Senecioylcholine in Heterometrus spinifer scorpion venom activates vertebrate nicotinic acetylcholine receptors

Scorpion venoms are natural sources of neurotoxins that primarily modulate the properties of neuronal ion channels, particularly voltage-gated sodium and potassium channels. However, they also contain a variety of small bioactive molecules that have been largely neglected in the omics era of toxinological research. In the present study, we employed a high-throughput FLIPR screen of arthropod venoms against nicotinic acetylcholine receptors (nAChRs) expressed in the human SH-SY5Y neuroblastoma cell line. The venom of the Malaysian forest scorpion Heterometrus spinifer (family: Scorpionidae) proved to be an agonist of nAChRs, and the small choline ester senecioylcholine was identified as being responsible for the observed effect. Synthetic senecioylcholine and its isomers tigloylcholine and angeloylcholine were not insecticidal when injected into sheep blowflies. Based on previous reports of senecioylcholine being toxic to vertebrates along with the agonistic effect we observed on human nAChRs, we postulate that senecioylcholine in scorpion venom is likely to assist with vertebrate prey capture and defense against vertebrate predators. Since senecioylcholine has only been previously reported in marine gastropods and moths, its presence in scorpion venom seems to represent an unusual case of convergent evolution.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. Each pentamer is assembled from a combination of four distinct categories of transmembrane subunits, namely α, β, γ, and δ (Changeux, 2020). The most abundant functionally characterized subtypes of nAChRs within the mammalian central nervous system (CNS) are α7 homomeric receptors that bind α-bungarotoxin, a peptidic snake neurotoxin, with high affinity, and α4β2 heteromeric nAChRs that have high affinity for nicotine (Ho et al., 2020). Acetylcholine (ACh), an excitatory neurotransmitter widely distributed across all the kingdom of life, is the endogenous agonist of nAChRs (Kawashima and Fujii, 2008), including those in prokaryotes (Fujita et al., 2024) and archaea (Yamada et al., 2005). As neurotransmitter, ACh mediates cholinergic transmission in the central and peripheral nervous systems (Wessler and Kirkpatrick, 2017). Physiological functions regulated by ACh include muscle contraction, immune responses, pain signaling, and reproduction in mammals and honeybees (Wessler and Kirkpatrick, 2017). nAChRs are found in neuronal and non-neuronal cell types of vertebrates and invertebrates. However, in insects the nAChRs are strictly located in the CNS while mammals also employ nAChR in the autonomic nervous system and the neuromuscular junction (Rosenthal and Yuan, 2021).

Animal venoms are rich sources of biologically active compounds, including peptides, proteins and small molecules, that are primarily used for capture prey or defense (Herzig et al., 2020). Given the important physiological functions regulated by ACh, it is unsurprising that many venomous organisms evolved toxins that interfere with cholinergic transmission to facilitate prey capture or defense against predators. For example, cone snail venoms contain a wide variety of toxins that modulate the activity of AChRs (Herrero et al., 1999; Jin et al., 2014; Rybin et al., 2020). Acylpolyamines from spider venoms have bene shown to inhibit AChRs (Radis-Baptista and Konno, 2024). Conversely, only one spider-venom peptide, κ-atracotoxin-Hv1c (κ-ACTX-Hv1C) from the Blue-Mountains funnel web spider Hadronyche versuta, has been reported as a positive allosteric modulator of insect nAChRs (Windley et al., 2017). In scorpions, there is evidence that the venom of Buthus occitanus tunetanus depolarizes muscle cells via modulation of nAChRs, but the active venom component has not been identified (Cheikh et al., 2007). The peptide toxins OSK-1 from the Central Asian scorpion Orthochirus scrobiculosus and HelaTx1 from the Vietnamese forest scorpion Heterometrus laoticus were reported to inhibit nAChRs (Kasheverov et al., 2019). Except for the presence of ACh in the venom of the Malaysian forest scorpion Heterometrus spinifer (Nirthanan et al., 2002), no other agonists of nAChRs have yet been identified from scorpion venoms.

In this study, a high-throughput fluorescent assay was used to screen 22 spider venoms and four scorpion venoms for modulators of human nAChRs expressed in SH-SY5Y neuroblastoma cells, which endogenously expressed α7 and α3* nAChRs (Lukas et al., 1993; Puchacz et al., 1994; Vetter and Lewis, 2010). We determined that venom from H. spinifer (family: Scorpionidae) caused an agonistic effect on the calcium response mediated by nAChRs. The active venom component was then isolated and identified as the choline ester senecioylcholine.

