Genomic dissection of behavioral maturation in the honey bee

Abstract

Honey bees undergo an age-related, socially regulated transition from working in the hive to foraging that has been previously associated with changes in the expression of thousands of genes in the brain. To understand the meaning of these changes, we conducted microarray analyses to examine the following: (i) the ontogeny of gene expression preceding the onset of foraging, (ii) the effects of physiological and genetic factors that influence this behavioral transition, and (iii) the effects of foraging experience. Although >85% of approximately 5,500 genes showed brain differences, principal component analysis revealed discrete influences of age, behavior, genotype, environment, and experience. Young bees not yet competent to forage showed extensive, age-related expression changes, essentially complete by 8 days of age, coinciding with previously described structural brain changes. Subsequent changes were not age-related but were largely related to effects of juvenile hormone (JH), suggesting that the increase in JH that influences the hive bee-forager transition may cause many of these changes. Other treatments that also influence the onset age of foraging induced many changes but with little overlap, suggesting that multiple pathways affect behavioral maturation. Subspecies differences in onset age of foraging were correlated with differences in JH and JH-target gene expression, suggesting that this endocrine system mediates the genetic differences. We also used this multifactorial approach to identify candidate genes for behavioral maturation. This successful dissection of gene expression indicates that, for social behavior, gene expression in the brain can provide a robust indicator of the interaction between hereditary and environmental information.

Fig. 1.

Division of labor in honey bee colonies and brain gene expression: age-related, behavior-related, and genetic differences. PCA using brain gene expression measurements for all 5,736 genes from all 72 bees in experiment 1 was performed. (A) Cumulative variance of PCs. For randomized data, gene expression levels were shuffled among genes (within sample). (B and C) Individual bees are plotted as a function of PC1 and PC2 (B) or PC1 and PC3 (C). Age/behavior group and subspecies are indicated in the key. See Table 4 for PCs 1–9.

Fig. 2.

Foraging experience and JH-target brain gene expression in honey bees. Gene expression ratios for forager/nurse bees from ref. are plotted on the x axes; ratios for forager/hive-restricted bees from experiment 3 are plotted on the y axes (log₂ values). (A) Behavior (nurse/forager) marker genes that were regulated by methoprene (P < 0.05; red triangle, up-regulated; blue square, down-regulated). (B) Behavior marker genes not regulated by methoprene (P > 0.2).

Fig. 3.

Physiological basis for subspecies differences in brain gene expression. (A) Correlation between PC3 (gene loadings from experiment 1) and physiological treatment effects (experiment 2) (rank-correlation analyses, Spearman's ρ). (B) Age-related differences in circulating titers of JH between ligustica and mellifera bees.

Fig. 4.

Candidate genes for honey bee behavioral maturation. Shown are a subset of the 100 behavioral marker genes for which we replicated the findings in ref. in the current study (hive-to-forager differences, P < 0.05) and which have functional (GO) annotation. Genes listed without parentheses are putative D. melanogaster orthologs based on reciprocal best BLAST match. Genes followed by parentheses are best Drosophila match (BLAST e value indicated). The gene listed in parentheses is a predicted gene with no Drosophila matches at BLAST e < 10⁻⁵. The color and letter indicate the direction of regulation: red, up-regulated (U); blue, down-regulated (D); red, higher in forager (F); blue, hive bee (HB); red, ligustica (L); blue, mellifera (M). ∗, P < 0.05; ∗∗, P < 0.001; ∗∗∗, P < 1 × 10⁻⁶; n.d., not determined; n.s., not significant. Marginally significant P values are indicated for experience-dependence. Statistical tests are from Table 1. A total of 15 of the 100 genes listed here met three of three predictions (see Results) for genes that could play causal roles in the hive-bee-to-forager transition.