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55 56 57 58 59 60 61 journal homepage: www.elsevier.com/locate/toxicon 62 63 64 65 66 67 68 69 Arie van der Meijden*, Pedro Coelho, Mykola Rasko 70 71 rio de Vaira ~o, Rua Padre Armando, Quintas, N 7, CIBIO Research Centre in Biodiversity and Genetic Resources, InBIO, Universidade do Porto, Campus Agra 72 ~o, Vila do Conde, Portugal 4485-661 Vaira 73 74 75 a r t i c l e i n f o a b s t r a c t 76 77 Article history: Scorpions have been shown to control their venom usage in defensive encounters, depending on the Received 12 December 2014 78 perceived threat. Potentially, the venom amount that is injected could be controlled by reducing the flow Received in revised form 79 speed, the flow duration, or both. We here investigated these variables by allowing scorpions to sting into 21 April 2015 an oil-filled chamber, and recording the accreting venom droplets with high-speed video. The size of the 80 Accepted 22 April 2015 spherical droplets on the video can then be used to calculate their volume. We recorded defensive stings 81 Available online xxx of 20 specimens representing 5 species. Significant differences in the flow rate and total expelled volume 82 were found between species. These differences are likely due to differences in overall size between the 83 Keywords: species. Large variation in both venom flow speed and duration are described between stinging events of Venom 84 single individuals. Both venom flow rate and flow duration correlate highly with the total expelled Venom metering 85 volume, indicating that scorpions may control both variables in order to achieve a desired end volume of Scorpions 86 venom during a sting. Defensive behavior 87 © 2015 Elsevier Ltd. All rights reserved. 88 89 90 91 92 defensive stinging behavior seems to be dependent on body mass, 1. Introduction 93 at least in Centruroides vittatus (Carlson et al., 2014). 94 For predation, the Arizona hairy scorpion, Hadrurus arizonensis, Most animal venoms consist of a complex mixture of peptides 95 only uses the venom to immobilize large or struggling prey and proteins in an aqueous medium. The venom components are 96 (Edmunds and Sibly, 2010). Also two Parabuthus species have been adapted to alter the target's physiology (Fry et al., 2009; McCue, 97 shown to minimize venom use if it is not necessary to immobilize 2005). These venoms are applied in defense, and for the incapaci98 struggling prey (Rein, 1993). It therefore seems that scorpions tation of prey. It is generally accepted that many venomous animals 99 optimize their venom expenditure for prey incapacitation. Scorwill use their venom frugally, as it can sometimes represent a large 100 pions that are regenerating their depleted venom have significantly metabolic investment, and can take a long time to replenish 101 increased metabolic rates, indicating that venom production is a (McCue, 2006; Nisani et al., 2007; Smith et al., 2014; Wigger et al., 102 costly process (Nisani et al., 2012, 2007). 2002). This venom optimization hypothesis has been tested in 103 Also defensive venom metering has been investigated in scordifferent groups of venomous animals; for a review, see 104 pions. By pushing the telson through parafilm, and comparing the Morgenstern and King (2013). 105 weight of the scorpion before and after venom expulsion, Nisani Scorpions use their venom defensively against predators, and to 106 et al. (2007) measured venom expenditure in spitting scorpions immobilize their prey. There are large differences in the defensive 107 (Parabuthus transvaalicus). Nisani and Hayes (2012) measured use of the stinger between species (Van der Meijden et al., 2013). An 108 venom mass and protein content of the expelled venom. Nisani and ontogenetic shift in stinger use for prey immobilization was re109 Hayes (2011) found that scorpions under high-threat conditions ported for two unrelated species of scorpions, Paruroctonus boreus 110 expel 2.2 times more venom than under low-threat conditions. The and Pandinus imperator (Casper, 1985; Cushing and Matherne, 111 scorpions also expelled more opalescent and milky venom, the 1980), with older specimens using the stinger less. Interestingly, 112 protein-rich venom that emerges after the clear and pain-inducing 113 “prevenom” is exhausted, under high threat conditions. Q1 114 To test the venom optimization hypothesis, the venom expen115 * Corresponding author. ded in defense or prey incapacitation must be quantified. 116 E-mail address: [email protected] (A. van der Meijden). 117 118 http://dx.doi.org/10.1016/j.toxicon.2015.04.011 119 0041-0101/© 2015 Elsevier Ltd. All rights reserved. Contents lists available at ScienceDirect