Venoms

Arachnid venoms were extracted by aggravation and/or mild electrical stimulation as previously described (Guo et al., 2018). For H. spinifer, the venom from 10 specimens of undetermined sex was pooled and used for all subsequent experiments.

Materials used

RPMI (Roswell Park Memorial Institute) medium, (+)-L glutamine; DPBS (Dulbecco’s Phosphate Buffered Saline) without CaCl 2 and MgCl 2; 0.25% Trypsin/EDTA were from Gibco® Life Technologies (Mulgrave, Vic, Australia). Fetal Bovine Serum (FBS) were from Scientific Life (Clayton, Vic, Australia). PNU-120596 (1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl) urea) was from Sigma-Aldrich. FLIPR TETRA pipet tips and Calcium-4 no-wash kit were from Molecular Devices (Sunnyvale, CA, USA). 384-Well cell/imaging microplates (flat-well, clear-bottom black polystyrene) and 384-Well reagent microplates (clear-well, round-bottom polypropylene) were from Corning (Lowell, MA, USA). Nicotine from Sigma-Aldrich (Castle Hill, New South Wales, Australia) was prepared as a stock solution of 100 mM in Milli-Q water. The composition of the Physiological Salt Solution (PSS) used in all the assays was NaCl 140 mM, glucose 11.5 mM, KCl 5.9 mM, MgCl 2 1.4 mM, NaH 2 PO 4 1.2 mM, NaHCO 3– 5 mM, CaCl 2 1.8 mM, HEPES 10 mM, and pH adjusted at 7.4 using NaOH (Vetter, 2012).

Cells lines and culture

SH-SY5Y human neuroblastoma cells (a kind gift from Victor Diaz, Max Planck Institute for Experimental Medicine, Göttingen, Germany) were maintained at 37°C with 5% (v/v) CO 2 in RPMI medium which contained 2 mM L-glutamine and 15% (v/v) FBS. Cells were passaged every 3–5 days using 0.25% trypsin/EDTA at a dilution of 1:7. The SH-SY5Y cells were passaged a maximum of 10–20 times in antibiotic-free medium, prior to use for assays.

High-throughput FLIPR calcium assay

Crude arachnid venoms at a concentration of 1 μg/30 μL were screened against SH-SY5Y cells using a FLIPR assay designed to measure changes in the intracellular calcium concentration. Agonistic and antagonistic activity at nAChRs was measured according to previous methods (Vetter and Lewis, 2010). Briefly, ~ 30,000 SH-SY5Y cells per well were seeded on uncoated black-walled 384-well imaging plates (Corning, Lowell, MA, USA) and maintained at 37°C and 5% CO 2 for 48 h before running the experiment. Once a 95% confluent monolayer was obtained, the cells were loaded with Calcium 4-no-wash-dye (Molecular Devices) diluted in PSS for 30 min at 37 °C and 5% CO 2. After loading the calcium-sensitive dye, the cell plate was transferred to a FLIPR TETRA (Fluorescent Imaging Plate Reader, Molecular Devices, Sunnyvale, CA, USA) and fluorescent calcium responses were recorded with excitation wavelength of 470–495 nm and an emission wavelength of 515–575 nm. A two-step addition protocol was used for all FLIPR measurements, with 300 s of data recorded after the first addition (PSS or crude venom or venom fractions) and another 300 s recorded after the second addition (30 μM nicotine). The fluorescent baseline was recorded for 5 s before the first addition. To test the activity of venoms or venom fractions, the venoms (1 μg) or venom fractions (5 μg venom equivalent for primary HPLC fractions and 14.4 μg venom equivalent for secondary HPLC fractions) were added first, followed by a second addition of 30 μM nicotine. To confirm if the calcium response induced by nicotine, crude venom or venom fractions was mediated by nAChRs, 30 μM d-tubocurarine, a potent nAChR inhibitor (Bowman, 2006), was included in the dye loading buffer.

