Intraspecific and Intracolonial Variation in the Profile of Venom Alkaloids and Cuticular Hydrocarbons of the Fire Ant Solenopsis saevissima Smith (Hymenoptera: Formicidae)

1 Laboratório de Entomologia Médica e Molecular, Instituto de Biof́ısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (IBCCF/UFRJ), Rio de Janeiro, RJ, Brazil 2 Instituto de Quimica, Universidade Estadual de Campinas (IQ/UNICAMP), 13083-862 Campinas, SP, Brazil 3 Instituto de Biociencias, Universidade Estadual Paulista (UNESP), Campus de Rio Claro and Centro de Estudos de Insetos Sociais (CEIS), 13506-900 Rio Claro, SP, Brazil 4 Laboratório de Mirmecologia, Centro de Pesquisas do Cacau (CEPLAC), Itabuna, BA, Brazil 5 Laboratório de Tecido Conjuntivo, Instituto de Bioquı́mica Médica, UFRJ, RJ, Brazil


Introduction
The fire ants of the genus Solenopsis Westwood include species considered pests of worldwide importance, especially Solenopsis invicta Buren, which were accidentally transported to other countries outside their native range in South America.To date, fire ant invasion is a major concern in the USA.These ants, particularly those belonging to the Solenopsis saevissima species group, react aggressively when their earthen nests are disturbed.Their stings, in addition to pain, can cause serious anaphylactic reactions to sensitive subjects (e.g., [1]).
The species Solenopsis saevissima Smith is native to South America and common in Brazil [2], wherein it is potentially responsible for over 35% of the registered accidents with insects (personal communication of Mário Sérgio Palma).

Collection of Samples.
Samples of workers of mixed sizes were obtained upon disturbing nests of S. saevissima in the field at Brasilia, Distrito Federal (15 • 48 00"S 47 • 51 50 W), Palmas, Paraná State (26 • 13 44 S 52 • 40 15 W), and Rio de Janeiro (22 • 51 45"S 43 • 13 26 W), Brazil (Figure 1).Additionally, samples of each individual caste were obtained from five whole fire ant nests collected from a house garden at the municipality of Pedro do Rio, Rio de Janeiro, Brazil (22 • 20 30 S 43 • 07 44 W) following the methods for handling and rearing these insects in the laboratory described in [20].
Species identification was based on the series of characters given in [5] and additional useful traits from [14]; the following diagnostic characters of major workers of S. saevissima were confirmed: no postpetiolar process, complete mandibular costulae, absence of a frontal medial streak or ocellus, and a poorly developed median clypeal tooth.Voucher specimens are deposited in the Adolph Hempel Entomological Collection of Instituto Biol ógico de São Paulo, SP, Brazil.

Sample Preparation.
Random worker samples obtained in the field were directly immersed in 100 µL of hexane.Workers from the whole nests were separated in the laboratory into different size classes (size interval, mean weight ± SD) as follows: minor workers (1-2 mm; 0.40 ± 0.08 mg), medium workers (3-4 mm; 0.9 ± 0.18 mg), and major workers (5-6 mm; 2.0 ± 0.52 mg).This division was merely analytical, as most authors only recognize a continuous size range from minors to majors.Males (1.9 ± 0.4 mg) and gynes (unmated queens; 3.1 ± 0.2 mg) were separately analyzed.
Venom Alkaloids.Females of different size classes and castes were cold-anesthetized and had their venom sacs dissected with a fine forceps and macerated in bidistilled ethyl acetate.The venom extracts were adjusted to a final extract concentration of 1 mg/mL.
Cuticular hydrocarbons from body wash: the bodies without venom glands were washed with distilled water   (in 5 mL for 10 min) three times, dried with a piece of filter paper, and then dipped into 2 mL of hexane for 5 min.The obtained body wash extracts were then adjusted to 1 mg/mL in hexane.
Cuticular Hydrocarbons from Crushed Heads.Several workers of different size classes, gynes, and males were cold-anesthetised and decapitated.The excised heads were crushed in 2 mL of bidistilled hexane, filtered, and dried, and the resulting wash was adjusted to 1 mg/mL in hexane.
For quantification of venom alkaloids, certain amounts of individuals (workers or gynes) were pooled, immersed in hexane for 10 min, and the final yield of dry extract weighed with an analytical scale, being divided by the number of individuals used.The same method for measuring cuticular hydrocarbons proved impracticable as the extracted amounts were too small to be accurately measured; thus, the relative amounts of cuticular hydrocarbons could be only estimated from comparing the peak area of n-alkane standards with obtained peaks from GC-MS chromatograms with known numbers of individuals and solvent volume.

