Research on the energetic demands of venom use have focussed on costs of production, rather than maintenance, as it is difficult to measure the energy used in maintaining and storing toxins experimentally. Nisani et al. (2007) showed that after depleting the venom glands of scorpions (Parabuthus transvaalicus), metabolic rates increased by 39% during the first 3 days of regeneration. A later study (Nisani et al., 2012) found milked P. transvaalicus had on average a 21% higher metabolic rate than un-milked scorpions during the first 8 days of regeneration, but in this second study the rate did not rise as high during the first 3 days (Nisani et al., 2007). Metabolic rates fluctuated throughout the experiment, and the authors suggested this likely reflected the asynchronous regeneration of toxins (Nisani et al., 2012). Whilst differences were observed between the studies, both identify a large increase in metabolic rate above baseline levels, indicating that in scorpions venom production is an important energetic expense (Nisani et al., 2007, 2012). These studies also likely underestimate total energetic costs, as venom regeneration can take longer than 8 days to complete (Carcamo-Noriega et al., 2019). Furthermore, scorpion venom varies between species in complexity, toxins utilized, and volume stored and injected (de la Vega et al., 2010; Sunagar et al., 2013; van der Meijden et al., 2015); all of which alter energy requirements. Energetic costs are also dependent on the metabolic rate, which is likely to vary between species adapted to different ecological niches. Furthermore, as scorpions are ectotherms their metabolic rate will vary with environmental conditions, as Nime et al. (2013) showed scorpion activity is positively correlated with temperature.

Indirect costs are associated with the ecological limitations arising from depleted venom supplies, such as increased predation risk or reduced ability to capture prey. Scorpions can store a limited volume of venom (van der Meijden et al., 2015), and regeneration of toxins can take at least 2 weeks to be complete (Carcamo-Noriega et al., 2019), reducing the ability to use venom for prey capture and predator defense until venom supplies are restored. Ecological costs of venom depletion cannot easily be quantified, as they will vary widely between species and environments with fluctuating selection pressures. Nonetheless, evaluation of behavioral changes that arise when venom stores are depleted may serve as evidence of ecological costs. Such a behavioral response has yet to be reported in scorpions, but some spiders with depleted venom supplies will adapt their hunting behavior to target easily caught prey, as has been found in the wandering spider Cupiennius salei (Wullschleger and Nentwig, 2002). Scorpions and spiders often share many of their natural predators and prey items, therefore the ecological costs of venom depletion in both organisms may bear some similarity. Comparable behaviors in scorpions might include a switch to smaller prey that can be successfully captured with only the pedipalps while venom is replenishing.

To reduce the costs associated with unnecessary venom use, scorpions adapt their hunting strategies to the particular prey items targeted (Simone et al., 2018), and are less likely to use venom when capturing small or easily subdued prey (Rein, 1993; Edmunds and Sibly, 2010). Spiders appear to target the injection of their venom toward the thorax or head of prey items to maximize venom efficiency (Wigger et al., 2002; Carlson et al., 2014). However, to our knowledge it is not known if scorpions seek to apply stings to an optimal location, or if an optimal sting location even exists, as one study on Bothriurus bonariensis found that sting location did not affect the time taken to subdue prey (Simone et al., 2018).

Both the size and activity of prey items can influence a scorpion's choice to sting, as Parabuthus liosoma, Parabuthus pallidus and Hadrurus spadix sting larger and more active prey items more frequently (Rein, 1993; Edmunds and Sibly, 2010). Rein (1993) observed that the scorpions did not use their sting immediately when encountering prey, but would rather grab with their pedipalps and apply stings if the prey continued to struggle, presumably to minimize venom use whilst ensuring predation success. Ontogenetic changes in stinging behavior can also be used to optimize venom use. Older Paruroctonus boreus and Pandinus imperator use their larger pedipalps to overpower prey and sting less often, avoiding using venom (Cushing and Matherne, 1980; Casper, 1985).

