Transcriptomics and neuroanatomy of the clonal raider ant implicate an expanded clade of odorant receptors in chemical communication

Antennal Morphology.

Silver staining and light microscopy of antennae was performed largely following Navasero and Elzen (47). In brief, isolated ant antennae were washed with ethanol and then washed 3 × 10 s in acetone. Samples were then incubated in 0.1 M silver nitrate solution for 5–10 min, washed 3 × 10 min in deionized water, incubated in Kodak D-76 photographic developing solution for 5 min, washed in 3% (vol/vol) acetic acid for 1 min, dehydrated serially in ethanol, and finally air dried and imaged in air on glass slides. Imaging was performed using an Olympus BX53 compound microscope with backlight plus epifluorescent illumination through a FITC filter at 100× and 200× magnification. As described by Renthal et al. (16), the green autofluorescence of the cuticle formed a bright background against which dark silver precipitate that forms within porous sensilla could be seen. Serial focus stacks were taken manually and then combined using Helicon Focus (Helicon Soft).

For scanning electron microscopy of antennae, whole heads were fixed in 4% (wt/vol) formalin in sodium cacodylate buffer, washed 3 × 5 min in deionized water, serially dehydrated in ethanol, and then mounted on carbon tape at various angles and sputter coated with 1–2 nm gold-palladium. Imaging was performed on a ZEISS LEO 1550 scanning electron microscope.

Gene Expression.

Tissue- and sex-specific gene expression data were obtained from McKenzie et al. (25). This study included one male antennal library and one female antennal library for the sex-specific analysis, as well as three libraries for each female tissue type examined [antennae, heads (minus antennae), bodies (thoraces and abdomens), and legs]. For gene expression of ventral vs. dorsal halves of the female antennal club, antennae were dissected from cold-anesthetized ants and positioned for cutting inside a droplet of OCT (optimal cutting temperature) medium (TissueTek) on an inverted Petri dish. A plaster insert that had been chilled on dry ice was then placed under the sample, freezing the OCT. The embedded antennal club was then split and separated from the antenna by hand using a cryostat razor blade under a dissecting microscope. To test the accuracy of this procedure, 10 antennae were silver stained and split following this protocol, and both halves imaged as described above. For sequencing, each antennal club half was immediately transferred to cold TRIzol reagent (Life Technologies) following dissection. Samples were then homogenized using a TissueLyser II (Qiagen), and the aqueous phase was recovered using Phase Lock columns (5Prime) and purified using the RNeasy kit (Qiagen) following the manufacturers’ protocol. Libraries were then constructed using the Clontech SMART kit and sequenced on an Illumina HiSeq2500. We pooled 20 antenna halves per library, and sequenced two libraries (one dorsal half and one ventral half) for each of three biological replicates (one per clonal lineage; MLL1, MLL4, and MLL6 in ref. 50). All libraries were run on a single HiSeq2500 lane, generating 38–43 million single-end 100-bp reads per library. Reads were aligned to the O. biroi reference genome (assembly V3.0) using Tophat (48) with prealignment to OGS 1.8.6 (42). Cufflinks (49) was then used to quantify gene expression, and CuffDiff (50) was used to test for significant differences in expression. RNAseq reads are deposited in the National Center for Biotechnology Information sequence read archive (Bioproject accession no. PRJNA345039).

Neuroanatomy.

For visualization and 3D reconstruction of the female antennal lobe, brains were dissected in PBS, pH 7.4, fixed in 1% glutaraldehyde in PBS solution for 7 d at room temperature, washed in PBS (three times for 10 min), and dehydrated in an ascending series of ethanol [30%, 50%, 70%, 90%, 95%, and 2 × 100% (vol/vol); 10 min each step]. Finally, brains were cleared in methyl salicylate (M-2047; Sigma Aldrich). The antennal lobe was imaged by using a Zeiss LSM 710 confocal microscope, with individual glomeruli visualized by autofluorescence. Males are produced exceedingly rarely in O. biroi (54), and we were unable to obtain a male brain for preparation in the same manner as the female brains. We thus used a phalloidin-stained male brain previously prepared for a separate experiment. The male brain was dissected in cold PBS, pH 7.4, fixed by incubation in 4% (wt/vol) paraformaldehyde solution in PBS overnight, washed in PBS and 0.5% Triton-X at room temperature (3 × 20 min), incubated in 1% BSA in PBS for 30 min, washed in PBS 0.01% Tween for 5 min, incubated overnight with a primary antibody containing solution in 1% BSA and 0.5% Triton-X in PBS solution, washed with PBS Tween (3 × 10 min), incubated with a secondary antibody solution containing Alexa Fluor 555 phalloidin (Thermo Fisher Scientific) 1 μL (of stock solution 6.6 µM) in 100 mL of 1% BSA and 0.5% Triton-X in PBS solution for 2 h, washed five times in PBS, and mounted with Dako mounting medium. The antennal lobe was imaged using a Zeiss LSM 710 confocal microscope. Only the phalloidin staining was analyzed in this paper.

