Novel alkaloids from the fire ant, Solenopsis geminata

South American fire ants, Solenopsis richteri and Solenopsis invicta, were accidently introduced into the southern USA in the 1900s and 1930s, respectively. The rapid spread and high population densities of S. invicta, and its potent sting, resulted in broad economic impacts and a variety of research efforts. In the 1970s, their venom alkaloids were identified as a complex blend of trans-2-methyl-6-alkyl- and alkenyl-piperidines. Solenopsis geminata is a worldwide tramp species but a native of the southern coastal regions of the USA. It was found to only produce cis- and trans-2-methyl-6-undecyl-piperidines. These alkaloids were considered the Solenopsis ancestral alkaloid profile since they were identified from female sexuals (potential queens) of all Solenopsis species in South and North America. The dramatic modification of alkaloids in Solenopsis invicta was attributed to their response to evolutionary pressure and the lack of change in S. geminata alkaloids due to no response to evolutionary pressure. Here we report the unexpected discovery of 6-undecyl-pyridine, 2-methyl-6-undecyl-pyridine and 2-methyl-6-(1)-undecenyl-pyridine as components of S. geminata worker venom, suggesting that S. geminata like its South American relatives have responded to evolutionary pressures. Our results will stimulate future research on S. geminata populations throughout the tropical/subtropical world.


Introduction
The fire ants, Solenopsis geminata and Solenopsis xyloni (members of the S. geminata species complex, Pitts et al. 2018), are native to the southern USA and have a long history of human interactions. While they have a significant sting, it was the accidental introduction of two South American fire ants into the southern USA, Solenopsis richteri (1920s) and especially Solenopsis invicta (1930s) (Solenopsis saevissima species complex, Pitts et al. 2018), which generated widespread attention. High population densities of Solenopsis invicta and a propensity for disturbed habitats led to frequent human interactions and broad economic impact. Five trans-2-methyl-6-alkyl-(C 11:0 , C 13:0 , C 15:0 ) and alkenyl-(C 13:1, C 15:1 )-piperidines were identified from S. invicta worker venom (MacConnell et al. 1971). Venom alkaloid research was extended to workers of the native fire ants, S. geminata and S. xyloni. They produce a mixture of cis-and trans-2-methyl-6-undecyl-piperidines (Brand et al. 1972, Fig. 1, compounds 2 and 4), in contrast to the variety of piperidine alkaloids that dominate the two invasive species. Since all female sexual forms (future queens) associated with the S. geminata and S. saevissima species complexes produce cis-and trans-2-methyl-6-undecyl-piperidines, these two alkaloids were considered the ancestral type. It was thought S. invicta and S. richteri workers evolved beyond the undecyl-piperidines, while S. geminata species complex workers maintained the ancestral alkaloid blend (Brand, 1978).
Alkaloid use in chemotaxonomy was illustrated by Brand et al. (1973), in that S. xyloni and S. geminata workers could be identified by their cis/trans 2-methyl-6-undecyl-piperidine ratio. A broad survey of Solenopsis venom alkaloids in North and South America also supported alkaloid use in chemotaxonomy (MacConnell et al.  Brand et al. (1972) found detectable amounts of 2-methyl-6-undecyl-Δ 1,2 -piperideine (see Fig. 1, compound (5), in S. xyloni venom) but suggested it was a precursor to the major piperidine venom components, as later verified by Leclercq et al. (1996). A reproductively viable and morphologically cryptic hybrid between S. invicta and S. richteri was initially detected based on their venom alkaloid profiles (Vander Meer and Lofgren, 1985).
Piperideine alkaloids from S. invicta were reported as minor components by two research groups Chen and Fadamiro, 2009). Both groups suggested that the piperideines likely functioned as piperidine precursors (Leclercq et al. 1996). A focused study of S. geminata venom alkaloids (Shi et al. 2015) isolated the expected cis-and trans-2-methyl-6-undecyl-piperidines as the major components. Pyridine alkaloids were not reported from Solenopsis venom until Chen et al. (2019) described trace amounts of ten pyridines from workers of S. invicta, S. richteri, and their hybrid. The identified pyridines were each < 1 ng/ant in both species and their hybrid. For perspective, each S. invicta worker produces about 18 μg of venom alkaloids (Haight and Tschinkel 2003).
Solenopsis geminata alkaloids were well established as cis-and trans-2-methyl-6-undecyl-piperidines when in 2019 three worker ants intercepted at a port in Hawaii, identified as Solenopsis, were sent to our facility to determine if they were S. invicta. Molecular analyses (mitochondrial COX1 gene) classified the ants as S. geminata. Chemical analysis unambiguously identified two major components as 2-methyl-6-undecyl-pyridine and trans-2-methyl-6-undecyl-piperidine. The normally dominant cis-2-methyl-6-undecyl-piperidine was a minor component (Ascunce et. al. 2021). This surprising result was the driving force for reinvestigation of S. geminata venom alkaloids.

