Scorpion venom is composed mainly of peptides and proteins and has high metabolic cost. Thus, individuals need to be economic in its use. This study aimed to evaluate the reactions of Tityus pusillus Pocock, 1893 scorpion to threats of different intensities. For the experiments, 28 males and 26 females were used; animals were gently touched five times on their mesosoma at different time intervals. Touches with five second intervals were considered as the high-intensity threats, while those with five-minute intervals were considered as the low-intensity threats. We found that scorpions, independently of sex, stung more often under high threat compared to low threat treatments. However, no significant differences in the defensive behavior of males and females were observed according to threat intensity. We can thus infer that regardless of sex, T. pusillus uses its stings and consequently venom only in situations of high threat.
Scorpions are preyed upon by more than 150 species of vertebrates and invertebrates (Mullen & Sissom 2019). Therefore, behaviors and mechanisms determining the survival of these arachnids are extremely important (Coelho et al. 2017). To reduce the likelihood of being preyed upon, scorpions employ different strategies. Of these, camouflage, which is facilitated by their similar color to the surrounding environment and avoidance of activities at times when chances of encountering predators are the highest, are classified as primary defense mechanisms (McCormick & Polis 1990; Coelho et al. 2017). If the primary mechanisms fail and the scorpion is forced to directly confront the predator, secondary mechanisms are used, such as thanatosis, metasomal autotomy, clamping with pedipalp pincers, venom spraying and finally stinging (Mattoni et al. 2015; Nisani & Hayes 2015; Coelho et al. 2017; Lira et al. 2019).
Morphological differences, such as those related to sexual dimorphism, may influence the defensive responses in these animals (Carlson et al. 2014; Lira et al. 2019). For example, female Centruroides vittatus (Say, 1821), are known to behave more defensively, and sting more often than males, while males, with their elongated appendages, have a higher escape velocity and reduced sting speed (Carlson et al. 2014). In addition, previous studies have shown that scorpions have control over venom use. For example, some species use venom very restrictively, using this feature only against resistant prey or when threat from a potential enemy is high (Nisani & Hayes 2011; Lira et al. 2017; Silva et al. 2019). The production and storage of venom requires high metabolic investment (Nisani et al. 2007; Morgenstern & King 2013; Evans et al. 2019). Thus, scorpions are extremely cautious in using their sting, and consequently the venom is metered through behavioral control (Inceoglu et al. 2003; Nisani et al. 2012; Silva et al. 2019). This type of mechanism in venomous animals, where venom is used only when necessary, has led to the venom optimization hypothesis. According to this hypothesis, venom has a high metabolic cost and animals use it economically (Morgenstern & King 2013; Evans et al. 2019).
A previous study on Tityus pusillus Pocock, 1893 behavior showed that this species exhibits a sex-based defensive strategy, with males performing more thanatosis and stinging while females performed fleeing, metasoma wagging, and stinging (Lira et al. 2019). These authors suggest that sexual dimorphism or different life strategies may explain their results. However, these authors performed their experiments with a similar threat intensity. Therefore, for expanding our knowledge about defensive strategies of scorpions, the present study aims to evaluate the defensive response of T. pusillus to threats of different intensities. We investigated the differences in the responses of males and females to these stimuli. We tested the following predictions: (1) during low-intensity threats, scorpions use their sting less frequently than during high-intensity threats; (2) females are more defensive than males, especially when subjected to high-intensity threats.
The scorpion species studied in this work, T. pusillus, is widely distributed across northeastern Brazil, and is considered to be the most abundant scorpion in the Atlantic Forest, where it predominantly occurs in the litter layers (Lira et al. 2013, 2018a). Individuals are approximately 30–35 mm long, with a reddish yellow color with brown spots over the entire body (Lourenço 2002; Lira et al. 2018b). This species exhibits sexual dimorphism (Fig. 1), with females being larger than males, while males have robust pedipalp chelae and elongated metasomal segments (Lira et al. 2018b). Females of this species can generate up to three litters per year, each consisting of 4–12 juveniles, which undergo 4–5 molts before reaching adulthood (Albuquerque & Lira 2016).
We collected 60 adult scorpions (30 males and 30 females) from an Atlantic forest remnant located in the municipality of Moreno (8°06′38.1″S; 35°06′56.4″W), Pernambuco state, Brazil. In the laboratory, the animals were maintained in individual plastic terraria (14 cm × 10 cm × 8 cm), provided with cardboard for shelter and moistened cotton wool as a water source. They were quarantined for 30 days before the start of the experiments to avoid sick animals and to detect signal of pregnancy (embryos visible through the ventral mesosoma). Scorpions were housed at 24°C ± 2°C and 80% ± 5% relative humidity, under a 12:12 h light:dark photoperiod. They were fed weekly with Nauphoeta cinerea (Oliver, 1789) cockroach nymphs. The test animals were not fed for 7 days before the experiments.
The tests were conducted at night, between 19:00–22:00, under red light. Red light was used because scorpions cannot see this spectrum, and thus, do not exhibit any behavioral changes (Machan 1968). Individuals were first separated based on sex, and then divided into four groups, with 15 individuals each (males under high threat, females under high threat, males under low threat, and females under low threat). The experiments were performed simultaneously for each threat level (high and low). All the procedures were performed by two researchers (KBCA and AFAL). Each scorpion was used only once to avoid bias in behavior and pregnant females were excluded. For each test, the scorpions were transferred to individual circular plastic terraria (9 cm high and 14 cm in diameter) for acclimatization for 15 minutes. Behavioral tests under high and low threat were then conducted, following Nisani & Hayes (2011). The high-threat tests consisted of 5 consecutive touches (1 second of duration) on the scorpion's mesosoma within 5 seconds of each other, while in the low-threat tests, 5 mesosomal touches were performed with 5-minute intervals. The stimuli were delivered on the mesosoma, using metal tweezers with rubber on the tips. At the end of each touch, the reaction of each individual was observed. These reactions included stinging and metasoma wagging, described as the defensive behaviors of T. pusillus by Lira et al. (2019).