Toxicon

Q8 Q7

Variability in venom volume, flow rate and duration in defensive stings of five scorpion species

Please cite this article in press as: van der Meijden, A., et al., Variability in venom volume, flow rate and duration in defensive stings of five scorpion species, Toxicon (2015), http://dx.doi.org/10.1016/j.toxicon.2015.04.011

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2

Species

Androctonus amoreuxi

Specimen

Sc2171

Sc2172

Sc2173 Sc2174

Androctonus bicolor Sc2175

Sc1188

Sc1189

Hadrurus arizonensis Sc1194

Sc2188 Sc1240

Sc1241

Sc1245

Sc1248 Sc1249

Hottentotta gentili

Smeringurus mesaensis

Sc1576

Sc2209

Sc1577

Sc2212

Sc2213

Sc2220

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Number of 5 6 1 3 4 2 2 5 4 2 3 3 3 2 4 3 4 3 1 4 stings 0.681 0.550 0.848 2.127 0.632 0.292 0.303 0.146 0.324 1.488 0.358 0.549 1.301 1.289 0.939 0.088 0.141 0.059 0.049 0.131 Mean venom volume (ml) Mean injection 269 196 620 583 244 105 158 151 94 197 145 199 307 394 300 150 395 145 142 250 duration (ms) 20 26 68 46 55 124 236 48 37 57 33 29 168 14 43 31 8 58 8 10 Mean delay before ejection (ms) 2.534 2.803 1.367 3.651 2.590 2.779 1.920 0.969 3.469 7.554 2.472 2.753 4.232 3.273 3.128 0.589 0.356 0.404 0.345 0.522 Mean flow rate (ml/s) 0.0020 0.0011 0.0018 0.0026 0.0014 0.0016 0.0010 0.0010 0.0013 0.0031 0.0018 0.0020 0.0013 0.0014 0.00018 0.00028 0.00020 0.00021 Mean venom duct area (mm2) 0.63 1.30 1.01 0.49 0.98 0.62 0.48 1.80 2.86 0.40 0.74 0.82 1.24 0.22 1.01 0.71 0.86 1.22 Mean estimated flow speed (m/s) Prosoma L 8.8 9.4 7.4 9.0 9.3 8.4 6.8 8.7 13.5 12.8 11.5 12.6 10.1 11.2 9.2 5.9 6.5 7.3 6.8 (mm) Total length 59.9 66.2 57.7 62.6 71.0 63.7 60.5 59.9 91.7 86.0 81.2 88.8 76.2 85.4 75.4 51.1 53.7 58.0 52.6 (mm) Telson length 9.9 9.8 9.7 10.1 9.2 8.4 6.5 7.3 14.5 12.2 11.1 12.8 11.3 11.2 10.4 6.3 7.0 7.6 7.2 (mm)

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Table 1

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2. Materials and methods 2.1. Animal selection and husbandry Five species of desert scorpions, Androctonus australis, Androctonus bicolor, H. arizonensis, Hottentotta gentili and Smeringurus mesaensis, representing three families were selected (Table 1). Live scorpions were obtained through field collecting or the pet trade, and in apparent good health upon acquisition as well as throughout the experimental period. Live scorpions were maintained individually in plastic boxes with dry substrate (cork granulate) and hiding structures (polyethylene tube) within a temperature- and lightcontrolled chamber (26  C; 12 h photoperiod). They were fed once every 3 weeks with live crickets (Acheta sp.). 2.2. Experimental procedure Fig. 1. Schematic of experimental setup. 1. LED array providing back illumination; 2. Glass chamber filled with mineral oil; 3. Metal plate with slit covered by parafilm. This is where the scorpion is allowed to sting; 4. Single layer mirror; 5. Dissecting microscope with high speed video mounted (only lower part shown).

