Toxicity and protein composition of venoms of Hottentotta saulcyi, Hottentotta schach and Androctonus crassicauda, three scorpion species collected in Iran

Abstract Background Scorpion stings comprise a serious problem throughout the globe, especially in regions where they are more frequent. Despite a recent upsurge of interest in scorpion venoms by various research groups, there remain many challenges. Objective Therefore, in this study, we aimed to study the toxicity and protein composition of venoms of Hottentotta saulcyi, Hottentotta schach and Androctonus crassicauda, three scorpion species collected in Iran. Materials and Methods Scorpion species were collected from Esfahan farm scorpion company and maintained in the laboratory in containers that mimic their natural habitat. Venom was extracted from A. crassicauda, H. schach and H. saulcyi by electrical stimulation of 8 and 10 V. The toxicity of each venom was established by using four groups of male Swiss albino mice aged 2 months (weighting 18–20 g) for testing each dose of venom. One group was used as a control. Venom was injected into mice by subcutaneous route. Then, animals were monitored for 24 h and LD50 was estimated by the graphic method of Miller and Tainter. Thus, high‐performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was used to determine amino acids in the venom, and protein concentrations were determined by the Biuret method. Results LD50 of scorpion venoms by subcutaneous route was found to be 1.70 mg/kg b.w (A. crassicauda), 1.47 mg/kg b.w (H. saulcyi) and 0.85 mg/kg b.w (H. schach). A. crassicauda, H. saulcyi and H. schach contain 26, 30, and 31 amino acids, respectively. A. crassicauda contains low concentrations of alpha‐aminoadipic acid, beta‐aminoisobutyric acid, beta‐alanine and citrulline. H. saulcyi contains a concentration of hydroxylysine, whereas H. schach has no such concentration. A. crassicauda also had the highest levels of tyrosine and threonine. Only A. crassicauda venom contains a low proportion of proteins (14.80%) compared with those of H. schach (16.26%) and H. saulcyi (16.20%). Albumin content in the venoms was 11.7% (H. saulcyi), 5.4% (H. schach) and 4.4% (A. crassicauda). Conclusion Scorpions venoms have a variable toxicity and an interesting composition in amino acids and proteins. Work on the development of anti‐venom is fundamental.

Scorpions are venomous arachnids with major medical health importance in Iran (Navidpour et al., 2020). They have acquired the ability to defend against predators and capture prey by producing toxin-laden venom secreted by specialised venomous glands found at the end of the scorpion telson (Sollod et al., 2005). Envenomation of scorpion is a public health problem worldwide and in particular in Iran (Yadav et al., 2020).
Throughout their long evolutionary existence on this planet, coupled with the selective pressure exerted on these organisms, the scorpions have succeeded in developing series of poisonous peptides that present various biological activities and pharmacological functions (de la Vega et al., 2010). Scorpions have always received attention in Iran because of their frequency and venom. High doses of venom can cause death and hypersensitive reactions (Saganuwan, 2018). In the past 50 years, many aspects of scorpion biology have been studied in Iran, including venom (Motevalli Haghi & Dehghani, 2017). Several scattered studies on the scorpion have been conducted, but the maintenance on the captivity of some species, venom extraction, toxicity and amino acid constituent is still not clear.
According to the literature, in Iran, Androctonus and Hottentotta genera contain dangerous and medically important species (Hauke & Herzig, 2017;Ward et al., 2018). Hottentotta saulcyi (H. saulcyi), Hottentotta schach (H. schach) and Androctonus crassicauda (A. crassicauda) are three species of scorpions belonging to the family of Buthidae (Dehghani et al., 2009). They are among the species most involved in scorpion stings in Iran (Dehghani & Fathi, 2012). There are insufficient data on the toxicity of their venom and its amino acid and protein composition.
Studying the different methods of captivity, venom extraction, its toxicity and its constitution is of paramount importance for researchers. This work aims to study the toxicity and protein composition of venoms of H. saulcyi, H. schach and A. crassicauda, three scorpion species collected in Iran.

Identification of scorpions
A stereomicroscope and a morphological identification key were used to identify the scorpion. Based on morphological characteristics such as body colour, pedipalp shape and size, sternum shape, telson colour and several pectin teeth, collected scorpions were identified at the species level.

