The anti-microbial properties of Australian scorpion (Urodacus elongatus) venom

The anti-microbial properties of Australian scorpion (Urodacus elongatus) venom

Nilan Kumerage

Barker College

Purpose: Existing research has shown that the venom of various species of scorpions from around the world has anti-microbial properties. This project aims to determine whether Australian scorpion species (which have so been under- studied) also display such properties. Design/methodology/approach: The experiment was conducted by first extracting venom from the scorpions using electronic stimulation and then applying it onto lawned agar plates of bacteria (Staphylococcus albus and Escherichia coli). This method was ethically conducted and gave promising results. Findings: The results of the investigation suggested that the venom inhibited the growth of E. coli. Research Limitations/implications: Insufficient venom volume and contamination of plates resulted in unreliable results. However, there is a strong suggestion that U. elongatus venom is antibacterial and this should be verified with further study using a larger sample size. Practical implications: Australian scorpions have been underrepresented in venom research. This report suggests that the venom of at least one species of Australian scorpion has antimicrobial properties. Originality/value: I undertook all the experimental work including venom harvesting and zone of inhibition testing. I was responsible for sourcing, housing, feeding, and caring for the scorpions. A laboratory assistant constructed the electro-stimulator and poured and prepared the agar plates. Keywords: Venom, Scorpion, Antibacterial Paper Type: Research paper

Literature Review

Venom Research

Research has shown venom from animals should not be feared but admired (Shaw, 2022.). Venom has been used in the formulation of medicine and the treatment of disease for thousands of years and is still being used currently (Thomas, 2022). Snake venom, for example, has been used in a variety of life saving medications such as Captopril (used for treating high blood pressure) and Tirofiban (used as an anticoagulant). Insect venoms are also used in medical applications. For example, honeybee venom has been commercialised in preparations such as Apitox (used to treat arthritis and chronic pain).

Many venoms have been shown to be antibacterial (Bocian and Hus, 2020; Perumal-Samy et al., 2007). In the context of antibiotic resistance (Frieri et al., 2017), new sources for antibiotic treatments are an extremely important focus for research to prevent even common infections becoming immune to treatment (Livermore, 2004).

Scorpion Venom

Scorpions are venom producing arthropods of the class Arachnida. There are approximately 1500 species (Culin and Polis, 2018) that are distributed on every continent except Antarctica and are found in a wide range of habitats from deserts to rainforests and even alpine climates (Putnam, 2009). Scorpions from around the world have different venoms to suit the environment and prey options, however, each one shares similarities. Scorpion venom contains a mixture of neurotoxins, cardiotoxins and nephrotoxins as well as many more (Cheng et al., 2018). This is used to incapacitate prey before consuming and also to be used as a defence measure against predators. All species of scorpions contain venom however, they greatly vary in potency. Venom is produced in the telson (venom bulb) and is delivered to its prey via the aculeus (stinger). Figure 1 contains an anatomical drawing of a scorpion.

Table 1: Effect of venom on anti-microbials Source: Perumal-Samy et al., 2007, p 652

Figure 1: Anatomy of a scorpion (Source: Kazilek, 2016)

Scorpion venom is a mixture of peptides, enzymes, lipids along with many other unknown substances (Oukkache et al., 2013). Peptides from many species of scorpions around the world have shown to be highly effective against bacterial and fungal infections. Hoffmannihadrurus aztecus is a Mexican species of scorpion which has the peptide hadrurin in its venom which has been shown to be antibacterial due to its ability to destabilise membranes (Yacoub et al., 2020). Heterometrus laoticus is a southeast Asian scorpion which has the peptide ‘Heteroscopine-1’ in its venom, this peptide can eradicate the bacteria Pseudomonas aeruginosa, which is known to cause urinary tract infections and dermatitis (Khalid et al., 2012). Numerous peptides (ahelical, cationic and pore-forming), which are found in the venom of scorpions (Opistophtalmus carinatus), also contain a broad spectrum of antimicrobial activity against bacteria and fungi (Moerman et al., 2002). Yacoub et al. 2020 concluded that all venomous animals to an extent contain antimicrobial properties, and for scorpions their venom tends to contain peptides which are antimicrobial and antifungal.