Measuring venom expenditure in prey incapacitation is particularly challenging, as the venom resides in the prey. Several methods have been used. The victim can be weighed before and after the envenomation event (Kochva, 1960). Also radioactively labeled venom can relatively easily be quantified in the prey (Allon and Kochva, 1974), but this method may run into prohibitive safety and ethical regulations and concerns. Enzyme-linked immunosorbent assays (ELISAs) have been more frequently used to quantify the venom content of homogenized prey, but require the production of antibodies (Holderied et al., 2011; Malli et al., 1998; Morrison et al., 1982; Wigger et al., 2002). Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) allows the identification of individual venom constituents, and can also show how far specific venom compounds have penetrated into the tissues of the victim (Francese et al., 2009). Young and Zahn (2001) used an ultrasound perivascular flow probe to measure venom flow directly during defensive and prey capture strikes, but implanting the flow probe requires invasive surgery, which could affect venom delivery. Measuring the amount of expelled venom during defensive behavior is more straightforward, and several direct and indirect methods have been used. In snakes, the amount of venom expelled during defensive bites has been quantified by allowing the harassed snake to bite into a piece of foam (Pe and Cho, 1986) and weighing the foam before and after the strike. Hayes et al. (2002) used a glove filled with warmed saline as an artificial human limb, and used a Bradford (1976) protein assay to quantify the amount of expelled venom. As noted above, Nisani et al. (2007) measured expelled venom directly after enticing the scorpion to defensively sting into a parafilm covered centrifuge tube (Nisani et al., 2012). Zlotkin (1969) used a similar method using a parafilm covered wire frame and measuring the volume of the expelled venom droplets using a glass capillary. Using these methods, it has been shown that snakes and scorpions control their venom expenditure, at least in some of the studied cases. Potentially, the amount of venom expelled during a sting could be controlled by varying the rate of venom expulsion, the duration of expulsion, or a combination of these two. Thus far, flow rate and duration have not been recorded in scorpions. We here introduce a new method of measuring expelled venom and flow rate for small venom volumes, and use it to identify differences between species in venom delivery during defensive stings.

All experiments were carried out in a climate-controlled room at 23 ± 1  C. Scorpions were taken from their enclosure and held by the 4th or 5th metasomal segment with rubber-tipped forceps, allowing free movement of the telson. The scorpion was held with the telson near a metal plate with a narrow slit, behind which a parafilm membrane was spanned. If scorpions were apathetic or otherwise unwilling to sting, they were enticed to do so by holding a pedipalp with another pair of forceps, thereby increasing the perceived level of threat. The scorpions made multiple searching movements with the telson, with the aculeus sliding across the metal plate. When the parafilm was touched, this caused an immediate stinging motion through the parafilm. The venom was injected into a small custom built glass chamber behind the parafilm, filled with mineral oil. The injection and subsequent accretion of venom droplets in the oil was recorded at 500 frames per second at a resolution of 1280 by 1024 pixels using a FasTec TS digital high speed camera (FasTec Imaging, San Diego, CA, USA) mounted on an Olympus SZX16 microscope. Backlight illumination was provided by a 100 W LED array. Videos were recorded at a spatial resolution of 56.9 pixels per mm. As the glass side of the oil chamber was perpendicular to the axis of the microscope, a single layer mirror was used to allow a side-view into the oil chamber from the vertically positioned microscope (Fig. 1). Specimens were allowed to sting up to three times subsequently, and the stings that resulted in venom release were further analyzed. Some specimens were tested more than once, and these events were separated by at least 8 days to allow replenishment of the venom (Nisani et al., 2012). 2.3. Video analysis For all videos, the time point of the entry of the stinger through the membrane was recorded, as well as the first appearance of the venom. As the mostly aqueous venom was injected into mineral oil, it immediately formed a spherical droplet. The videos were digitized frame by frame in ImageJ (Schneider et al., 2012) by manually drawing a circle around the accreting droplet. The surface area of the circle was used to calculate the radius of the spherical venom droplet, which was in turn used to calculate its volume. If multiple droplets formed, the volumes were summed. Occasional momentary deviations from a spherical droplet shape were caused by the jet of venom or movement of the aculeus. These frames were not digitized. Gaps in the volume data due to a momentary nonspherical shape of the droplet were filled by linear interpolation between known volumes. Thus we obtained the volume of the accreting venom droplets at a temporal resolution of 500 Hz. The videos were digitized until no more venom accreted for several frames. The last frame at which venom was being ejected was noted. This allowed us to calculate the total duration of the venom

