Introduction

Sexual receptivity in females varies over time, generally increasing in the period of increased availability of resources. This is particularly true in scorpions, where, for example, species of the Brazilian semiarid region have their reproductive period during the rainy season (Lira et al. 2018a, b). Females are often the decisive sex when it comes to choosing reproductive partners due to their greater investment in offspring. Therefore, scorpion females tend to resist a male sexual initiative (e.g., Peretti 2001; Chantall-Rocha and Japyassú 2017), primarily outside the reproductive period. In these arachnids, unreceptive females may perform antagonistic interactions against male sexual initiative, such as pushing and stinging (Polis and Sissom 1990; Peretti 2001; Peretti and Carrera 2005; Carlson et al. 2014). Because of the aggressive and predatory tendency of females, males reduce their aggression through sequence of specific movements (Polis and Farley 1979; reviewed in Simone and van der Meijden 2021). For example, males may perform behavioral actions such as sexual stings, juddering, and cheliceral massage to induce females to cooperate during courtship (e.g., Polis and Sissom 1990; Peretti and Carrera 2005; Foerster et al. 2021).

Courtship and mating behavior of scorpions consists of ritualistic processes composed of four phases: initiation, promenade à deux, sperm transfer and termination (e.g., Polis and Sissom 1990; Tallarovic et al. 2000; Pordeus 2016). The initial contact by the couple, which consists of a sequence of behaviors that either stimulate the female or decrease her aggressiveness (Polis and Farley 1979). The promenade à deux also inhibits aggressive female tendencies, and allows the couple to find an ideal substrate for the deposition of the spermatophore (Alexander 1956; Polis and Sissom 1990). Finally, in the sperm transfer phase the semen in the spermatophore is transferred to the genital tract of the female and each individual goes to one side (Polis and Sissom 1990; Tallarovic et al. 2000; Peretti 2003).

In the Atlantic Forest of Northeastern Brazil, there are not well-determined seasons, a dry and a wet period (Dias et al. 2006) that directly influence scorpion foraging activity (Lira et al. 2018a, b). For example, an increase of foraging activity in Tityus pusillus Pocock, 1893 occurs during the dry season, mainly in November and January (Lira et al. 2013; Dionisio-da-Silva et al. 2018). Dionisio-da-Silva et al. (2018) based on increases in male foraging activity suggests that the reproductive period of T. pusillus occurs during the dry season. Therefore the lack of well-defined seasons and a broad reproductive season may indicate that the sexual receptivity of female T. pusillus varies over time. In this way, our goal was to investigate the receptivity of females of T. pusillus to male reproductive advances over time. Therefore, the following hypothesis was tested: females will be more receptive to male advances during the reproductive period.

Materials and methods

Scorpion sampling

Individuals of T. pusillus individuals were collected from the Tapacurá Ecological Station (8º 2′28''S, 35º11′46''W), an Atlantic Forest fragment with approximately 400 ha, located in the municipality of São Lourenço da Mata, Pernambuco, Brazil. The animals were collected for five months (November 2019 to March 2020) at night (19:00–22:00 h), using ultraviolet light flashlights and tweezers measuring approximately 20 cm. The scorpions were kept in the laboratory under natural conditions with a mean temperature of 26.7 ± 0.2 °C, 73.4 ± 2.4% relative humidity, and a 12:12 h light:dark photoperiod. Before the experiments, the scorpions were quarantined for 7 days in individual plastic terraria with cardboard as shelter and wet cotton as a water source. During this period, the T. pusillus scorpions were fed a cockroach nymph N. cinerea.

Female receptivity essays

For behavioral analyzes, 300 adult individuals of T. pusillus were used, 150 males and 150 nonpregnant females (no embryos visible through the mesosoma). Behavioral essays were carried out monthly during five months (November 2019 to March 2020) in the laboratory, at night (19:00–22:00 h) under red light (Machan 1968). In this way, 30 wild-caught couples were used each month, without repeating individuals. To simulate what happens in nature, the couples were randomly paired. The females were individually placed in circular arenas of 12 cm in diameter, containing a sheet of filter paper as substrate, and after 10 min of acclimatization, the males were inserted, one in each arena. The activities of each couple were observed for 20 min, in which the following parameters were analyzed: the number of reproductive attempts by the male, the female's body reaction and the occurrence or not of copulation. We considered each male action of holding a part of the female's body (e.g., legs, metasoma, or pedipalp) with the pedipalp as a reproductive attempt. The reaction of the female was noted and specified individually among the six categories observed (Table 1).

