J Arthropod-Borne Dis, June 2022, 16(2): 84–96 MB Ghavami et al.: Molecular Survey of … 84 http://jad.tums.ac.ir Published Online: June 30, 2022 Original Article Molecular Survey of Mitochondrial Genes in Different Populations of the Black Fat-Tailed Scorpion, Androctonus crassicauda *Mohammad Bagher Ghavami1, Zohreh Alibabaei1, Fatemeh Ghavami2 1Department of Medical Entomology and Vector Control, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran 2Department of Electronics, Faculty of Electrical and Computer Engineering, Tarbiat Modares University, Tehran, Iran *Corresponding author: Dr Mohammad Bagher Ghavami, E-mail: ghavami@zums.ac.ir (Received 18 May 2019; accepted 18 Dec 2021) Abstract Background: Androctonus crassicauda is the most medically relevant scorpion and understanding its genetic forms is essential for improvement of anti-venom sera, and risk management of scorpionism. Present study was designed to identify the variations of mitochondrial genes in different populations of A. crassicauda. Methods: Adults of A. crassicauda were collected from Zanjan Province during 2016–2017. Genomic DNA of samples was extracted and fragments of mitochondrial 16S, COI and ND1 genes were amplified and some of the amplicons were sequenced. Haplotype of samples were identified by multiple alignment of sequences, then phylogenetic trees of haplo- types were constructed. Results: Fragments of 352bp, 618bp and 680bp were amplified from 16S, COI and ND1 genes respectively. Nucleotide sequence in COI fragments was conserved, however, five haplotypes with some specific polymorphic sites were detect- ed in 16S and ND1 fragments. Haplotype I was dominant and found in all areas. Other haplotypes were rare and limited to specific regions. Analysis of the phylogenetic trees inferred from 16S and COI genes, confirmed a strong positive correlation between geographic and genetic distance. Conclusion: Mitochondrial COI, 16S and ND1 genes were detected suitable for identifying the population structure. Five genotypes were found using 16S and ND1 genes. To prepare and improve the anti-venoms quality, additional stud- ies are necessary to identify the toxin electrophoretic profile and geographical/ecological niche models of these geno- types in future. Keywords: Androctonus crassicauda; 16S; ND1; COI; Mitochondrial genotypes Introduction Scorpions are potentially fatal venomous animals whose venom consist of a variety of toxic compounds specifically target ion chan- nels and other cellular receptors (1). Scorpion envenomation is a major global health prob- lem and anti-venom is still widely used for its treatment because there is no vaccine or other effective agents (2). Among different families of the scorpions, Buthidae with the universal distribution is the greatest and the most dan- gerous scorpions in this family belong to the Androctonus (2). Androctonus, is a typical Saharo-Sindian taxon that occurs frequently from the Atlantic coast of North Africa through western India. This genus includes the largest known buthid scorpions that some of them have extremely toxic venom to humans (2). The taxonomic re- lationship of Androctonus types is not well re- solved. This genus was described by Vachon in 1952, but since then it has been catalogued first by Fet et al. (3) and last by Teruel et al. (4). After these revisions, many species were de- scribed as new, resurrected from synonymy, or Copyright © 2022 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/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, June 2022, 16(2): 84–96 MB Ghavami et al.: Molecular Survey of … 85 http://jad.tums.ac.ir Published Online: June 30, 2022 raised from subspecies to species level (4–8). Nowadays there are correctly seven known spe- cies and 40 formerly valid subspecies in this ge- nus (9). The black fat-tailed scorpion, A. cras- sicauda, is the most dangerous species of An- droctonus due to causing several human deaths and antivenom production. This species is abun- dant in various natural habitats ranging from subtropical to temperate regions mostly in the Middle East and Africa (3). From 33 provinc- es of Iran, the black fat-tailed scorpion has been found in 28 provinces (10) Extensive distribu- tion of this species