Perumal-Samy et al. (2007) studied a range of different venoms and determined that the North African species ‘Buthotus hottenota hottenota’, showed only a limited antimicrobial effect against Staphylococcus aureus. They tested venoms against five microorganisms (Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, Proteus mirabilis,

Pseudomonas aeruginosa and Staphylococcus aureus) and their results are summarised in Table 1. According to their findings, scorpion venom was effective against S. aureus and E. aerogenes but not against any of the other microorganisms. The two species of scorpions that they investigated were B. h. hottenota and Buthus martensii karsch, which are considered to have a low venom toxicity, which is similar to the Australian species U. elongatus (Liu et al., 2002)

Australian Scorpions

Australian scorpions are relatively understudied in terms of the capabilities of their venom in medicine. Anecdotally, Australian scorpion venom that is known is considered mild compared to scorpions from other parts of the world. Within Australia, there are currently 43 known species of scorpion, which are found in all states and territories. The relative abundance of species and distribution warrants investigation of the utility of their venom.

An important aspect of this research is compliance with the ethical and risk protocols of the high school environment. The two species of Australian scorpions selected (U. elongatus and Urodacus manicatus) were identified as being readily suitable due to their low cost, availability, and low venom toxicity. Luna-Ramirez et al. (2017) investigated the peptides from the Australian scorpion genus Urodacus which showed to have a strong antimicrobial effect against P. aeruginosa. Due to the species selected in this investigation being in the same genus and showing strong antimicrobial properties it supported the hypothesis that U. elongatus venom would create a zone of inhibition against the bacterium. Smith et al. (2012) found that the venom from Australian scorpions contained less peptides than many other species and this could be due to the toxicity of individual peptides compared to other species relying on a concoction of enzymes and peptides.

Similarly, with respect to microbiology in school, many micro-organisms are not deemed safe in student laboratories. For this reason, a denatured strain of E. coli (K-12) and a S. albus strain are available for use in schools and were considered appropriate candidates against which to test the antimicrobial properties of scorpion venom.

Scientific Research Question

To what extent does Australian scorpion (U. elongatus and U. manicatus) venom inhibit the growth of E. coli and S. albus?

Scientific Hypothesis

Flinders Ranges Scorpion (U. elongatus) venom will produce a small zone of inhibition on the bacterium S. albus and E. coli

Method

Keeping Scorpions

Scorpions are eminently suitable for secondary school research. Firstly, they are invertebrates and so they do not fall under the restrictions of Animals in Schools. Secondly, Australian Scorpions have only a mildly toxic sting (Isbister et al., 2004). Thirdly they are abundant and relatively in expensive to purchase and house.

Originally it was intended to compare two species of Australian Scorpion (U. elongatus and U. manicatus), however, due to unforeseen circumstances, only U. elongatus survived until the experimental phase. Originally six individuals had been purchased, three of each species. Within the first two weeks of owning them, a small male U. manicatus managed to escape and was never retrieved. The scorpions then missed being fed and watered for one week due to a COVID-19 outbreak, this resulted in one U. manicatus and one U. elongatus dying. Finally, as seen in Figure 2, a female U. manicatus had given birth, but unfortunately it died shortly after due to dystocia. By the time of the experiment commencing, only two U. elongatus remained.

Scorpions were housed in plastic food safe containers that had the lids perforated for oxygen exchange. This was selected due to them being sealable, able to hold substrate and keep moisture in the environment. For the substrate a mix of Australian red desert sand and an organic, chemical free potting mix were combined. This was selected as it closely matched the soil type to where the scorpions are naturally found. Having the potting mix also allowed for better water retention than just sand. In order for the scorpions to feel secure, cardboard from a paper roll was cut in half lengthwise and this acted as a hide. They were also provided with an upside-down plastic bottle lid which was used as a water dish. For feeding, the scorpions were fed live crickets once every two to three days or until they were all consumed. The crickets had to be live as firstly it was mental stimulation for the scorpions to hunt and secondly scorpions only react to live prey items. Ventilation was provided to the scorpions by putting small holes in the lid of the container which allowed for airflow while maintaining heat and humidity.

Due to the experiment being conducted with live venomous animals, proper risk assessment had to be addressed. Any contact with the scorpions occurred in the presence of another person to ensure if someone was to get stung, they could receive assistance. Direct contact with the scorpions was minimised by handling the scorpions in a high walled container using forceps and gloves. The containers housing the scorpions were labelled with the species name as well as this they were sealed so that anyone unaware of what was being housed in them would not leave the lid open.

