Acetylcholinesterase inhibitory potential of scorpion venom in Aedes aegypti (Diptera: Culicidae)

Scorpion venom contains a variety of neurotoxins which interact with ion channels and affect their activities. The present study was designed to evaluate the potential of scorpion venom as acetylcholinesterase (AChE) inhibitor by using Aedes aegypti as model organism. Venoms of two species, Hottentota tamulus (Fabricus, 1798) and Androctonus finitimus (Pocock, 1897) were selected for this study. Two peptides (36 kDa from H. tamulus and 54 kDa from A. finitimus ) were separated from scorpion venom by using HPLC. Selected peptides caused significantly higher mortality in larvae and adults of Aedes aegypti than control (no mortalities were observed in control groups). Significant acetylcholinesterase (AChE) inhibitory potential of both peptides was recorded by spectrophotometer. The peptide of A. finitimus caused significantly higher mortality (95±1.53% in larvae and 100% in adults) than the peptide of H. tamulus (84.33±2.33% in larvae and 95.37±1.45% in adults). While H. tamulus peptide was more efficient in reducing AChE activity (0.029±0.012 in larvae and 0.03±0.003 in adults) than the peptide of A. finitimus (0.049±0.005 in larvae and 0.047±0.001 in adults). It was concluded that H. tamulus venom peptide was more efficiently reducing AChE activity, thus it could be a potential bio-insecticide which can be synthesized at industrial scale for the control of harmful insects. (95 ± 1,53% em larvas e 100% em adultos) que o peptídeo de H. tamulus (84,33 ± 2,33% em larvas e 95,37 ± 1,45% em adultos). Enquanto o peptídeo de H. tamulus foi mais eficiente na redução da atividade da AChE (0,029 ± 0,012 em larvas e 0,03 ± 0,003 em adultos) do que o peptídeo de A. finitimus (0,049 ± 0,005 em larvas e 0,047 ± 0,001 em adultos). Concluiu-se que o peptídeo do veneno de H. tamulus foi mais eficiente na redução da atividade da AChE, podendo ser um potencial bioinseticida que pode ser sintetizado em escala industrial para o controle de insetos nocivos. Palavras-chave: escorpiões, peptídeos, HPLC, atividade da AChE, Aedes aegypti.


Introduction
Being ecofriendly in nature, bio-pesticides are getting popular for the control of target insect populations (Ortiz and Possani, 2015). Scorpion venom, due to its specific toxins against insects, has become the remarkable candidates for the production of bio-pesticides (Tahir et al., 2015). Scorpions use their venom for self-defense and to subdue prey. Scorpions of Buthidae family contain distinctive bio-active neurotoxins (Gwee et al., 2002;Tan et al., 2006;Radha, 2014;Diaz-Garcia et al., 2015). Many neurotoxins of venom target the nervous system of insects, disturbing their ion channels and neural activity (Radha, 2014). The excitatory insect toxins may be helpful O veneno do escorpião contém uma variedade de neurotoxinas que interagem com os canais iônicos e afetam suas atividades. O presente estudo foi desenhado para avaliar o potencial do veneno de escorpião como inibidor da acetilcolinesterase (AChE) usando o Aedes aegypti como organismo modelo. Venenos de duas espécies, Hottentota tamulus (Fabricus, 1798)  Acetylcholinesterase inhibitory potential of scorpion venom in Aedes aegypti (Diptera: Culicidae) kept in plastic cups containing 7 ml water. Larvae were divided into seven groups, each group containing 10 larvae. No treatment was applied to the control group; however, the larvae 1 st , 2 nd and 3 rd experimental groups were treated by mixing 10µg/ml, 20 µg/ml and 30 µg/ml of venom of H. tamulus in the water, respectively. Similarly, larvae of experimental groups 4 th , 5 th and 6 th were respectively treated with 10µg/ml, 20 µg/ml and 30 µg/ml of A. finitimus venom. The mortality rate in control and experimental groups was assessed after 24 h post treatment. After that, each larvae was homogenized in 600 µl sodium phosphate buffer (0.1 M; PH 7.0) containing 0.01% (w/v) of Triton X-100. This homogenate was centrifuged at 13500 rpm for five minutes. The supernatant was collected and used further for enzyme analysis.
Ae. aegypti adults (n=70) were divided into one control and six experimental groups. Control group was left untreated however, 1 st , 2 nd and 3 rd experimental groups was topically treated with 10µg/ml, 20 µg/ml and 30 µg/ml venom of H. tamulus and experimental groups 4 th , 5 th and 6 th were topically treated with 10µg/ml, 20 µg/ml and 30 µg/ ml venom of A. finitimus. The mortality rate was assessed after 24 h post treatment. For enzyme estimation the head of each adult mosquito was removed and homogenized in 600 µl sodium phosphate buffer (0.1 M; PH 7.0). Further processing was same as described above.