Please cite this article in press as: van der Meijden, A., et al., Variability in venom volume, flow rate and duration in defensive stings of five scorpion species, Toxicon (2015), http://dx.doi.org/10.1016/j.toxicon.2015.04.011

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Fig. 2. Video stills showing significant events during a typical sting (event 39, specimen Sc1248, Hadrurus arizonensis). At 0 ms, the aculeus can first be seen penetrating the parafilm membrane. At 38 ms, the first venom can be seen. At 76 ms, the venom droplet is deformed due to movement of the aculeus. At 116e256 ms, the venom droplet is accreting. Background has been subtracted from the images.

Table 2 Means per species. Values ± S.D. Standard deviations are based on the sting events, not individuals. Species

Specimens

Events

Volume (ml)

Androctonus amoreuxi Androctonus bicolor Hadrurus arizonensis Hottentotta gentili Smeringurus mesaensis

5 4 5 2 4

19 13 13 7 12

0.867 0.248 0.937 0.574 0.109

± ± ± ± ±

0.798 0.292 0.710 0.962 0.117

Duration (ms) 309 127 220 236 263

± ± ± ± ±

233 71 133 213 265

Flow rate (ml/ms) 2.81 1.94 4.27 2.44 0.41

± ± ± ± ±

1.04 1.53 2.62 1.73 0.67

Delay (ms) 36 85 64 38 21

± ± ± ± ±

22 109 119 22 23

Flow speed (m/s) 0.88 0.83 1.04 0.89 1.39

flow, the volume ejected per second, the delay between the penetration of the parafilm by the aculeus and the first appearance of venom, and the total venom volume ejected during the event.

were used to estimate the speed of the venom flow as it is ejected by dividing volume per second by the area of the two venom duct furrows for each specimen.

2.4. Morphological measurements

2.5. Data analysis

The following linear measurements were made of each specimen to the nearest 0.01 mm: prosoma length and width, total length and telson length. As scorpions can vary in length, body mass and girth considerably due to differences in feeding state, prosoma length, width or area are most often used as a proxy for overall size. We here used area, the product of prosoma length and width, to correct for overall size. We also measured the diameter of the openings of the paired venom ducts at the tip of the telson under a dissecting microscope. When the openings of the venom ducts were not clearly visible, we measured the width of the furrow in which the venom duct empties to the nearest 0.001 mm, as this is approximately the width of the opening of the venom duct. We used these as the estimated diameter of the end of the venom duct in order to calculate the cross-sectional area of the end of the venom duct. Since ejection rate (ml/s) can be measured from the video footage, the cross section of the ends of the venom ducts

A ShapiroeWilk normality test was performed on the variables for each event in R version 2.14.0 (R Development Core Team, 2012), and the results were found to not be normally distributed. We therefore performed the analyses of variance with permutation tests rather than parametric ANOVA's. A non-parametric ANOVA was performed to test significant differences between the amounts of venom expelled on the first, second, third consecutive injection event on a single day. Contrary to the findings of Nisani and Hayes (2011), there was no significant difference between the amount of expelled venom per consecutive event (p ¼ 0.15), and all events were therefore included in subsequent analyses. In order to identify differences between the tested species in sting variables, such as duration, volume and flow rate, while accounting for possible differences between specimens, we performed nested permutation tests for each of these sting variables in R using the Adonis function in the Vegan package (Oksanen