exhibits its adaptation to dif- ferent ecological conditions and probably pos- sess different types (11). Diagnosis of taxonom- ic groups in this species is traditionally based mainly on morpho-sculpture and coloration char- acters (12). Although few comparative morpho- logical studies have been done on the black fat- tailed scorpion, no modern precious analyses method exists to differentiate various types and the only existing key is out of date and its types remain largely unresolved. Despite the wide dis- tribution of this species in different regions, its ecological requirement brings it into close con- tact with human settlements regarded as a po- tential threat then responsible for several deaths (11). Given its medical importance, understand- ing the intraspecific diversity and their spatial distribution are essential for, production of ef- fective anti-venoms, and treatment of enven- omation. Over the last four decades, mitochondrial DNA (mtDNA) has been the most popular mark- er in the study of intra and interspecies varia- tions (13). The genetic information of these markers including gene arrangement patterns, coding usage patterns, nucleotide sequence from protein-coding genes, amino acids and A+T con- tents (skewness) could be applied as a powerful tool for taxonomy and identification of some species/subspecies. These markers also have been efficiently applied to detect introgression be- tween taxa, answer fundamental questions of the population structure, and resolve their phyloge- netic problems (14). Moreover, they are assumed to be clock-like and could generate some sig- nals about population history and could reflect its divergence time (13). Recently molecular tools have been used to assess the phylogeny of Androctonus using nu- clear DNA ITS regions (15), barcoding mark- ers for species determination using 16S-rDNA (16) and clade identification of species with COI genes (17). In recent years also two genetic groups were found in studied populations of A. crassicauda from Turkey according to the se- quence analysis of 16S gene (18). In addition, the phylogeographical patterns in six species of Androctonus were exhibited in North Africa by COI, 16s and ND1 mtDNA markers (19). More- over, COI as a molecular tool has been used for phylogeny of different species of scorpions (20– 23). Black fat-tailed scorpion consists of the main species of scorpions in Zanjan Province, north- west of Iran (24, 25). This species is the most significant scorpion species in Iran and other countries of the Middle East and includes many cases of scorpionism in Iran (26–28), Turkey (29) and Egypt (30). This paper is a part of an ongoing study of A. crassicauda biodiversity. We report the first pilot data based on the anal- ysis of COI, 16S and ND1 mtDNA genes from different populations of this scorpion in Zan- jan Province. The aims of the current report are identification of intraspecific variations of mi- tochondrial genes in populations of A. crassi- cauda and expression of relevant molecular markers in order to select of candidate groups for improving anti-venom quality. Materials and Methods Sampling A total of 84 adult specimens of the black fat-tailed scorpion were collected from twenty localities of Zanjan Province, northwest of Iran, from spring 2016 to autumn 2017 (Fig. 1). The study areas have different ecological conditions and include low (below 700 meters above sea level (masl)), semi-high (700–1500masl) and http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 84–96 MB Ghavami et al.: Molecular Survey of … 86 http://jad.tums.ac.ir Published Online: June 30, 2022 high (above 1500masl) lands. Sample size from each locale were rather low (n< 12) because we found the scorpions in general at extremely low densities. Main morphological characters used in identifying this species include carapacial and metasomal carination and coloration, and absence of trichobothria under pedipalp patella (Fig. 2). Scorpion samples were stored at -20 °C pending molecular analysis. The sex of the scor- pions was assessed by the general sexual dimor- phism, male having significantly higher num- ber of pectinal teeth than females (Fig. 2, d, e). DNA extraction Total DNA was extracted from muscle of