Keeping of scorpions
In the laboratory, scorpions were placed in a large glass aquarium (diameter: 54 cm, height 22 cm) on top layer of soil about 3 in. in depth, above which proper size stones were kept, imitating their natural habitat.
As a source of drinking water, a petri dish lid containing tap water was kept in the centre of the glass aquarium. The scorpions were fed with crickets and worms of flour. At 3-day intervals, the water content of the petri dish was checked and, if necessary, filled.
Scorpions were only handled on the day of venom extraction and during soil replacement, which was done once every 2 months. The venom was not removed the day immediately following the feed and no food was added up to 3 days after the venom was removed.
The tail was held firmly wing forceps and electrically stimulated by pointing two electrodes connected to a step-down transformer at junctions between the tail segments, one next to the telson. The other at the junction between IV and V metasomal segment. A. crassicauda, H.
schach and H. saulcyi were stimulated twice, at 8 and 10 V, respectively, for 2-3 s with short intervals between two stimulations.
The venom was allowed to secrete over a piece of parafilm placed at the base of its tail and then transferred to a vial. The venom obtained in a single episode from many scorpions of a species were pooled, mixed with excess double distilled water and centrifuged at 15,000 rpm for 20 min to remove the mucus; the supernatant was lyophilised and stored at 20 • C until used for protein estimation and toxicity studies; venom was resuspended in phosphate-buffered saline (PBS) PH7.

Determination of amino acids
The high-performance liquid chromatography-tandem mass spec- The mass spectrometer was carried out in both positive and negative ionisation multiple reaction monitoring mode. The source parameters were as follows: the capillary voltage set at 300 V for positive ionisation mode and −300 V for negative ionisation mode, the drying gas temperature was 320 • C, the flow was 11 L/min and nebulising gas pressure was 30 psi.

Liquid chromatography with tandem mass spectrometry
All reagents were of analytical grade; the solvents were of chromatographic purity and the water was purified by deionisation (Milli  were typically washed with a 1 mol L -1 NaOH solution (high pressure, 5 min), followed by deionised water (5 min) and electrolyte (30 min).

Concentration of proteins
In between runs, the capillaries were usually reconditioned by a flush with the electrolyte solution (high pressure, 2 min). At the beginning of the day, the coated capillary was conditioned by a flush of methanol followed by deionised water (1.38 × 10 2 kPa for 10 min, each flush).
In between runs, the coated capillary was just replenished with fresh ampholyte working solution (2 min flush). At the end of the day, the coated capillary was washed with deionised water, methanol and dried with a flush of nitrogen (1.38 × 10 2 kPa, 5 min each flush).

Statistical analysis
No statistical analysis was performed.

Identification of scorpions
Based on morphological characteristics such as body colour, pedipalp shape and size, sternum shape, telson colour and several pectin teeth, identified scorpions were presented in Table 1.

Determination of amino acids and low concentration amino acid
None of the venoms studied contain argininosuccinic acid and sulfo-

High concentration amino acid
Except for serine, arginine, aspartic acid, glutamine and threonine, H. A. crassicauda also had the highest levels of tyrosine and threonine (Table 6).  were active and healthy with a 100% survival rate. The use of a Petri dish as a water trough makes the water easily accessible to all scorpions as also reported (Nagaraj et al., 2015). According to Nagaraj et al. (2015), it is an effective and easy method to use moist cotton balls placed in a container (Candido & Lucas, 2004;Whittemore et al., 1963). Water-efficient spraying restores soil moisture content and contributes to scorpion moulting (Rubio, 2008).

Concentration of proteins
The stimulator used in this study was the one used by Nagaraj et al. (2015) for venom extraction and was easier to use than the one used by Lowe and Farrell (2011). The venom of A. crassicauda, H. schach and H. saulcyi was extracted by electrical stimulation (8 and 10 V). This voltage variability was noted by Nagaraj et al. (2015) who reported that the voltage required to stimulate scorpions varies according to species and size (Nagaraj et al., 2015). The venom of the species Androctonus mauretanicus and Buthus occitanus tunetanus was extracted by stimulation at 12 V, whereas Parabuthus sp., Tityus serrulatus and Heterometrus gravimanus were stimulated at 40, 12.5 and 8 V, respectively (Candido & Lucas, 2004;du Plessis, 2005;Oukkache et al., 2013;Whittemore et al., 1963). The study by Nagaraj et al. (2015) revealed that Hottentotta rugiscutis and Hottentotta tamulus secrete venom when stimulated at 8 V and that Heterometrus swammerdami secretes venom only at 10 or 12 V depending on the size of the species. These variations can also be related to the conditions and variability of the stimulators used.
Therefore, a study conducted in Pakistan on buthids also reports that 25 V voltage is best suited for venom extraction in scorpions (Yaqoob et al., 2016). The use of an electrical signal remains an interesting technique to extract scorpion venom through the muscles of the venom gland (Lowe & Farrell, 2011).

