi (synonym of P. mesopotamicus) in the prov- ince. This discrepancy may be due to the col- lection method we used in this study. The va- lidity of our findings requires a thorough pro- cess from design to using various sample col- lection methods. However, the present study http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2023, 17(1): 94–104 A Heydari et al.: Molecular Characterization of … 100 http://jad.tums.ac.ir Published Online: March 31, 2023 failed to consolidate presence of other five species in the province. Thus, P. fuscipes can be regarded as the main local causative agent of linear dermatitis. It is worth mentioning that P. kalalovae (one of the five species mentioned above) and P. fuscipes is treated as synonym spe- cies in the modern systematics of Staphylini- dae (45, 46). Paederus fuscipes has been found in north, centre and south of Iran (22, 40, 47‒54). It has also an extensive geographical dispersal globally in the old-world countries (20, 55, 56). Species classification of the rove beetles is mostly dependent on the examination of the gen- italia and demands a distinguished level of pro- ficiency particularly for doubtful species. This results in struggle to identify and confines their examination, management, and control. In cur- rent study the ITS2 of rDNA region was used for species identification of the rove beetle. This locus is a recognized powerful molecular in- dicator for species diagnostic assay. It is prov- en that inter-specific variation in this moder- ately fast evolving region is higher than intra- specific sequence variation (38, 57‒60). Further studies are now necessary to clari- fy the Paederus species composition of in the country using type specimens or morphologi- cally distinguished species. Nikbakhtzadeh et al. (2012) reported 14 subspecies and species of the genus Paederus, belonged to five sub- genera in the country (22). It is also suggested to examine sequence variation of ITS2 region or other genes such as mitochondrial DNA cy- tochrome oxidase subunit one (mtDNA COI), 28S, Topoisomerase I, and wingless, in Pae- derus spp to provide a molecular key for dis- tinguishing of the Iranian Paederus species. There is a substantial report on application of COI in species identification, systematics, and population genetics of beetles in the literature (32, 36, 57, 61, 62). There are a scarce existing COI sequence data of P. ruficollis (JX416589), P. littoralis (ANs: DQ155980; JX416591), P. fuscipes (DQ156010), and P. riparius (JX 416588) in Genbank database. Species identi- fication can be performed by using RFLP out- lines on PCR amplicons or developing species- specific primers to supply species-specific band/ s. Bazrafkan et al. (2016) demonstrated that mo- lecular typing using mtDNA COI gene followed by RFLP was useful to differentiate between two species P. littoralis (=syn: P. lenkoranus, P. ilsae) and P. fuscipes, and recommended PCR-RFLP of mtDNA COI for distinguishing of other Paederus species, which morphologi- cally are identical or very challenging to be distinguished (63). Conclusion The composition of the local Paederus population in Mazandaran Province in north of Iran consist typically of P. fuscipes and seems to be the main source of linear dermati- tis in the region. According to our estimation, this dermatitis is probably seen in almost eve- ry place of MP at Caspian Sea coast and in adjacent regions during hot and humid sum- mer months, it is confused with various der- matoses thus detailed research must be done. Due to difficulties and doubtful morphologi- cal identification of Paederus species, more detailed morphological and molecular systematic and faunal investigations need to be done in the future to determine the Paederus fauna of MP and Iran more accurately, and the number of species confirmed. Acknowledgements This work was supported financially by Tehran University of Medical Sciences, Iran. Ethical considerations This study was performed based on Teh- ran University of Medical Sciences (TUMS) Ethical Committee Guideline. http://jad.tums.ac.ir/ http://www.ncbi.nlm.nih.gov/nuccore/JX416589.1 http://www.ncbi.nlm.nih.gov/nuccore/JX416591.1 http://www.ncbi.nlm.nih.gov/nuccore/JX416588.1 http://www.ncbi.nlm.nih.gov/nuccore/JX416588.1 J Arthropod-Borne Dis, March 2023, 17(1): 94–104 A Heydari et al.: Molecular Characterization of … 101 http://jad.tums.ac.ir Published Online: March 31, 2023 Conflict of interest statement The authors declare there is no conflict of interests. References 1. 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