dissected tissue of coxa III–IV after two washes in distilled water. To extract genomic DNA (gDNA), samples were transferred to 1.5mL Ep- pendorf tubes and suspended in 500mL of TENS lysis buffer (10mM Tris-HCl (pH 8), 1mM EDTA, 150mM NaCl, 0.5% SDS) in the presence of 0.1g of acid-washed glass beads (400–600μm in diameter) and two stainless steel ball bearings (5mm in diameter). The extraction mixture was centrifuged at 10,000g for 5min. The upper phase was extracted and added with 100µl of 5M potassium acetate, and an equal volume of ethanol incubated for 1h at -20 °C. After incubation the extracted suspension was centrifuged at 17,000g for 15min, washed twice with cold 70% ethanol, dried and resuspended in a 50μL nuclease-free TE buffer (SinaClon, Iran). Evaluation of DNA quantity and quality DNA samples were analyzed by electropho- resis in 1.2% agarose gels containing safe stain, nucleic acid gel stain (SinaClon) in Tris-bo- rate-EDTA buffer and visualized with UV light using a Gel Doc system (Uvitec, Cambridge United Kingdom). A spectrophotometer (NanoDrop; Thermo Fisher Scientific Inc.) was also used to measure absorbance at 260, 280 and 230nm and estimate concentration and con- tamination with protein. Amplification of mtDNA fragments The 16S, COI and ND1 fragments were am- plified in all samples using specific primers (Ta- ble 1). The amplifications were performed in a final volume of 25µL containing 1U taq DNA polymerase (SinaClon), PCR master mix, 10 pM each primer and 1µL purified DNA. All amplification conditions were slightly modi- fied for the DNA polymerase requirements and consisted of 1min at 94 °C; followed by 40 cy- cles of 94 °C for 30s; 53 °C for COI and 48 °C for 16S and ND1 genes for 60s and 72 °C for 60s; and a final step of 72 °C for 10min. Amplicons from the PCR were separated by electrophoresis as described previously and a 100bp DNA ladder (Invitrogen) was used to es- timate amplicon size. Great precautions were taken to minimize the risk of DNA contami- nation of PCR amplification. PCR amplifica- tions, DNA extractions and electrophoresis were set up in separate areas and using specific sets of materials including gloves, pipettes, filter tips and laboratory coat. Working positions were repeatedly cleaned with 10% NaOCl to dena- ture potential contaminating nucleic acids. Am- plified products were analyzed by electropho- resis and single bands of the expected size were sequenced with both forward and reverse pri- mers by Seqlab (www.Seqlab.dc). Analysis of sequences and identification of genotypes Chromatograms of nucleotide sequences were checked by eye using Chromas Pro soft- ware (http://www.technelysium.com.au) and multiple sequence alignment was done with ClustralW. All sequences were aligned and ed- ited by using BioEdit software (https:// bi- oedit.software.informer.com) and checked in- dels and single nucleotide polymorphisms with- in homologous groups. Some of the sequences of this study have been deposited in GenBank. The resulting alignments were checked by eye and phylogenetical relationships were performed using Maximum Likelihood (ML), Neighbor Joining (NJ) and Minimum Evolution (ME) and http://jad.tums.ac.ir/ http://www.seqlab.dc/ J Arthropod-Borne Dis, June 2022, 16(2): 84–96 MB Ghavami et al.: Molecular Survey of … 87 http://jad.tums.ac.ir Published Online: June 30, 2022 Maximum Parsimony (MP) methods with MEGA X software by using Bootstrap with 1000 replications and Kimura-2 parameter model (33). The alignments were also applied to con- struct haplotype networks using the transitive consistency score (TCS) method (34). Addi- tionally, the alignments were checked against COI, ND1 and 16S sequences available in ge- netic data banks and consensus of nucleotide and amino acid sequences were achieved by BLAST software. Androctonus australis was included in phylogenetic analysis as an out- group. This species was the most appropriate species in the buthid taxa phylogeny and has been repeatedly used in previous studies (14). Results The gDNA was extracted from 86 samples of adult black fat-tailed scorpions. The quality of purified DNA was an A260:A280 ratio of 1.8–2.1 and A260:A230 ratio of 1.3–2.0 for scor- pion DNA extracts. The concentration of