Olfactory discrimination tasks can provide useful information about how olfaction may have evolved by demonstrating which types of compounds animals will detect and respond to. Ants discriminate between nestmates and non-nestmates by using olfaction to detect the cuticular hydrocarbons on other ants, and Camponotus floridanus have particularly clear and aggressive responses to non-nestmates. A new method of adding hydrocarbons to ants, the “Snow Globe” method was further optimized and tested on C. floridanus. It involves adding hydrocarbons and a solvent to a vial of water, vortexing it, suspending hydrocarbon droplets throughout the solution, and then dipping a narcotized ant in. It is hoped this method can evenly coat ants in hydrocarbon. Ants were treated with heptacosane (C27), nonacosane (C29), hentriacontane (C31), a mixture of C27/C29/C31, 2-methyltriacontane (2MeC30), S-3-methylhentriacontane (SMeC31), and R-3-methylhentriacontane (RMeC31). These were chosen to see how ants reacted in a nestmate recognition context to methyl-branched hydrocarbons, R and S enantiomers, and to multiple added alkanes. Behavior assays were performed on treated ants, as well as two untreated controls, a foreign ant and a nestmate ant. There were 15 replicates of each condition, using 15 different queenright colonies. The Snow Globe method successfully transfers hydrocarbons, as confirmed by solid phase microextraction (SPME) done on treated ants, and the behavior assay data shows the foreign control, SMeC31, and the mixture of C27/29/31 were all statistically significant in their differences from the native control. The multiple alkane mixture received a significant response while single alkanes did not, which supports the idea that larger variations in hydrocarbon profile are needed for an ant to be perceived as foreign. The response to SMeC31 shows C. floridanus can respond during nestmate recognition to hydrocarbons that are not naturally occurring, and it indicates the nestmate recognition process may simply be responding to any compounds not found in the colony profile and rather than detecting particular foreign compounds.

Oxymonas is a genus of Oxymonad protist found in the hindgut of drywood termites (family Kalotermitidae). Many genera of drywood termites are invasive pests globally. The hindgut microbiome of Cryptotermes brevis, the West Indian drywood termite, has not been described in detail, and only one published sequence exists of Oxymonas from C. brevis. This study aims to analyze Oxymonas sequences in C. brevis from whole gut genetic material, as well as to dissect its place in phylogenetic trees of Oxymonas and how it fits into specific and evolutionary patterns. To amplify the 18S rRNA gene Oxymonas from C. brevis, the MasterPure DNA extraction kit was used, followed by PCR amplification, followed by agarose gel electrophoresis, followed by purification of the resulting gel bands, followed by ligation/transformation on to an LB agar plate, followed by cloning the resulting bacterial colonies, and topped off by colony screening. The colony screening PCR products were then sequenced in the Genomics Core, assembled in Geneious, aligned and trimmed into a phylogenetic tree, along with several long-read amplicon sequences from Oxymonas in other drywood termites. All whole gut sequences and one amplicon from C. brevis formed a single clade, sharing an ancestor with a sister clade of Oxymonas sp. from C. cavifrons and Procryptotermes leewardensis, but the other long-read fell into its own clade in a different spot on the tree. It can be conjectured that the latter sequence was contaminated and that the C. brevis clones are a monophyletic group, a notion further corroborated by a distantly related clade featuring sequences from Cryptotermes dudleyi, which in turn has a sister taxon of Oxymonas clones from C. cavifrons and P. leewardensis, pointing toward a different kind of co-diversification of the hosts and symbionts rather than cospeciation.