Identification of scorpions
Based on the morphological characteristics such as body colour, the shape and size of pedipalps, shape of the sternum, the colour of telson and several pectine teeth, the collected scorpions were identified to the species level according to Nagaraj et al. (2015), Sureshan et al. (2007) and Veronika et al. (2013).

Lethality of scorpion venom
The toxicity evaluation of scorpion venom is a critical step for an efficient determination of the venom activity (Oukkache et al., 2014). The most common model for venom toxicity analyses is the LD 50 value determination in mice (Charman et al., 2000;Charman et al., 2001;Dzikouk et al., 2002;Krifi et al., 1998 Venom toxicity varies with several factors such as genus, species, members of one species, age, physiology, feeding state and region of the scorpion (Hassan, 1984;Krifi et al., 1998;Ozkan et al., 2006;Padilla et al., 2003;Theakston et al., 2003). Therefore, these parameters must be specified for any venom LD 50 values reported. Ismail et al. (1994a,b) found that the LD 50 of A. crassicauda venom obtained by electric stimulation was 0.64 mg/kg, whereas Latoxan Laboratory reported an LD 50 of 0.87 mg/kg for A. crassicauda venom obtained by the same method (Ismail et al., 1994a;Ismail et al., 1994b).
However, Altinkurt and Altan (1980) reported that the LD 50 of A. crassicauda venom, from the Sanliurfa region, was 11.5 mg/kg by the maceration method. The LD 50 of scorpion venom must vary even if the venom was extracted by using a single method. Venoms from species of the Androctonus genus are highly toxic (Ozkan & Filazi, 2004). A. crassicauda venom has a 0.32 mg/kg LD 50 when intravenously injected into mice, which makes this scorpion species one of the most toxic in the world (Bonnet, 1997;Ismail et al., 1994a;Ismail et al., 1994b). Also, Ismail (1993) reported that the LD 50 of Leiurus quinquestriatus venom varies from 0.23 to 6.5 mg/kg (Ismail, 1993). Induced by the subcu-taneous route, LD 50 calculated by probit analysis was 1.1 mg/kg for venom obtained by electric stimulation (Ozkan et al., 2007).
According to Yagmur et al. (2015), the lethal assay of the H. saulcyi venom was 0.73 mg/kg in mice (Yagmur et al., 2015). This lethal dose 50 is lower than that obtained in the present study by the probit method.
These results confirm those of Altinkurt and Altan (1980) and Dittrich et al. (2002) which state that the same species present a range of LD 50 values in the lethality test of venoms. The low LD 50 obtained for Hottentotta species agrees with a report indicating that scorpions of the Buthidae family are of medical importance (Ozkan et al., 2011).  (Altinkurt & Altan, 1980). The study of Caliskan et al. (2006) showed that A. crassicauda has more than 58 amino acids (Caliskan et al., 2006). Mohamadpour et al. (2008) also reported that H. schach has more than 20 chains of amino acids of different molecular weights (Mohamadpour et al., 2008).

Determination of amino acids
Scorpion venom is a complex combination of proteins, peptides, amino acids and other biomolecules as well as certain minerals. This combination makes the venom a potential biological drug and therefore has a lot of therapeutic potentials to be exploited (Goudarzi & Salehi Najafabadi, 2019).
The lower albumin level recorded for A. crassicauda indicates its higher level of purity compared with other venoms. He showed a higher level of purity followed by those of H. schach and H. saulcyi, respectively. The low presence or absence of albumin in the venom expresses its higher level of purity (Ismael et al., 2018 Similar results were obtained by Ozkan et al. (2007) for A. crassicauda which states that protein bands of the venom sample obtained by electric stimulation were between 12 and 53 kDa (Ozkan et al., 2007).
The method adopted in this study for the maintenance of scorpions in the laboratory is efficient.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author.