nu- cleic acids ranged between 50 and 100ng/µl. Fragments of 325bp, 618bp, and 680bp were produced by PCR from 16S, COI and ND1 mi- tochondrial genes, respectively. Nucleotide contents A total of 80 high-quality amplicons of the 16S, COI and ND1 fragments were subjected to sequencing. The representative sequences of each group were deposited in GenBank with ac- cession numbers MH352581- MH352611. From twenty-five subjected sequences of 16S rRNA fragments, 16 samples were select- ed for analysis. These sequences were depos- ited in GenBank with accession numbers of MH352581- MH352596. In these samples the PCR product size was 325bp and the A+T con- tent reached a range of 72.84-73.54%. The nu- cleotide alignment of these samples showed five haplotypes that differed in eight single nucle- otide polymorphic sites (SNPs) (Fig. 3). Hap- lotype I, distributed in all regions, was the dom- inance form and comprised 82% of samples (Fig. 4). BLAST analysis of this haplotype along with sequences available in GenBank databases showed 100% identity with the sample of West Azerbaijan (Sardasht, accession no AJ277598) and 93.4% with the sample of south Anatolia from Turkey (accession no EJ217735). Like the 16S gene, 25 samples of PCR prod- ucts of the ND1 gene which showed a sharp band were selected randomly for sequencing. The nucleotide sequences of six samples of these products were submitted in the GenBank with accession numbers MH352597 to MH352602. Multiple alignments of the sequences showed a length of 680 base pairs (bp) with 17 SNPs (Fig. 5). The A+T content in these sequences were in the range of 70.91–71.18%. In the align- ments of the deduced sequences 415 nucleo- tides decode ND1 and the remaining (265 nu- cleotides) involve tRNA genes BLAST analy- sis of these sequences showed 89% identity with ND1 gene of A. australis (KJ538181) and 87% similarity with Buthus occitanus ND1 gene. Five haplotypes were obtained from TCS net- work tree of the ND1 gene sequences (Fig. 6). Haplotype I was a dominant group which con- tained 75% of samples. This haplotype is dis- tributed in all areas. From 17 SNP sites of ND1 fragments, 2 sites were parsimonious informative including one transversion and one transition substitution. In the 15 remaining sites, 4 muta- tions were transition substitutions (G↔A, and T↔C) and 11 mutations were transversion sub- stitutions (A↔T, A↔C, G↔C, and G↔T). Multiple alignment of decoded amino acids in ND1 peptide showed the study populations clus- tered in 3 isoforms. Nucleotide sequencing of 20 randomly se- lected COI PCR products showed the prod- ucts with 618bp. The deduced nucleotides in this fragment decode 206 amino acids. Data analysis revealed that all sequences have unique nucleotide arrangement, without any variation. The A+T content in these sequences was the lowest amount (59.51%) in the mtDNA frag- ment. Nine representative samples of these se- quences were deposited in GenBank with ac- http://jad.tums.ac.ir/ https://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-020-04364-z#ref-CR46 J Arthropod-Borne Dis, June 2022, 16(2): 84–96 MB Ghavami et al.: Molecular Survey of … 88 http://jad.tums.ac.ir Published Online: June 30, 2022 cession numbers of MH352603–MH352611. Analysis of the BLAST showed 99.4% identi- ty with the A. crassicauda sample of Makoo (accession no MH814933) and 98.1% with sample of Sardasht (accession no MK814934) West Azerbaijan, northwest of Iran. These se- quences also shared 99.4% and 98.1% simi- larity with COI fragments of A. crassicauda from Iraq (accession no MT2298940) and Egypt (accession no MT636858), respectively (Fig. 4). Phylogenetic analysis Phylogenetic analysis of COI and 16S se- quences of A. crassicauda retrieved from the GenBank database revealed a close genetic re- lationship among studied populations and the samples of Makoo and Sardasht, northwest of Iran, Turkey, Iraq and Egypt. Constructed trees by ML, NJ, ME and MP methods strongly sup- port the monophyly of black fat-tailed scorpi- on populations in the Middle East. Analysis of the phylogeny indicates that the haplotypes of this scorpion in Zanjan and northwestern Iran, Makoo and Sardasht districts, form a clade that its sister group is the populations of Iraq, Tur- key