Isolation of the active compound from H. spinifer venom

Crude lyophilized H. spinifer scorpion venom (1 mg) was dissolved in 1 mL of 5% of solvent B (90% acetonitrile containing 0.05% TFA) and centrifuged at 5,000 g for 15 min at 4°C. After centrifugation, the supernatant was loaded onto a Phenomenex (Sydney, Australia) C18 reversed-phase (RP) HPLC column (Jupiter 250 x 4.60 mm; 5 µm particle size; 300 Å pore size) and fractionation performed using a Shimadzu Prominence HPLC system (Shimadzu Scientific Instruments Oceania, Rowville, VIC, Australia). Venom fractionation was performed using a flow rate of 1 mL/min with the following gradient of solvent B in solvent A (Milli-Q water + 0.05% TFA): 0–5 min: 5%; 5–10 min: 5–20%; 10–80 min: 20–50%. The elution profile was recorded at 214 nm and fractions were collected manually. Venom fractions were then lyophilized, dissolved in Milli-Q water, and stored at –20°C until required.

Active fraction 10 from H. spinnifer venom (F10, 720 μg venom equivalent) was subjected to a second round of HPLC purification using a shallower acetonitrile gradient on a Vydac ® 218TP C18 analytical RP-HPLC column (250 x 4.60 mm; 5 µm particle size; and 300Å pore size; Grace, Columbia, MS, USA) and processed through a Shimadzu Prominence HPLC system at a flow rate of 1 mL/min using the following gradient of solvent B in solvent A: 0–5 min: 5%; 5–10 min: 5–10%; 10–50 min: 10–30%; 50–55 min: 30–80%.

Determination of molecular mass of active venom component

The active component isolated from H. spinifer venom was dissolved in 5% HPLC-grade acetonitrile and analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS)-TOF performed on a Model 4700 Proteomics Bioanalyser (Applied Biosystems) operating in the positive ion linear mode. The sample (0.4 µL) was mixed on a MALDI plate with 0.4 µL of α-cyano-4-hydroxycinnamic acid (CHCA) matrix (6 mg/mL) dissolved in acetonitrile/Milli-Q water/formic acid (50:45:5, v/v). The MALDI-TOF spectra did not reveal any masses higher than 1 kDa. Thus, the active fraction was further analyzed by LCMS using an Agilent 1100 series LCMS (Agilent Technologies Australia, Mulgrave, VIC, Australia) comprised of HPLC modules equipped with diode array detector and single quadrupole mass detector in both positive and negative ion modes. The sample was run on an Agilent Zorbax-C 8 column (4.6 mm x 150 mm, 5 μm particle size) using a gradient of 10–100% acetonitrile in Milli-Q water (with constant 0.05% formic acid modifier) over 15 min, using a flow rate of 1 mL/min.

Identification of active venom components using HRMS and LCMS

High-resolution (HR) electrospray ionization (ESI) MS measurement were obtained on a Bruker microTOF (Bruker Pty Ltd, Preston, VIC, Australia) with an ESI probe by direct infusion in acetonitrile at 3 μL/min using sodium formate clusters as an internal calibrant. LCMS was performed on native compound co-injected with synthetic choline esters using an Agilent 1100 series LCMS with an Agilent Zorbax-C 18 aqua column (4.6 x 150 mm, 5 μm particle size). Isocratic elution was performed using 4% acetonitrile/H 2 O (with constant 0.05% formic acid modifier) at a flow rate of 1 mL/min, with single ion extraction at 186 [M] +.

Chemical synthesis of choline esters

General experimental details: Nuclear magnetic resonance (NMR) spectra were acquired on an Avance 600 MHz spectrometer (Bruker, Preston, VIC, Australia) equipped with a 5 mm PASEL 1 H/D- 13 C Z-Gradient probe. The spectra were acquired at 25 °C in CD 3 OD and referenced to residual signals (δ H 3.30 and δ C 49.15 ppm) in deuterated solvents. High-resolution ESIMS measurements were obtained on a Bruker micrOTOF mass spectrometer by direct infusion in MeCN at 3 μL/min using sodium formate clusters as an internal calibrant. HPLC-grade chromatography solvents from Labscan (ChemSupply Australia, Gillman, SA, Australia) were filtered/degassed through a 0.45-μm polytetrafluoroethylene (PTFE) membrane prior to use. Deuterated solvents were purchased from Cambridge Isotopes (Novachem, Heidelberg West, Vic, Australia). All chemicals used in the synthesis were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia).

Synthesis of chlorocholine chloride

Choline chloride (279 mg, 2 mmol) was dried under high vacuum at 200 °C for 2 h. Thionyl chloride (SOCl 2, 2 mL) was added, and the solution was heated under reflux for 1 h. After cooling, the solvent was evaporated to dryness. Solid residue was redissolved in methanol (3 x 2 mL), and re-evaporated to dryness. The resultant white solid was dried under high vacuum at 80 °C for 2 h.

