Cuticular hydrocarbons from ants (Hymenoptera: Formicidae) Odontomachus bauri (Emery) from the tropical forest of Maranguape, Ceará, Brazil

Semiochemicals, 5-methyl-nonacosane, Alkanes, Chromatography.Ants are eusocial organisms with great relative abundance and species richness. Studies on these organisms are scarce, especially in the high altitude humid forest environments of the state of Ceará. In view of this condition, an evaluation of the chemical composition of cuticular hydrocarbons (CHCs) of the species Odontomachus bauri found and recorded for the first time in this study in the tropical forest of Maranguape was carried out, based on the hypothesis that different nests have different compositions of CHCs. For this purpose, collection incursions were carried out in the region to obtain individuals from three different nests. In the laboratory, the extraction of CHCs was performed by immersing the ants in hexane for 30 minutes. After removing the ants, the extracts were subjected to drying to assess the yield of the samples. Then, the extracts were subjected to a silica gel column, before performing an analysis by gas chromatography coupled with mass spectrometry. The chromatograms obtained were analyzed and demonstrated a great similarity, showing longchain saturated hydrocarbons, mainly represented by alkanes and methyl-alkanes. 5-Methyl-nonacosane was the component with the highest relative abundance. Statistical analysis of similarity and correlation between samples was performed using non-parametric tests. These analyzes provided sufficient statistical evidence to confirm the existence of a strong positive correlation between the samples and also a significant difference in the composition of HCCs in two of the three paired analyzes performed, thus confirming the study hypothesis.


Introduction
Ants are organisms that play important interactions in ecosystems, acting as fundamental biological indicators in the monitoring of degraded areas thanks to their great diversity, numerical representation and biomass in ecosystems (Lutinski et a., 2017), contributing, for example, to greater quality and reduction of the number of pests in landscapes dedicated to monoculture (Helms et al., 2020;Fernandes et al., 2021).
Ants of the genus Odontomachus are predators that build their nests in different strata of the environment such as soil (Cerquera & Tschinkel, 2010), as roots of epiphyte plants (Oliveira et al., 2011) and in rotten branches and trunks deposited in the soil (Brown, 1976). Odontomachus ants are commonly found in the oriental (69.2%) and neotropical (30.6%) regions of the world (Ward, 2000), and are known for the rapid closing of their jaws (Gronenberg et al., 1993) and for having a sting pain associated with several cases of urticaria and anaphylaxis (Rodriguez-Acosta et al., 2010).
In Odontomachus ants, as well as in other social insects, maintenance and survival of the nests depends on the communication between their individuals. This communication is accomplished through chemical compounds that can trigger various types of behaviors such as recruitment, alarm, defense and nestmate recognition (Wilson, 1965;Holldobler & Wilson, 2009). Recognition between individuals depends on chemical compounds present in the exoskeleton of these animals, the cuticular hydrocarbons (CHCs) and, in some cases, depend on the proportion of volatile compounds present in specific body regions, such as the head and abdomen (Jaffe & Marcuse, 1983).
The cuticle (skin) of all insects is covered with a very thin epicuticular layer of wax (lipid). This cuticle protects the insects from various conditions (e.g., pathogens), but one of the most important features is the protection against transcuticular water loss. In the majority of the insects, the main compounds in the wax layer are hydrocarbons (Drijfhout et al., 2009). These compounds are mainly formed by straight or branched chain alkanes that have a regulating function of cuticular permeability, protecting the animal against dehydration and giving greater adaptability to ambient temperature variations (Blomquist & Bagnères, 2010). CHCs also contribute to the determination of caste and fertility status of individuals (Dietemann et al., 2003;Abdalla et al., 2003), presenting chemical profiles that vary between different species and even between nests of the same species. This intraspecific diversification is indirectly related to the type of feeding available for the nest (Richard et al., 2004).
Monomethyl alkanes are related to communication signals and may be linked to the formation of dimethyl alkanes, specific chemicals that may be characteristic of a species or nest (Martin & Drijfhout, 2009). These characteristics show us the importance of chemical compounds for communication in social insects.
In this work, the species Odontomachus bauri (Emery), collected in the tropical forest of Maranguape, was investigated in relation to the composition of CHCs. The tropical forest of Maranguape is located approximately 25 km away from Fortaleza (capital of Ceará) with 920 m high, presenting an area of significant landscape importance, where there are crystalline rocks, including granites, granodiorites, gneisses and migmatites (Tavares et al, 2018). The type of vegetation is characterized as a remnant of tropical forest, as it is formed by a humid and evergreen forest located in the semiarid region of northeastern Brazil. These rainforest enclaves have a positive correlation between the abundance of predators and prey in the region with changes in dominant arthropod species between the rainy and dry seasons. There is also an influence on the abundance and richness of species in response to late rains (Azevedo et al., 2020).
The tropial forest of Maranguape is a rocky grassland mountain which is inserted in a matrix of sandy and stony grasslands, including several vegetation types, such as Cerrado (Brazilian savanna), Atlantic Forest, and Caatinga. The Myrmicinae and Formicinae are the most representative subfamilies of ants in rocky grassland mountains, with 53% and 18% of the total species richness, respectively, but few data were reported about Ponerinae sub-family, which O. bauri is included (Costa et al., 2015). Some representatives of Myrmicinae from rocky grassland mountains are the leaf cutters ants Atta sexdens Linnaeus (Giesel et al., 2020; and as example of ants with sub-family Formicinae, Nakano et al. (2013) reported the occurrence of Myrmelachista Roger in the Atlantic Forest of Southeastern Brazil and Lachnomyrmex nordestinus was found in tropical forest of Maranguape (Feitosa & Brandão, 2008). The characterization of the types of ants that inhabit this mountain is scarce. Ants of the genus Odontomachus have already been identified in humid highland forests of Ceará, having been seen in the tropical forest of Baturité (Azevedo Filho et al., 2003;Hites et al., 2003). This will be the first report of the presence of ants of the genus Odontomachus in the tropical forest of Maranguape. Therefore, this work aimed to evaluate the hypothesis that ants from different nests have different compositions of CHCs and, for that, O. bauri ants were collected in the northeastern region of Brazil to obtain the characterization of their CHCs.