Table 1 Ethogram for the behavioral categories exhibited by the female scorpion Tityus pusillus during the mating ritual
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Statistical analyses

Tityus pusillus females presented only one behavioral act in response to males reproductive attempts or approaching. Therefore, their reactions were converted into binomial response variables (1 = occurred, 0 = did not occur). A similar procedure was adopted to the probability of courtship (complete or incomplete). To analyze the sexual receptivity of T. pusillus females to male reproductive advances through time, we used linear models (LM) and generalized linear models (GLM). Then, models were made in which we placed the number of male attempts and the probabilities of courtship (complete or incomplete) and female reactions as response variables. In these models, the month in which the tests were performed was placed as a fixed effect. Furthermore, in the courtship (complete or incomplete) occurrence models, the number of male attempts and female reaction as explanatory variables. The normality, homoscedasticity, and overdispersion of the variables were evaluated graphically using the fitdistrplus package (Delignette-Muller and Dutang 2015). When the assumptions were not met, the variables were modeled according to their best distribution. For data with binomial distributions, the 'glm' function from the stats package was used (R Core Team 2018). Variables with negative binomial distribution were modeled with the 'glm.nb' function of the MASS package (Ripley et al. 2013) and the statistical significance of the GLMs was calculated with the 'ANOVA' function of the car package (Fox et al. 2012). A posteriori tests were carried out with the 'glht' function of the multcomp package (Hothorn et al. 2009). All analyzes were performed on the R software v. 3.3.3 (R Core Team 2018).

Results

The females of Tityus pusillus exhibited differences in behavior in relation to the male approach between the months (Table 2). Overall, 14.8% of females show a nonreceptive reaction (tail wagging or escape) in male approaching; however, when males attempt to reproduce ca. 92.7% of females exhibits nonreceptive reactions (Online resource: Table S1). These scorpions were more prone to mobility in January and less in March (χ2 = 14.120; p = 0.007) (Fig. 1a). However, in February and March, T. pusillus females pushed more than the males (χ2 = 16.445; p = 0.003) (Fig. 1b) than December and January. Other behaviors also varied temporally (sting: χ2 = 12.642; p = 0.013; tail wagging: χ2 = 12.874; p = 0.012), although trends were less clear. The escape and avoid males occurred in a low proportion and did not vary temporally (escape: χ2 = 3.705; p = 0.447; avoid: χ2 = 7.678; p = 0.104). Males also differed in the number of attempts to court females over the months (χ2 = 13.595; p = 0.009) (Fig. 2a). During January, the males made significantly fewer attempts than in February and March (Fig. 2a). Female behaviors of T. pusillus were related to the number of attempts (χ2 = 13.153; p = 0.001). When males made more attempts, the most common female reactions were push and sting (Fig. 2b). However, the number of attempts increases the probability of successful mating (χ2 = 4.206; p = 0.040) (Fig. 2c), and was marginally related to the probability of courting (χ2 = 3.672; p = 0.055). However, 41.67% of the couples resulted in unsuccessful mattings. The probability of successfully courting or mating did not differ significantly between months (courting: χ2 = 2.26; p = 0.587; mating: χ2 = 6.582; p = 0.160).