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Fig. 3. Flow profiles of four events in two specimens, showing different flow rates and durations. Data was filtered with a Butterworth low-pass noise filter at a cut-off frequency of 100 Hz. Open markers indicate linearly interpolated data due to the venom droplet being deformed.

et al., 2013) with specimens as a nested factor in species. In addition, to identify size-independent species differences, we scaled the variables ‘venom volume’ and ‘flow rate’ by regressing against the product of prosoma length and prosoma width to the 3/2 power, as area relates to volume2/3. The residuals were then used in a permutation test for each variable, with specimens as a nested factor in species, in order to identify size-independent differences in venom volume and flow rate between species. Zlotkin (1969) identified differences in the final volumes expelled in defensive stings between clear, opalescent and milkyviscous venom. We categorized the venom type expelled from the video footage as clear, opaque, or going from clear to opaque. Then we performed a nested permutation test on venom volume with species and venom type as nested categories.

A multiple regression with volume as the dependent, and flow rate and duration as independent variables was performed in R. The data were first standardized to obtain standard partial regression coefficients. 3. Results and discussion A total of 64 events were analyzed. When presenting the metasoma to the plate, the scorpions did not eject venom when the aculeus was touching the metal plate, but only released venom after penetrating the parafilm membrane. The delay between penetrating the membrane and the first visible appearance of venom from the aculeus was on average 49 ± 7.6 ms. This is an order of magnitude longer than the delay observed in snakes,

Table 3 Results of nested ANOVA's on permuted data with specimens as a nested factor within species. P-values below 0.05 are in bold typeface.

Total expelled venom Total expelled venom, size corrected residuals Average flow rate Average flow rate, size corrected residuals Flow duration Delay between telson penetration and venom expulsion Total expelled venom

Factor

Sum sq.

D.f.

Mean sq.

F

R2

p

Species Specimens Species Specimens Species Specimens Species Specimens Species Specimens Species Specimens Species Venom type

7.31 9.81 2.80 10.43 86.82 53.52 12.40 34.06 0.27 0.68 0.03 0.12 7.31 3.49

4 15 4 14 4 15 4 14 4 15 4 15 4 9

1.8 0.7 0.7 0.7 21.7 3.6 3.1 2.4 0.1 0.0 0.0 0.0 1.8 0.4

5.57 1.99 2.13 2.27 8.94 1.47 1.26 0.98 1.88 1.28 1.74 1.67 4.40 0.93

0.23 0.31 0.10 0.38 0.35 0.22 0.08 0.22 0.11 0.28 0.09 0.33 0.23 0.11

0.001 0.050 0.099 0.034 0.001 0.168 0.342 0.496 0.117 0.254 0.136 0.131 0.008 0.463

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Fig. 4. Boxplots of expelled venom volume per event, average flow rate, duration of flow, and the delay between the penetration of the aculeus through the parafilm membrane, and the first visible expulsion of venom. Positions of outliers above the dashed line are not to scale. Top and bottom of the boxes represent the 25 and 75 percentile, the whiskers represent the most extreme data points that are not considered outliers. For full species names, refer to Table 2.

4.8 ± 2.7 ms (Young and Zahn, 2001). The flow of venom then continued, forming a spherical droplet or multiple such droplets in the oil chamber (Fig. 1). Often, two droplets formed, one for each venom duct. These often quickly merged into a single droplet. The venom flow continued for an average of 0.24 s, but in a small number of events we found pauses in the accretion of the venom (Fig. 2d). Venom flow ceased before the aculeus was retracted. An injection event, from aculeus penetration to cessation of venom flow, took 286 ± 209 ms. We found large differences in flow rates and flow duration, even between stings of the same specimen (Fig. 2a and b). As a result, the end volume varied considerably between different stings of the same specimen. We found differences between species in several of the variables (Table 2; Fig. 3). Total injected venom and the average flow rate differed significantly between species, but no significant differences were found between specimens (Table 3) in these variables. We found no significant difference between species in the delay between penetration of the parafilm membrane and the first visible expulsion of venom. The species we tested in this study injected 0.109 ± 0.117 ml (S. mesaensis) to 0.937 ± 0.710 ml (H. arizonensis) on average per defensive sting. This is in the same order of magnitude of the findings on P. transvaalicus, which inject 1.38 ± 0.82 ml in high threat stings, and 0.62 ± 0.38 ml under low threat conditions (Nisani

