Venom composition
Scorpions belonging to the subgenus Archaeotityus have been portrayed as having relatively simple venoms and –perhaps‒ being less relevant from a medical perspective (Albuquerque et al., 2009). However, results from our analyses show that in T. ocelote, venom is more complex than expected and has a composition at least comparable to that found in other Tityus species. It combines a variety of proteins and a group of cysteine-containing peptides that potentially act as modulators of ion channels, inducing neurotoxic effects (Guerrero-Vargas et al., 2012; de Oliveira et al., 2015; 2018; Ortiz and Possani, 2018; Kalapothakis et al., 2021). It also has particularities that make it worth mentioning.
Enzymatic effects were almost exclusively associated with proteolytic and hyaluronidase activities. Proteinases are the leading group of medium-sized proteins found in Tityus species (Ortiz et al., 2014). Among them, serine proteases are frequently present, but in a low proportion, in the toxic secretions of these and other buthid scorpions (Estrada-Gómez et al., 2017). This situation contrasts with their predominant presence in the venom of non-buthid species (Carmo et al., 2014; Lok So et al., 2021).
The presence of metalloproteinases in T. ocelote venom confirms their ubiquity in Buthidae secretions. Multiple paralogs are expressed in different Tityus species (Carmo et al., 2014; Ortiz et al., 2014; de Oliveira et al., 2015; 2018), reinforcing the notion that these proteinases may be under evolutionary pressure for diversification. Despite this variation, the proteolytic effect attributable to these enzymes on gelatin was weak in T. ocelote venom. However, this could be the result of the substrate commonly utilized for testing the activity, since, invertebrate venom proteolytic enzymes could probably be related to toxin processing and their target would not be necessarily the extracellular matrix, as in the case of vertebrate venom proteases (Langenegger et al., 2018). In any case, the low proteolytic activity of Tityus venoms has been previously reported (Carmo et al., 2014).
Scorpion venom metalloproteinases are relatively shorter and do not have the multiple cysteine-rich domains found in snake venoms, and it has been postulated that this type of proteinase evolved from an ancestor of the ADAM Arachnida type, which lost the disintegrin and cysteine-rich domains (Carmo et al., 2014; de Oliveira et al., 2018).
Although we could not detect any enzymatic activity, partial sequences identified as belonging to PLA2s were expressed at the protein level in T ocelote. The consistent records of PLA2 in the venom gland transcriptome of several scorpion species (Rodríguez de la Vega et al., 2010; Ma et al., 2012) suggest that these enzymes are also ubiquitous in the different lineages of the Order, although in Tityus they represent less than 1% of the transcripts (de Oliveira et al., 2018). Thus, most of these phospholipases may have lost their enzymatic activity or the remaining scaffolds could have developed new functions associated with, for instance, the induction of inflammation with reactions that often accompany envenomings induced by scorpions and other arthropods (Lok So et al., 2021).
As in all members of the Buthidae family, cysteine-containing peptides dominate in T. ocelote venom. These molecules are responsible for the neurotoxic effects commonly associated with the sting of buthids, modifying the function of ion channels in excitable and non-excitable tissues (Lok So et al., 2021). A diversity of peptides affects different ion channels, but those that modulate sodium (NaTxs) and potassium (KTx) channels, prevail in Buthidae (Lok So et al., 2021).
At least six of the β-NaTx type and two of the α-NaTx type, are displayed in T. ocelote venom. These two types of sodium-channel modulators differ in their effects on membrane potentials and channel binding sites (Cid-Uribe et al., 2020). There is a differential distribution of these NaTx-types in Buthidae, with α-NaTxs predominating in the venom of Old World species, while the β-NaTx prevailing in the New World forms. β-NaTxs are remarkably diverse in Tityus and other American buthids and probably precede the appearance of the α-NaTxs, whose origin could have occurred before the separation of South America from Africa (Guerrero-Vargas et al., 2012). The loss and subsequent modifications of the genes that code for these peptides would explain the existence, and lower prevalence, of α-toxins in American buthids, including in T. ocelote. Zhu and Gao (2006) suggested that the ancestor of the NaTxs was probably a β-toxin, a variant of a lipolysis activating peptide (LAP) α- subunit, which displays 7 cysteines instead of 8, contrary to most of the NaTxs. Interestingly, two peptides with homology to LAP were present in T. ocelote venom, a finding that, in the case of New World scorpions, has only been reported for species from the T. (Tityus) subgenus and Centruroides (Zhu and Gao, 2006; de Oliveira et al., 2018; Kalapothakis et al., 2021). On the other hand, the presence of potassium channel toxins in T. ocelote venom is expected, considering that transcripts of these peptides have been found in every studied scorpion species to date, including non-buthid families (Zhu et al., 2011; Cid-Uribe et al., 2020).