Synthesis of tigloylcholine

K 2 CO 3 (138 mg, 1 mmol in 10 mL water) was added to a solution of tiglic acid (200 mg, 2 mmol) in a dioxane–water (3:2) mixture (10 mL). After stirring for 10 min, the mixture was evaporated to dryness and again dried under high vacuum at 70 °C for 1 h. Chlorocholine chloride and anhydrous DMSO were added and the mixture heated at 60 °C for 48 h. DMSO was removed by vacuum distillation and the residue was purified by using reversed-phase column chromatography to afford tigloylcholine as a light brown hygroscopic solid. 1 H NMR (600 MHz, CD 3 OD): δ H 6.92 (qq, J = 7.1, 1.3 Hz, H-3), 4.59 (m, H 2 -1'), 3.75 (m, H 2 -2'), 3.22 (s, 9H, NMe 3), 1.85 (dq, J = 1.3, 1.1 Hz, H 3 -5), 1.82 (dq, J = 7.1, 1.1 Hz, H 3 -4) (see Supplementary Figure 1); 13 C NMR (150 MHz, CD 3 OD): δ C 168.4 (C-1), 140.3 (C-3), 129.1 (C-2), 66.4 (C-2'), 59.1 (C-1'), 54.60 (NMe), 54.58 (NMe), 54.55 (NMe), 14.7 (C-4), 12.3 (C-5) (see Supplementary Figure 2); HRMS calc. for C 10 H 20 NO 2 + 186.1489, found m/z = 186.1491.

Synthesis of senecioylcholine

As described for the synthesis of tigloylcholine, starting with choline chloride (2 mmol, 279 mg) and 2-methyl butenoic acid (2 mmol, 200 mg), senecioylcholine was synthesized and purified using reversed-phase column chromatography, to afford senecioylcholine as a white hygroscopic solid. 1 H NMR (600 MHz, CD 3 OD): δ H 5.76 (qq, J = 1.1, 1.1 Hz, H-2), 4.54 (m, H 2 -1'), 3.73 (m, H 2 -2'), 3.22 (s, 9H, NMe 3), 2.18 (d, J = 1.1 Hz, H 3 -4), 1.93 (d, J = 1.1 Hz, H 3 -5) (see Supplementary Figure 3); 13 C NMR (150 MHz, CD 3 OD): δ C 166.8 (C-1), 161.2 (C-3), 115.8 (C-2), 66.4 (C-2'), 58.1 (C-1'), 54.66 (NMe), 54.63 (NMe), 54.61 (NMe), 27.6 (C-4), 20.6 (C-5) (see Supplementary Figure 4); HRMS calc. for C 10 H 20 NO 2 + 186.1489, found m/z = 186.1485.

Synthesis of angeloylcholine

As described for the synthesis of tigloylcholine, starting with choline chloride (2 mmol, 279 mg) and angelic acid (2 mmol, 200 mg), angeloylcholine was synthesized and purified using reversed-phase column chromatography, to afford angeloylcholine as a white hygroscopic solid. 1 H NMR (600 MHz, CD 3 OD): δ H 6.23 (qq, J = 7.2, 1.3 Hz, H-3), 4.60 (m, H 2 -1'), 3.75 (m, H 2 -2'), 3.22 (s, 9H, NMe 3), 2.00 (dq, J = 7.2, 1.3 Hz, H 3 -4), 1.91 (dq, J = 1.3, 1.3 Hz, H 3 -5) (see Supplementary Figure 5); 13 C NMR (150 MHz, CD 3 OD): δ C 167.9 (C-1), 141.8 (C-3), 128.0 (C-2), 66.4 (C-2'), 58.6 (C-1'), 54.59 (NMe), 54.56 (NMe), 54.54 (NMe), 20.8 (C-4), 16.2 (C-5) see Supplementary Figure 6); HRMS calc. for C 10 H 20 NO 2 + 186.1489, found m/z = 186.1483.

Analyses of FLIPR data

Crude venom or venom fractions were tested in triplicate and all data were analyzed using ScreenWorks ® software, version 3.1 (Molecular Devices). Raw fluorescence values obtained after addition of nicotine, venom, or venom fractions were normalized to baseline fluorescence values measured during the period between 0 and 5 s after starting the assay. Once the data were corrected for the baseline fluorescent reads, the response to nicotine was normalized to 100% and data were exported and analyzed using Microsoft Excel for Mac 2011 v14.4.3. Data are expressed as mean ± standard error of the mean (SEM) from n = 3 independent experiments.