Glomeruli were reconstructed from image stacks of a representative female and male brain by manually outlining each glomerulus in the segmentation editor of the Fiji distribution of ImageJ (NIH) (51). Glomeruli were sorted into input tracts by following input fibers through focus stacks. In some cases innervating input tracts were unclear and we then relied on visible segregation and clustering of glomeruli. Assignment of glomeruli on the borders of the clusters is thus likely imprecise, especially for input tracks T1, T3, and T4. In the female, a visible sulcus separated the T5 and T6 clusters from the remaining clusters; thus, the separation of T6 glomeruli was clear except for a few glomeruli bordering the T5 cluster. These glomeruli were assigned to the T5 cluster to provide a conservative estimate of T6 glomeruli numbers. In the male, T6 glomeruli were visibly separated from the remaining glomeruli in 3D renderings.

Tracing was performed by immobilizing ants at 4 °C in viscoelastic liquid silicone (Silly Putty) with one antenna stretched out against double-sided tape. Either the dorsal or the ventral surface of the antennal club was then scraped with a razor to break sensilla, and then a drop of microruby (rhodamine dextran with biotin, 3,000 MW, D7162; Thermo Fisher Scientific) in distilled water was applied over the scraped area. Ants were incubated like this at 4 °C in a humid box for 15 min and then allowed to move freely in a humid dish for 8–12 h at room temperature. Brains and antennae were then dissected, and the antennae were visually inspected to confirm sensilla breakage. Brains were then fixed in 4% (wt/vol) paraformaldehyde for 4 h, dehydrated in an ascending ethanol series (50%, 70%, 90%, 95%, and 3 × 100% (vol/vol)], cleared and mounted in methyl salicylate, and imaged as described above. Innervated glomeruli were counted manually from the confocal image stacks. Fluorescent quantification was performed using the MeasureStack pluggin in ImageJ. Fluorescence of each ipsilateral antennal lobe compartment was normalized to the entire contralateral antennal lobe to account for baseline fluorescence.

Evolutionary analyses.

Odorant receptor gene sequences from five ant species (Harpegnathos saltator, Ooceraea biroi, Linepithema humile, Camponotus floridanus, and Pogonomyrmex barbatus) and two hymenopteran outgroups (Nasonia vitripennis and Apis mellifera) were obtained from Robertson et al. (55), Zhou et al. (27), Oxley et al. (42), Smith et al. (28), and Smith et al. (29). Odorant receptors from Solenopsis invicta, Atta cephalotes, and Acromyrmex echinatior were manually annotated as part of the current study following Oxley et al. (42). Briefly, genomes were downloaded from hymenopteragenome.org, and existing OR protein sequences from other ant species aligned to the genomes using Exonerate (56) and tblastn (57). Models were then constructed manually in the Apollo genome editor (58), guided by alignment evidence, and visually compared with other models using Muscle alignment (59) when necessary. Protein sequences for all newly annotated genes can be found in Dataset S3, and gff3 lines for the annotations are in Dataset S4. For all ant species, nonpseudogenized sequences were aligned by OR subfamily (sensu 27) using the Mafft algorithm (version 7) with linsi parameter settings (local pairwise alignment, maximum of 1,000 iterations, all other parameters default) (60). Then all subfamily alignments were profile aligned by using the Mafft seed alignment algorithm, again using the linsi parameters.

For copy number analyses, pseudogenes were added to the previously described alignment with the Mafft add algorithm with linsi parameters. A maximum likelihood phylogenetic hypothesis was then constructed using RAxML default tree search algorithm with the LG model of protein evolution (61) and gamma-distributed substitution rates. Node support was tested using the RAxML rapid bootstrap algorithm (62) with 100 rapid bootstrap replicates.

Existing model-based tools for statistical estimation of gene birth and death rates either fail to take gene trees into account [e.g., computational analysis of gene family evolution (CAFE)] (63) or are computationally infeasible for such large gene expansions (e.g., jprime delirious) (64). We thus reconstructed birth and death events by calculating the most parsimonious reconciliation of the gene tree with the species tree using Notung (v2.8.1.7) (40). Because many nodes of the gene tree are poorly resolved, we used a 70% bootstrap consensus gene tree resolved using Notung’s “resolve” algorithm, with duplication and loss costs set to defaults. Birth and death event counts are therefore conservative estimations. Ant species relationships in the species tree followed Brady et al. (65),and the BEAST-estimated topology of Moreau and Bell (66). Birth, death, and net expansion rates were calculated as the number of times the repertoire has doubled (birth and net expansion rates, calculated including or excluding lineages that subsequently went extinct, respectively) or halved (death rate) along a branch or set of branches divided by branch length. Thus, the birth rate along a specific branch is calculated aslog2((parent node repertoire+duplications)/parent node repertoire)/branch length.Aculeate divergence times were taken from Moreau and Bell’s divergence dates using their BEAST estimated tree topology (66), whereas the Nasonia-Aculeata divergence time followed Ronquist et al. (67).