To evaluate the effects of the threat level (low vs. high and the frequency of stimulus one to five) on the defensive behavior of male and female T. pusillus, we used a 2×5 repeated measures ANOVA. For this analysis, the behaviors were ranked as follows: 1 = no reaction or cessation of action, 2 = metasoma wagging, and 3 = sting. They were considered as sting when the scorpion managed to insert its stinger into the rubber. The level of defensiveness between the sexes in response to the stimulus level was analyzed using a contingency table G test. In this analysis, we considered individuals that exhibited at least one sting across all five of their trials as being in the “sting” category; all other individuals were grouped in a “no sting” category. All analyses were performed using BioEstat 5.3 software (Ayres et al. 2007).
In total, behaviors of 54 individuals were observed: 30 (15 males and 15 females) under high threat, and 24 under low threat (13 males and 11 females). Six scorpions (2 males and 4 females) died during the quarantine period. Both sexes were more defensive in the high threat tests, with 86% of individuals stinging, while in the low threat tests, just over half (58.3%) of the individuals resorted to stinging. It was also observed that stinging was preceded by metasoma wagging in most cases, especially in high-intensity situations (Fig. 2), with individuals showing an increase in their defensive behaviors (males: F1,120 = 32.17; P < 0.0001 and females: F1,100 = 10.59; P = 0.0019) between high- and low-intensity stimuli. Both sexes stung more in high intensity (males = 2.92 ± 1.44 and females = 3 ± 1.67) than low-intensity (males = 1.07 ± 1.18 and females = 1.90 ± 1.92) situations.
Scorpions under high intensity situations escalated their sting behavior compared to low intensity stimuli (males: F4,120 = 6.04; P = 0.0004 and females: F4,100 = 7.35; P = 0.0001). Around 46% of the males stung on the second stimulus under high-intensity threat, while under low threat stimuli, the animals resorted to stinging only on the third trial (Fig. 2). A similar pattern was observed for females, with 53% of individuals stinging on the second trial under high-intensity threat, and 45% stinging on the third stimulus under low-intensity threat (Fig. 2). Defensive behavior levels of males and females did not differ significantly in either low threat intensity (G2x2 = 0.73; P = 0.3901) or high threat intensity (G2x2 = 0.45; P = 0.4979), where both sexes used stinging in similar way (Fig. 3).
In this study, we investigated the defensive behavior of T. pusillus in high- and low threat situations. Our results indicated that this species regulates the use of stinging according to the threat level to which the animal is exposed, since there was a significant difference in the defensive behavior of individuals with escalation to stinging more likely during high-threat encounters. Previous studies have already reported such behavioral characteristics of scorpions exhibiting a more rapid escalation of defensive behaviors with a more persistent threat (Nisani et al. 2011; Lira et al. 2017; Rasko et al. 2018).
Tityus pusillus individuals were more defensive in the high-intensity tests, with most of the scorpions stinging at least once. Lira et al. (2017) obtained similar results for high threat tests in Tityus stigmurus (Thorell, 1876), where more than half of the individuals (83%) resorted to stinging. This more defensive response observed in T. pusillus, with a larger number of stinging individuals, is quite common in species of the Buthidae family, as they have less robust pedipalps than members of other families, such as Liochelidae and Scorpionidae, which may explain the greater use their stinger-tipped metasoma (Warburg 1998; van der Meijden et al. 2013).
Based on the sex-related defensive responses, our results showed that both male and female T. pusillus react in similar way, independently of threat. This reaction was previously reported for these species during prosoma touches (Lira et al. 2019). According to these authors, T. pusillus mostly reacted with stings, independently of sex. Therefore, our findings suggest that sexual dimorphism may not influence this species' reaction to imminent danger. This is in contrast to the observation made in C. vittatus, where females were found to be more defensive than males (Carlson et al. 2014). Interestingly, before stinging, individuals of both sexes performed metasoma wagging. This type of behavior has commonly been associated with reproduction (e.g., Melville et al. 2003; Gaffin & Brownell 2010; Taylor et al. 2012; Pordeus et al. 2019), although recently, Lira et al. (2019) linked such behavior to self-defense. According to these authors, this behavior can act as a warning to potential predators. Our results showing that metasoma wagging occurred during simulated encounters with predators supports this earlier suggestion.
In addition, the reluctance of T. pusillus to escalate to stinging under low-threat situations may be supported by the venom optimization hypothesis, where scorpions appear to use their stings only when required by the attacks of a persistent predator (Nisani et al. 2011; Lira et al. 2017; Rasko et al. 2018). In conclusion, our results suggest that T. pusillus individuals have the ability to modulate their reaction according to level of threat. Although animals reacted defensively at both threat levels (high and low), they exhibited this behavior more often, and stung more often in high-threat situations.
ACKNOWLEDGMENTS
We are grateful to Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) for granting a postdoctoral scholarship (BFP-0010-2.05/19) to AFA Lira. We are also indebted to the editor and anonymous reviewers for their comments to improve the presentation of the manuscript. We declare no conflict of interest.