Extracting venom Manual

A review of literature had indicated that manual venom extraction would be appropriate. The first method of venom extraction used was through manual stimulation. This was done by using a pipette and forceps to irritate the scorpions which for some species of scorpions is an effective method of venom extraction for research (Oukkache et al., 2013). The intention was to aggravate the scorpion and entice them to sting into a dry sterile cotton tip, from which venom could be rinsed with sterile water. This, however, was ineffective due to Australian scorpions having a non-aggressive demeanour compared to other species from countries around the world (Volpe, 2016). The next attempt to milk the scorpions was by enticing them with a prey item to excite them. This was also ineffective as they were timid if people were around them and would prefer to hide than hunt.

Electrical

Due to the manual extraction method not working, the only other option was to use an electrical stimulator which would cause the scorpion to release venom. Lowe and Farrell (2011) provided precise instructions on the construction of such a device using inexpensive components. Their device was constructed exactly following their instructions and the pulse was verified using an oscilloscope. This device could send a precise pulse of electricity to the telson of the scorpion. It was manufactured by a laboratory technician using the design in Figure 3, the stimulator used is seen in Figure 4. Using the modified forceps, a small amount of pressure was applied to either side of the telson and a current of up to 45 volts was applied. However, when applied to the Australian scorpions no venom was extracted.

Figure 3: Electrical stimulator design (Source: Lowe and Farrell, 2011)

Figure 4: Electrical stimulator built for this experiment

A further review of literature indicated that brine could be applied to the telson to improve the electrical conductivity of the circuit. Oukkache et al. (2013), for example, applied brine to the telson which improved conductivity between the forceps and the scorpion. Thus, a supersaturated sterile aqueous solution of NaCl was applied to the telson using an

eye dropper. Each scorpion’s telson was then gripped, using the modified forceps in order for the venom extraction to occur, as seen in Figure 5. The electrical stimulator caused it to release venom from the telson (stinger) and milky white venom was released into the Pasteur pipette. Doing it this way also ensured it was still ethically harvested and that it yielded results. This caused venom to be injected into the end of the pipette so that it could be administered onto the filter paper discs, seen in Figure 6.

Figure 5: Scorpion venom harvesting

Figure 6: Filter discs being treated with venom

Zone of inhibition testing Pilot test

A very small amount of venom was extracted from the scorpions using the method outlined above. To avoid issues with storage, the Pasteur pipettes containing the tiny fraction of venom were moved to the sterile fume cupboard and immediately prepared for use.

Full-Scale Study

Following the encouraging results from the pilot study, the experiment proceeded to the full-scale experiment.

The intended experimental design was to prepare 12 agar plates in sterile conditions and according to the manufacturer’s direction. Six plates were to be lawned with a culture of E. coli K-12 (debilitated strain) and the remaining six with a culture of S. albus. Disks containing venom from U. elongatus would then be applied to the lawns along with a sterile water control disk.

However, by the time this experiment was ready to begin, only two individuals of one species (U. elongatus) remained alive. This was very disappointing but could not be avoided given all the parameters of school research.

Additionally, it was also determined that only a very small amount of venom could be obtained from the scorpions and that they could not be harvested too frequently. It was initially intended to use S. albus for half the plates but when it was determined that only a very small volume of venom was able to be extracted from the scorpion, it was decided that it would be better to have more replicates on one microorganism to yield reliable results. Accordingly, 4 plates were lawned with E. coli (since this had shown promise in the pilot study).

Once the venom had been collected, a small droplet of water was placed onto a sterile agar plate and a tiny amount was drawn up into the Pasteur pipette to rinse the venom from the pipette. The venom would be diluted by far less than the 1:1 - 1:5 maximum ratio advised by Khalid et al. (2012). This slightly diluted venom was then expelled onto sterile filter paper disks which had been placed onto the inoculated plates. The Pasteur pipette only contained enough venom to saturate two paper disks.

Once this had occurred, all plates had one half with the venom treatment and the other with the distilled water which acted as a control. The plates were incubated at 37 degrees for 48 hours and the zone of inhibition on the bacterial growth was then measured using vernier callipers.

Results

Pilot Study

The results of the pilot study are recorded in Table2, Figure 7 and Figure 8 and this shows a photograph of the plate with the measured zone of inhibition for each of the two individual scorpions, which clearly demonstrates that a zone of inhibition had been created by the venom on the E. coli inoculated plate.