Sampling
The tropical fire ant, S. geminata, inhabits the southern tier of the USA and can be found in the Gainesville, FL, area, normally in dry, well-drained areas that are less likely to be occupied by the invasive fire ant, S. invicta. Solenopsis geminata is unique in that it can be readily differentiated from other Solenopsis species by the presence of a worker caste that has a disproportionately large head. All samples of S. geminata workers (20-50) were collected from individual colonies (replicates) and placed in a vial containing enough hexane (Fisher Optiva, Cole Palmer, Vernon Hills, IL) to cover the ants. After 24 h, the hexane was pipetted into a clean vial in preparation for GC-MS analysis to determine tentative component identification and relative component percentages. Collection site A was sampled in 2015. At that time, cuticular hydrocarbon patterns were of prime interest since the alkaloids were assumed to be cisand trans-2-methyl-6-undecylpiperidines. The GC-MS temperature program targeted separation of cuticular hydrocarbons. The two major alkaloid peaks (2) and (4)  The total ion chromatogram is shown for an extract of S. geminata workers (Gainesville, FL) immersed in hexane for 24 h. The seven peaks were demonstrated to be composed of single components rather than mixtures of co-eluting compounds (MassWorks, Cerno Bioscience, https:// cerno biosc ience. com). The structures for six of the seven alkaloids were determined by mass spectral fragmentation patterns, synthesis, and co-injection and are inserted into the chromatogram The alkaloids are antibiotic and very stable and have been recovered after decades of room temperature storage (see method for synthetic component 3). In view of the Hawaii results (Ascunce et al., 2021), the 2015 samples (site A, n = 9) were reconstituted in hexane, vortexed, and analyzed using GC-MS program b. Fragmentation patterns (Fig. 2) were compared to synthetic standards. Components 1 to 5 and 7 (Fig. 1) were identified. The TIC (total ion chromatogram) was used to integrate the areas under each peak, and the peak percent composition was calculated. Twelve additional samples were collected and analyzed in 2019 from Gainesville, FL, site B, which was approximately 25 km from site A. Graphical representations of alkaloid peak proportions were prepared (Fig. 3, site A and site B) using GraphPad Prism, version 9.01 (GraphPad Software Inc., San Diego, CA).

Gas chromatograph-mass spectroscopy (GC-MS) analysis
Mass spectral data were obtained from (a) Shimadzu QP-2020 GC-MS (Palo Alto, CA) equipped with an RTX-5 ms, 30 m × 0.25 mm i.d. column. The instrument was temperature programmed from 60 to 250 °C at 10°/min and Fig. 2 The mass spectral fragmentation patterns and associated structures are shown for each of the 6 identified S. geminata alkaloid components from an extract of S. geminata workers (Gainesville, FL) immersed in hexane for 24 h. The structures for six of the seven alkaloids were determined by mass spectral fragmentation patterns, synthesis, and co-injection   Fig. 3 The mean + SE of the percent composition for the 7 alkaloid components based on the integration values for each sample (total ion chromatogram, TIC: (1) collection site A, 2015, n = 9, and (2) collection site B, 2019. Locations A and B are approximately 25 km apart. Each replicate represents a unique S. geminata colony held there for 20 min (Virginia Military Institute, all synthetic samples) or (b) Agilent Intuvo 9000 GC system (Santa Clara, CA) equipped with an HP-5 MS ultra-inert nonpolar column, 30 m × 0.25 m i.d. column, coupled to a 5977 B mass spectral detector and a MassHunter Data Acquisition Workstation version 10.0.368 (Santa Clara, CA). Injector temperature was set at 250 °C. The oven temperature was programmed at 40 °C for 2 min and then to 285 °C at 5 °C/ min, followed by a 10 min hold at 285 °C (USDA, Gainesville, Florida). Agilent's MassHunter and NIST MS Library Database (2020) was used to compare venom component mass spectral fragmentation signatures (see Fig. 2). Mass-Works software (Cerno Bioscience, https:// cerno biosc ience. com/) determined the purity of each peak and their retention index (RI) relative to a series of normal hydrocarbons.

Identification of compounds 1-5 and 7
See Fig. 2 for the mass spectral fragmentation pattern of each compound.

2-undecyl-pyridine (1)
Venom component 1 (Fig. 1) was tentatively identified as 2-undecyl-pyridine through a search and match of the NIST (2020) mass spectrum library. To verify the structure assignment, the known compound was synthesized by alkylation of 2-picolyl magnesium bromide with 1-bromodecane (Proffe and Linke, 1960). The synthetic product was distilled by Kugelrohr distillation and had a mass spectral fragmentation pattern that matched the reported fragmentation pattern (Heller et al. 2002) for 2-undecyl-pyridine (1). This pattern was identical to that of natural S. geminata component (1). In addition, synthetic 2-undecyl-pyridine co-eluted with natural component (1) when co-injected on the GC-MS, verifying the structural assignment.