J Arthropod-Borne Dis, March 2022, 16(1): 13–25 MB Ghavami et al.: Morphometric Indices and … 13 http://jad.tums.ac.ir Published Online: March 31, 2022 Original Article Morphometric Indices and Venom Protein Profile in Different Populations of 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, Email: Ghavami@zums.ac.ir (Received 28 Jan 2019; accepted 27 Oct 2021) Abstract Background: Androctonus crassicauda is the most medically relevant animal and understanding its morphological characteristics is essential in the production of antiscorpion sera. Methods: Adults of A. crassicauda were collected from different areas of Zanjan Province and the morphometric pa- rameters and the cuticular fluorescence patterns of samples were studied. The crude venom of samples was extracted by electric stimulation, and their biochemical properties were analyzed by the SDS-PAGE method. Results: Values of the morphometric parameters depended on sex and altitude of the area. Except for values of the pec- tinal organ, these parameters in females were higher than in males. No significant difference was in the number, shape, and intensity of cuticular fluorescence patterns. The body length of males in high and lowlands was 72.53±1.53 and 77.33±2.70mm, respectively. Females' body lengths in that area were 81.66±2.19 and 86.55±2.33mm, respectively. Analysis of toxin proteins showed two isotypes that the 12, 13, 15, 16, 18, and 19kDa proteins were in all areas. How- ever, the 41 and 74kDa proteins, and 46 and 63kDa proteins were detected in low and highlands, respectively. Conclusion: Black fat-tailed scorpion has a considerable dominancy and developing preventive programs and providing treatment facilities in studied areas are necessary. Values of the morphological parameters and venom electrophoresis patterns depended on the geographical location. Therefore, pool crude toxin is suggested for the production of effective antivenoms. Moreover, additional field complementary works in the geographic information system based niche model- ing and mass fingerprinting of scorpion venoms are suggested for screening effective isotypes. Keywords: Androctonus crassicauda; Black fat-tailed scorpion; Morphometric parameters; Scorpion venom SDS-PAGE; Scorpion venom extraction Introduction Scorpions are potentially fatal venomous animals, and their envenomation is a major public health problem in the world. Buthidae is the largest family of scorpions with world- wide distribution and contains the most dan- gerous species (1). The Black fat-tailed scor- pion, Androctonus, is a large and ancient group of Buthidae scorpions that has successfully adapted to various ecological conditions and occurs in different habitats. The overall body plan of this group has changed slightly and its systematics at the species level remains poorly defined. At present, eight species and 17 sub- species are reported within the Androctonus genus and among them, A. crassicauda is the most dangerous, causing many human deaths (2-5). Populations of this scorpion have a widespread distribution in the Middle East and Africa. The wide distribution of this species mostly in subtropical and temperate regions, between 23o–38o latitudes, indicates it's adapt- ing to various ecological conditions and has different types (6). Despite intensive research performed on external morphology in differ- 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/ mailto:Ghavami@zums.ac.ir https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, March 2022, 16(1): 13–25 MB Ghavami et al.: Morphometric Indices and … 14 http://jad.tums.ac.ir Published Online: March 31, 2022 ent species of scorpions, few investigations have been conducted on A. crassicauda, and the characterization of some parameters of this species such as reproductive apparatus, is still unknown (7, 8). In the past decades, scorpion biology has been revolutionized using ultraviolet (UV) lamps (black lights) for their detection in the field and morphological surveys (9, 10). These animals are strongly fluorescent when illumi- nated with long UV light. Molecules associ- ated with the cuticular fluorescence of scorpi- ons have been identified as the beta-carbon and 4-methyl, 7-hydroxy coumarin. The fluo- rescence exhibited by the tegument and the intensity of the emitted light is depended on the amount of these compounds in the cuticle. However, the biological function of fluores- cence has not been definitively demonstrated (6, 11, 12). No difference in