Figure 7: Zone of inhibition created by scorpion

Table 2: Table of results showing zone of inhibitions created by water and venom treatments

Female Male

Water: 0mm Water: 1.9mm Venom: 1mm Venom: 2.4mm

Figure 8: Zone of inhibition created by female scorpion

Full-scale Study

Following 48 hours of incubation the plates were carefully examined and found to contain a white opaque filamentous growth that was assumed to be fungal contamination. The filamentous fungal growth obscured clear observation of the plate although, moving the plate in different directions near the window allowed glimpses of the lawn of E. coli underneath. Due to vision being obstructed, it was too hard to get an accurate measurement of the zone and inaccurate results would have been unreliable for the research. It appeared that a zone of inhibition may have been present but it was impossible to measure accurately the size due to the obscured view and so the experiment was abandoned.

Intended Data Analysis

Due to complications in the full-scale study, complete data analysis could not be conducted. However, it had been intended to use two T tests, one comparing the means of the zone of inhibition for E. coli against the means of the water control and then a second T test for the S. albus against the water control. The T test would have allowed a conclusion to be drawn about whether there was a significant difference between the test conditions and the control and if a difference had been found, it could have been concluded that the venom had antimicrobial properties. Promising results were seen on the plates, however, due to the fungal contamination, accurate results were not measurable. Though both groups of means could not be collected, significant zones of inhibition were observed on the full-scale study as well as on the pilot study indicating there was an antimicrobial relationship between the venom and the bacterium that it was tested against.

Discussion

Lessons from the Pilot Study

Much was learned from the pilot study and a great deal of effort and time was invested in overcoming the problems of extracting the scorpion venom and modifying the procedures in the literature for Australian species. Whilst it was not the intended outcome of the research, it is possible to now report a methodology that works for the successful extraction of scorpion venom within a secondary school laboratory and this was a significant undertaking.

There were too few data points in the pilot to allow for means to be calculated and a T-test to be performed but the pilot study confirmed that the methodology is appropriate and can be used to answer the research question.

Lessons from the Full-scale Study

There was insufficient evidence from the abandoned experiment to draw any conclusions in relation to the

research hypothesis. There was promising observation from the contaminated plates that there appeared to be a zone of inhibition and so it is determined that further venom collection and experimentation is both warranted and desirable.

Limitations

This study investigated to what extent would Australian scorpion venom effect the zone of inhibition on bacterial growth. This was carried out by contrasting the zones created from venom of (Urodacus elongatus) and distilled water which acted as the control. The experiment used the bacterium S. albus and E. coli. Data was then recorded over 48 hours for each plate. This supported the hypothesis that the venom would create a zone of inhibition.

One of the most significant hurdles for this research was venom volume. The scorpion specimens did not yield large amounts of venom and it took a long time to regenerate after each milking. Nisani et al. (2012) investigated the regeneration of venom from the South African species of scorpion, Parabuthus transvaalicus and concluded that the scorpion requires at least four days for venom levels to go back to normal after milking. It was observed that U. elongatus took much longer to regenerate than this and it is hypothesised that this may be due to its lower toxicity.

Implications/ Directions for Future research

This research along with other similar studies has shown the importance into studying venom and its properties. With only one species of scorpion (U. elongatus) being used in the experiment against only one microbe (E. coli), the evidence showed that the venom created a visible zone of inhibition. Future research should be conducted to determine if any other species of Australian scorpions possess antimicrobial properties in their venom. For this experiment to be conducted more successfully, a larger collection of scorpions would be required due to the low venom yield per scorpion. A freeze-drying storage facility would also aid in the research in order for venom to be collected and stored for longer periods of time. Tobassum et al. (2018) suggested cold weather resulted in comparatively lower venom yield than in warm weather. In the future conducting the research in summer months would allow for more venom to be collected, therefore, more samples could be tested against.

Conclusion

The research project was to determine if the venom from Australian scorpions would inhibit the growth of microbials. Through extensive research and experimentation, the venom from U. elongatus was successfully harvested to be used in the treatment. This extraction proved out the methodology used to complete the task was successful and replicable for research going forward. Though a limited amount of data was collected, these initial findings have been extremely positive in determining that Australian scorpion (U. elongatus) venom is most likely antimicrobial after creating an obvious zone of inhibition on the E. coli lawned plates in the pilot study (Figure 7, Figure 8). By expanding the number of scorpions in the experiment, using a broader range of microbials and conduct milking during summer months, enough data will be collected to confirm the hypothesis. The implications of this experiment could lead to further research and then the development of new antibiotics in order to respond to the ongoing antibiotic resistance crisis.

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