and Hayes, 2011). Also flow speeds were similar to those reported by Nisani and Hayes (n.d.) who measured a flow speed of 0.758 m/s in P. transvaalicus. We found average flow speeds of 0.83 m/s to 1.39 m/s in the different species (Table 2). There seems to be some relationship between the average amount of expelled venom and the LD50 of that species. Although no LD50 is available for S. mesaensis or H. gentili, the LD50 of the venoms of A. bicolor (0.31 mg/kg; Zlotkin et al., 1971), Androctonus amoreuxi (0.75 mg/ kg; Zlotkin et al., 1971) and H. arizonensis (168 mg/kg; Johnson et al., 1966) mirror their injected venom volumes in relative order, but not in magnitude. However, no conclusions can be drawn from the current dataset, and a larger sample size and LD50 values obtained using standardized tests (see Van der Valk and van der Meijden, 2014) will be needed to statistically test this relationship (Fig. 4). Q3 The difference between species in volume and flow rate can be due to the differences in size, as total available venom volume and venom duct diameter likely vary with overall size. The sizecorrected volume and flow data do not show significant differences between species (Table 3), but a difference existed between specimens in total expelled venom. The disappearance of a significant difference between the species after size correction indicates that these species differences may be mainly due to overall size. A similar relationship between the amount of injected venom and body size has been found in snakes (Hayes and Mackessy, 2010). Scorpions consecutively expel different types of venoms, clear prevenom, followed by opalescent venom and finally viscous venom (Yahel-Niv and Zlotkin, 1979; Zlotkin, 1969). The two latter types contain more proteins (Inceoglu et al., 2003). Out of the 64 events recorded, 20 stings consisted of only clear venom, while 30 had only opaque venom. In 14 events, we noted a transition from clear to opaque venom. This shows that venom type is not a property that remains constant per sting. Zlotkin (1969) reported a volume per sting of 0.23e0.45 ml in Leiurus quinquestriatus depending on venom type. We did not find an association between venom type and the total volume of expelled venom (Table 3). The multiple regression showed both of flow rate and duration to correlate with the total injected venom volume (multiple R2 ¼ 0.83, p for both < 2e-15). The standard partial regression coefficients were 0.69 for flow rate and 0.57 for flow duration, indicating that flow rate and flow duration contribute similarly to determine the total amount of expelled venom. Venom expulsion duration took 28e1068 ms, with an average 237 ± 199 ms. Most expulsion durations were therefore longer than recorded for the spitting spider, Scytodes, which takes less than 30 ms (Suter and Stratton, 2009). However, Scytodidae are highly specialized, and may not represent expulsion durations for spiders in general. For rattlesnakes, a flow duration of 205.8 ± 25 ms for defensive bites against rats, and 228.5 ± 28.5 ms against mice was reported (Young and Zahn, 2001). The total amount of venom that a scorpion injects during defensive strikes is an important variable that, in combination with the LD50, can provide insight into the ecological relevance of scorpion venom for defense. The variance in defensively injected venom is also important for the understanding of scorpionism. Using high-speed video recordings of accreting venom droplets allows the quantification of expelled venom at a high temporal resolution. In addition, the injected venom is fully available for further molecular analysis as isolated droplets suspended in oil. However, this method will likely not work for larger volumes, such as those expelled by large snakes, as the large venom stream will likely break up into a very large number of small droplets, making quantification difficult. The method could be optimized by varying the viscosity of the oil used to control the time in which an injected volume forms a spherical droplet, and thus reduce the occurrence of non-spherical droplets. The amount of venom measured with