fluorescence spectra between genders of species is detect- ed. However, statistical significance between the two sympatric species and subspecies of some scorpions was observed previously (13). Black scorpions have a protected body form with black coloration and current morphologi- cal methods, using white light, cannot be ac- curate enough to identify the minor morpho- logical differences in these scorpions. There- fore, studying fluorescence patterns could pro- vide complementary information to the mor- phological study of A. crassicauda. Scorpion venom is the other most important item in the study of scorpions. It is a secretion compound of water, salt, and low molecular weight peptides with 13–76 amino acid resi- dues. It resembles typical short and long-chain toxins specific for ion channels and receptor target cells. Biochemical analyses of scorpion toxins show about 44 different eluting frag- ments, from which 30 fragments are com- pletely separated. These fractions are unique defense, feeding weapons, and effective sem- iochemicals that modulate their behavior. Scor- pions apply these compounds in several so- phisticated ways, for subduing prey, and de- terring predators, probably during mating and frequently for deferring against humans (6). Molecular and biochemical characterization of A. crassicauda venom in Anatolia, south- east of Turkey, represented at least 44 differ- ent fractions with toxicity to mice and insects. The analysis of toxin in this scorpion identi- fied 80 distinct molecular mass compounds varying from 267–44551 Da peptides that could modify K and Na channels. To date, only eight peptides have been identified from A. crassicauda venom in that region, three of which have been fully sequenced (Acra1, Acra3, Acra4), and one (Acra2) is partially sequenced, and the other four are putative (Acra5-8) (14, 15, 16). However, due to the increment of new strategies of proteome anal- ysis and gene cloning from transcriptomes, the venom variation in different areas, and the number of identified components may increase significantly (17). The Black fat-tailed scorpion is the most significant scorpion species in Iran and the Middle East countries. It is the main scorpion species in Zanjan Province, northwest of Iran, causing many cases of scorpionism in this ar- ea (18, 19). Due to the multifunctional role of venom in scorpions, its analysis could exhibit the variation of population groups in A. cras- sicauda. Moreover, detection of venom com- ponents in A. crassicauda is extremely im- portant to produce effective antivenom and understanding of clinical symptoms of pa- tients. Although A. crassicauda causes many human fatalities, the knowledge of this spe- cies is restricted and limited to past decades. In addition, despite the long history of venom research in the world, the venom of a few Ira- nian scorpions has investigated. Therefore, it is important to study the polypeptide electro- phoretic patterns, and morphometric indices of A. crassicauda in distributed areas, as these characteristics, may have been applied in ther- apeutic management. Therefore, the aims of this study were to characterize the morpho- metric indices, the fluorescence pattern of ex- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 13–25 MB Ghavami et al.: Morphometric Indices and … 15 http://jad.tums.ac.ir Published Online: March 31, 2022 ternal anatomy, and the polypeptide electro- phoresis pattern of venom in different popula- tions of A. crassicauda in Zanjan Province. Materials and Methods Study area Zanjan Province is located in northwest Iran, from 35°35' to 37°15'N and 47°15' to 49°25'E (Fig. 1). The altitude in this region varies from 270m to 3400m above sea level (asl). The average maximum temperature in this area is around 27 °C, whereas the average minimum temperature stands at -19 °C. Mean- while, the temperature rises to 32 °C on hot days; it drops to -27 °C on icy days. The av- erage annual rainfall in the first month of spring stands at 72 millimeters, while in the second month of summer, it slips to a meager 3.6mm. The rate of humidity in the morning stands on average at 74% and at noon at 43% (https://en.climate- data.org/asia/iran/zanjan/zanjan-764536/). Sample collection Adults of A. crassicauda specimens were collected in different periods between Jun and September 2015–2016 from twenty-three lo- calities of Zanjan Province (Table 1, Fig. 1). The collection sites are grouped in two low- lands (under 700m asl) and highlands (above 1200m asl). The geographical characteristics of sampling stations were determined by the Global Positioning System (GPS). Scorpions were caught at night by using ultraviolet light, a portable flashlight equipped with 3W 375– 380nm Ultraviolet LED, detection method, and a few were captured in the daytime by rock rolling in the field. Avoiding scorpion cannibalism, captive specimens were housed in individual plastic boxes. They were kept alive in laboratory conditions (40% relative humidity, 12:12 L: D, and 24±2 °C) and fed with ground meat and living crickets and re- ceived daily water ad labitum. Food was giv- en a weak after venom extraction in order to allow time for the animals to recover from stress. Dead specimens were transferred into 75–96% ethyl alcohol and kept at -20 °C for further studies. They were deposited in the scorpion collection at the Department of Med- ical Entomology, Zanjan University of Medi- cal Sciences, Iran. Morphological survey In the laboratory, sexing and taxonomic study of the samples were conducted with the help of stereomicroscope (Olympus, SZX9) and the keys suggested by Farzanpey (20). Exam- ination of cuticular sculpture and morphology were facilitated using white light and ultravio- let fluorescence photomicrography. Preserved specimens were attached to a dark, non-flu- orescent plastic surface. Images were acquired when the light was excited by the 3W Indium Gallium Nitride (InGaN) light emitting diodes (LEDs) emit light within a narrow band in the near UV wavelength range (395–410nm) and 455–470nm in white light posited at the dis- tance of 0.25m from the specimen. Imagines were produced using a digital camera (Nikon DS Camera DS-Fi1) fitted on a Nikon SMZ 1500 stereomicroscope. Biometric measure- ments were taken with a >0.001mm accuracy by using Digimizer software. (https://www. digimizer.com). Measurements of 27 morpho- metric characters of adult specimens were adapted from Mirshamsi et al. (21) for subse- quent statistical analysis. Morphometric variables Abbreviations of morphometric characters include Bl: body length, Ca_L: carapace length, Ca_Aw: anterior width of carapace; Ca_Pw: posterior width of carapace, X: distance be- tween anterior margin of the carapace and an- terior margin of median eyes; Y: distance be- tween anterior margin of median eyes and pos- terior margin of carapace; Mt_L (I–V): length of metasomal segments I–V; Mt_W (I–V): width of metasomal segments I–V; Mt_H (I– http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 13–25 MB Ghavami et al.: Morphometric Indices and … 16 http://jad.tums.ac.ir Published Online: March 31, 2022 V): height of metasomal segments I–V; Tl_L: telson length; Tl_W: telson width; Tl_H: telson height; Ch_L: chela length; M_L: manus length; Mf_L: movable finger length; Pl-L: pectinal left lamella length, and Pr-L: pectinal right lamella length. Venom extraction and preparation The crude venom was obtained by electri- cal stimulation of the telson of scorpion using an electro-pulse stimulator. In summary, one person held the pre-abdomen and telson of a scorpion individual with tweezers to keep it stationary and another person collected the venom from the aculeus of the scorpion. For electrical stimulation of the membrane anteri- or to the telson, we used the transcutaneous electrical nerve stimulation Tensmed 400 (Ar- man Poya Co, Iran) connected with modified tweezers to apply power. To improve stimula- tion and enhance electrical contact, the twee- zers were lubricated with glycerin saline gel. The intensity of the stimulation current was adjusted in pulse width from 200–250µs, a pulse rate of 60–80Hz, and amplitude of 12– 16v. The extracted venom was collected with a microcapillary glass tube (inner diameter 1.5mm) that was attached to a flexible silicon tube and disposable insulin syringe and trans- ferred into a 1.5ml centrifuge tube. Crude ven- om was suspended in deionized water, centri- fuged (12,000×g, 4 °C, 10min), and the super- natant that contained soluble venom proteins were transferred to a clean 1.5mL tube. Fi- nally, the taken supernatants were immedi- ately lyophilized and stored at -20 ºC until use according to their geographical origins. After milking venom (venom collection), the ani- mals were housed in individual boxes for an- other milking process. Measurement of protein concentration Protein concentrations was determined by absorbance measurements at 280nm and ex- pressed as mg protein/ml. For each venom type, we prepared a solution of venom with a final concentration of 5mg/mL. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of venoms Electrophoretic analysis of venoms was performed on 18% polyacrylamide gel in the presence of SDS. All samples were dissolved in a sample loading buffer (50mM Tris–HCl, pH 6.8, 1% β-mercaptoethanol, 6% glycerol, 2% SDS, and 0.01% bromophenol blue). For the separation of proteins, samples were run on running buffer (25mM Tris, 192mM gly- cine, 0.1% SDS, pH 8.3). The molecular mass standard (SinaClon, PR911654) was run par- allel to calculate the molecular weights of pro- teins. A constant electric current of 80mA was applied for 4–5 hours for the migration of proteins. After migration, the gel was stained with 0.1% Coomassie Blue R-250. The gel was then scanned, and the molecular weights of the proteins were calculated. Results A total of 98 samples of adult scorpions comprising A. crassicauda, Mesobuthus eupeus Odontobuthus doriae, and Scorpio maurus town- sendi were collected from the studied areas. The black fat-tailed scorpion was the most frequent species and included about 70 % (68 scorpions) of the samples. The frequency of the rest scorpions, M. eupeous, Od. doriae, and Scorpio maurus, was 19%, 4%, and 3%, re- spectively. Samples of A. crassicauda were col- lected mainly in the summer months (Table 1). Morphological survey The descriptive statistics (minimum and maximum values) of morphometric characters for each sex of A. crassicauda in study groups are presented in Table 2. The schematic view of representative males and females in low and highlands are illustrated in Fig. 2. Analy- sis of morphometric parameters showed a sta- tistical difference between sexes for the length http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 13–25 MB Ghavami et al.: Morphometric Indices and … 17 http://jad.tums.ac.ir Published Online: March 31, 2022 of pedipalps, carapace, telson, and pectinal or- gans. A univariate comparison of morphologi- cal parameters showed that these parameters also vary with the local altitude. Female scor- pions were presented as larger than males. The values of some characters were generally large in females except for the pectinal fea- tures. Small spectrum variation for long, wide, and high metasomal 1-V characters is consid- ered to have minimal scalar overlapping with their homologous character. The body length values of male scorpions in high and lowlands were 72.53±1.53 and 77.33±2.70mm, respec- tively. This parameter also varied in females in different areas and average body length reached 81.66±2.19 and 86.55±2.33mm in low and highlands, respectively. A comparison of these values showed that the body length of female scorpions in highlands overlapped with the size of males in lowlands. The values of the pectinal organ as other morphometric parameters depended on scor- pion sex and local altitude. Male scorpions showed pectinal lengths of 9.25±0.25 and 9.43±0.32mm in high and lowlands, respec- tively. Despite the body length, the length of the pectinal organs in female scorpions did not vary and its value was 8.04±0.18mm in both study areas. A Survey of the genital operculum of scorpions showed that it is more oval in males and slightly triangular in fe- males. In addition, the genital operculum is fused in females and split among males. Carination The emitted florescent light from different parts of scorpions and cuticular florescent pat- terns are illustrated in Fig. 3. No significant difference was seen in the number, shape, and intensity of emitted light from carina in the carapace, prosoma metasomal segments, tel- son, and pedipalps of specimens. Scorpion venom All scorpions were observed as being very aggressive during all keeping milking time. A total of 75µl crude venom was extracted from each scorpion sample. A colorless watery se- cretion was obtained during capturing fol- lowed by more viscous milky droplets or ejac- ulate (mucous accompanied with the venom) during stimulation and did not turn blue after milking. After centrifugation of the whole ven- om, the supernatant was of a more viscous form. SDS-PAGE Analysis of the Venom The protein profiles of A. crassicauda ven- oms were analyzed by SDS-PAGE followed by Coomassie blue staining. In these analyses, 2 different isotypes, each with a different mo- lecular mass, were detected. The number of protein bands for the investigated scorpions in two groups was 9. Out of all protein bands, the bands of 12, 13, 15, 16, 18, 19 and 57kDa consistently appeared in all venom samples (Fig. 3). In addition to the above share pro- teins, two protein bands of 47kDa in lowlands and 63kDa in highlands were noticed unique in each group, and two bands of 41kDa in lowlands and 46kDa in highlands were not clearly different between two groups (Fig. 4). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 13–25 MB Ghavami et al.: Morphometric Indices and … 18 http://jad.tums.ac.ir Published Online: March 31, 2022 Table 1. Frequency of collected Androctonus crassicauda from different areas of Zanjan Province, northwest of Iran Studied Areas Coordinates Latitude and Longitude Altitude (Meters asl) Time of collection Frequency District Locality Zanjan Subtotal Zanjan 36.698138 N 48.514163 E 1600–2000 Sept, 2016 4 4 4 3 4 3 3 24 Gavazang 36.719335 N 48.521419 E Aug, 2016 Valarood 36.716381 N 48.372671 E Aug, 2016 Sarimsaghlu 36.759271 N 48.371128 E Sept, 2016 Do Asb 36.706016 N 48.566930 E Sept, 2015 Sayan 36.647160 N 48.541718 E Sept, 2015 Khodabandeh Subtotal Khodabandeh 35.940912 N 48.144053 E 1600–1800 July, 2016 1 1 1 1 2 2 8 Tatardeh 35.956463 N 48.102211 E Sept, 2015 Khalife 35.994119 N 47.993913 E July, 2016 Sohrevard 36.074354 N 48.432798 E July, 2016 Qeshlaq Vakil 36.144121 N 48.046618 E July, 2016 Garmaab 35.850285 N 48.198390 E July, 2016 Mahneshan Subtotal Mahneshan 36.768985 N 47.668715 E 1200–1500 Jun, 2016 5 4 3 2 14 Sari Aghol 36.823192 N 47.629671 E Jun, 2016 Sahand E Sofla 36.773948 N 47.541359 E Jun, 2016 Sahand E Olia 36.777957 N 47.519815 E Sept, 2015 Tarom Subtotal Chavarzagh 36.994055 N 48.777488 E 400–700 July, 2016 2 5 4 3 2 4 2 22 Daraam 37.024636 N 48.778250 E Aug, 2016 Haronabad 36.834549 N 49.025299 E Aug, 2016 Abbar 36.921868 N 48.960540 E Aug, 2016 Dastjerdeh 36.849681 N 48.943995 E Sept, 2016 Sansooz 36.835585 N 48.943389 E Sept, 2016 Tashvir 36.789542 N 49.002457 E Sept, 2016 Total 68 Fig. 1. Map of study areas in Zanjan Province. Locality of collection sites ( ), district center ( ) lowlands ( ) and highlands ( ) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 13–25 MB Ghavami et al.: Morphometric Indices and … 19 http://jad.tums.ac.ir Published Online: March 31, 2022 Table 2. Minimum (min) and maximum (max) values of the morphometric indices (with 95% confidence interval) in specimens of Androctonus crassicauda collected from low and highlands of Zanjan Province Parameter Lowlands (400-700m asl) Highlands (1200-2000m asl) ♂(n=27) ♀(n=19) ♂(n=12) ♀(n=10) min max min max min max min max BL 72.03 82.63 81.98 91.12 69.53 75.53 77.36 85.96 Ca- L 8.50 9.32 9.29 10.93 8.38 9.01 9.29 10.49 Ca-Aw 4.23 5.08 4.87 5.62 4.44 4.93 5.04 5.55 Ca-Pw 8.60 9.69 9.99 11.96 8.55 9.13 9.98 11.21 Mt(I)-L 5.51 6.22 5.85 7.06 5.56 6.24 5.70 6.47 Mt(I)-W 3.58 4.27 3.78 4.80 3.70 4.37 4.05 4.41 Mt(I)-H 4.20 5.10 4.11 5.27 4.16 4.74 4.40 4.94 Mt(II)-L 6.47 7.14 6.86 7.79 6.34 6.97 6.75 7.45 Mt(II)-W 4.17 5.17 4.48 5.67 4.47 5.12 4.69 5.21 Mt(II)-H 4.31 5.13 4.29 5.66 4.28 4.86 4.42 4.99 Mt(III)-L 6.66 7.48 7.14 8.01 6.49 7.06 6.94 7.62 Mt(III)-W 5.11 6.13 5.14 6.29 5.13 5.68 5.33 6.07 Mt(III)-H 4.59 5.34 4.73 5.94 4.56 5.20 4.90 5.50 L Mt(IV)- 7.52 8.59 7.88 9.22 7.62 8.17 7.89 8.60 Mt(IV)-W 5.66 6.67 5.46 6.85 5.54 6.16 5.64 6.43 Mt(IV)-H 4.77 5.42 4.73 5.74 4.56 5.19 5.14 5.48 Mt(V)-L 7.33 8.48 8.37 9.64 7.58 8.21 7.65 8.68 Mt(V)-W 5.26 6.20 5.19 6.57 5.28 5.85 5.42 6.13 Mt(V)-H 2.97 3.46 3.05 3.69 2.92 3.40 3.29 3.72 Tl-L 8.01 9.32 9.23 9.81 7.71 8.72 8.83 9.98 Tl-W 2.94 3.51 3.38 4.22 3.02 3.39 3.63 3.91 Tl-H 2.82 3.33 3.04 3.64 2.64 3.06 3.19 3.58 X 3.44 3.82 3.86 4.49 3.47 3.73 3.94 4.41 Y 5.12 5.73 5.47 6.67 5.01 5.44 5.49 6.18 Ch- L 14.69 15.64 16.41 18.60 14.64 15.93 15.83 17.79 M-L 6.36 7.01 6.91 8.16 6.49 7.24 6.81 7.71 Mf-L 10.43 11.37 11.99 13.02 10.19 11.13 11.56 12.59 Pl-L 8.64 9.89 7.11 8.83 8.94 9.92 7.35 8.36 Pr- L 8.52 9.77 7.25 8.90 8.94 9.88 7.69 8.39 Fig. 2. Schematic view of the representative male and female of Androctonus crassicauda in study areas. Male (A, C) and female (B, D) samples in lowland (A, B) and (C, D) highland http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 13–25 MB Ghavami et al.: Morphometric Indices and … 20 http://jad.tums.ac.ir Published Online: March 31, 2022 Fig. 3. Schematic presentation of external body parts in Androctonus crassicauda under UV fluorescence. Carapace (A); tergite (B); ventral aspects of prosoma in male (C, E, G) and female (D, F, H); legs and pedipalp (I, J, K, L); meta- somal segments