and Egypt. This analysis confirms the re- lationship between geographic distance and ge- netic diversity; as the geographical distance in- creases, the genetic diversity also increases (Figs. 6, 7). Fig. 1. Distribution map of sampling sites of Androctonus crassicauda in Zanjan Province http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 84–96 MB Ghavami et al.: Molecular Survey of … 89 http://jad.tums.ac.ir Published Online: June 30, 2022 Table 1. Details of the primers used in this study Gene Nucleotide sequence (5’- Reference 16S Forward CGATTTGAACTCAGATCA (29) Reverse GTGCAAAGGTAGCATAATCA ND1 Forward CGACCTCGATGTTTGAATTAA (31) Reverse TCGTAAGAAATTATTTGAGC COI Forward GGTCAACAAATCATCATAAAGATATTG (32) Reverse TAAACTTCAGGGTGACCAAAAAATCA Fig. 2. Schematic presentation of external body parts in Androctonus crassicauda. Dorsal view of carapace (a) and metasomal segments (b), pedipalp patella without ventral trichobothria (c), pectinal area of male (d) and female (e), and lateral view of telson (f) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 84–96 MB Ghavami et al.: Molecular Survey of … 90 http://jad.tums.ac.ir Published Online: June 30, 2022 Fig. 3. Multiple alignment of 16S fragments in different haplotypes of Androctonus crassicauda. The numbers in the parentheses after the name of each haplotype denote the representative accession no of sample that each haplotype belongs to Fig. 4. TCS network tree of the 16S (a) and ND1 (b) mitochondrial gene haplotypes of Androctonus crassicauda in Zanjan Province. Each dash represents one single nucleotide difference between two neighboring haplotypes. The num- bers in the parentheses after the name of each haplotype denote the representative accession no of sample http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 84–96 MB Ghavami et al.: Molecular Survey of … 91 http://jad.tums.ac.ir Published Online: June 30, 2022 Fig. 5. Nucleotide alignments of ND1 mtDNA gene in different haplotypes of Androctonus crassicauda in Zanjan Province. The numbers in the parentheses after the name of each haplotype denote the representative accession number of sample that each haplotype belongs to http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 84–96 MB Ghavami et al.: Molecular Survey of … 92 http://jad.tums.ac.ir Published Online: June 30, 2022 Fig. 6. Phylogenetic tree of COI haplotypes in different populations of Androctonus crassicauda in Zanjan Province, northwest of Iran, based on Maximum Likelihood, Neighbor-Joining, Minimum Evolution (a) and Maximum Parsimony (b) analyses methods. The numbers in the parentheses denote the accession no. The number on each branch represents percentage of tree containing that branch. The scale bar at the bottom of the trees shows the amount of genetic changes (the number of changes per 100 nucleotide sites) Fig. 7. Phylogenetic tree of 16S mtDNA fragments of Androctonus crassicauda in Zanjan Province, northwest of Iran. The constructed tree is inferred by using the Maximum Likelihood, Neighbour-Joining and Minimum Evolution (a), and Maximum Parsimony (b) analyses methods. The numbers in the parentheses denote the accession no. The value on each branch demonstrates the possibility of branching. The scale bar at the base of trees indicates the genetic diversity (the number of changes per 100 nucleotide sites) Discussion The most dangerous scorpion species, A. crassicauda, has been recorded from low to high areas with various climatic conditions in present study. These findings reflect wide range adaptation and tuning of optimal ecological con- ditions necessary to survive for this scorpion in different areas. The remarkable distribution of this scorpion in these areas calls for the author- ities to take the necessary measures to prevent and cure envenomation. The results of the present study showed that the A+T content in COI fragments of fat-tailed scorpion is 59.51%. It is slightly lower compared to those of other studied genes, 16S and ND1, in this species. Multiple alignments of COI frag- ments in study samples indicated that nucleo- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 84–96 MB Ghavami et al.: Molecular Survey of … 93 http://jad.tums.ac.ir Published Online: June 30, 2022 tide