In terms of the Na + channel modulating peptides, the proteomic analysis of T. ocelote venoms from three populations shows some geographic variation between Sarapiquí and the other two locations. Conversely, the profiles of Carara and Barú, both on the Central Pacific coast of the country, did not register significant variation. In general terms, T. ocelote from Sarapiquí only showed Tityus subgenus Na + channel modulating homologs in its venoms, whereas specimens from the Costa Rican Central Pacific coast showed peptides with similarity to both subgenera, Atreus and Tityus. The same two main K + modulating toxins identified in this study appear in all the analyzed venoms.
The intraspecific divergence in venoms between populations on both sides of the Costa Rican central mountain axis has been recorded in vipers (Alape-Girón et al., 2008; Madrigal et al., 2012), elapids (Mena et al., 2022) and dart-frogs (Mebs et al., 2014). In these cases, quantitative differences in venom components could translate into variation at the level of their pathophysiological effects (Gutiérrez et al., 1980). The final uplift of the central mountainous axis, particularly the Cordillera de Talamanca, during the Pliocene (MacMillan et al., 2004) constitutes one of the most recognized cladogenic events that shaped the biogeography of the region (Daza et al., 2010). This final uplift promoted the population structure and restricted gene flow between populations that, like T. ocelote, are distributed in the humid lowlands of Lower Central America (Wang et al., 2008; Saldarriaga-Córdoba et al., 2017).
Evolutionary significance of T ocelote venom composition
Until recently, subgenera of Tityus were considered natural groups (Lourenço 2006). Furthermore, the basal position of members of the subgenus Archaeotityus had been assumed by most researchers mainly based on a few putative plesiomorphic morphological characters (Lourenço 1999; 2002; Borges et al., 2010). However, this view is changing due to new phylogenetic evidence based on the analysis of molecular characters at various substitution rates (Borges et al., 2010; Ojanguren-Afilastro et al., 2017; Román et al., 2018; Moreno-González et al., 2021).
In independent studies, Ojanguren-Afilastro et al. (2017) and Moreno-González et al. (2021) presented their hypotheses of the evolutionary relationships among species that make up the most influential groups within Tityus. The most important point for our discussion is the finding that T. (Archaeotityus) turns out to be the sister clade of a subgroup of Tityus (Tityus), composed of the T. bahiensis, T. stigmurus, and T. trivitattus species complex (the “T. (Tityus) bahiensis clade,” sensu Ojanguren-Affilastro et al. 2017). The analyses of Borges and Graham (2016) and Román et al. (2018) also support these findings, although none of the presented reconstructions show high support for this branch.
According to Ojanguren-Affilastro et al. (2017), deep divergence within Tityus occurred about 30 Mya ago (95% CI: 22.9–37.6), rendering the mentioned major division within the genus, while the T. (Archaeotityus) and the T. (Tityus) bahiensis clades might have split from a common ancestor around 24.2 Mya (95% CI: 16.6–30.6) during the Oligocene. Following this proposition, members of the T. (Tityus) bahiensis clade and those included in T (Archaeotityus) would exhibit closer relationships in gene expression with each other than with other Tityus clades. In support of this prediction, Borges et al. (2012) described the sequence homologies of two NaTx, Tcl1, and Tcl2, obtained from the venom gland transcriptome of T. clathratus, the type species of Archaeotityus. According to these authors, both toxins are more closely related to members of the T. (Tityus) bahiensis clade, showing up to 87% sequence identity. A similar conclusion was presented by de Oliveira et al. (2021) concerning two T. (Archaeotityus) mattogrossensis toxins, Tm1 and Tm2, that show similarity (> 83% identity) to T. (Tityus) fasciolatus and T. (Tityus) bahiensis sequences. To this combined evidence, we could add our present finding of Toc1 and Toc2 (and Toc8) that also show a high level of identity with the toxins expressed in those Brazilian scorpions and that –in the case of the first two– also share significant sequence similarities to the T. (Archaeotityus) toxins previously reported.
The aforementioned phylogenetic hypothesis would also predict the segregation between NaTxs from T. (Tityus) bahiensis and those from the subgenus T. (Atreus). In that regard, Guerrero-Vargas et al. (2012) analyzed 75 peptides from 10 nominal species of both groups and, in their reconstruction, found 13 well-supported NaTxs clusters. Except for one (NaTx3), each cluster included toxins from members of only one subgenus. However, even in NaTx3 (composed of toxins from both groups), they occupy different positions in the cluster, highlighting the segregation between toxin homologs produced in these phylogenetically distant groups.