in ventral (M, N), dorsal (O, P), and lateral (Q, R) aspects; metasomal segment V and telson in lateral (S) and ventral (T) aspects http://jad.tums.ac.ir/ https://www.researchgate.net/figure/Schematic-representation-of-external-and-internal-body-parts-of-octopus_fig14_305329627 J Arthropod-Borne Dis, March 2022, 16(1): 13–25 MB Ghavami et al.: Morphometric Indices and … 21 http://jad.tums.ac.ir Published Online: March 31, 2022 a b Fig. 4. Electrophoretic profile of Androctonus crassicauda venoms on poly acryl amide gel in the presence of SDS. Part a: venom obtained from captured scorpions in highlands and part b: venom obtained from collected scorpions in lowlands. Lanes 6 in highlands and 7 in lowland are molecular weight (kDa) markers, and the others are venom samples Discussion The results of the present study showed that A. crassicauda is a dominant scorpion in studied areas and occurs in different condi- tions. The appearance of this species in study areas reflects the tuning of optimal ecological conditions necessary for its survival in these areas. The presence of this scorpion in these areas calls for the authorities to take the nec- essary measures to prevent and cure enven- omation. The findings of this study showed that the body size of the specimens is depended on gender and local conditions (altitude). Local dependence on morphometric indices causes both sexes to have a wide range of body lengths. Although body size can distinguish the sex of the same population, geographical distance, and the overlap of the sizes of both sexes can challenge gender discrimination. Therefore, it is suggested that the values of the morphological parameters of each region must be used to analyze the samples of that region. Our data allowed us to evaluate the size variation of A. crassicauda with respect to local conditions. Season length is an im- portant ecological factor that may influence the variation of deduced parameters. In high altitudes the average ambient temperature is lower, and the duration of the activity period is shorter than the lowlands. Because the black fat-tailed scorpion is a slowly developing an- imal, therefore, its encounter in short activity season reduces the growth period, resulting in reduced body size in highlands. Moreover, the patterns in body size variation we found in A. crassicauda might be adaptive under the giv- en ecological conditions and food availability in these two lands. In the current study sexual dimorphism among scorpions was performed by morphometric parameters; length of body, carapace, pedipalp, and structure of the pecti- nal organ. These findings are similar to the results of studies conducted in Turkey and Fars province, southern Iran (7, 8). Many studies have been carried out on the use of UV light to the finding of scorpions (9, 10), but the present study is the first study, in collecting of scorpions and determining the pattern of carina in different parts of the scor- pion. Therefore, the protocol of this study is recommended for the study of scorpions, es- pecially those that have dark tegument. The findings of this study revealed that A. crassicauda in the study areas represented a http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 13–25 MB Ghavami et al.: Morphometric Indices and … 22 http://jad.tums.ac.ir Published Online: March 31, 2022 homogenous species and all the samples had one fluorescent pattern. This process may be related to the presence of selective pressure or specific ecological behavior (sex finding and mating) of specimens. Further studies could solve this problem in different situations. In the current study, the electrophoretic protein patterns of the black fat-tailed scorpi- on venom were presented in the range from 12 to 74kDa. Among these proteins, six bands 12, 13, 15, 16, 18 and 19kDa appeared in all samples. Analysis of electrophoretic patterns also indicated that this scorpion possesses other proteins whose molecular weight was more than 40kDa and varied in different strains of a scorpion. Previews studies indicated that ven- om proteins of A. crassicauda, collected from Sanliurfa and Mardin provinces (Southeastern Anatolia) of Turkey, were in six bands with 12, 15, 29, 35, 53, and 58kDa. They showed that 35, 53, and 58kDa proteins played an im- portant immunogenic role in the production of antivenom against this scorpion (22, 23). Scorpion venoms in the present study were documented into two groups according to their molecular sizes. These groups