Insect materials
A qualitative-quantitative case study (Pereira et al., 2018) was carried out and, for that, O. bauri ants were collected in an area of the tropical forest of Maranguape (CE, Brazil) (3°52'03.4"S 38°41'44.1"W; MMA/ICMBIO/SISBIO authorization: 58972-1). Ants were collected during the 2016 rainy season, in March and April. The rainfall and thermal average of the period was 176 mm and 23 ºC, respectively. The nests were located at an altitude of 300 m and at least 300 m apart. Ants from three different nests were collected by aspiration and stored in separate containers which were then taken directly to the laboratory for CHCs extraction. Two hundred individuals from each container were selected for the extraction of cuticular hydrocarbons, thus composing three distinct groups. Only workers were used in the study, providing a sufficient number of individuals of the same caste to remove the extract. The extraction of CHCs by immersion in solvent was performed based on the studies carried out by Menzel et al. (2017), with modifications. Each group was separately immersed in hexane for 30 minutes at room temperature (25°C). After this time, the ants were removed by filtration, leaving behind a hexane extract. The extract was subjected to rotary evaporation for complete solvent removal, forming a dry mass for analysis by gas chromatography coupled to mass spectrometry (GC-MS).

Analysis of Extracts
The obtained extracts from ant samples were passed through a silica gel (60 mesh) chromatography column, being eluted with hexane, to exclude polar minor compounds before being sent for CG-MS. The analysis was performed in a Shimadzu QP-5050 instrument with a DB-1 fused silica capillary column with dimethylpolysiloxane (30m x 0.25mm ID x 0.25μm film); Injection initial temperature: 25° C; Interface temperature: 230° C; Control Mode: Split; Split Ratio: 1:27; Column inlet pressure: 100 kPa; Column flow: 1.7 mL/min; Total Flow: 50.0 mL/min; Equilibrium Time: 1.00 min; Linear Velocity: 47.4 cm/sec; Temperature was 35 to 180° C at 4° C /min, then 180 to 280° C at 17° C /min and 280° C for 10 min; The mass spectrum was obtained by electron impact at 70 eV. Hydrocarbons were identified using a library search (NIST08), the diagnostic fragmented ions and the Kovats indices.

Statistical Analysis
The relative abundance data of the CHCs were organized in a matrix, thus allowing a comparative statistical analysis.
The analyzes were performed using the JASP Team software (2021, version 0.14.3). The normality of the data was assessed by the Shapiro-Wilk test (α = 0.05). The Shapiro-Wilk test of three samples showed significant values (p < 0.001) and, for this reason, the null hypothesis (H0) was rejected. Therefore, the relative abundance data of the samples do not have a normal distribution. In view of this condition, statistical analysis was performed based on non-parametric tests. To this end, the Wilcoxon signed-rank (paired) and Spearman (correlation) tests were conducted. The three samples were considered as replicates. The Wilcoxon signed-rank test proved to be significant when comparing samples I-II (p = 0.003) and II-III (p < 0.001), thus rejecting the H0. Therefore, there is sufficient statistical evidence to affirm that there are differences in the comparisons between samples I-II and II-III ( Figure 1). The comparison between samples I-III was not significant (p = 0.254), that is, there was no rejection of H0. Therefore, there is sufficient statistical evidence to state that samples I and III are the same. The Spearman's test ( Figure 2) showed a strong positive correlation between the samples (Samples I-II: 0.926; Samples II-III: 0.871; Samples I-III: 0.876, p < 0.001).