Please cite this article in press as: van der Meijden, A., et al., Variability in venom volume, flow rate and duration in defensive stings of five scorpion species, Toxicon (2015), http://dx.doi.org/10.1016/j.toxicon.2015.04.011

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this method may not be an accurate representation of the amount of venom injected into a living organism, as blood pressure and resistance to expansion of the tissues of the sting victim may constitute a counter pressure to venom flow, and thus reduce the total amount of expelled venom. Ethical statement The manuscript and data were not previously or simultaneously submitted elsewhere. All experiments in this paper were carried out under the standard procedures of scientific ethics, including the care of experimental animals. All authors have read the manuscript and agree to its publication in Toxicon and agree that it has followed the rules of ethics presented in the Elsevier's Ethical Guidelines for Journal Publication. Acknowledgments

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We thank Arendo Flipse for his help in obtaining some of the specimens, and Sergio Henriques, Diana Pedroso, Ana Neto and Pedro Sousa for their help in the field. Thanks to Antigoni Kalontzopoulou for helpful discussions, and two anonymous reviewers for helpful comments on the manuscript. AvdM is supported by a grant ~o para a Cie ^ncia e a Tecnologia (FCT, Portugal) under the by Fundaça ^ncia Programa Operacional Potencial Humano e Quadro de Refere gico Nacional funds from the European Social Fund and Estrate rio da Educaça ~o e Cie ^ncia (SFRH/BPD/101057/ Portuguese Ministe 2014). This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors e COMPETE and by National Funds through FCT e Foundation for Science and Technology under PTDC/BIA-EVF/2687/2012 and FCOMP-01-0124FEDER-028340. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2015.04.011. References Allon, N., Kochva, E., 1974. The quantities of venom injected into prey of different size by Vipera palaestinae in a single bite. J. Exp. Zool. 188, 71e75. Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Carlson, B.E., McGinley, S., Rowe, M.P., 2014. Meek males and fighting females: sexually-dimorphic antipredator behavior and locomotor performance is explained by morphology in bark scorpions (Centruroides vittatus). PLoS One 9, e97648. Casper, G.S., 1985. Prey capture and stinging behavior in the emperor scorpion, Pandinus imperator (Koch) (Scorpiones, Scorpionidae). J. Arachnol. 13, 277e283. Cushing, B.S., Matherne, A., 1980. Stinger utilization and predation in the scorpion Paruroctonus boreus. Gt. Basin Nat. 40, 193e195. Edmunds, M.C., Sibly, R.M., 2010. Optimal sting use in the feeding behavior of the scorpion Hadrurus spadix. J. Arachnol. 38, 123e125. Francese, S., Lambardi, D., Mastrobuoni, G., la Marca, G., Moneti, G., Turillazzi, S., 2009. Detection of honeybee venom in envenomed tissues by direct MALDI MSI. J. Am. Soc. Mass Spectrom. 20, 112e123. Fry, B.G., Roelants, K., Champagne, D.E., Scheib, H., Tyndall, J.D., King, G.F., Nevalainen, T.J., Norman, J., Lewis, R.J., Norton, R.S., Renjifo, C., de la Vega, R.C.R., 2009. The toxicogenomic multiverse: convergent recruitment of proteins into animal venoms. Annu. Rev. Genomics Hum. Genet. 10, 483e511.

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Please cite this article in press as: van der Meijden, A., et al., Variability in venom volume, flow rate and duration in defensive stings of five scorpion species, Toxicon (2015), http://dx.doi.org/10.1016/j.toxicon.2015.04.011

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Variability in venom volume, flow rate and duration in defensive stings of five scorpion species.

Scorpions have been shown to control their venom usage in defensive encounters, depending on the perceived threat. Potentially, the venom amount that ...
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