Fig 1.
Phylogenetic analysis of S. invicta and other hymenopteran hexamerin candidate proteins under Bayesian inference.
Node value posterior probability was rounded to two significant figures. Scale bar indicates branch length. The two hexamerins of the termite R. flavipes were used as the outgroup. The four clade names represent the four A. mellifera hexamerin proteins. The hexamerins of S. invicta (boxed in red) clustered as follows: Hexamerin 1 (XP_025995150.1) with Hex 70b, Hexamerin 2 (XP_011171862.1) with Hex 110, Arylphorin subunit alpha-like (XP_011155485.1) with Hex 70c, and Arylphorin subunit beta (XP_011155484.1) with Hex 70a. Alignment of the S. invicta hexamerin proteins can be found in supplementary information (S2 Fig).
Table 1.
Composition of the predicted S. invicta hexamerin proteins: number of residues typically enriched in insect hexamerins [38] and their corresponding percentage (in parenthesis).
Table 2.
Percentage sequence identities and similarities among the S. invicta hexamerins determined by BlastP analyses.
Identities are shown above, similarities below the diagonal line represented by shaded cells.
Fig 2.
Expression analysis of hexamerin transcripts among medium-sized foragers, medium-sized nurses, alate virgin queens, and dealate mated queens.
Relative expression levels of (A) hexamerin 1, (B) hexamerin 2, (C) arylphorin subunit alpha-like, and (D) arylphorin subunit beta transcripts. Gene expression was quantified using RT-qPCR and analyzed using the ΔΔCt method. For each gene, mRNA expression level was normalized relative to rp18. Bars represents mean ± SEM fold change relative to foragers (n = 6). Different letters indicate significant differences in gene expression as determined by one-way ANOVA with Tukey’s post hoc analyses (p < 0.05). The fold change in the Y-axis is in log-scale.
Fig 3.
Expression analysis of hexamerin transcripts in medium-sized foragers 12 h following topical application of S-hydroprene.
Relative expression of (A) hexamerin 1, (B) hexamerin 2, (C) arylphorin subunit alpha-like, and (D) arylphorin subunit beta transcripts. Gene expression was quantified using RT-qPCR and analyzed using the ΔΔCt method. For each gene, mRNA expression level was normalized relative to rp18. Bars represents mean ± SEM fold change relative to the untreated control (n = 6). Statistical relationships between groups were assessed using one-way ANOVA. Significant differences in gene expression (p < 0.05) were determined for hexamerin 2 and arylphorin subunit beta. For those genes, different letters indicate significant differences in gene expression as determined with Tukey post hoc analyses.
Fig 4.
Expression analysis of hexamerin transcripts in medium-sized nurses 12 h after topical application of S-hydroprene.
Relative expression of (A) hexamerin 1, (B) hexamerin 2, (C) arylphorin subunit alpha-like, and (D) arylphorin subunit beta transcripts. Gene expression was quantified using RT-qPCR and analyzed using the ΔΔCt method. For each gene, mRNA expression level was normalized relative to rp18. Bars represents mean ± SEM fold change relative to untreated control (n = 6). Different letters indicate significant differences in gene expression as determined by one-way ANOVA with Tukey’s post hoc analyses (p < 0.05).
Fig 5.
Expression analysis of hexamerin transcripts in alate virgin queens 12 h following topical application of S-hydroprene.
Relative expression of (A) hexamerin 1, (B) hexamerin 2, (C) arylphorin subunit alpha-like, and (D) arylphorin subunit beta transcripts. Gene expression was quantified using RT-qPCR and analyzed using the ΔΔCt method. For each gene, mRNA expression level was normalized relative to rp18. Bars represents mean ± SEM fold change relative to untreated control (n = 6). Significant differences in gene expression (p < 0.05) were determined for hexamerin 1 and arylphorin subunit alpha-like. For those genes, different letters indicate significant differences in gene expression as determined with Tukey post hoc analyses.
Fig 6.
Proposed two modules regulating hexamerin expression in S. invicta: queens and workers.
In workers, hexamerin expression is regulated by JH. The expression of the four hexamerins decreases as workers transition from nursing to foraging tasks, which supports the existence of changes in JH as the workers age (represented in dashed). In queens, only hexamerin 1 and arylphorin subunit alpha-like are regulated by JH. Both genes are down-regulated in mated queens compared to virgin queens.