An Engineered orco Mutation Produces Aberrant Social Behavior and Defective Neural Development in Ants

Abstract

Ants exhibit cooperative behaviors and advanced forms of sociality that depend on pheromone-mediated communication. Odorant receptor neurons (ORNs) express specific odorant receptors (ORs) encoded by a dramatically expanded gene family in ants. In most eusocial insects, only the queen can transmit genetic information, restricting genetic studies. In contrast, workers in Harpegnathos saltator ants can be converted into gamergates (pseudoqueens) that can found entire colonies. This feature facilitated CRISPR-Cas9 generation of germline mutations in orco, the gene that encodes the obligate co-receptor of all ORs. orco mutations should significantly impact olfaction. We demonstrate striking functions of Orco in odorant perception, reproductive physiology, and social behavior plasticity. Surprisingly, unlike in other insects, loss of OR functionality also dramatically impairs development of the antennal lobe to which ORNs project. Therefore, the development of genetics in Harpegnathos establishes this ant species as a model organism to study the complexity of eusociality.

Keywords: CRISPR; Harpegnathos saltator; Orco; ant; eusocial insect; glomerulus; neural development; odorant receptor; odorant receptor neurons; pheromone; reproduction; social behavior.

Figure 1. Harpegnathos life cycle, early embryo development, and propagation of the mutant allele

(A) The female embryos develop into larvae. At the 4^th instar (Penick et al., 2012), the larvae differentiate into queen vs. worker larvae. The former develop into callow virgin queens with wings that can found a new colony, while the latter become workers in the nest and, when isolated in group or individually, can become gamergates and lay eggs. Certain cuticular hydrocarbons (CHCs) of a queen or a gamergate act as queen pheromones that inhibit worker reproduction. (B) Cryosections and DAPI staining of young embryos harvested at different time points after egg deposition (AED, hours below each image). (C) The steps to propagate mutant ants to develop hemizygous males (F3/F5) and homozygous females (F4) for phenotypic analyses. Of note, the F3 mutant males mated with F2 heterozygotes to generate F4 ants, which were half homozygotes and half heterozygotes (Table S1). The F4 heterozygotes, like F2s, also produced male mutants (F5). See also Figure S1.

Figure 2. Development of somatic and germline mutations

(A) The structure of Orco protein with 7 transmembrane domains, labeled 1 to 7. The mutation is at the N-terminus of the protein, indicated by the red mark. Extra and Intra: extra- and intra-cellular domains. (B) Microinjections of orco CRISPR into embryos generated multiple types of short deletions. The target site in the first exon of the orco gene, indicated by an arrow, is located 3 nucleotides upstream of the PAM (TGG) sequence. The small guide RNA (sgRNA1) target sequence is underlined. (C) The germline mutation used for this study was a 2-nucleotide AT deletion, as indicated in the sequencing chromatogram (+: WT; −: mutation). The mutation caused a frame shift at codon 17 and a premature stop codon, giving rise to the truncated protein of 31 amino acids. (D) Mass spectrometry indicates the abundance (peak area in inset) of detected peptides from Orco protein in the WT female ant (+/+) compared to the F4 orco homozygous mutant ant (−/−). Each row represents a fragment of the WT Orco protein sequence with its start and end amino acid positions. See also Figure S2.

Figure 3. Reduced antennal responses to odorants in orco homozygous mutant ants

(A) The differential score (Y-axes) of each heterozygous (+/−, n=8) or homozygous ant (−/−, n=6) relative to its paired WT ant was plotted and the p value indicated. Mann-Whitney test was used for statistical analyses. Bars and error bars represent mean ± SEM (standard error of the mean). (B) The cumulative differential scores in response to 7 odorants were separated into heterozygous and homozygous groups. Each ant was named by its painted color combination (See Methods). Of note, YGPi was paired with an outlier, a WT ant with low responses to odorants. See also Figure S3 and Movie 1.