Ellman's assay and statistical analysis
The AChE activity was estimated following the method of Ellman et al. (1961). In this method, DTNB was used as a reagent and Acetylthiocholine iodide as substrate. The absorbance was recorded at 412 nm for 3 min using spectrophotometer (APELPD-303S). The whole experiment was performed in triplicate.
Normality of the data was assessed before data processing. One way-ANOVA followed by Tukey`s test was applied to compare the AChE activity among different groups. Probit analysis was used to compute LC 50 values. Statistical Package for Social Sciences (SPSS version 16.0) and Minitab (13.4) were used for statistical analyses.

Results
The recorded molecular weights of peptide fractions separated from the venoms of H. tamulus and A. finitimus were 36 KDa and 54 KDa respectively. With different concentration of these peptides, the significant mortalities were observed in Ae. aegypti larvae and adults (Table 1). Venom concentration-dependent mortality was observed. Maximum mortality rate in larvae (84.33±2.33%) and in adult (95.37±1.45%) were recorded with 30 µg/ml of venom peptide of H. tamulus. While 95±1.53% and 100% deaths were recorded with the same concentration of venom peptide of A. finitimus in larvae and adults of Ae. aegypti respectively.
A significant decline was found in AChE activity in Ae. aegypti larvae in venom treated groups than control (P< 0.05; Figure 1). It was depicted from the results that A. finitimus venom peptide has more inhibitory potential than H. tamulus venom peptide (Figure 1). The significant in designing new insect selective bio-pesticides because of their special modes of action like they target only the nervous system of insects. The neurotoxins present in the scorpion venom are also involved in disturbance of neurotransmitters released at synaptic junctions of nerves (Theakston et al., 2003;Ozkan and Kat, 2005).
A variety of compounds such as hyaluronidase, lipids hydrolyzing enzymes, mucopolysaccharides, serotonin, histamine, proteinases, histamine releasers and different polypeptide compounds like discreplasminin are present in the scorpion venom (Valdez-Cruz et al., 2007;Feng et al., 2008). Scorpion venom also contain PLA2 (phospholipase A2), phosphatases and acetylcholinesterase inhibitors (Jalali et al., 2012). These enzymes play a major role in various morbid alterations in circulatory system, central nervous systems and skin (Seyedian et al., 2010). The estimated number of components in scorpion venom range from 72 in Androctonus spp. to 600 in Hottentota spp. (Batista et al., 2007;Oukkache et al., 2008). The neurotoxins in venom divert the action potential, resulting in the release of neurotransmitters from cholinergeic and adrenergic neurons (Theakston et al., 2003). They also block the neuromuscular transmission by stopping the ACh release (Gwee et al., 2002;Ozkan and Kat, 2005;Cordeiro et al., 2015).
Keeping in view, these specific properties of scorpion venom, the current study was intended. The plan was to separate the peptides from the venom of H. tamulus and A. finitimus venom and use them against Aedes aegypti, not only to evaluate their mortality but also their AChE inhibitory potential.

