Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee (Apis mellifera) and shed new light on the molecular evolution of the circadian clock

Abstract

The circadian clock of the honey bee is implicated in ecologically relevant complex behaviors. These include time sensing, time-compensated sun-compass navigation, and social behaviors such as coordination of activity, dance language communication, and division of labor. The molecular underpinnings of the bee circadian clock are largely unknown. We show that clock gene structure and expression pattern in the honey bee are more similar to the mouse than to Drosophila. The honey bee genome does not encode an ortholog of Drosophila Timeless (Tim1), has only the mammalian type Cryptochrome (Cry-m), and has a single ortholog for each of the other canonical "clock genes." In foragers that typically have strong circadian rhythms, brain mRNA levels of amCry, but not amTim as in Drosophila, consistently oscillate with strong amplitude and a phase similar to amPeriod (amPer) under both light-dark and constant darkness illumination regimes. In contrast to Drosophila, the honey bee amCYC protein contains a transactivation domain and its brain transcript levels oscillate at virtually an anti-phase to amPer, as it does in the mouse. Phylogenetic analyses indicate that the basal insect lineage had both the mammalian and Drosophila types of Cry and Tim. Our results suggest that during evolution, Drosophila diverged from the ancestral insect clock and specialized in using a set of clock gene orthologs that was lost by both mammals and bees, which in turn converged and specialized in the other set. These findings illustrate a previously unappreciated diversity of insect clockwork and raise critical questions concerning the evolution and functional significance of species-specific variation in molecular clockwork.

Figure 1.

Phylogenetic relationships of the Cycle/Bmal, Tango/Arnt, and Clock protein family. We used the Clock protein as an outgroup to root the tree based on its divergence from the Cycle/Bmal and Tango/Arnt sister proteins. Support levels are shown only for the main protein lineages, which are separated slightly vertically for visual clarity (percentage of trees showing a branch in distance and parsimony bootstrapping, followed by percentage of maximum likelihood quartet puzzling steps). Distinct font styles are used to highlight major taxonomic lineages. Bold for insects, italics for vertebrates, underline for the sea urchin, and plain for all the others. The honey bee Apis mellifera is highlighted with an asterisk.

Figure 2.

Phylogenetic relationships of the Timeless and Timeout proteins. We used the plant TIMELESS protein as an outgroup to root the tree. For additional details, see legend to Figure 1.

Figure 3.

Phylogenetic relationships of animal Cryptochrome and related Photolyase proteins. We used the CRY-DASH protein family as an outgroup to root the tree (based on its position in a larger tree that was rooted with another distantly related photolyase protein lineage that is present in insects and vertebrates) (data not shown). We termed the mammalian-like CRY proteins “CRY-m” and the Drosophila-like proteins “CRY-d.” For additional details, see legend to Figure 1.

Figure 4.

Schematic presentation of putative functional domains and motifs on Cycle/Bmal and Clock proteins from the honey bee Apis mellifera, the fruit fly Drosophila melanogaster, the giant silk moth Anthereae pernyi, and the mouse Mus musculus. See legend for domain and motif identity and text for additional details on each domain. Numbers below domains indicate identity/similarity with corresponding sequences on the honey bee ortholog. The numbers at the end of each diagram indicate protein size (number of amino acid residues). (A) Cycle/Bmal proteins. Inset shows a CLUSTALW multiple sequence alignment of a putative domain that is thought to be necessary for apBMAL and mBMAL transcriptional activity (see text for details). bmCYC is the CYC ortholog from the moth Bombyx mori. Alignments were generated with CLUSTALW and colored with JalView according to the default CLUSTALX convention. (B) Clock proteins.

Figure 5.

Schematic presentation of putative functional domains and motifs in Cryptochrome proteins from mouse (mCRY1 and mCRY2), honey bee (amCRY), and fruit fly (dCRY). See legend for domain/motif identity and text for additional details on each domain. Phosphorylation sites for MAPK (Sanada et al. 2004) are marked with asterisks. The blue “b” and “m” mark the location of cry^b (D401N mutation) and cry^m (truncation of the last 19 residues in Drosophila) mutations in dCRY. Low panels show multiple sequence alignments of a putative nuclear localization signal (NLS) in the RD2b domain (left) and the coiled-coil region (right). Aligned are mammalian-type (mouse, mCRY1 and mCRY2; Zebrafise, zCRY1–3; mosquito, agCRY-m; honey bee, amCRY) and Drosophila-type (Drosophila, dCRY; mosquito, agCRY-d) CRY proteins. zCRY3 is a mammalian-type CRY protein but has no transcription repressing function in vitro according to the method of Hirayama et al. (2003).

Figure 6.

Brain transcript abundance over time in foragers entrained and collected in LD illumination regime. The plots show the correlation between average (±SE) relative mRNA levels for each time point (filled circles and bars) and a cosinus model with a cycle of 23–25 h (continuous line) for bees from colony S1. (A) amPeriod; (B) amClock; (C) amCrypto-chrome; (D) AmCycle; (E) AmTimeout. In parentheses are the adjusted R² and period of the cosinus model for each gene. Time points with different letters are significantly different (ANOVA, P < 0.05; LSD post hoc test, P < 0.05). In two additional experiments, each with bees from a different, independent colony, we obtained similar results (see text for details). The bars at the bottom of plots indicate the illumination regime during sample collection. Black bar indicates dark; open bar, light. Sample size = 6.

Figure 7.

Brain transcript abundance over time in foragers entrained in LD and collected in DD illumination regime. Details of plots as in Figure 6, but show correlations with a cosinus model with a cycle of 22–26 h for bees from colony S11. (A) amPeriod; (B) amClock; (C) amCryptochrome; (D) AmCycle; (E) AmTimeout. Dashed regression line indicates that a co-sinus model accounts for <1% of the variation over time in amClk mRNA levels. We obtained similar results in two additional experiments, each with bees from a different, independent colony. The bars at the bottom of plots indicate illumination regime during sample collection. Black bar indicates dark; striped bar, subjective day. Sample size = 6.

Figure 8.

The relationships between brain transcript abundance over time for five putative honey bee clock genes in foragers from free-flying colonies. Brain mRNA levels for all genes were measured from the same RNA sample. (A) Foragers entrained and collected in light: dark illumination regime (LD). Colony S1, n = 6 bees/time point (same data as in Fig. 6). (B) Foragers entrained in LD and collected in constant darkness. Colony S11, n = 6 bees/time point (same data as in Fig. 7). (C) Schematic representation of the oscillations of clock genes in the honey bee brain in LD and DD illumination regimes. The phase of mRNA cycling is shown for amPer,amCry, and amCyc for which there is a strong correlation with a cosinus model with about a 24-h cycle. The phase of amCyc transcript is almost in antiphase to that of amPer and amCry. For clarity, the model does not include amClk that does not oscillate and amTim for which the pattern of mRNA variation over time was not consistent across experiments. Amplitudes for the various genes are not to scale. For additional details, see Figures 6 and 7.