Table 2 Temporal variation of sexual patterns in Tityus pusillus scorpions over a five-month period
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Fig. 1
figure 1

Temporal variation of female response behaviors to males’ reproductive approach. a Occurrence of immobility throughout the months. b Occurrence of pushing throughout the months. Different letters indicate significant differences between groups

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Fig. 2
figure 2

a Number of male reproductive attempts throughout the months. b Relationship between female response behavior and the number of male attempts. c Effect of the number of male attempts on mating probability. Different letters indicate significant differences between groups

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Discussion

In the present work, we investigated the sexual receptivity of females scorpion Tityus pusillus to male reproductive advances over a 5-month period. In our work, females of T. pusillus were more receptive to males’ sexual advances in February and March months, suggesting that this is a breeding season. However, our results contrast with the suggested breeding season for T. pusillus that occurs between the October and January months (Dionisio-da-Silva et al. 2018). It is important to note that our study was performed in a different forest fragment distant ca. 30 km from the studied area analyzed by Dionisio-da-Silva et al. (2018). Although the two study areas are classified as semi-deciduous seasonal forests (Dionisio-da-Silva et al. 2018), the two locations where the studies were carried out differ in some characteristics. Our study area consists of a 400 hectare fragment immersed in a sugar cane matrix (Moura 2019). The study area of Dionisio-da-Silva et al. (2018) corresponds to a fragment with 7,324 hectares immersed in a matrix mosaic of uses. These differences in fragment size and surrounding matrix are important modulators of the population dynamics of Atlantic forest scorpions, especially T. pusillus (Lira et al. 2015, 2021). Therefore, our findings indicate that different populations of T. pusillus may have different reproductive periods depending on the habitat.

The females of T. pusillus reacted in different ways according to the number of sexual attempts made by their partner, increasing the probability of pushing and stinging the males. Similar female reactions were described in the buthid Zabius fuscus (Thorell, 1876) (Peretti 2001). According to the author, this type of aggressive reaction reflects the resistance of females to sexual courtship, indicating an intermediate receptivity. Chantall-Rocha and Japyassú (2017) show that unsuccessful courtships in Jaguajir rochae (Borelli, 1910) are characterized by reduced complexity in interactions, with females refusing the male approximation during the initiation phase. Here we found that T. pusillus male reproductive success increased in more insistent individuals, suggesting that repeated actions are able to decline female aggressiveness. Furthermore, males did not exhibit greater insistence with all females, indicating that males of T. pusillus may select their partner. However, this hypothesis needs to be tested in future studies.

Our results also showed that, in pairs in which males made few attempts, females reacted more aggressively. In these couples, the T. pusillus females usually performed a tail wagging behavior. Although this behavior commonly occurs in males at the time of spermatophore deposition (e.g., Alexander 1959; Chantall-Rocha and Japyassú 2017; Lira et al. 2018a), in females this is related to aggressiveness and little receptivity (Outeda-Jorge 2010). According to Lira et al. (2020), moving the metasoma before stinging may be considered as a warning of the level of aggressiveness of the scorpion against potential predators or competitors. Considering that T. pusillus females react more aggressively after male touch may be indicative of the quality of their potential pair. The ability to overcome the aggressive behavior of females can be an indication of the quality of males. Low-quality males are less vigorous in their attempt to reproduce (Nobile and Johns 2005; Olivero et al. 2019). Thus, suggesting that T. pusillus females showed low receptivity to males that performed few reproductive attempts. It is important to note that, although we did not measure the individual body size, this functional trait may play an important role in sexual interaction. In scorpions of the genus Tityus, males of different sizes are common to occur (e.g., Lourenço 1979; Lourenço and Cloudsley-Thompson 2010), some due to the occurrence of an extra molt (Albuquerque and Lira 2016). The body size of males may directly influence reproductive behavior, since larger males may be more insistent than smaller males (Olivero et al. 2019).

Conclusions

The higher incomplete mattings reported in this study, is a common event in scorpions in which after the male deposits the spermatophore, the female refuses it and the individuals separate, with no passage of sperm to the female's genital operculum (e.g., Alexander 1956; Peretti 1996, 2001, 2003; Tallarovic et al. 2000; Briceño and Bonilla 2009). Females present a greater parental effort in terms of reproductive investment, as they are responsible for generating and caring for their offspring (Polis and Sissom 1990). In addition, the number of female gametes is produced in small quantities, having a high metabolic cost in reproduction (Trivers 1972). This leads us to believe that there is some criterion for choosing partners in T. pusillus, however this hypothesis will be tested in future works. In summary, our study reported two main findings, I) scorpion females showed differences in their sexual receptivity to males advances, II) male insistence increases the chances of a successful matting decreasing female aggressivity.