Venom collection and characterization
Scorpions (total 60 scorpions) were collected from district Sargodha, Punjab, Pakistan and kept in the laboratory at Department of Zoology, University of Sargodha. Two species of scorpions, Hottentota tamulus (30 scorpions) and Androctonus finitimus (30 scorpions) were used for this study. These scorpions were maintained in the laboratory following the method described in Yaqoob et al. (2017). Venom was collected by electrical stimulation method described by Ozkan and Filazi (2004) and Yaqoob et al. (2017). From this venom, 6 mg venom was dissolved in 0.05% trifloroacetic acid (TFA) in graded water of HPLC. It was centrifuged for 15 mins at 14000 rpm and supernatant was collected. The peptide fractions were separated by HPLC (LC.20 AT SPD-M20A) from the crude venom on C18 column at the flow rate of 1ml/minute. Fractions were collected manually and stored at -20°C. The fractions with the highest peak and in more amount were selected. Approximate molecular weights of two selected fractions were determined by SDS-PAGE by comparing with standard protein markers.

Model organisms and toxicity assay
Aedes aegypti (larvae and adults) were collected from Insectary, GC University, Lahore, Pakistan. The larvae were Brazilian Journal of Biology, 2024, vol. 84, e259506 3/5 Acetylcholinesterase inhibitory potential of scorpion venom difference in AChE activity between venom treated adults and control in Ae. aegypti was also observed (P< 0.05; Figure 2). Figure 2 showed that the least AChE activity was observed with 20 µg/ml of A. finitimus and with 30 µg/ml of H. tamulus venom peptide in Ae. aegypti adults. The comparison between the activities of AChE in larvae and adults, when treated with 30 µg/ml venom peptide respectively, was depicted in Figure 3. A significant difference between the activities of AChE was observed in the control and experimental groups while H. tamulus was found to be more effective in terms of reducing AChE activity.

Discussion
Scorpions with their neurotoxic venom also contain certain insect specific peptides thus behaving as bio-pesticide (Ghane et al., 2008). In view of this capability of scorpion venom, current study was focusing to control the population of Ae. aegypti by using scorpion venom. The idea was to target the AChE of Ae. aegypti that leads to accretion of ACh at synapse, thus upsetting the nerve impulse transmission. In the present study, the peptide fraction of A. finitimus seems to be more potent killer of Ae. aegypti as compared to H. tamulus venom peptide. It was found that maximum deaths in larvae were recorded against 30 µg/ml venom of H. tamulus and A. finitimus venom. Similarly, 30 µg/ml dose of each venom peptide caused maximum mortality in adult mosquitoes. Riaz et al. (2017) also reported that H. tamulus crude venom is highly effective against Rhopalosiphum erysimi. Selected scorpion venom peptides have potential to act as an insecticidal agents and showing significant mortalities as compared to control group. Tahir et al. (2015) also reported higher mortality in organism treated with venom than control. Riaz et al. (2019) reported the efficacy of A. finitimus and H. tamulus venom peptides against M. domestica.
The current results suggested that with increased venom concentration, AChE activity decreases. Basically, neurotoxins in venom disturbing the voltage dependent sodium channels that results in repetitive firing of somatic, sympathetic and para-sympathetic neurons. It further leads to abnormal nerve impulse transmission and enhanced the levels of neurotransmitters like adrenaline, nor-adrenaline, aspartate and acetylcholine. As AChE enzyme is involved in the hydrolysis of ACh, so inhibition of AChE causing the accumulation of ACh at nerve synapses. According to the results presented here, scorpion venom showing its anti-AChE activity effectively. Ozkan et al. (2007) found the sharp decline in AChE level after 12 th hour of venom introduction in rats as compared to control group. They used the venom of Androctonus spp. and found it to target the ACh receptors and increasing ACh concentration, thus acting as neurotoxic agent. These results are in accordance to our present findings that AChE activity was significantly lower in venom treated group as compared to the control. Likewise, Vatanpour (2003) and Abdel-Rahman et al. (2010) reported reduction in AChE activity in chicks and cockroaches when exposed with scorpion venom respectively.

Conclusions
Both peptide fractions due to their bio-insecticidal properties have potential to kill the target insects. The venom has have potential to target the ion-channels and to release the neurotransmitters like ACh. It was observed that H. tamulus venom peptide was more efficiently reducing AChE activity, thus can be used to control the harmful insects by targeting their neuromuscular junctions.