include the short and long chain neurotoxins with molecular size below 20kDa and the target aging pep- tides with up 40kDa weight. Among these groups more studies are conducted about neu- rotoxins, however, there is scant information on the other study groups, and the studies have been limited to their toxicity and immunogen- ic. The low molecular weight biogenic amines (histamine, dopamine, nor-adrenalin, etc.) found in samples are involved in local reactions and their release from a single sting can lead to systemic reactions. They can act on blood ves- sels and nerve endings inducing swelling, red- ness, pain, and itching. The major toxic ef- fects of venom are attributed to the presence of large peptides (4, 6). These peptides can cause damage to the cell membrane, leading to the release of enzymes from lysozymes and mast cell granules, resulting in cytolysis. Ad- ditionally, they can act as neurotoxic provok- ing hyper-excitability. Thus, the individual vari- ability in venoms is extremely important for evaluating the venom yield and the resulting toxicity after a scorpion sting. Further studies are needed to evaluate the toxicity of venoms and the median effective dose (ED50) of scor- pion anti-venom against envenomation (24) in various populations of this scorpion in the future. Analysis of electrophoresis peptide patterns in the present study indicated that A. crassi- cauda venom possesses different peptides both with neurotoxins and target aging according to the electrophoretic protein patterns. Poly- morphisms of protein contents also have been observed in the venom of individual A. aus- tralis hector and A. mauretanicus (25, 26). In addition, differences in venom content have been described in other species of scorpions; M. tamulus (27), and Leiurus quinquestriatus (28). Abdel-Rahman et al. (29) suspected that a combination of local environmental condi- tions, geographical separation, and genetic sep- aration may play a major role in the intra-spe- cific variation of venom of Scorpio maurus palmatus. Moreover, Ozkan and Ciftci (22) in- dicated that variation of protein bands detect- ed in the venom of captive male M. gibbosus from the same biotope in Turkey might result from the physiological condition of scorpions. The peptide variation in different populations of deduced scorpions might result from adap- tive radiation to environmental conditions. Nev- ertheless, this might represent only the tip of the iceberg and the number of novel toxins will still be expanding in the future. Moreo- ver, differences in the band pattern of sepa- rated protein in venom samples clearly sug- gest the existence of genetic variation among the scorpion strains of different regions in the study areas. Results of this study showed that the mo- lecular pattern of the venom proteins varies in geographical areas. It should be noted that this variation may have a significant role in the venom toxicity of geographical or ecolog- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 13–25 MB Ghavami et al.: Morphometric Indices and … 23 http://jad.tums.ac.ir Published Online: March 31, 2022 ical groups of scorpions. Due to the effective- ness and toxin neutralizing capacity depend- ence on intraspecific variations of this scorpi- on, it is recommended that pooling of the nu- merous venoms from population groups is used in the production of antivenoms. Obvi- ously, before mixing the crud venoms, it is necessary to identify the biochemical frac- tions and toxic and immunogenic proteins of each venom in different populations. Moreo- ver, further studies are recommended for the mass fingerprinting of scorpion venoms for barcoding, chemotaxonomy, and screening of effective isotypes in the future. Conclusion Different populations of black fat-tailed scorpions have the same fluorescence patterns. However, morphometric parameters and ven- om electrophoretic patterns of this scorpion vary in different situations. Based on these findings the morphometric parameters of each region are suggested to analyze the samples of that area. Moreover, additional complemen- tary field works in the GIS ecological niche model together with the mass fingerprint of scorpion venoms are recommended for a screen- ing of effective isotypes in future studies. Acknowledgment This work was supported by research pro- ject no. A-12-84-8 offered by Vice-Chancel- lor for Research and Technology of Zanjan University of Medical Sciences. 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