Figure 4. Behavioral phenotypes in orco homozygous mutant ants

(A) The time for homozygous young workers (−/−, n=6) vs. heterozygous (+/−, n=6) and WT (+/+, n=16) young workers to wander outside of the nest (Movie 3): p<0.001 between homozygotes and heterozygotes; p<0.0001 between homozygotes and WTs; p=0.9335 between heterozygotes and WTs (Two-way ANOVA with Tukey’s multiple comparisons test). (B) The wandering homozygous ants did not forage. The foraging scores are indicated in the heat map for old WT, young WT, heterozygous, and homozygous workers (Day: test day, also age of young workers). p<0.001 between young homozygous and old WT workers (Friedman test with Dunn’s multiple comparisons test). (C) The average dueling times in 11 heterozygous duelers (left) vs. 3 homozygous duelers (right) in the first 9 dueling days. The 6 constant duelers and 4 gamergates are indicated under the individual ants. See also Tables S2 and S3. (D) Abnormal antennal twitching behavior (Movie 4) was found in 6 out of 12 individually isolated homozygotes, but not in any of the 12 heterozygotes, under the same conditions: p<0.05 (Mann-Whitney test). Data are represented as mean ± SEM.

Figure 5. Defective reproduction and nursing in orco homozygous mutant females, but no change in lifespan of orco mutant males

(A) When individually isolated, the heterozygous ants (+/−, n=10) laid the first egg at day 29.4 ± 1.1, while the homozygous mutant ants (−/−, n=9) did so at day 45.6 ± 7.1: p<0.05 (unpaired t test). (B) The heterozygous ants (n=10) laid 8.0 ± 1.2 eggs before being 50 day old, while the homozygous ants (n=10) laid 1.2 ± 0.4 eggs before 50 days old: p<0.0001 (unpaired t test). (C) While all heterozygous ants (n=10) produced larvae, only 1 homozygous ants (n=10) did so: p=0.0001 (Fisher’s exact test). See also Table S4. (D) The WT (+, n=32) and hemizygous mutant F3 male ants (−, n=31), which lived with individually isolated female ants, had average lifespans of 13.19 ± 1.76 days and 12.26 ± 1.58 days, respectively: p>0.05 (unpaired t test). Data are represented as mean ± SEM.

Figure 6. Neuroanatomical phenotypes in female and male mutant ants

(A,E) The ant brain samples were stained with Phalloidin (green) and counter-stained with DAPI (blue). Confocal images of two WT (+/+) vs. two homozygous (−/−) old female ants (~3 months old) are shown in (A): 10× objective (top panel) and 20× objective (bottom panel). Confocal images of WT (+) vs. mutant (−) male ants using 20× objective are shown in (E). The glomerular areas of antennal lobes are indicated by arrows in (A) and (E). (B) The average glomerular volume of left and right antennal lobes in WT (n=8): 2.13 ± 0.12 × 10⁶ µm³, heterozygous (+/−, n=3): 2.15 ± 0.08 × 10⁶ µm³, and homozygous female ants (n=5): 1.30 ± 0.09 × 10⁶ µm³. (C) The number of glomeruli in WT: 275.2 ± 4.0, heterozygous: 270.2 ± 11.2, and homozygous female ants: 61.8 ± 1.9. (D) The diameter of glomeruli in WT: 20.8 ± 0.4 µm, heterozygous: 24.4 ± 0.6 µm, and homozygous female ants: 38.0 ± 1.4 µm. (F) The glomerular volume of each antennal lobe in WT (n=8): 1.41 ± 0.06 × 10⁶ µm³, and mutant male ants (n=8): 0.93 ± 0.06 × 10⁶ µm³. (G) The number of glomeruli in WT: 78.8 ± 1.0, and mutant male ants: 31.3 ± 1.0. Female: one-way ANOVA with Tukey’s multiple comparisons test; male: unpaired t test. Data are represented as mean ± SEM. See also Figures S4 and S5, Movies 6 and 7.

Figure 7. Loss of olfactory receptor neurons in female mutant ants

(A) Single focal plane images of antennal flagellum (F10, the most distal flagellomere (Ghaninia et al., 2017)) in the young female ants (WT vs. homozygous, <1 day old): DAPI (blue), Phalloidin (green). (B) Images of F7 in old ants (WT vs. homozygous, >3 months old). The red circles in WT panels indicate clustered nuclei. (C) A summary of Orco function in ants: In orco mutant female ants, reduced number of ORNs causes a series of morphological phenotypes in the antennal lobe (AL). However, it is not clear how these changes affect the projection neurons (PNs) that innervate to the mushroom body (MB) and the lateral horn (LH). Eventually, defective olfactory sensing results in a range of abnormal behaviors and reduced capability of egg-laying and brood care. OL: optic lobe.