GC-MS Analyses.
The obtained extracts-venom alkaloids and body and head hydrocarbons-were analyzed by gas chromatography and mass spectrometry (GC-MS) by injecting 1 µL of each extract in a GCMS-QP2010 (SHI-MADZU) system equipped with a RTX-5MS silica capillary column (30 m × 0.25 mm × 0.25 µm).Helium was the carrier gas, used at a flow rate of 1.0 mL/min on split mode.The MS were taken at 70 eV, and scanning speed was set to 1228, from m/z 50 to 700.The interface temperature was maintained at 280 • C. The injector temperature was 250 • C. Oven temperature was programmed to increase at 12 • C/min from 50 • C to 330 • C, with a final hold time of 1 min.

Derivatization by
Dimethyl Disulfide/Iodine.Alkenes were derivatized according with the methodology described in [31].

Samples from Different Geographical Localities.
The samples with workers of mixed sizes revealed the existence of two radically different chemical profiles (see Figure 2) within S. saevissima, which shall be referred heretofore as variety A and variety B. The venom alkaloids of both varieties were composed by isomers of 2-methyl-6-undecylpiperidine (C11) and 2-methyl-6-tridecenylpiperidines (C13), but isomers of C13 prevailed in the venom profile of variety A (with only trace amounts of 2-methyl-6-pentadecylpiperidines), while isomers of C11 prevailed in variety B. Cuticular hydrocarbons were also different to the same extent (see Figure 2).A complete list of identified venom alkaloids is given in Table 1, and a complete list of the cuticular hydrocarbons observed is given in Table 2 (shorter names for each compound are given in these tables).Relative proportions of the main cuticular hydrocarbons identified in field samples of S. saevissima varieties A and B are given in Table 3. Variety A was found in Brasilia and Paraná, while variety B was retrieved from Rio de Janeiro.Molecular evidence (not shown) confirmed that the obtained varieties correspond to two consistent and distinct mtDNA haplotypes within the S. saevissima clade-that is, they are cryptic species.These molecular results will be dealt with in a separate publication.Preliminary tests (not shown) with 100% ethanol demonstrated that this solvent is also useful for recovering hydrocarbons and alkaloids from fire ants, as long as the mixture is injected in the GC-MS no later than within 2 weeks of collection.

Whole-Nest
Samples.Further field inspections (not shown) revealed that S. saevissima varieties A and B were abundant at the mountains of Rio de Janeiro; thus, a strategic collection point at the city of Pedro do Rio was elected for obtaining whole nests.Solenopsis saevissima was the only fire ant species found around that area, and nests of the two cryptic varieties were present.For sorting in the field between the varieties, we devised the following method: (i) a 15 cm wide carton circle (the "arena") was placed in a plastic or glass bowl; (ii) a sample of one nest was collected with a spoon and placed on one side of the paper arena; (iii) another spoonful sample, from the other nest, was placed on the opposite side of the paper arena; (iv) the degree of aggressiveness of the ants was observed for a few moments.Fighters invariably proved being of different varieties when later analysed by GC-MS.An example of the test can be seen online at http://archive.org/details/AggressivenessTestsFire-Ants.

Venom Alkaloids.
Minor workers yielded ∼7 µg per venom sac (N = 2 groups of 1,000 ants), while media workers yielded ∼12 µg per venom sac (N = 1 group of 1,000 ants), and major workers yielded ∼25 µg per venom sac (N = 2 groups of 400 ants).Gynes yielded ∼90 µg of venom alkaloids per venom sac (N = 15).Table 4 illustrates the relative amounts of each venom alkaloid among workers of different sizes and gynes.Relative proportions of alkaloid isomers of workers varied with size: minor workers always had higher proportions of trans isomers (Table 4), while greater size classes had increased amounts of cis isomers; cis isomers were always predominant in the venom of gynes.Venom alkaloids were not detected in neither body wash nor head extracts of males.Larvae of both varieties contained detectable amounts of the same venom alkaloids of workers (not shown).
Hydrocarbons obtained from the hexane extracts of head and body extracts were always the same (not shown), thus these results were qualitatively equivalent.A comparison of the head extracts of major workers of S. saevissima varieties A and B is shown in Figure 3. Main cuticular hydrocarbons of S. saevissima variety A were 13-heptacosene, 13-methylheptacosane, and 3-methyl-pentacosane, while the main cuticular hydrocarbons of S. saevissima variety B were tricosane, 3-methyl-tricosane, 10-pentacosene, pentacosane, and 3-methyl-pentacosane (see Figures 2 and 3).
The relative proportions of cuticular hydrocarbon obtained from body extracts of workers of different sizes and castes of S. saevissima varieties A and B are shown in Table 5.The relative amounts of cuticular hydrocarbons varied sensibly among workers of different sizes and castes.A full chromatogram illustrating the differences between cuticular Psyche  hydrocarbons and venom alkaloids of major and minor workers of S. saevissima variety B is given in Figure 4.