The extent of divergences translates to the immunochemical level since Borges et al. (2020) found that scorpion venoms belonging to subgenera T. (Atreus) and T. (Tityus) display high antigenic diversity against several commercial antivenoms. Based on the differences in immunoreactivity and peptide venom composition, these authors recognize the existence of four “toxinological regions”: (1) Lower Central America-Amazonia, (2) Venezuela, (3) Southeastern South America, and (4) a region seemingly centered in the Andean foothills. The first two regions comprise T. (Atreus) species, while the third includes those of the T. (Tityus) bahiensis clade. The fourth region contains species of T. (Tityus) bolivianus clade (sensu Moreno-Gonzáles et al., 2021) together with T. cerroazul from Lower Central America, suggesting a previously unrecognized similarity. Interestingly, the immunogenicity of the T. (Archeaotityus) group was previously evaluated through the type species T. clathratus, showing poor recognition by commercial antivenoms (Borges et al., 2010), which suggests some evolutive separation, either from regions II (T. discrepans) and III (T. serrulatus) groups. Based on clinical evidence, Borges and collaborators (2020) even included a member of the Archaeotityus subgenus, Tityus silvestris, in region I, suggesting close similarity of Archaeotityus with some Atreus species such as T. obscurus, for instance.
Our phylogenetic analyses also show strong homologies between T. (Archaeotityus) NaTxs and those from the T. (Atreus) clade. Five T. ocelote toxins (Toc3, Toc4, Tc5, Toc6, and Toc7) shared well-supported phylogenetic proximity with several species included in T. (Atreus) group. Toc5 sequence is identical to that of TpaCR2, a toxin first identified in T. (Atreus) jaimei (formerly, T. pachyurus) from Costa Rica (Borges et al., 2020). In addition, in our phylogenetic analysis, and the one presented by Borges et al. (2012, their consensus tree), Tcl1 is included in a clade composed of toxins of the subgenus T. (Atreus). Likewise, we were not able to resolve the relationship of T. mattogrossensis Tm1, and Tm2 with toxin homologs from either the T. (Tityus) bahiensis clade or T. (Atreus), a situation also evidenced in the de Oliveira et al. (2021) analysis (their Fig. 7). Thus, the claim that these same toxins are more closely related to the T. (Tityus) bahiensis clade is, at best, weak. Increasing the number of terminal groups in a phylogenetic analysis can reduce the effects of long branches, which might otherwise "attract" and erroneously group, terminal nodes (Wiens, 2005). However, this issue is commonly ignored during reconstructions of phylogenetic relationships instead of increasing the sequence length or the total number of characters in the analysis. Taxon sampling also improves estimates of evolutionary parameters derived from phylogenetic trees and is therefore crucial for optimizing applications of phylogenetic analyses (Heath et al., 2008).
Since, according to previous molecular phylogenetic evidence, Archaeotityus and Atreus are not considered sister groups, the closeness we found of some NaTxs from these subgenera homologs is intriguing. One possible explanation is that these are paralogs that conceivably arose by gene duplication before the divergence of the most recent ancestor between these subgenera. These paralogs must have been lost later, during the divergence of the T. (Tityus) bahiensis clade. How ancestral toxins were maintained in Archaeotityus is not entirely clear. Both positive and purifying selection has allowed the development and maintenance of venoms adapted to their ecological functions: selecting toxins that are more sensitive to specific groups of predators or toxins that allow arthropods to be immobilized (Sunagar et al. al., 2013; Van der Meijden et al., 2017). The evolution of α-NaTx in Old World scorpions probably resulted from defensive pressure, as predator's sodium channels show greater variability than their insect counterparts (Zhang et al., 2015). Indeed, multiple independent origins of mammalian-specific NaTxs occurred in separate lineages of medically important buthids, and this diversification chronologically coincides with the appearance of their mammalian predators (Santibáñez-López et al., 2022). Thus, increased toxicity in some Tityus lineages (i.e., T. discrepans, T. bahiensis, T. pachyurus) may result from greater selective pressure. Unlike other members of the genus, Archaeotityus scorpions have a small body size, reduced telson, and are primarily of reserved arboreal habits. These characteristics suggest that these animals are probably not frequently targeted by mammalian or other vertebrate predators (but see Gabriel et al., 2021 for a documented predation case from a lizard). Therefore, they might experience less selective pressure on toxic components targeting mammals than the more conspicuous and toxic species of the genus. In a relaxed selection scenario, the pressure to maintain a trait is removed or reduced, but the result does not necessarily lead to a clear predictive pattern (Lahti et al., 2009). Thus, a trait maintained by several factors of selection (such as the joint action of trophic and defensive pressures) could retain its advantage even when one of the sources of selection declines. In the present case, relaxed selection could have supported the maintenance of toxins derived from different ancestral lineages in scorpions of the genus Archaeotityus by reducing the pressure to maintain toxins with a specific function.