Discussion
The comparative analysis of the obtained compounds of O. bauri ants revealed the existence of a great variety of  (Martin & Drijfhout, 2009).
The high proportion of n-alkanes can be interpreted as consistent according to the species' nesting pattern, known for building nests under branches and trunks deposited in the soil (Brown, 1976), which favors greater susceptibility of individuals to climatic conditions. The northeastern region of Brazil is known for its semi-arid condition, with dry characteristics and high temperatures (Marengo et al., 2018). High temperature and low humidity contribute to a differentiation of the cuticular layer, promoting a greater relative abundance of n-alkanes in its composition. (Wagner et al., 2001).
Six of the 12 methyl alkanes found are exclusive of sample II (Table 2), demonstrating the existence of an intraspecific variation in the population, which is in accordance with the statistical results obtained. This distinction is possibly related to the geographic distance of the nests and the availability of resources for them. The intraspecific differences of CHCs have already been observed in several insect species and may be related to feeding variations and nest substrate type (Richard & Hunt ,2013). The diet of ants has the ability to change the composition of CHCs, which affects the recognition ability between individuals (Liang & Silverman, 2000).
The recognition ability is related to the aggressive behavior presented against individuals that do not belong to the nest. This aggressiveness can be reduced when neighboring nests are submitted to the same diet (Buczkowski, 2004). The similarities between samples I and III may be a consequence of geographical proximity or the same availability of food in the environment in which the nests are found, thus contributing to the recognition and reduction of aggressiveness among individuals. Studies carried out with the species Pachycondyla analis (Latreille) have shown that n-alkanes remain consistent in the cuticle profiles of different nests, while methyl alkanes and alkenes are possibly related to the ability to recognize among individuals (Yusuf et al., 2010).
In this study, the 8-heptadecene component was observed in the three samples, indicating that the extraction methodology obtained organic compounds from other regions of the animal's body besides the cuticle. This component has been previously identified in the Dufour gland of ants O. bauri  and also in ants of the genus Diacamma as a recruitment pheromone (Fujiwara-Tsujii et al., 2012). Nevertheless, the Odontomachus ants do not form trails and the search for food occurs in a solitary way with the orientation based on the canopy of the trees (Oliveira & Hölldobler, 1989), then the component 8-heptadecene is possibly related to recruitment capacity among the nest individuals.
Taken into account a wide spectrum of studies, it appears clearly that the main role of the n-alkanes is to control transcuticular water movement while the unsaturated compounds and methyl-branched hydrocarbons are more likely to be involved in communication (Drijfhout et al., 2009). CHCs profiles are well known in the literature as a method of differentiating varieties into insects and may vary quantitatively and qualitatively in species (Blomquist & Bagnères, 2010). In ants of the genus Odontomachus is similar. The major component of the cuticle of these animals acts as an indicator of fertility.
For example, in O. brunneus (Patton) ants, a component that acts as a fertility signal in the species, the (Z)-9-nonacosene was identified. This component is used to differentiate reproductive and non-reproductive females in the nest. (Smith et al., 2012).
A pattern that has been conserved in the species despite the existence of intraspecific variations of cuticular profiles of the population (Smith et al., 2013). Males of the species have another signaling component, (Z)-9-pentacosene (Smith et al., 2014).
In species O. ruginodis (Smith), queens differ from workers due to the presence of a specific cuticular component,  (Smith et al., 2016).
In O. bauri samples the 5-methylnonacosane component showed a high relative abundance and probably play a role in the communication as a sex attractant of the species, contributing to the nest caste characteristic. In addition, two other molecules with chains of the same length and different branch points were observed, 3-methyl-and 2-methylnonacosane. This variation in the methylation points may be related to the intensification of the ants' signaling capacity. A study with the ant Linepithema humile (Mayr) showed that the species is able to distinguish different branching points in molecules with chains with same length more easily than in molecules with chains of different sizes and equal branching points (van Wilgenburg, 2012).
The 5-methylnonacosane component was found in less relative abundance in the cuticular profile of the species O.
relictus (Smith et al., 2016;Smith, 2019). However, the component indicated as a marker of fertility of the species was (Z)-9nonacosene (Smith et al., 2016). This same component was identified in the post-pharyngeal gland secretions of O. bauri ants collected in Venezuela, also showing a small relative abundance (Sainz-Borgo, 2011). Given this information, this is the first report of this cuticular component with high relative abundance in the species, as well as the record of its cuticular profile in northeastern Brazil.

Conclusion
The extract samples show great similarity in the composition of the CHCs, a condition reinforced by the Spearman correlation analysis. Wilcoxon's paired analysis between the extracts confirmed the hypothesis that the nests have different compositions in two of the three pairings. Pairings I-II and II-III proved to be statistically distinct. However, the same did not occur in the pairing between samples I-III, being therefore considered statistically equal. This condition may be a reflection of the proximity between the nests and the availability of food in the region. Future studies can be conducted to assess the effect of distance on the composition of CHCs between nests. The 5-methyl-nonacosane component had the highest relative abundance in all samples, being this the first representative record of this component in the species. Based on information obtained from several studies on ant CHCs, it is observed that the major components found in the cuticle of these animals act as chemical signals that identify the fertile and non-fertile individuals in the nest. Thus, the 5-methyl-nonacosane component is possibly the chemical signal for infertile females in the nest. However, future studies must be carried out to confirm this hypothesis.