Samples of Different Geographical
Localities.Random samples of mixed worker sizes suggested that the described cryptic varieties are widespread and common over Brazil, in agreement with the scenario predicted by [7] using other tools.It remains to be investigated if the cryptic species appointed by [7] would correspond to similar chemical profiles.A complete scenario can be easily obtained by further sampling from other localities.Other varieties within nominal S. saevissima are probably also present.The practice of testing workers for aggressiveness was very helpful in locating a nest of the less prevalent S. saevissima variety A within the study area, as it dispensed the need for returning several samples back to the laboratory for GC/MS analyses.

Psyche
Table 5: Relative abundance (%) of cuticle hydrocarbons from small workers (SWs), medium workers (MWs), and major workers (LW), gynes (Gs) and males (Ms) of Solenopsis saevissima varieties A and B, collected in Pedro do Rio, Rio de Janeiro, Brazil.The fact that ethanol provided good chemical profiles of samples collected in the field is interesting, as it is a more common, cheaper, and less volatile solvent.This opens the possibility of checking the alkaloids present in long-standing fire ant samples deposited in wet collections.It should be noted, however, that care should be taken in such cases as polar solvents can be harmful to some analytical systems, for they can interact with the column matrix.

Venom Alkaloids and Cuticular Hydrocarbons.
The reported amount of cuticular hydrocarbons extracted from fire ants is dramatically superior-sometimes >200 timesto other much larger ants such as Ectatomma ruidum Roger [32] and Ectatomma brunneum Smith F. and Atta laevigata Smith F. (unpublished results of E.G.P.F.).This disparate abundance of waxes on the body of fire ants must have some biological implication, which probably explains why they can conveniently be separated by nest debris by flotation [20].In comparison with previous studies with fire ants, in the present report we found they were in agreement with cuticular hydrocarbons reported by [13] from similar extractions with S. invicta and S. richteri, yet another study [33] found as little as 300 ng per ant.We think that such difference could be explained by either populational or specific differences, but the fact that the reported values proved also quite variable between different studies with the same species indicates that the matter would merit further comparative investigation.We ought however to emphasize that our chromatograms with different samples within the same nest suggest that there is considerable intraspecific variation in the alkaloids/hydrocarbons ratio.
The abundance of venom alkaloids in the body wash extracts is also elevated, and it should be noted that these ants apparently discharge the contents of their venom glands upon immersion in organic solvent.However, we herein recovered about one-third of the amount reported by [22] with S. invicta.Again, it is possible that such difference be due to specific variations, but it should be minded that these authors employed different methods for their venom estimates.The fact that venom alkaloids were also recovered from bodies with excised abdomens indicates that they accumulate for some time on the exoskeleton of the insects.This alkaloidal covering might serve as armor against entomopathogenic fungi and predators, or even have some important role in nestmate recognition, given their abundance.

Venom Alkaloids and Cuticular Hydrocarbons as
Taxonomic Tools.The sampled regions of the present study, from being distant from each other, present marked differences in climate, soil, and vegetation.The different patterns of venom alkaloids and cuticular hydrocarbons of the collected samples indicate they belong to two different cryptic species (compare Tables 1, 2, and 4).These patterns were yet markedly different from reports for S. saevissima by [14] based on samples from São Paulo, thus indicating the existence of other cryptic species in Brazil.The additional profile published by [19] further indicates the existence of another cryptic species in French Guyana.
The existence of cryptic species in this clade was solidly demonstrated in the broad study of [7], using other methods.Our finds with chemical characters thus add evidence to the conclusions of Ross et al. ( 2009) that different evolutionary Psyche 9 entities were grouped in the nominative species S. saevissima because of morphological similarity.The actual number of cryptic species and the range of their variation could only be established by investigations employing further different approaches (e.g., isoenzymes, larval characters) based on extensive sampling from other regions of South America.This is an issue of major interest as it pertains the biological boundaries of individual species, how many actually exist, and how they are set in nature and thus warrants immediate attention.
Moreover, the present report also imposes exceptions to the belief (e.g., [10,34]) that venom alkaloidal composition in fire ant venoms is species specific.Based solely on the pattern of venom alkaloids and following the results in these studies, S. saevissima variety B would have been assumed to be S. geminata or some close relative, like Solenopsis xyloni McCook; variety A would have been likewise mistaken for S. richteri.
It seems unlikely that venom alkaloid composition of the different species could be interpreted as remissive of the phylogenetic relationships among fire ants, as first proposed by [29] and discussed in [18].These authors suggested that the chemical structure of venom alkaloids from different species might reflect evolutionary relationships within fire ants; species with most diverse venom alkaloids would stand a step higher in the taxonomic history of the group.As remarked by [18], this assumption was based on studies of the few North American species.From regarding the herein described patterns of S. saevissima in face of preterit reports, it follows that there are at least three completely different alkaloidal patterns recorded within this nominal taxon and that such patterns are strikingly similar to the venom patterns of other species considered distant from each other as, for example, S. geminata versus S. invicta, which belong to different groups of fire ants species.