Statistical analyses

Statistical analysis of the mean FLIPR calcium responses were performed according to two-way ANOVA followed by Šídák’s multiple comparisons test of dTb, or venom fraction treatments against the respective nicotine control group using (Prism v11.0, GraphPad Software, San Diego, CA, USA).

Insecticidal toxicity screens of choline esters in blowflies

Sheep blowflies (Lucilia cuprina) were injected intrathoracically with 3 to 4 different doses of the synthetic choline esters senecioylcholine, tigloylcholine, and angeloylcholine and their behavior monitored at 15 min, 30 min, 1 h, 5 h and 24 h after injection according to our previously published methods (Guo et al., 2018).

We conducted a high-throughput FLIPR screen of 22 spider and four scorpion venoms against nAChRs endogenously expressed in human neuronal SH-SY5Y cells (Figure 1). Several venoms caused to an increase in fluorescence, consistent with an increase in intracellular calcium (Figure 1A), but only the calcium response evoked by H. spinifer venom was inhibited by tubocurarine, a potent nAChR inhibitor (Figure 1B). The venom of the scorpion H. spinifer was therefore selected for the subsequent experiments to identify the venom component responsible for increasing the nAChR-mediated activity.

Isolation of the bioactive venom component

We next performed activity-guided fractionation of H. spinifer venom to isolate the component responsible for agonism of human nAChRs. Fractionation of the venom using C18 RP-HPLC revealed a complex venom profile (Figure 2), but only fraction 10 eluting at 13.5 min activated nAChRs in the FLIPR assay. F10 increased nAChR activity by 43.1% compared to control, while co-addition of tubocurarine reduced the activity to 50.8% of the control value (Figure 3). Thus, fraction 10 recapitulates the nAChR agonistic activities of crude H. spinifer venom.

Fraction 10 was therefore further fractionated using a shallower acetonitrile gradient on C18 RP-HPLC, which yielded the active subfraction 4 (SF4) eluting at 11.1 min (Figure 2 inset). LCMS analysis showed that SF4 only contained one major compound, with a mass of 186.1 Da in positive mode (see Supplementary Figure 7). SF4 increased nAChR activity by 73.4% compared to control, while addition of tubocurarine reduced the activity almost completely to 2.0% (Figure 3).

Identification of the active venom component by HRMS

Analysis of SF4 using MALDI-TOF-MS revealed no masses larger than 1 kDa. Due to the noise caused by the matrix (CHCA), it is difficult to detect masses smaller than 1 kDa by MALDI-TOF-MS. Therefore, SF4 was further analyzed using QTOF high-resolution mass spectrometry (HRMS), which revealed a single compound attributed to a molecular formula C 10 H 20 NO 2 (m/z 186.1504 [M] +, Δmmu –1.5) (see Supplementary Data Supplementary Figure 7, S8). Database searches revealed that the mass formula of C 10 H 20 NO 2 corresponds to a previously described choline ester named senecioylcholine, isolated from the hypobranchial gland of several molluscan species in the Muricoidea family (Roseghini et al., 1996). However, there are two isomers of senecioylcholine, namely tigloylcholine (TGCh) and angeloylcholine (ANCh), with identical mass formula, and based on HRMS data alone it is impossible to determine which of these isomers corresponds to the native venom-derived compound. Thus, all three isomers were chemically synthesized and LCMS co-elution studies were performed to identify the native compound. We found that only senecioylcholine co-eluted with the active nAChR agonist purified from SF4 of H. spinifer venom (Supplementary Figure 9). We therefore conclude that the nAChR agonist in H. spinifer venom is senecioylcholine.

Insect toxicity

At the highest doses injected into sheep blowflies, senecioylcholine (10.8 μmol/g) caused no mortality. The maximum extent of induced paralysis was 20% at 30 min, but this reduced to 0% at 24 h (Supplementary Table 1). Tigloylcholine (10.1 μmol/g) caused no paralysis or mortality (Supplementary Table 2). Angeloylcholine (9.0 μmol/g) induced 10% paralysis and mortality at 24 h (Supplementary Table 3).