In addition to trade-offs between the use of venom and pedipalps for prey capture, there may be trade-offs in venom use and mobility when avoiding predation, as faster scorpions appear less likely to sting predators (Carlson et al., 2014; Miller et al., 2016). For example, female Centuroides vittatus scorpions that are heavier and less mobile, are more likely to sting a potential predator than males, which are more likely to sprint to safety (Carlson et al., 2014; Miller et al., 2016). Furthermore, within each sex, sprint speed decreases and sting rate increases with mass, indicating that higher rates of aggression are associated with reduced mobility (Carlson et al., 2014; Miller et al., 2016). Through fleeing, the males and smaller scorpions are not only able to avoid being eaten, but they also save their venom supply for future encounters and do not need to expend energy regenerating toxins, thus reducing ecological and energetic venom costs.

Unlike other scorpions, seven species of Parabuthus spray venom defensively, which may cause irritation to the sensitive tissues, such as eyes, of predators (Newlands, 1974; Nisani and Hayes, 2015). P. transvaalicus defensively spray when presented with both air-flow and touch stimuli simultaneously, suggesting the behavior may be optimized toward high-threat scenarios where defensive tactics must be implemented before a predator gets close enough to sting (Nisani and Hayes, 2015). These scorpions are often attacked by predators such as grasshopper mice, which disarm scorpions by biting off their tails, and therefore place their face in close proximity to the telson (Nisani and Hayes, 2015). Compared with injection, sprayed venom has a higher risk of missing its target, likely increasing the costs of venom necessary to deter predators. However, these costs are likely offset by the advantage of deterring predators while the predator is still at a distance.

Scorpions can still avoid venom use when stinging in defensive encounters by employing “dry” stings, where no venom is injected. Scorpions readily utilize dry stings in defensive situations (Nisani and Hayes, 2011; Lira et al., 2017; Rasko et al., 2018). The factors that determine whether venom is injected in a sting, however, are currently unclear. In P. transvaalicus, dry stinging behavior is correlated with threat level, with the scorpions employing dry stings more frequently when the threat level is low (Nisani and Hayes, 2011). Additionally, this study suggests P. transvaalicus, when induced to sting multiple times in succession, use dry stings more frequently early in the stinging sequence and are more likely to inject venom as the threat persists (Nisani and Hayes, 2011). The combination of these results provides evidence that scorpions use dry stings in low threat situations to optimize their venom use. In contrast to the findings by Nisani and Hayes (2011) studies on dry stinging behavior in other scorpion species have produced differing results. Lira et al. (2017) presented Tityus stigmurus with the same “low-threat” and “high-threat” stimuli described by Nisani and Hayes (2011), and found no correlation between dry sting rate and threat level. Furthermore, it has been shown that repeated simulated attacks against Hadrurus arizonensis lead to an increase in dry sting rate, despite venom remaining in the gland (Rasko et al., 2018). This latter result seems counter-intuitive, as scorpions might be expected to increase their defensive investment as a threat persists. Whilst the studies investigating the factors influencing dry stinging behavior in scorpions are limited, the evidence supports the idea that at least some scorpion species utilize dry stings as a means to optimize their venom in defensive contexts (Nisani and Hayes, 2011), while others may not (Lira et al., 2017; Rasko et al., 2018). Further research should aim to identify whether interspecific differences are truly occurring, or if methodological differences between the studies are responsible for the observed differences. Furthermore, it is not currently known if scorpions utilize dry stings as a tactic to save venom when capturing prey, as spiders use dry bites (Malli et al., 1998; Wigger et al., 2002). Spiders, however, have the ability to masticate their prey with their fangs, but the dry sting of a scorpion provides comparatively little aid in the incapacitation of prey. It is therefore unlikely that scorpions use dry stings to save venom when targeting prey.

While scorpion stinging behavior involves a dichotomy between dry stings vs. stings with venom injected, scorpions also have the ability to vary the volume of venom they inject, both within each sting and through the application of multiple stings (Nisani and Hayes, 2011; van der Meijden et al., 2015). P. transvaalicus injected twice as much venom per single sting in high-threat situations compared with low-threat situations, indicating scorpions may use this tactic to vary their defensive investment in response to perceived threat level. The defensive sprays of P. transvaalicus display variable duration and flow rate suggesting the volume expelled could be controlled by contraction of the venom gland, but it is not currently known if the volume sprayed is influenced by threat level (Nisani and Hayes, 2015).