Caste Variations.
As mentioned, workers of fire ants range in size (polymorphism) over a broad continuum [35], and thus we decided to adopt an arbitrary division into minor, medium, and major workers.Moreover, worker size also depends on the physiological status and age of the nest [35,36].This implies that the arbitrary size class of media workers includes specimens of minor and major workers.Considering this aspect, intermediary workers were predominant in the collected samples.The obtained results confirm previous observations that the worker venom alkaloid trans/cis isomeric ratio is dependent on the ant size, with minor workers having higher trans/cis isomeric ratios (Tables 1 and 2), and are suggestive that the trans/cis ratio gradually changes at some point within the intermediary size range.A clear trend of ever-increasing relative amounts of venom cis-piperidinic alkaloids is perceived towards larger females, queens.Similar trend was observed with other species of fire ants, including S. invicta and S. geminata [4,22,29,37].Venom alkaloids were not detected in the male body nor head extracts.
The pattern of cuticular hydrocarbons of S. saevissima variety A was quite different from the one obtained for S. saevissima variety B.Moreover, intercaste differences in the patterns of cuticular hydrocarbons of S. saevissima were detected in both varieties, illustrated by small variations in the relative amounts of the main compounds.For instance, there was a clear tendency for reduction in the relative amounts of C 23 from minor workers towards major workers (Table 2).Gynes presented a wider range of different cuticular hydrocarbons, whist males presented marked proportion alterations on the same compounds (Table 2).
The present study generally depicts the knowledge gap about fire ant populations in their native South American range.Most of what is currently assumed about the group was extrapolated from laboratory experimentation with North American S. invicta and based on a few scattered observations with other species in South America.The validity of currently accepted fire ant species must be revisited, as should their diagnostic features.Chemical characters are indeed valuable parameters that aid species identification, but given the similarity between the obtained chromatograms and those of other distinct species (S. saevissima versus S. geminata or S. xyloni) and the considerable dissimilarity among nominal S. saevissima populations, at present chemical characters have to be employed with great caution and also in association with other characters and techniques (e.g., mtDNA).We also hope to have contributed with practical methods and guidance to expand the survey of these characters with other distant fire ant populations.

Figure 2 :
Figure 2: Total ion current chromatograms of (top) field worker sample of Solenopsis saevissima variety A from Brasília, Distrito Federal, Brazil; (below) field worker sample of S. saevissima variety B from Rio de Janeiro, Brazil.For further information on the chemical fingerprints, refer toTable 1 and narrative.

Notes:Figure 3 :
Figure 3: Total ion current chromatograms of hydrocarbons from head extracts of major workers of Solenopsis saevissima varieties A (top) and B (below) from Rio de Janeiro, Brazil.

Figure 4 :
Figure 4: Total ion current chromatograms of body washes of workers of Solenopsis saevissima variety A from Rio de Janeiro, Brazil: (top) Minor workers; (below) Major workers.

Table 1 :
Venom alkaloids identified from hexane extracts of Solenopsis saevissima from different regions within Brazil.

Table 2 :
Cuticular hydrocarbons from hexane extracts of fire ants Solenopsis saevissima obtained from different regions of Brazil.

Table 3 :
Relative abundance (%) of cuticle hydrocarbons from random worker samples of Solenopsis saevissima varieties A (n = 5) and B (n = 13) obtained from different nests from three different states in Brazil (see Figure1).Values given in bold were tested for each species variety using Students' t-test, and differing means were attributed to different letters.
Notes: (-): not found; (tr): trace amounts.For information on the compounds, collection sites, and species varieties, refer to narrative.

Table 4 :
Relative abundance (%) of piperidinic alkaloids obtained from hexane extracts of small workers (SWs), medium workers (MWs), and major workers (LWs) and gynes (Gs) from different nests of Solenopsis saevissima varieties A (n = 3) and B (n = 4) from Pedro do Rio, Rio de Janeiro state, Brazil.