The only previous reports of senecioylcholine in Nature are in the hypobranchial glands of marine gastropods in the Muricidae family (Roseghini et al., 1970, Roseghini et al., 1996; Turner et al., 2018) and the defensive glands of the tiger moth Arctia caja (Bisset et al., 1960; Morley and Schachter, 1963). In addition to senecioylcholine, the related choline esters murexine and dihydromurexine are commonly found in the hypobranchial glands of murucid gastropods, and they are often present at very high concentration (Roseghini et al., 1970, Roseghini et al., 1996).

Muricid gastropods are drilling predators that feed on bivalves and snails by rasping a hole and opening their shells. The function of their hypobranchial gland is to secrete mucus for trapping and cementing particulate matter sucked into the mantle cavity with the respiratory water current prior to its expulsion and to produce Tyrian blue dye (Benkendorff, 2013; Fretter and Graham, 1994; Yonge, 1947). A precursor of the Tyrian blue is stored in the hypobranchial gland as a choline ester (Benkendorff, 2013). While the ecological role of these choline esters in predatory gastropods is uncertain, the mucus secretions from these molluscs are known to deter predators such as crustaceans, fish, and star fish (Derby, 2007). Similarly, senecioylcholine is found at high concentration in the paired prothroracic defensive glands of the tiger moth (Bisset et al., 1960; Morley and Schachter, 1963), and it is presumably present in the irritant fluid ejected from these glands to deter close-range attackers, primarily birds and bats.

So what is the ecological role of senecioylcholine in the venom of H. spinifer ? One possible role is incapacitation of insect prey. However, high doses of senecioylcholine did not induce significant insecticidal activity when injected in sheep blowflies, which are commonly used as a susceptible model organism for examining the insecticidal effects of venom toxins (Guo et al., 2018). Moreover, H. spinifer is a very large scorpion (10–15 cm length) (Kawai et al., 2023) with massive pedipalps (pincers) that are used to crush insect prey without the need for venom. While we examined the activity of senecioylcholine against sheep blowflies as the only insect species that was tested (albeit at very high doses), it seems highly unlikely that senecioylcholine is used for capture of insect prey.

Another possible function is defense against pathogens. In captivity, buthid scorpions have been observed to spread venom across their dorsal surface, possibly for hygienic purposes. Consistent with such a role, antimicrobial peptides have been purified from their venoms (Harrison et al., 2014). Within the genus Heterometrus, the antimicrobial peptide heteroscorpine-1 was isolated from venom of the forest scorpion H. laoticus, which also causes abdominal paralysis in crickets (Uawonggul et al., 2007). We did not perform any antimicrobial assays, so we cannot rule out a role for senecioylcholine or its isomers in defense against environmental pathogens. However, to date, there is no evidence that senecioylcholine or any of the related choline esters found in the hypobranchial gland of gastropods are antimicrobial.

A more likely role for senecioylcholine in H. spinifer venom is defense against vertebrate predators, consistent with the defensive role that this choline ester appears to play in marine gastropods and the tiger moth, and the fact that H. spinifer uses venom predominantly in a defensive capacity. The common physiological property of the choline esters found in muricid gastropods, including senecioylcholine, is their ability to evoke excitatory paralysis of the skeletal musculature, an nAChR-mediated action that is suppressed by the anticholinergic agent atropine (Roseghini et al., 1970, Roseghini et al., 1996). Murexine causes muscle paralysis in dogs, fish, frogs, and octopods, and severe muscle contracture in birds (Roseghini et al., 1970, Roseghini et al., 1996). The latter activity is particular relevant given that birds are major predators of scorpions and the tiger moth.

Forest scorpions are opportunistic predators that feed on diverse prey, including small vertebrates such as frogs, lizards, and small rodents. Murexine induces muscle paralysis in vertebrates, and high doses are lethal to birds and mice (Roseghini et al., 1970, Roseghini et al., 1996), and thus we cannot rule out the possibility that senecioylcholine is also used in prey capture. Heterometrus venom also contains high concentrations of acetylcholine (~80 µM), which was reported to cause reversible venom-induced contractions in the chick biventer cervicis muscle (Nirthanan et al., 2002). These effects of acetylcholine would be potentiated by the nAChR agonistic activity of senecioylcholine, thereby facilitating the incapacitation of vertebrates.

In conclusion, based on our data and previous reports, the most likely role of senecioylcholine in H. spinifer venom is for defense against vertebrate predators and to facilitate capture of small vertebrate prey. Given that senecioylcholine has previously only been found in moths and molluscs, its presence in the venom of scorpions appears to represent an interesting case of convergent evolution. It remains to be determined whether choline esters are ubiquitous in scorpion venoms or limited to specific families or genera.