The volume injected in single stings may be limited by morphological constraints, or the time that the aculeus is pierced into the target (van der Meijden et al., 2015). When scorpions are faced with repeated attacks from predators, they will continue to defensively sting as the attacks continue. Experiments into the defensive investment of scorpions in response to predation threat suggest that scorpions will repeatedly sting predators as the threat persists (Lira et al., 2017), but it is unclear whether the investment per attack increases or decreases with sting number (Nisani and Hayes, 2011; Rasko et al., 2018). Targeting prey, scorpions often hold on with their pedipalps and judiciously apply stings as the prey continues to struggle (Casper, 1985; Rein, 1993). The rate of stings increases with both prey size and activity (Edmunds and Sibly, 2010), suggesting that scorpions are being frugal with their venom, and only apply extra stings as necessary.

In addition to reducing venom costs by metering the volume of venom they inject, scorpions are able to alter the composition of venom injected into their target and avoid unnecessarily injecting costly venom components. Scorpion venom is heterogeneous and changes in composition as it is expelled from the aculeus (Figure 2). As the venom is secreted from the aculeus tip, the initial expulsion is a clear liquid, followed by an opalescent liquid, and finally turns milky colored and viscous (Yahel-Niv and Zlotkin, 1979). These different secretions also vary in toxicity (Yahel-Niv and Zlotkin, 1979). Inceoglu et al. (2003) found that the initial clear secretion in P. transvaalicus constituted around 5% of the total venom volume within the gland, and they termed this “prevenom”. Prevenom and main milky venom have distinct compositions and modes of action in both invertebrate and vertebrate targets (Inceoglu et al., 2003). The different mode of actions of prevenom and main venom may act together to induce greater toxicity, in a similar way to the toxin cabals employed by cone snails which target different pathways simultaneously (Olivera et al., 1999; Inceoglu et al., 2003). P. transvaalicus prevenom was found to contain six-times less peptide and protein concentration compared to the main venom, but a 16-fold higher potassium (K⁺) salt concentration (Inceoglu et al., 2003). The authors suggested this extremely high K⁺ concentration causes large and rapid depolarization of nerves in the target, causing quick paralysis in insects and pain in vertebrates (Inceoglu et al., 2003). Prevenom not only contains a much lower peptide/protein concentration, but a comparatively simplistic composition (Inceoglu et al., 2003). It is therefore expected that prevenom is metabolically cheaper to produce than the main venom, as K⁺ salt likely requires less energy to be replenished than peptides requiring production and folding (Inceoglu et al., 2003). The relative costs of prevenom vs. main venom have not been calculated experimentally, but in P. transvaalicus prevenom components appear to regenerate quickly and at little metabolic cost compared with other toxins (Nisani et al., 2012). Prevenom may therefore have evolved as a mechanism to avoid injecting larger volumes of peptide rich mixtures, thereby minimizing both metabolic and ecological costs of depletion, although this connection is difficult to test experimentally (Nisani et al., 2012).

By using prevenom first, scorpions can save their main peptide-rich venom for high-threat situations or when initially low-threat situations escalate. This hypothesis is supported by evidence that the composition of venom (prevenom vs. main venom) scorpions inject is context dependent. In low-threat situations, scorpions are able to avoid injecting their metabolically “expensive” mixtures of toxins, by injecting only prevenom (Nisani and Hayes, 2011). P. transvaalicus inject their main venom more frequently in high-threat situations, and in later stings when induced to sting repeatedly at both low and high threat levels (Nisani and Hayes, 2011). Furthermore, in low-threat situations, T. stigmurus injected prevenom in all trials, but when faced with the high-threat treatments most of the scorpions injected their main milky venom secretion (Lira et al., 2017). The use of prevenom in low threat situations not only minimizes metabolic costs but also reduces ecological costs, as prevenom appears to regenerate faster than the main venom components (Nisani et al., 2012).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.