sequences in this fragment are conserved. In previous studies phylogenetic analysis of COI gene in Mesobotus eupeus showed clear di- vergence between Northern and Southern clades in different areas of Iran (21). High levels of genetic diversity that represent some geograph- ical coherences also were found in subspecies of Scorpio mourus from Moroco and Turkey (20, 23). In addition, gene diversity of Maghre- bian Hottentottus is accessed by COI gene (22). Despite genetic diversity of COI gene in dif- ferent species of scorpions, findings of present study suggest propriety of this molecular mark- er for accurate species diagnosis and interspe- cific taxonomic relationships of A. crassicauda. Despite the stability of nucleotide sequences in the COI gene, the genetic diversity of 16S and ND1 genes was observed from different pop- ulations of fat-tailed scorpions in the study ar- eas. These findings provide considerable detail on the diversity and valuable information about the population structure of this scorpion. Dif- ferent reports have revealed diversity of 16S gene in this scorpion and other species (35–38); however, to the best of author’s knowledge, this study represents the first evidence of ND1 diversity in the black fat-tailed scorpion. Significantly genetic diversity representing 5 haplotypes have been found with 16S and ND1 gene analyses of study populations. More than half of study groups clustered in one clade. According to phylogenetic analyses, all the de- tected haplotypes in the present study were shared with isolates of worldwide origin, An- atolia, West Azerbaijan, and Northwest of Iran. The recorded high genetic variation within 16S sequences suggests the hypothesis that multi- ple introductions have occurred in populations of this species. Therefore, accurate population analysis and determination of the source of gen- otypes would be necessary to confirm this hy- pothesis in further studies. Phylogenetic analysis results in this study showed that the haplotype I of this scorpion is a dominant group that is represented in all ar- eas, and the other haplotypes which were sep- arated from this haplotype distributed in spe- cial localities. Finding of this analysis sug- gests that one monophyletic lineage exists within study populations. Further detailed in- vestigations should be carried out to test this hypothesis and many more populations should be tested for other data tests (toxin structure, geographic information system/ecological niche model and nuclear genes) to establish the true genetic structure of populations currently ex- isting in this species. Conclusion The black fat-tailed scorpion was recorded from different altitudes in studied areas. This call for authorities to take the necessary pre- ventive and envenomation measures for this scorpion. Among molecular markers, COI gene is suitable for determining the interspecific re- lationship of this scorpion. However, ND1 and 16S genes were suggested to identify the inter- species and population structures in this scor- pion. Additional complementary works in fur- ther studies are needed to elucidate the facts behind the different haplotypes and popula- tion groups in this species. Acknowledgements This work was financially supported by the Deputy for Research and Technology, Zan- jan University of Medical Sciences (ZUMS) with reference number A-12-84-8. The authors are grateful to Mr Taghiloo and Mr Torabi for their valuable assistance during surveys and samplings. Ethical considerations This study was approved by the Ethics Com- mittee in Biomedical Research (ZUMS.REC. 1394.141) of Zanjan University of Medical Sci- ences, Zanjan, Iran. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, June 2022, 16(2): 84–96 MB Ghavami et al.: Molecular Survey of … 94 http://jad.tums.ac.ir Published Online: June 30, 2022 Conflict of interest statement Authors declare that there is no conflict of interest. References 1. Martin-Eauclaire MF, Abbas N, Ce´ard B, Rosso JP, Bougis PE (2015) Androctonus Toxins Targeting Voltage-Gated Sodium Channels. In: Gopalakrishnakone P, Pos- sani LD, Schwartz EF, Rodríguez de la Vega RC (Eds) Scorpion Venoms. Spring- er, London, pp. 441–471. 2. 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